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Optimization of Medication Delivery Drone with IoT-Guidance Landing System Based on Direction and Intensity of Light
# Introduction
Unmanned air vehicles (UAV), which are commonly known as drones, are classified globally based on their weight, flight range, flight speed, and payload. Drone applications are becoming an increasingly important part of many services. Drones are playing a significant role with the advances in technologies in the Fourth Industrial Revolution (4IR). The investment in the drone industry reached 127 billion USD and is expected to offer a hundred thousand job opportunities in 2025 [bib_ref] Unmanned Aerial Vehicles (UAVs): A Survey on Civil Applications and Key Research..., Shakhatreh [/bib_ref].
Drone applications in the medical field to enhance healthcare services to patients have grown rapidly. Recently, the spread of COVID-19 has resulted in the application of drones in the medical field, such as street disinfection to stop the spread of the virus. In addition, a thermal camera has also been used to detect the abnormal temperature of an infected person in a crowded area and lead to action to isolate him [bib_ref] Do drones have a realistic place in a pandemic fight for delivering..., Euchi [/bib_ref].
Homecare, telemedicine, and lockdowns have encouraged medicine delivery to patients using drones. Social distancing is one of the most important factors in stopping the spread of the virus, and drones are playing a significant role because their cameras can monitor any violations; loudspeakers attached to a drone can also be used to encourage people to keep a safe distance [bib_ref] Do drones have a realistic place in a pandemic fight for delivering..., Euchi [/bib_ref].
Research from Beck has also focused on drones delivering anti-allergy injections that help in saving lives. The study focuses on the effect of the injection ingredients in different scenarios, such as frequencies of vibration, environmental temperatures, and flight time.
(EBGM) is the best algorithm for the posing challenge, but at the same time, it is sensitive to light. Finally, the Neural Networks is the most accurate algorithm for facial recognition, but it requires more data compared to most algorithms. This paper states that three out of five algorithms are sensitive to light. The process of the facial recognition system is divided into three parts. The first step is to start with face detection by using many techniques such as the Viola-Jones detector and Principal Component Analysis (PCA) [bib_ref] Algorithms for face recognition drones. Mater, Shanthi [/bib_ref]. Second is the extraction of the face's features such as face's shape, mouth, nose, and eyes by extraction tools such as Eigenface and Local Binary Pattern (LBP). Third, the extracted face's features are compared with faces in the database [bib_ref] Algorithms for face recognition drones. Mater, Shanthi [/bib_ref].
The Internet of Things (IoT) was invented in 1999 by Kevin Ashton. He added RFID to all lipsticks to communicate together. Today, companies and communication service providers are competing to offer IoT solutions to individuals and corporations. Students, researchers, and entrepreneurs are exploring the challenges and opportunities to develop new innovative products and solutions. Researchers have reviewed IoT applications for smart home, industry, healthcare, agriculture, environment, and transportation [bib_ref] Internet of Things (IOT): An overview and its applications, Dudhe [/bib_ref]. IoT mobile apps are believed to bring assistance and are able to facilitate access to information and organize processes and activities [bib_ref] Health management and user protection: An analysis of gamification elements in applications..., Monte [/bib_ref]. Developing research in recent years has been the pursuit of designing smart drones with the addition of IoT sensors. Drones embedded with IoT may be made more beneficial and effective by using an assembly of technologies such as sensors, transmitters, and cameras for an array of various and advanced applications [bib_ref] Smart Cybersecurity Framework for IoT-Empowered Drones: Machine Learning Perspective, Aldaej [/bib_ref].
The availability of easy-to-use IoT modules, a variety of online platforms for IoT, and availability of Internet connectivity indoors and outdoors increase the demand for IoT technology globally. Technology improves the battery's lifetime to more than 10 years like the Narrow Band IoT (NB-IoT) and eMTC [bib_ref] Power Consumption Analysis of Nb-IOT and Emtc in Challenging Smart City Environments, Jorke [/bib_ref].
IoT applications on drones are used for remote sensing and sending the data by Internet. The 5th generation of mobile technology known as 5G also has many advance features that support IoT applications. 5G provides a better data transfer rate up to 100 times compared to previous network generations and download speed up to 20 Gbps plus a very low latency such as 1 ms latency which is useful for autonomous drones [bib_ref] Unmanned Aerial Vehicles for Post-Disaster Communication Networks, Saif [/bib_ref].
Monitoring the vital signs of the human body in the healthcare sector is required to save a life, better diagnose, and provide better treatment to the patient. IoT helps healthcare providers to enhance the quality of their services which reflects in a healthy community and reduces the risk of medical error. On the other hand, we should not neglect the privacy of the patient's data by keeping critical data in an online database [bib_ref] Internet of Things for Smart Healthcare: Technologies. Challenges, and Opportunities, Baker [/bib_ref].
The basic structure of IoT system is to connect the IoT module with a sensor to send data over the Internet. The collected data by drone's sensors are processed either on the board or sent by the Internet to the base station. The drone's IoT sensors are divided into flight control, data collection, and communication sensors [bib_ref] UAV IoT Framework Views and Challenges: Towards Protecting Drones as, Lagkas [/bib_ref]. Medicine Box is developed by a group of students who connected by IoT to the Internet. This Intelligent Box sends a notification to the mobile application in the patient mobile phone to take his medicine on time.
Today, researchers are monitoring data from sensors on the mobile phone application. Groups of researchers are monitoring and storing the pressure on the heel from sensors in shoes. The pressure values are sent by PIC Microcontroller via Bluetooth [bib_ref] Development of in-shoe wearable pressure sensor using an Android application, Mostfa [/bib_ref]. A recent study at Taif University in Saudi Arabia monitored the acquired data from weather sensors on the Blynk app. The BME280 weather station sensors are mounted on a drone to collect pressure, temperature, altitude, and humidity as well as a transmitter to send a video from a thermal camera. Blynk is IoT mobile app that works on mobile phones and tablets for Android and iOS [bib_ref] Modifying Hata-Davidson Propagation Model for Remote Sensing in Complex Environments Using a..., Almalki [/bib_ref].
The standard Huskylens facial recognition camera has a Kendryte K210 processor with a 2 megapixel camera; the company stated that their system uses the You Look Only Once (YOLO) algorithm which works between one to two frames per seconds. The Huskylens camera is thirty time faster than YOLO but the company did not share any details about it [bib_ref] Epidemic Prevention System Based on Voice Recognition Combined with Intelligent Recognition of..., Zhou [/bib_ref]. Kendryte K210 is a low-power consumption system on chip (SoC) and powerful for facial recognition and detection as well as image classification and object detection. It uses the Convolutional Neural Network (CNN) for machine vision. It performs a real-time facial recognition up to 60 fps. Redmon et al. introduced his YOLO for object detection in 2016. YOLO is widely used for object detection. He stated that the fast YOLO can work up to 155 fps while the regular YOLO works up to 45 fps [bib_ref] You Only Look Once: Unified, Real-Time Object Detection, Redmon [/bib_ref].
Thus, the objective of this paper is to optimize the facial recognition ability of a developed medical delivery drone based on the direction and intensity of light. The main challenge for the drone's facial recognition occurs when the sun has a sharp angle during the sunrise in the early morning and from afternoon to sunset, as well as during the night when there is insufficient light, especially when the light intensity is between 20 and 50 Lux. The light intensity and direction of the light affect the accuracy of our facial recognition. The enhancement includes the development of a Guidance Landing System (GLS) incorporate with IoT mobile apps.
# Background
From the literature review, most of the facial recognition algorithms are developing their software to overcome illumination issues. Our contribution in this paper is to develop a hardware system along with an IoT mobile app to enhance the efficiency of medicine delivery.
A medical delivery drone with a facial recognition ability has been developed as shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref]. The medical delivery drone is equipped with a secure mechanical delivery box (DB) inclusive of a sanitizing unit (SU) that sprays sanitizer to avoid the risk of the spread of the COVID-19 virus during the delivery. The developed drone can deliver medicine in a very secure way by using facial recognition to identify the user [fig_ref] Figure 2: Facial recognition to identify specific user on the medical delivery drone [/fig_ref]. The drone was designed to be interactive with the user and uses voice commands to communicate with the user. The drone is equipped with a Huskylens facial recognition artificial intelligence (AI) camera that is used for the facial recognition process to identify the patients and to proceed with the delivery process according to the user profile. The accuracy of the delivery is based on the accuracy of the facial recognition, which is affected by the distance of face detection (the distance between the face and camera). A positive relationship exists between the light intensity and detection distance, the detection distance increases when the light intensity increases and vice versa. To solve this issue, this paper presented the design of the GLS to help the operator or autonomous system to select the best landing area. Five light sensors were assembled on GLS to detect the light direction and intensity and evaluate the system's accuracy.
A multi-rotor drone has the ability to land at the same location with freedom of rotates (yaw) 360 - around the vertical axis as shown in [fig_ref] Figure 3: Drone rotates [/fig_ref]. Outdoor facial recognition has extreme challenges based on the variation of environmental conditions. A set of experiments were conducted to improve the quality of facial recognition and detection. This paper presents a precision medication delivery system using drones and IoT GLS as shown in [fig_ref] Figure 2: Facial recognition to identify specific user on the medical delivery drone [/fig_ref]. The drone uses facial recognition to identify the user as shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref]. In this paper, Section 1 covers a literature review about drones, IoT, and facial recognition. Then, Section 2 focuses on the hardware and software of the developed drone's system. Next, Section 3 presents the result. After that, Section 4 is for the discussion. Finally, Section 5 concludes the findings of the study.
# Materials and methods
## Development of facial recognition system
The Huskylens facial recognition camera (DFRobot Electronics, Shanghai, China) has a Kendryte K210 processor. It is a very light camera that has a facial recognition function and does not require any Internet connection. It is compatible with the Arduino development board (Arduino S.R.L., Via Andrea Appiani, 25, 20900 Monza MB, Italy) via Universal Asynchronous Receiver Transmitter (UART) or Inter-Integrated Circuit (I2C). The first development board reads from the Huskylens facial recognition via UART serial communication at a 9600 baud rate as shown in [fig_ref] Figure 4: Circuit connections diagram for the Huskylens facial recognition camera [/fig_ref]. The development board reads the user ID from the camera and matches it with a pre-defined user's identity. It operates by 5 volts. Digital pins 10 and 11 are connected to Rx and Tx of the Huskylens camera, respectively. Huskylens has a Multiple Faces Learning function. Thirty photos were obtained from our volunteers. Faces were given ID numbers 1 to 3. ID = 0 is given for any undefined faces. ID = 0 code is helping us in the detection of any face presence. Accordingly, a specific response was programmed in Arduino Uno to the user ID. The delivery box (DB) will open for the defined ID 1, 2, and 3 to deliver the medicine, while the Voice Guiding System (VCGU) will play a greeting voice message by his/her name for ID 1, 2, and 3 or will play a voice message for ID 0 to move away from the drone.
## Development of guidance landing system (gls)
To improve the medical delivery drone with GLS, the system was enhanced with the ESP32 DevKitC V4 board (Espressif Systems Co., Ltd., Shanghai, China). This microcontroller board has a dual-core processor, 12-bit ADC pins, and Wi-Fi which is useful for IoT applications. In addition, four MH Light-Dependent Resistor (LDR) sensors are connected to 12-bit ADC pins [fig_ref] Figure 5: Medical delivery drone enhanced components for Guidance Landing System [/fig_ref]. A Motion Detection Unit (MDU) was also implemented with the GLS to save the power of the drone and ensure the safety of users [fig_ref] Figure 6: Guidance Landing System [/fig_ref]. The medical delivery drone is set to be in standby mode when no motion is detected beside the drone and disable the process of the delivery box when there is human movement beside the drone, and back to standby mode when the person moves away from the drone. The take-off mode will start when there is no presence of a human close to the drone. The MDU combines a Huskylens camera and Passive Infrared (PIR) motion detector sensors. The GLS is equipped with a Global Positioning System (GPS) to guide the drone to the outdoor landing position close to the patient's home. The patient receives a notification on the estimated delivery time during the process of scheduling a delivery. Before the delivery, SMS and emails notification will be sent to the patient's registered mobile phone number and email. Delivery is based on the patient's identity. In the case of an elderly, sick, or busy patient, the database of faces must be updated with the facial recognition details for two of the patient's relatives. When the drone reaches the patient's location, the SMS and email will be sent to the patient's registered mobile phone number and email, along with a siren alarm.
The drone is equipped with a Voice Guiding System (VCGU) to be an interactive system with the user, as shown in [fig_ref] Figure 5: Medical delivery drone enhanced components for Guidance Landing System [/fig_ref]. A Serial MP3 player model HW-311 is used to give a voice command to the user. Serial communication is used to send an array of 8 bytes to play a specific audio file. Each array contains starting and ending bytes, version, array size, command, enable and disable feedback, and two bytes of data. For instance, the first audio message is for greeting the user, and the second message will request that the patient stand in front of the camera.
## Development of iot mobile app for guidance landing system (gls)
LDR sensors are used to measure the light intensity from four directions as shown in . A light intensity sensor gives a numerical value of the light intensity in Lux. The sensor is aimed opposite to the facial recognition camera to measure the light intensity that falls on the subject's face. A Blynk mobile app was developed to show the real-time light intensity and compare the light intensity from all directions in terms of percentages. The values of the five sensors are saved on five virtual pins after the calculations. In the mobile apps, the five virtual pins are selected to display the values of the five sensors.
## Experimental methods to study the effect of light direction using uncontrolled light source
Multiple scenarios were simulated to determine the relationship and effect of light direction on facial recognition quality using an uncontrolled light source. Lux (lx) indicates the amount of light intensity in Lumen per square meter.
The distance between the LED light source and drone was fixed at 111 cm, and the LED light source intensity was fixed at 580 Lux.
Scenario A:
In a dark room with a single LED light source where the light falls on the back of the face, as shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref].
## Scenario b:
In a room with a light intensity of 27.5 Lux with a single LED light source where the light is falling on the back of the face, as shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref].
Scenario C: A dark room with a single LED light source when the light is falling on the face, as shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref].
Scenario D:
A room with a light intensity of 27.5 Lux with a single LED light source when the light is falling on the face, as shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref]. The detection distance of the subject's face using the drone camera was observed for all four scenarios.
## Experimental methods to study the effect of light intensity and color using controlled light source
In this experiment, a controlled light source was used to control light intensity and light color. The controlled values were 3200 K and 5500 K. Two types of light sources were used [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref] , a light source with a diffuser and a light source without a diffuser. The Kelvin (K) is a light color temperature. Lower light temperature indicates reddish color while the higher light temperature is bluish. The controlled light direction was aimed at the backside of the drone's camera; thus, the sensor (Lux) was moved to the backside of the drone, as shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref]. The first group of tests was conducted using a controlled light source with a diffuser, as shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref] ,B. The light intensity and distance were measured when the subject's face was recognized at five levels. The five levels of tests were repeated for 3200 K and 5500 K.
The tests were repeated for the second group of tests using a controlled light source without a diffuser, as shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref] ,D, and again the light intensity and distance were measured when the subject's face was recognized at five levels.
The following Pearson correlation coefficient was used to find the correlation between the distance of detection and intensity of the light in different scenarios.
[formula] r = ∑ n i=0 (x i −x)((y i −ȳ)) ∑ n i=0 (x i −x) 2 ∑ n i=0 (y i −ȳ) 2(2) [/formula]
# Results
By enhancing the IoT-GLS system, the medical delivery drone's operator or autonomous system was able to select the best angle of landing based on the light direction and intensity to ensure the optimal efficiency of facial recognition. The drone was able to land and switch off all rotors in the patient's location based on the GPS coordinates. Then, the drone was able to detect any person near the drone's landing area through the motion detector and facial recognition camera and can request the patient to stand in front of the camera to verify the patient's identity. For an easier user experience, a controllable laser light was used to point a red laser light to the ground in front of the camera and guide the patient to the right location in front of the camera, which is close to the delivery box. Then, according to the patient's identity, the drone was able to greet the patient by his/her name using a voice. Then, the delivery box was opened for the patient to collect his/her medicine. Finally, the box closed, and the sanitizing process started to ensure that the spread of any viruses and bacteria to the patients and the operators can be eliminated. In case of detecting of any unauthorized person in front of the camera, the drone starts with a warning message that requests the person to move away. In case the violator does not respond within 10 s, the alarm sounds and a photo is sent to the operation center.
The accuracy of the Huskylens facial recognition camera was tested on three volunteers as well as 5000 faces from the Flickr-Faces-HQ dataset (Q) were used to check the accuracy of the system. The majority of the faces were undefined to calculate the accuracy of face detection. The photos of the three volunteers, representing 0.596% of the total number of 5030 photos, were identified.
The Huskylens camera has two features; the first feature is detecting the presence of a human face, while the second is identifying the identity of the face. The first test included 5000 photos from the FFHQ dataset for human face detection by the Huskylens camera. The system successfully detected the presence of 4997 faces with 99.94% accuracy. Only 3 faces out of 5000 were not detected. Two faces were not detected because the hair was covering a part of the faces, while the third face was covered with a hat. The false positive ratio for face detection is 0.06%.
In the second experiment, 30 face pictures of the three volunteers were used to check the accuracy of the system. The facial recognition camera detected 29 faces. The one undetected face had a low-quality picture. As a result, the percentage of error reached 3.33%.
The first two tests were conducted with the three volunteers' photos plus 5000 photos from the FFHQ dataset. The overall percentage of our two tests' accuracy reached 99.92% in a controlled environment with perfect lighting conditions. The light percentage from four directions was then sent to the IoT mobile application to compare the light intensity from all angles, so the best angle for landing could be chosen. The developed IoT mobile app is shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref]. The upper numerical value shows the light intensity at the backside [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref] , the light intensity percentage of the front-side sensor [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref] , the light intensity percentage of the right-side sensor [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref] , the light intensity percentage of the left-side sensor [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref] , and the light intensity percentage of the back-side sensor [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref].
In the study of the effect of light direction using uncontrolled light source tests, in scenario A, where the facial recognition efficiency was tested in a dark room with a single LED light source where the light direction is falling on the back of the face, the camera did not detect the face even from a very close distance. In scenario C, where the facial recognition efficiency was tested in a dark room with a single LED light source, when the light direction fell on the face of the subject, the camera was able to detect the subject's face from 192 cm.
In scenarios B and D, the facial recognition efficiency was tested in the room with the light at 27.5 Lux, and the LED light source was kept at the same position. When the light direction fell on the back of the subject's head, the camera detected the face from 60 cm (scenario B). However, in scenario D, when the light direction fell on the subject's face, the camera detected the subject's face from 207 cm. The results of the study of the effect of light direction using uncontrolled light source scenarios show that the IoT-GLS improved the distance of detection by 192% in a dark environment with a single light source when the IoT-GLS chose the best angle when the direction of light fell to the face of the subject. Furthermore, the test was repeated in a room with low light intensity (27.5 Lux) plus a single LED light source. The measurement exhibited an improvement in face detection distance up to 147 cm by using the IoT-GLS.
To improve the measurement of the light intensity that falls toward the face, the direction of the sensor changed from upward to the back of the drone [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref]. The second test was conducted to study the effect of light intensity and color by controlling the light source.
The first group of controlled light source tests was conducted using a controlled light source with a diffuser. The light intensity was measured at five levels. The five levels of tests were repeated for 3200 K and 5500 K. The distance when the subject's face was recognized and measured at each test is shown in [fig_ref] Table 1: Compare the relation among light intensity [/fig_ref]. [fig_ref] Table 2: Compare the relation among light intensity [/fig_ref] shows the collected data for the light sources without diffuser tests. The highest light intensity was at 5500 K direct light, which reached to 500.83 Lux and the detection distance reached 184 cm. Then, a set of four experiments was conducted to measure the effect of changing the light directions from the right and left of the drone and face. The third group of controlled light source tests was conducted using a controlled light source with a diffuser from the right side of the drone and face. The light intensity was measured at five levels. The five levels of tests were repeated for 3200 K and 5500 K. The distance when the subject's face was recognized was measured at each test.
The third group of controlled light source tests was conducted using a controlled light source with a diffuser from the right side of the drone and face. The light intensity was measured at five levels. The five levels of tests were repeated for 3200 K and 5500 K. The distance when the subject's face was recognized and measured at each test is shown in [fig_ref] Table 3: Compare the relation among light intensity [/fig_ref]. The actual and sidelight intensities were measured when the drone's sensor faced the light source and rotated to 90 - for the right side. The fourth group of controlled light source tests was conducted using a controlled light source without a diffuser from the right side of the drone and face. The light intensity was measured at five levels. The five levels of tests were repeated for 3200 K and 5500 K. The distance when the subject's face was recognized and measured at each test is shown in [fig_ref] Table 4: Compare the relation among light intensity [/fig_ref]. The actual and sidelight intensities were measured when the drone's sensor faced the light source and when rotated to 90 - for the right side. The fifth group of controlled light source tests was conducted using a controlled light source with a diffuser from the left side of the drone and the face. The light intensity was measured at five levels. The five levels of tests were repeated for 3200 K and 5500 K. The distance when the subject's face was recognized and measured at each test is shown in [fig_ref] Table 5: Compare the relation among light intensity [/fig_ref]. The actual and sidelight intensities were measured when the drone's sensor faced the light source and rotated to 90 - for the left side. The sixth group of controlled light source tests was conducted using a controlled light source without a diffuser from the left side of the drone and face. The light intensity was measured at five levels. The five levels of tests were repeated for 3200 K and 5500 K. The distance when the subject's face was recognized and measured at each test is shown in [fig_ref] Table 6: Compare the relation among light intensity [/fig_ref] ; the actual and the side light intensities were measured when the drone's sensor faced the light source and when rotated to 90 - for the left side.
Finally, a set of two experiments was conducted to measure the effect of changing the light directions from the front, right, and left of the drone and face at a fixed distance. The light intensity was measured from three directions. The tests were repeated for 3200 K and 5500 K with and without a diffuser. The distance was fixed to 130 cm when the subject's face was recognized as shown in [fig_ref] Table 7: Comparison [/fig_ref]. The actual and sidelight intensities were measured when the light sensors faced the light and when the drone's sensor rotated to 90 - for right and left. The actual and sidelight intensities were measured when the light sensors faced the light and when the drone's sensor rotated to 90 - for right and left.
# Discussion
The last two sets of experiments were conducted to measure and compare the effects of changing the light directions from the front, right, and left of the drone and face with a fixed distance. The light intensity was measured from three directions. The tests were repeated for 3200 K and 5500 K with and without a diffuser. The distance was fixed to 130 cm when the subject's face was recognized, as shown in Tables 7 and 8. The results clearly showed the impact of changing light direction from the two sides to the front side. The light intensity required to recognize the face identity at 130 cm was almost equal when the light was from the left and right with/without a diffuser. The 3200 K light tests that were conducted without a diffuser showed a huge difference of more than 85% of the light intensity required for the same distance when the light direction changed from the front side to right and left. The light intensity required from the front was 99.17 lux, while from the two sides was 185 lux, as shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref]. For the 3200 K without a diffuser, the light intensity required from the front was 95 lux while from the two sides was 154.17 lux which is around 62.3%. Then, the 3200 K light tests conducted with a diffuser showed a difference of more than 31.4% of the light intensity required for the same distance when the light direction changed from the front side to the right and left sides. The light intensity required from the front was 114.17 lux, while from the two sides was 150. For the 3200 K with diffuser, the light intensity required from the front was 97.5 lux while from the two sides was 133.33 lux, which is around 36.7%. From the above results, it shows clearly that for light without a diffuser, the differences are huge between the front side and two sides, while the light source with a diffuser has a smaller difference compared to without light without a diffuser. Since a significant correlation was found between facial recognition's detection distance, light source direction, light intensity, and light color (p < 0.05), as shown in [fig_ref] Table 9: Results of Pearson correlation coefficient between the 3200 K and 5500 K... [/fig_ref] , optimal efficiency of facial recognition for medication delivery could be achieved using the IoT-GLS where it can help the operator or autonomous system to select the best angle of landing based on the light direction and intensity. The highest correlation was found at 5500 K, where the soft light had a longer detection distance, as shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref]. For the 3200 K direct light, the detection distances were almost the same, as shown in [fig_ref] Figure 1: Drone with facial recognition and medical delivery box [/fig_ref]. The main challenge is to use a very lightweight facial recognition camera without Internet connectivity due to the drone's payload. The Huskylens camera has an on-chip facial recognition feature that fits the requirements and shows a significant accuracy in identifying the patient that exceeds 98% in our experiments in perfect environmental conditions. The facial recognition efficiency was improved by our proposed GLS system. The controllable laser light helps the patient in finding the accurate location in front of the camera.
In our design, there are four limitations. The first limitation is during the day when the sun has an angle of less than 45 degrees during sunrise and from afternoon to sunset. The second limitation is during the night in dark areas with a single LED light source where the light intensity is between 20 and 50 Lux. The third limitation is in a dark environment without any source of light or an insufficient light source where the light intensity is less than 20 Lux. Finally, the fourth limitation is when the user wears a face mask where our camera cannot detect faces.
The proposed solutions to overcome the first and second limitations were to introduce GLS, which provides the best landing angel. For the third limitation, a mini-LED light source can be installed and attached to the drone to flash the light when it detects a dark environment. For the fourth limitation, a Voice Guiding System (VCGU) was added to the system, which, when activated, will request the users to stand in front of the camera and remove their masks. In case of a further failure of detection, we propose to add a contactless NFC card reader RC-522 to the drone, as shown in [fig_ref] Figure 5: Medical delivery drone enhanced components for Guidance Landing System [/fig_ref] , as well as a keypad. For a more secure experience, the drone's camera will send a photo of an unauthorized person to the control center and send a video of the delivery process.
Most of the published studies to develop and improve the facial recognition algorithms are focused on developing software to overcome the illumination issues. Our proposed system is developed to be assistive to facial recognition algorithms, especially in an outdoor environment where it is difficult to control environmental conditions. The majority of the published research discusses indoor controlled environments.
# Conclusions
A contactless medical delivery drone was developed and tested successfully to deliver medicine to the patient in a safe mode environment in response to the COVID-19 pandemic. A facial recognition camera was used to detect users' identity. To overcome the current shortcomings of the medical delivery drone during sunrise, from afternoon to sunset and during night in dark areas where the light intensity is between 20 and 50 Lux, an IoT-GLS enhancement was introduced. The developed IoT mobile app is able to show the percentage of light intensity from four directions and light intensity value from the backside of the camera lens which indicates the light intensity that falls on the patient's face. Furthermore, results from the experiments found that there is significant correlation between the light direction and intensity with detection distance (p < 0.05). The finding enabled the developed IoT-GLS to assist the autonomous system or drone's operator to select the best angle of landing which consequently increased the distance of face detection to more than two meters. Future work will focus on the effect of a wide range of light color temperatures and improve the processes of medicine delivery, as well as test the GLS on different facial recognition algorithms.
[fig] Figure 1: Drone with facial recognition and medical delivery box. [/fig]
[fig] Figure 2: Facial recognition to identify specific user on the medical delivery drone. [/fig]
[fig] Figure 3: Drone rotates (yaw) 360 • around vertical axis at the same location. [/fig]
[fig] Figure 4: Circuit connections diagram for the Huskylens facial recognition camera. [/fig]
[fig] Figure 5: Medical delivery drone enhanced components for Guidance Landing System. [/fig]
[fig] Figure 6: Guidance Landing System (GLS) and Motion Detection Unit (MDU). [/fig]
[fig] Figure 7, Figure 8: Guidance Landing System (GLS) hardware components. The IoT development board reads from the sensors and sends the data to IoT mobile applications as shown in Figure 8. The IoT development board connects to the Wi-Fi network. The development board reads from five sensors as shown in the flowchart in Figure 9. The first light intensity sensor sends light intensity in lux to the development board. Four photoresistors compare the light percentage from four directions. The light percentage Equation (1) from four directions is then sent to a mobile application to compare the light intensity from all angles. % o f the light = Analog − to − Digital Converter (ADC) Part of Guidance Landing System (GLS) circuit connections diagram. [/fig]
[fig] Figure 9: Flowchart of Guidance Landing System. Notes: LDR-Light-Dependent Resistor; V1, V2, V3, V4, and V5 are the virtual pins in the IoT mobile apps. [/fig]
[fig] Author: Contributions: Investigation, M.O.B.; Supervision, F.I. and M.S.M. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: No funding for the research and APC is partially supported by grant no. PPSI-2020-INDUSTRI-03. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. [/fig]
[table] Table 1: Compare the relation among light intensity (Lux), light color (Kelvin), and detection distance (cm) for light source with a diffuser. [/table]
[table] Table 2: Compare the relation among light intensity (Lux), light color (Kelvin), and detection distance (cm) for light source without diffuser. [/table]
[table] Table 3: Compare the relation among light intensity (Lux), light color (Kelvin), and detection distance (cm) for light source with diffuser from right side. [/table]
[table] Table 4: Compare the relation among light intensity (Lux), light color (Kelvin), and detection distance (cm) for light source without diffuser from right side. [/table]
[table] Table 5: Compare the relation among light intensity (Lux), light color (Kelvin), and detection distance (cm) for light source with diffuser from left side. [/table]
[table] Table 6: Compare the relation among light intensity (Lux), light color (Kelvin), and detection distance (cm) for light source without diffuser from left side. [/table]
[table] Table 7: Comparison [/table]
[table] Table 8: Comparison [/table]
[table] Table 9: Results of Pearson correlation coefficient between the 3200 K and 5500 K with and without diffuser. [/table]
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10.1186/s12896-015-0191-3
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CCBY
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4535744
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26272331
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s2orc_pubmed_articles
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Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices
Background: In order to provide a system fully integrated with qPCR screening, usually used in GMO routine analysis, as well as being able to detect, characterize and identify a broad spectrum of GMOs in food/feed matrices, two bidirectional DNA walking methods targeting p35S or tNOS, the most common transgenic elements found in GM crops, were developed. These newly developed DNA walking methods are completing the previously implemented DNA walking method targeting the t35S pCAMBIA element. Methods: Food/feed matrices containing transgenic crops (Bt rice or MON863 maize) were analysed using the integrated DNA walking system. Results: First, the newly developed DNA walking methods, anchored on the sequences used for the p35S or tNOS qPCR screening, were tested on Bt rice that contains these two transgenic elements. Second, the methods were assessed on a maize sample containing a low amount of the GM MON863 event, representing a more complex matrix in terms of genome size and sensitivity. Finally, to illustrate its applicability in GMO routine analysis by enforcement laboratories, the entire workflow of the integrated strategy, including qPCR screening to detect the potential presence of GMOs and the subsequent DNA walking methods to characterize and identify the detected GMOs, was applied on a GeMMA Scheme Proficiency Test matrix. Via the characterization of the transgene flanking region between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette, the presence of GMOs was properly confirmed or infirmed in all tested samples. Conclusion: Due to their simple procedure and their short time-frame to get results, the developed DNA walking methods proposed here can be easily implemented in GMO routine analysis by the enforcement laboratories. In providing crucial information about the transgene flanking regions and/or the transgenic cassettes, this DNA walking strategy is a key molecular tool to prove the presence of GMOs in any given food/feed matrix.
# Background
In 2014, 181.5 million hectares of genetically modified organisms (GMOs) have been planted in 28 countries. On the European Union (EU) market, the commercialization of GMOs in the food/feed chain is subject to the EU legislation, which is becoming more and more complex to implement due to the increasing number and diversity of GMOs [bib_ref] The global pipeline of new GM crops: Implications of asynchronous approval for..., Stein [/bib_ref]. The majority of EU-authorized GMOs (78.6 %) harbours the transgenic p35S element (Cauliflower mosaic virus (CaMV) 35S promoter), the transgenic tNOS element (Agrobacterium tumefaciens nopaline synthase terminator) or both of them, with an occurrence respectively reported of 60.7, 53.6 and 35.7 % [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref] [bib_ref] How to deal with the upcoming challenges in GMO detection in food..., Broeders [/bib_ref].
To ensure the correct enforcement of the EU legislation, several GMO detection methods have been developed, mainly based on SYBR®Green and TaqMan® real-time PCR technologies. Usually, a screening is first performed with qPCR methods targeting the most common transgenic elements present in genetically modified (GM) crops (e.g. p35S and tNOS). These strategies, covering a broad spectrum of GMOs, allow to indicate the potential presence of GMOs in tested samples [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref] [bib_ref] How to deal with the upcoming challenges in GMO detection in food..., Broeders [/bib_ref] [bib_ref] Development of a molecular platform for GMO detection in food and feed..., Broeders [/bib_ref] [bib_ref] Combinatory SYBR® Green real-time PCR screening approach for tracing materials derived from..., Kluga [/bib_ref] [bib_ref] SYBR Green qPCR methods for detection of endogenous reference genes in commodity..., Mbella [/bib_ref] [bib_ref] A testing cascade for the detection of genetically modified rice by real-time..., Reiting [/bib_ref]. In case of positive responses, EU-authorized GMOs are subsequently identified and quantified using EU event-specific methods. If some observed positive screening elements, like p35S and tNOS, are not explained by these event-specific methods, the presence of EU-unauthorized GMOs can be indirectly suspected [bib_ref] How to deal with the upcoming challenges in GMO detection in food..., Broeders [/bib_ref]. However, as most of the targeted elements originate from natural organisms (e.g. p35S from CaMV and tNOS from Agrobacterium tumefaciens), the confirmation of their presence can be irrefutably provided only by the characterization of the transgene flanking regions between the plant genome and the integrated cassette [bib_ref] How to deal with the upcoming challenges in GMO detection in food..., Broeders [/bib_ref] [bib_ref] Development of a molecular platform for GMO detection in food and feed..., Broeders [/bib_ref] [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref]. To this end, DNA walking strategies have notably been proposed in order to get this crucial information allowing to identify GM crops [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref] and references therein, [bib_ref] Development of an event-specific Real-time PCR detection method for the transgenic Bt..., Babekova [/bib_ref] [bib_ref] Flanking sequence determination and specific PCR identification of transgenic wheat B102-1-2, Cao [/bib_ref] [bib_ref] Validation of a sensitive DNA walking strategy to characterize unauthorised GMOs using..., Fraiture [/bib_ref] and references therein, [bib_ref] Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR..., Liang [/bib_ref] [bib_ref] A novel T-DNA integration in rice involving two interchromosomal translocations, Majhi [/bib_ref] [bib_ref] Flanking sequence determination and event-specific detection of genetically modified wheat B73-6-1, Xu [/bib_ref] [bib_ref] Event-specific quantitative detection of genetically modified wheat B72-8-11 based on the 3′..., Zhang [/bib_ref]. However, these metods are not usually used in GMO routine analysis because they are not easily implementable by the enforcement laboratories. Recently, an integrated DNA walking strategy, better corresponding to the need of the enforcement laboratories, was developped to rapidly detect and identify EU-unauthorized GMOs, without significant additional cost and equipment [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref]. This method targets the t35S element from the pCAMBIA vector, which is frequent in transgenic plants and is absent in EUauthorized GMOs. This DNA walking approach, based on PCR, has the advantage to be fully integrated into the initial qPCR analysis as the same primers are used for the qPCR screening (detection) and the DNA walking (identification) [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref] [bib_ref] Current status of binary vectors and superbinary vectors, Komori [/bib_ref]. In addition, this approach was assessed as highly sensitive and able to deal with rice based mixtures and processed products, which is essential in GMO routine analysis [bib_ref] Validation of a sensitive DNA walking strategy to characterize unauthorised GMOs using..., Fraiture [/bib_ref].
Here, the concept of this integrated PCR-based DNA walking strategy has been adapted to also target p35S and tNOS, the most common transgenic elements found in GMOs, in order to characterize a broader spectrum of GMOs as well as to strengthen the initial DNA walking system targeting t35S from pCAMBIA. For each element, two DNA walking directions, starting from a position anchored on the sequences used for the p35S or tNOS SYBR®Green qPCR screening, have been established [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref]. First, the p35S and tNOS bidirectional DNA walking methods were developed on Bt rice, as previously used for the t35S pCAMBIA method. Second, these DNA walking methods were assessed using the certified reference material (CRM) of the GM maize MON863 (9,85 %), which represents a more complex matrix due to its large genome and its low target content. Finally, in order to illustrate its applicability in routine analysis, a GeMMA Scheme Proficiency Test food matrix was submitted to the entire integrated strategy, including the qPCR screening using the p35S, tNOS and t35S pCAMBIA markers to detect the presence of GMOs and, then, the DNA walking methods, corresponding to the qPCR positive responses, allowing to characterize them.
# Results and discussion
In order to characterize a broad spectrum of both EU authorized and unauthorized GMOs, two novel DNA walking methods, based on the p35S and tNOS transgenic elements, have been developed. These methods were designed similarly to the t35S pCAMBIA DNA walking method targeting only EU unauthorized GMOs [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref]. In the interest to provide an integrated approach, for each DNA walking method, the same primers allow the detection of the potential presence of GMOs containing the targeted elements (qPCR screening) as well as their characterization and identification insofar as possible (DNA walking).
## In silico study
Since the DNA walking approach is integrated into the screening step, the SYBR®Green primers published by Barbau-Piednoir et al., 2010 were used to target the p35S and tNOS elements [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref].
As three primers are required by the DNA walking method for each targeted element, an additional primer (b) intermediate to the screening primers (a and c) was designed [fig_ref] Table 1: Oligonucleotide primers used for the real-time PCR assays, the DNA walking approaches... [/fig_ref]. The specificity of these primers was successfully assessed in silico, against all EU-authorized GMOs, LLPs (Low Level Presence) and corresponding WTs (Wild-Type), using the software wEMBOSS (data not shown) . Moreover, for each of the targets, two walking directions were established (p35S-F, p35S-R, tNOS-F and tNOS-R) in order to extend the GMO coverage of the integrated DNA walking strategy.
## Development of the dna walking methods
## Assessment of p35s dna walking methods
For the p35S approach, several amplicons were observed from 100 % Bt rice, corresponding to 200 000 HGEs (Haploid Genome Equivalent), for the four different degenerated random tagging (DRT) primers (A-D), including 18 amplicons for the p35S-F DNA walking method (amplicons n°1 to 18) and 12 amplicons for the p35S-R DNA walking method (amplicons n°24 to 35) [fig_ref] Figure 1: Development of the bidirectional p35S and tNOS DNA walking methods on 100... [/fig_ref]. The size range of these amplicons was approximately from 100 bp to 1 Kbp and from 250 pb to 2 Kbp for the p35S-F and p35S-R DNA walking method, respectively. All these amplicons were consecutively analysed by sequencing to evaluate the specificity of the methods (Additional file 1).
All these characterized sequences corresponded specifically to the position of the p35S element in the transgenic cassette [fig_ref] Figure 1: Development of the bidirectional p35S and tNOS DNA walking methods on 100... [/fig_ref] [bib_ref] Bt rice harbouring cry genes controlled by a constitutive or woundinducible promoter:..., Breitler [/bib_ref]. As expected, these sequences present the continuity of the p35S element [GenBank:AF234296] for the p35S-F DNA walking method (amplicons n°1 to 18) and the p35S promoter [GenBank:AF234296] regulating the hygromycin resistance gene (hpt) [Gen-Bank:AAF65337] for the p35S-R DNA walking method (amplicons n°24 to 35) [fig_ref] Figure 1: Development of the bidirectional p35S and tNOS DNA walking methods on 100... [/fig_ref] and Additional file 1).
For the WT rice sample, few amplicons (amplicons n°19 to 23 for the p35S-F method and amplicons n°36 to 42 for the p35S-R method) were observed [fig_ref] Figure 1: Development of the bidirectional p35S and tNOS DNA walking methods on 100... [/fig_ref] and identified as corresponding to the rice genome (Additional file 2). They are probably due to the use of DRT primers which can potentially generate a background of aspecific products, especially in absence or in low amounts of targeted sequences [bib_ref] Genome walking in eukaryotes, Leoni [/bib_ref].
## Assessment of tnos dna walking methods
The use of the tNOS DNA walking approach with the two walking directions using the four different DRT primers (A-D) on the 100 % Bt rice sample produces several amplicons, including 17 amplicons for the tNOS-F DNA walking method (amplicons n°43 to 59) and 22 amplicons for the tNOS-R DNA walking method (amplicons n°66 to 87) [fig_ref] Figure 1: Development of the bidirectional p35S and tNOS DNA walking methods on 100... [/fig_ref]. The tNOS-F and tNOS-R DNA walking methods gave respectively amplicons with a size range of approximately 100 bp to 1.5 Kbp and 200 pb to 2 Kbp. To assess the specificity of the methods, all these PCR products were examined by sequencing (Additional file 1).
On the one hand, as expected, regarding the tNOS element localisation in the transgenic cassette, 100 % of the analysed amplicons coming from the tNOS-F DNA walking method have allowed to characterize the transgene flanking regions between the rice genome and the right border of the integrated pCAMBIA cassette via the amplicon sequences containing both the tNOS element and the rice genome [fig_ref] Figure 1: Development of the bidirectional p35S and tNOS DNA walking methods on 100... [/fig_ref] and Additional file 1). None of the obtained amplicons presented an unexpected sequence. As the Bt rice presents two transgenic insertions, two types of transgene flanking regions were characterized: one localised between the transgenic cassette [GenBank:AY836546.1] and a genomic sequence from chromosome II of Oryza sativa japonica Group [GenBank:OSJNBa0016G10] identified using the amplicons generated by the DRT C primers (amplicons n°51 to 56) and one situated between the pCAMBIA cassette [GenBank:AY836546.1] and a genomic sequence from chromosome III of Oryza sativa japonica Group [Gen-Bank:OSJNBb0111B07] identified using the amplification coming from the DRT A, B and D primers (amplicons n°4 3 to 50 and n°57 to 59) [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref] [bib_ref] Validation of a sensitive DNA walking strategy to characterize unauthorised GMOs using..., Fraiture [/bib_ref]. These results yet clearly demonstrate the importance to use four different DRT primer mixes. Indeed, the difference in affinity of these DRT primers allows increasing the likelihood to successfully characterize all targets [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref] [bib_ref] Validation of a sensitive DNA walking strategy to characterize unauthorised GMOs using..., Fraiture [/bib_ref]. In addition, the right border of the pCAMBIA cassette on chromosome II was shorter of two base-pairs compared to the one on chromosome III (Additional file 1). These two transgene flanking regions were also properly confirmed p35S R GGGTCTTGCGAAGGATAGTG [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref] SYBR®Green qPCR tNOS F GATTAGAGTCCCGCAATTATACATTTAA 69 [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref] tNOS R TTATCCTAGKTTGCGCGCTATATTT [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref] SYBR®Green qPCR t35S pCAMBIA c-F CGGGGGATCTGGATTTTAGTA 137 [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref] t35S pCAMBIA a-R AGGGTTCCTATAGGGTTTCGCTC [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref] DNA Walking p35S-F a (p35S R) GGGTCTTGCGAAGGATAGTG [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref] p35S by sequencing of PCR products obtained in using primers annealing to the pCAMBIA cassette and chromosome II or III [fig_ref] Table 1: Oligonucleotide primers used for the real-time PCR assays, the DNA walking approaches... [/fig_ref].
On the other hand, as expected, all PCR products generated from the tNOS-R DNA walking method allow to characterize the continuity of the tNOS element (amplicons n°71, 82 and 87) as well as, for the longer ones, the flanking region between the tNOS element [GenBank:HQ593861.1] and the Cry1B gene [GenBank:KC 414884.1] conferring an insect resistance (amplicons n°66 to 70, n°72 to 81 and n°83 to 86) [fig_ref] Figure 1: Development of the bidirectional p35S and tNOS DNA walking methods on 100... [/fig_ref] and Additional file 1). 100 % of the analysed amplicons corresponded to the expected sequences.
Similarly to the p35S DNA walking methods, the bidirectional tNOS approach presents uniquely specific amplifications further to the analysis of the Bt rice sample while few aspecific amplicons [fig_ref] Figure 1: Development of the bidirectional p35S and tNOS DNA walking methods on 100... [/fig_ref] , corresponding to the rice genome, were generated from the WT rice material (amplicons n°60 to 65 for the tNOS-F method and amplicons n°88 to 94 for the tNOS-R method) (Additional file 2).
## Practical application of the dna walking methods
## Analysis of gm maize
To test the developed p35S and tNOS bidirectional DNA walking methods on a more complex food matrix than rice in term of genome size and target amount, GM maize MON863 9.85 % (ERM-BF416c), corresponding to 3 788 HGEs, was selected as it possesses both the p35S and tNOS elements in its transgenic cassette [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref].
First, the presence of these elements in the tested CRM sample was confirmed by SYBR®Green qPCR screening (Additional file 4). Then, several amplicons were generated by each DNA walking method with a size ranging from approximately 200 bp to 4 Kbp [fig_ref] Figure 2: Application of the bidirectional p35S and tNOS DNA walking methods on GM... [/fig_ref]. In order to obtain the most informative sequences, the amplicon with the highest size for each DRT primer mix in each applied DNA walking method was selected to be sequenced (Additional file 5).
Most of the selected amplicons from the p35S-F DNA walking method present the 5' transgene flanking region between the maize genome [GenBank:DQ490951.2] and the p35S promoter [GenBank:KJ608136.1] from the transgenic cassette of MON863, as previously published [fig_ref] Figure 2: Application of the bidirectional p35S and tNOS DNA walking methods on GM... [/fig_ref] and Additional file 5) [bib_ref] Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified..., Yang [/bib_ref] [bib_ref] A specific qualitative and real-time PCR detection of MON863 maize based on..., Zhu [/bib_ref]. This transgene flanking region, confirming the presence of GM maize MON863, is also targeted by the EU event-specific qPCR method to identify and quantify this GMO. Only one tested amplicon (n°3) showed an aspecific sequence corresponding to the WT maize genome [GenBank:AC196084; Zea mays BAC clone CH201-52A17 from chromosome 5] [fig_ref] Figure 2: Application of the bidirectional p35S and tNOS DNA walking methods on GM... [/fig_ref] and Additional file 2). A possible explanation is that the tested sample contains primarily WT maize material and only a relative low amount of the target.
For the p35S-R DNA walking method, two different types of sequences were observed due to the presence of two p35S promoters in the transgenic cassette of GM MON863 maize [fig_ref] Figure 2: Application of the bidirectional p35S and tNOS DNA walking methods on GM... [/fig_ref] [bib_ref] Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified..., Yang [/bib_ref] [bib_ref] Event-specific qualitative and quantitative PCR detection of MON863 maize based upon the..., Pan [/bib_ref]. On the one hand, the continuity of the p35S promoter [GenBank:KJ608136.1; Zea mays transgenic line MON863 promoter region] regulated the neomycin phosphotransferase gene (nptII) from A. tumefaciens [GenBank:AAF65400.1] which confers a resistance to kanamycin [fig_ref] Figure 2: Application of the bidirectional p35S and tNOS DNA walking methods on GM... [/fig_ref] Most of the tested amplicons (81.25 %) derived from all the DNA walking methods presented a sequence corresponding to the GM targets. Based on these data, the presence of GM MON863 maize in the tested sample was clearly identified by isolation and sequencing of its junction between the maize genome and the transgenic cassette. In addition, this strategy allows to reconstruct 2.727 Kbp of the integrated transgenic cassette, going from the left border to a part of the gene Cry3Bb1, in agreement with the published information [fig_ref] Figure 2: Application of the bidirectional p35S and tNOS DNA walking methods on GM... [/fig_ref] and Additional file 5) [bib_ref] Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified..., Yang [/bib_ref] [bib_ref] A specific qualitative and real-time PCR detection of MON863 maize based on..., Zhu [/bib_ref] [bib_ref] Event-specific qualitative and quantitative PCR detection of MON863 maize based upon the..., Pan [/bib_ref]. These results also highlight that the proposed DNA walking strategy is able to identify GMOs from different plant species.
## Analysis of the food matrix
In order to illustrate its applicability in GMO routine analysis by the enforcement laboratories, the entire workflow of the integrated system was applied on a food matrix (GeM SU34-A) from a GeMMA Scheme Proficiency Test containing 1.2 % of GM maize MON863 event, corresponding to 461 HGEs.
First, similarly to the GMO routine analysis, the GeMMA food matrix was submitted to the SYBR®Green qPCR screening using the p35S, tNOS and t35S pCAMBIA For each DNA walking method, a schematic representation of the potential start position and direction, applied on the transgenic cassette of the GM maize MON863, is illustatred by the black arrows. Below the transgenic cassette, the sequence covering of the selected amplicons from the GM MON863 maize CRM (9.85 %) and the GeMMA proficiency test food matrix (GeMMA SU35-A) is schematically represented by rectangles. The corresponding amplicon numbering is indicated in the [fig_ref] Figure 2: Application of the bidirectional p35S and tNOS DNA walking methods on GM... [/fig_ref] and Additional file 6. LB (left border); p35S (CaMV 35S promoter); nptII (neomycin phosphotransferase II gene); tNOS (Agrobacterium tumefaciens nopaline synthase terminator); p4-AS1 (modified CaMV 35S promoter); wtCAB (Wheat major chlorophyll a/b binding protein gene); rAct (Rice Actin intron); Cry3Bb1 (synthetic Cry3Bb1 gene); tahsp17 (Wheat heat shock protein terminator); RB (right border); maize (maize genome) [Schema adapted from screening markers allowing to detect the potential presence of GMOs [fig_ref] Table 1: Oligonucleotide primers used for the real-time PCR assays, the DNA walking approaches... [/fig_ref]. As expected, a positive signal was observed for the p35S and tNOS screening markers while the t35S pCAMBIA screening marker gave a negative signal (Additional file 4), suggesting the potential presence of GMOs in the tested food matrix.
Second, based on the positive signals obtained from the screening qPCR analysis, the bidirectional p35S and tNOS DNA walking approaches were selected to be applied on the sample. In doing so, the potential presence of GMOs will be confirmed by the characterization of their sequences.
All applied DNA walking methods were able to produce amplicons in a size range from approximately 200 bp to 1.5 Kbp (Additional file 6). In order to follow an efficient workflow suitable for GMO routine analysis, only one amplicon, chose for its large size as well as for its ease to be selected on an electrophoresis gel, was sequenced for each DNA walking method (Additional file 6).
With all these DNA walking methods, 100 % of the analysed amplicons presented sequences specific to the GM target. Indeed, when using p35S-F DNA walking, the transgene flanking region between the maize genome All these sequences indubitably prove the presence of the GM MON863 maize event in the GeMMA food matrix sample though the identification of its junction between the maize genome and the transgenic cassette as well as the partial reconstruction of its transgenic cassette, in agreement with the published information [bib_ref] Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified..., Yang [/bib_ref] [bib_ref] A specific qualitative and real-time PCR detection of MON863 maize based on..., Zhu [/bib_ref] [bib_ref] Event-specific qualitative and quantitative PCR detection of MON863 maize based upon the..., Pan [/bib_ref].
Similarly to the t35S pCAMBIA DNA walking method, the good specificity of the newly developed DNA walking methods (p35S-F, p35S-R, tNOS-F, tNOS-R) was illustrated in this study since almost all of the sequences from the analysed amplicons generated from the Bt rice (100 %), MON863-9.85 % (81.25 %) and Gemma proficiency test (100 %) matrices corresponded to the GM targets [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref] [bib_ref] Validation of a sensitive DNA walking strategy to characterize unauthorised GMOs using..., Fraiture [/bib_ref]. The success of this strategy is mainly due to the specificity of the target-specific primers, allowing to initially amplify the targets by PCR and, then, to enrich them by two successive semi-nested PCRs [fig_ref] Table 1: Oligonucleotide primers used for the real-time PCR assays, the DNA walking approaches... [/fig_ref].
# Conclusion
In order to provide an integrated system able to detect, characterize and identify a broad spectrum of both EU authorized and unauthorized GMOs in food/feed matrices, two bidirectional DNA walking methods targeting p35S or tNOS, the most common transgenic elements, were developed to be anchored on the sequences used for the p35S or tNOS qPCR SYBR®Green screening described by Barbau-Piednoir et al., 2010 [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref]. These DNA walking methods also allow to strengthen the previously published t35S pCAMBIA DNA walking method in order to currently target around 75 % of the GM crops [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref] [bib_ref] Validation of a sensitive DNA walking strategy to characterize unauthorised GMOs using..., Fraiture [/bib_ref] , personal communication.
First, the p35S and tNOS bidirectional DNA walking methods were developed and assessed for their specificity using 100 % Bt rice. These methods were evaluated as highly specific since no aspecific amplifications were generated in presence of the target. Second, the developed DNA walking methods were tested on a more complex maize food matrix, in term of genome size, containing approximately 10 % of the GM maize MON863 event. Finally, the entire workflow of the integrated system, including the detection of the potential presence of GMOs by qPCR screening with the p35S, tNOS and t35S pCAMBIA markers and, subsequently, the confirmation of their presence using the DNA walking methods corresponding to the previously obtained qPCR responses, was applied on a GeMMA Scheme Proficiency Test matrix, containing 1.2 % of the GM maize MON863 event, to illustrate its applicability in GMO routine analysis by the enforcement laboratories. For all tested matrices, the p35S and the tNOS bidirectional DNA walking methods were successfully applied as the GMO presence was proven via the characterization of the junction between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette.
In addition to its clear benefit in GMO detection, this integrated system has the advantage to present a simple procedure and a short time-frame to get the results. However, in order to analyse even more easily the PCR products derived from the DNA walking methods, some adaptations in the entire DNA walking workflow could be done regarding the purification of the generated amplicons excised from the electrophoresis gel and the subsequent sequencing using Sanger technology. Indeed, even if the initial DNA walking workflow remains simple, in case of matrices containing several GMOs, harbouring the same targeted element, the purification of the potential numerous amplicons excised from the electrophoresis gel and the subsequent Sanger sequencing could be cumbersome. This situation could be for instance encountered with matrices presenting a low amount of EU-unauthorized GMOs mixed with EUauthorized GMOs harbouring the elements p35S and/or tNOS, very frequently observed in GM crops. In this scenario, the obtained amplicons will present different sequences, representing potentially one GMO per observed DNA fragment. Therefore, the simplified workflow, consisting in selecting the largest size amplicons to obtain the most informative sequences, does not guarantee the entire representativeness of GMOs present in the tested sample. Consequently, it's preferable to analyse all amplicons observed on the electrophoresis gel and to eventually them using Sanger technology, which may be a quite laborious work. In the future, this difficulty could be circumvented in replacing the step related to the purification of the amplicons excised from the electrophoresis gel and the subsequent Sanger sequencing by a high-throughput Next-Generation-Sequencing approach, as suggested by .
# Methods
# Plant material
Grains of an insect resistant transgenic Bt rice (Oryza sativa L. Japonica cv Ariete), transformed by Agrobacterium tumefaciens with the binary vector pCAMBIA 1300 containing the synthetic Cry1B gene from Bacillus thuringiensis, and its corresponding wild-type (WT) were used in this study [bib_ref] Bt rice harbouring cry genes controlled by a constitutive or woundinducible promoter:..., Breitler [/bib_ref]. The CRM of the GM maize MON863 9.85 % (ERM-BF416c) in the form of seed powder was obtained from the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium). The food matrix (GeM SU34-A), coming from a GeMMA Scheme Proficiency Test, is a maize flour, tumble blended for 50 h, containing 1.2 % (w/w) of 100 % GM maize MON863.
## Dna extraction, concentration and purity
Using a CTAB-based procedure (ISO 21571) in combination with the Genomic-tip20/G (QIAGEN, Hilden, Germany), DNA was extracted from a homogenous powder of rice grain obtained by manual grinding. Adapted from the EU-RL GMFF validated method, this DNA extraction method was carried out by four main successive steps: Extraction of proteins, polysaccharides and organic components, precipitation of DNA in the presence of C-hexadecyl-Trimethyl-Ammonium-Bromide (CTAB), purification of DNA using a tip20 column and precipitation of DNA with isopropanol. DNA concentration was measured by spectrophotometry using the Nanodrop® 2000 (ThermoFisher, DE, USA) device and the DNA purity was evaluated using the A260/A280 and A260/A230 ratios. DNA extraction, concentration and purity of the CRM and the food matrix (GeM SU34-A) were carried out as previously described [bib_ref] New SYBR®Green methods targeting promoter sequences used for screening of several GM..., Broeders [/bib_ref].
## Qpcr sybr®green technology
All qPCR assays were performed as described in [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref] [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref] using the primers indicated in [fig_ref] Table 1: Oligonucleotide primers used for the real-time PCR assays, the DNA walking approaches... [/fig_ref] [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref] [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref]. More precisely, a standard 25 μl reaction volume was applied containing 1X SYBR®Green PCR Mastermix (Diagenode, Liège, Belgium), 250 nM of each primer and 5 μl of DNA (10 ng/μl). The qPCR cycling program consisted of a single cycle of DNA polymerase activation for 10 min at 95°C followed by 40 amplification cycles of 15 s at 95°C (denaturing step) and 1 min at 60°C (annealing-extension step). The program for melting curve analysis was performed by gradually increasing the temperature from 60 to 95°C in 20 min (±0.6°/20 s). All runs were performed on an iQ™5 real-time PCR detection system (BioRad, Hemel Hempstead, UK). For each assay, a "No Template Control" (NTC) was included.
## Dna walking approach development and assessment of oligonucleotide primers
Two DNA walking approaches have been developed to target the p35S or tNOS elements. For each method, three target-specific primers are required to carry out first the DNA walking (a) and then the first (b) and the second (c) semi-nested PCR rounds. To provide an integrated approach, the design of the target-specific primers a and c is based on the sequences from the SYBR®Green real-time PCR screening markers p35S or tNOS published by Barbau-Piednoir et al., 2010 [bib_ref] SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS..., Barbau-Piednoir [/bib_ref]. An intermediate primer, corresponding to the target-specific primer b, was additionally designed. From each targeted transgenic element, two walking directions, called forward (F) and reverse (R) methods, have been performed [fig_ref] Figure 1: Development of the bidirectional p35S and tNOS DNA walking methods on 100... [/fig_ref]. Using the program "wprimersearch" from the software "wEMBOSS", that mimics PCR amplification, the specificity of oligonucleotide primers was initially assessed in silico [23].
## Dna walking strategy
## Dna walking and double semi-nested pcr reactions
The DNA walking strategy previously described by [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref] was adapted in this study to target the transgenic element p35S or tNOS [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref]. Similarly, a first reverse target-specific primer (a) and one kind of the degenerated random tagging primer (DRT) mix (A-D) were applied, in a first step, followed by two semi-nested PCR rounds using target-specific primers (b and c), that are each time nested to the previous reverse target-specific primer, combined to universal tagging primers (UAP-N1 and UAP-N2) [bib_ref] An innovative and integrated approach based on DNA walking to identify unauthorised..., Fraiture [/bib_ref]. All these methods were applied on 100 ng of DNA from 100 % of Bt rice and its corresponding WT as well as on 100 ng of DNA from the food matrix (GeM SPU34-A) and its corresponding CRM (GM maize MON863 9.85 %). Moreover, a NTC was included for each assay. PCR mixes and conditions were carried out according to the manufacturer's instructions (APAgene™ GOLD Genome Walking Kit from BIO S&T, Montréal, Canada). The final PCR products were analysed by electrophoresis on a 1 % agarose gel (INVITROGEN, CA, USA) (100 V, 400 mA, 60 min) in view to further analysis allowing to identify the generated sequences.
Analysis and workflow In order to assess the specificity of the developed p35S and tNOS bidirectional methods, all the visualized amplicons produced from the 100 % Bt rice and WT rice were excised from agarose gel and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, WI, USA) to then be sequenced and identified.
Next, to test the developed methods on a maize matrix, the CRM of maize MON863 (9,85 %) was used and a workflow convenient for the GMO routine analysis was followed. For each DNA walking method, only the longest and easily selectable amplicon observed for each DRT primer mix was excised from agarose gel and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, WI, USA) to be sequenced.
To test the applicability of the entire integrated system, a simplified workflow was used for the food matrix (GeM SPU34-A). Only the longest and easily selectable amplicon observed for each DNA walking method was excised from agarose gel and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, WI, USA) to be sequenced.
## Cloning and sequencing
Three different sequencing approaches were used to obtain the sequence of the selected amplicons. First, a direct sequencing was applied using the corresponding target-specific c primer or the UAP-N2 primer. Second, in case of an unsatisfying size or quality of the obtained sequences, two other sequencing approaches were carried out. On the one hand, a cloning strategy was performed. The amplicons were cloned into the pGEM®-T Easy Vector Systems (PROMEGA, WI, USA), according to the manufacturer's instructions. A PCR was carried out on colonies using pGEM®-T Easy Vector primers (T7: TAATACGACTCACTATAGGG; SP6: ATTTAGGT GACACTATAGAAT) and was analyzed by electrophoresis on a 1 % agarose gel (INVITROGEN, CA, USA) (100 V, 400 mA, 60 min). The colonies presenting a fragment of the correct size were then sequenced. On the other hand, an "enrichment" strategy, based on a PCR amplification using the corresponding targetspecific c primer and the modified UAP-N2 primer coupled to the T7 sequence (UAP-N2_T7: TTTAATAC GACTCACTATAGGGGGAAGCAGTGGTATCAACG), was used. To this end, a standard 25 μl reaction volume was applied containing 0.625 U of DreamTaq™ DNA Polymerase (Fermentas, CA, USA), 1X DreamTaq™ Buffer (Fermentas, CA, USA), 0.2 mM of dNTPs, 250 nM of each primer and 5 μl of the purified amplicon. The PCR program consisted of a single cycle of 3 min at 95°C (initial denaturation) followed by 45 amplification cycles of 30 s at 95°C (denaturation), 30 s at 50°C (annealing) and 4 min at 72°C (extension) and finishing by a single cycle of 10 min at 72°C (final extension). The run was performed on an iQ™5 real-time PCR detection system (BioRad, Hemel Hempstead, UK). The PCR products were analysed by electrophoresis on a 1 % agarose gel (100 V, 400 mA, 60 min; INVITROGEN, CA, USA) and purified using USB® ExoSAP-IT® PCR Product Cleanup (Affymetrix, CA, USA), according to the manufacturer's instructions, to be then sequenced via the T7 primer.
All sequencing reactions were performed on a Genetic Sequencer 3130XL using the Big Dye Terminator Kit v3.1 (Applied Biosystems, CA, USA). The sequences were aligned and analysed using "ClustalW2" software and "Nucleotide BLAST NCBI" software, respectively.
## Verification of the transgene flanking regions by pcr amplification
The two different transgene flanking regions between the right border of the pCAMBIA cassette and the rice genome identified by the tNOS-F DNA walking method were verified by PCR amplification using the tNOS-F c primer combined to a primer designed on the rice chromosome II or III [fig_ref] Table 1: Oligonucleotide primers used for the real-time PCR assays, the DNA walking approaches... [/fig_ref]. These oligonucleotide primers were initially evaluated in silico using the program "wprimersearch" from the software "wEMBOSS" [23]. A standard 25 μl reaction volume was applied containing 0.625 U of DreamTaq™ DNA Polymerase (Fermentas, CA, USA), 1X DreamTaq™ Buffer (Fermentas, CA, USA), 0.2 mM of dNTPs, 250 nM of each primer and 5 μl of Bt rice DNA (5 ng/μl). The PCR program consisted of a single cycle of 3 min at 95°C (initial denaturation) followed by 35 amplification cycles of 30 s at 95°C (denaturation), 30 s at 55°C or 60°C respectively for the rice chromosome III or II (annealing) and 1 min at 72°C (extension) and finishing by a single cycle of 10 min at 72°C (final extension). The run was performed on an iQ™5 real-time PCR detection system (BioRad, Hemel Hempstead, UK). The PCR products were analysed by electrophoresis on a 1 % agarose gel (100 V, 400 mA, 60 min; INVITROGEN, CA, USA) and purified using USB® ExoSAP-IT® PCR Product Cleanup (Affymetrix, CA, USA), according to the manufacturers' instructions, in order to be sequenced. All sequencing reactions were performed on a Genetic Sequencer 3130XL using the Big Dye Terminator
[fig] Figure 1: Development of the bidirectional p35S and tNOS DNA walking methods on 100 % Bt rice. a Visualisation of the obtained amplicons, numeroted from 1 to 94, using the p35S and tNOS DNA walking methods applied on 100 ng of 100 % Bt rice and WT rice. For each method, four different DRT primer mixes (A-D) have been used. b For each DNA walking method, a schematic representation of the potential start position and direction, applied on the transgenic cassette of the Bt rice, is illustatred by the black arrows. Below the transgenic cassette, the sequence covering of the obtained amplicons from the 100 % Bt rice is schematically represented by rectangles. The corresponding amplicon numbering is indicated in thea. LB (left border); t35S (CaMV 35S terminator); hpt (hygromycin phosphotransferase gene); p35S (CaMV 35S promoter); lacZ (LacZ alpha fragment); pUBI (maize ubiquitin promoter); Cry1B (synthetic Cry1B gene); tNOS (Agrobacterium tumefaciens nopaline synthase terminator); RB (right border); rice (rice genome) [Schema adaptedfrom 24] [/fig]
[fig] Figure 2: Application of the bidirectional p35S and tNOS DNA walking methods on GM maize matrices. a Visualisation of the obtained amplicons using the p35S and tNOS DNA walking methods applied on 100 ng of the GM MON863 maize CRM (9.85 %). For each method, four different DRT primer mixes (A-D) have been used. The analyzed amplicons are indicated by a numerotation going from 1 to 16. b [/fig]
[table] Table 1: Oligonucleotide primers used for the real-time PCR assays, the DNA walking approaches and the PCR confirmation of the transgenic junctions [/table]
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10.14336/AD.2017.0110
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CCBY
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5614320
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28966800
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s2orc_pubmed_articles
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Parity History Determines a Systemic Inflammatory Response to Spread of Ovarian Cancer in Naturally Aged Mice
Aging intersects with reproductive senescence in women by promoting a systemic low-grade chronic inflammation that predisposes women to several diseases including ovarian cancer (OC). OC risk at menopause is significantly modified by parity records during prior fertile life. To date, the combined effects of age and parity on the systemic inflammation markers that are particularly relevant to OC initiation and progression at menopause remain largely unknown. Herein, we profiled a panel of circulating cytokines in multiparous versus virgin C57BL/6 female mice at peri-estropausal age and investigated how cytokine levels were modulated by intraperitoneal tumor induction in a syngeneic immunocompetent OC mouse model. Serum FSH, LH and TSH levels increased with age in both groups while prolactin (PRL) was lower in multiparous respect to virgin mice, a finding previously observed in parous women. Serum CCL2, IL-10, IL-5, IL-4, TNF-α, IL1-β and IL-12p70 levels increased with age irrespective of parity status, but were specifically reduced following OC tumor induction only in multiparous mice. Animals developed hemorrhagic ascites and tumor implants in the omental fat band and other intraperitoneal organs by 12 weeks after induction, with multiparous mice showing a significantly extended survival. We conclude that previous parity history counteracts aging-associated systemic inflammation possibly by reducing the immunosuppression that typically allows tumor spread. Results suggest a partial impairment of the M2 shift in tumor-associated macrophages as well as decreased stimulation of regulatory B-cells in aged mice. This long term, tumor-concurrent effect of parity on inflammation markers at menopause would be a contributing factor leading to decreased OC risk. [bib_ref] Menopause and ovariectomy cause a low grade of systemic inflammation that may..., Abu-Taha [/bib_ref] [bib_ref] Effects of aging and menopause on serum interleukin-6 levels and peripheral blood..., Kim [/bib_ref] [bib_ref] Factors associated with inflammation markers, a cross-sectional analysis, Clendenen [/bib_ref] [bib_ref] Proinflammatory and anti-inflammatory cytokine changes related to menopause, Malutan [/bib_ref] [bib_ref] Changes in serum cytokine concentrations during the menopausal transition, Yasui [/bib_ref] [bib_ref] Effects of menopause and postmenopausal tibolone treatment on plasma TNFalpha, IL-4, IL-10,..., Vural [/bib_ref] [bib_ref] Cytokine pattern in postmenopause, Cioffi [/bib_ref] [bib_ref] Postmenopausal changes in production of type 1 and type 2 cytokines and..., Deguchi [/bib_ref] [bib_ref] Different circulating levels of monocyte chemoattractant protein-1 and interleukin-8 during the menopausal..., Tani [/bib_ref] , which acting in combination, could lead to the observed low-grade systemic inflammatory state.
Ovarian cancer (OC) incidence and mortality significantly increases with age, both parameters reaching a peak during post-menopause. Elevations of some of the above mentioned cytokines have been shown a direct correlation with increased OC risk [bib_ref] Inflammatory Markers and Risk of Epithelial Ovarian Cancer by Tumor Subtypes: The..., Ose [/bib_ref] [bib_ref] Pre-diagnostic serum levels of inflammation markers and risk of ovarian cancer in..., Trabert [/bib_ref] [bib_ref] The immune system in the pathogenesis of ovarian cancer, Charbonneau [/bib_ref] [bib_ref] Circulating interleukin-8 and plasminogen activator inhibitor-1 are increased in women with ovarian..., Aune [/bib_ref] , while others have been linked to reproductive hormones known to be altered during the menopausal transition. The gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) typically increase at menopause due to impairment of the hypothalamic-pituitary-gonadal (HPG) axis and, importantly, have been proposed to play a role in OC pathogenesis [bib_ref] Gonadotropin signalling in epithelial ovarian cancer, Mertens-Walker [/bib_ref]. Extending this link, plasma levels of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α were positively correlated with circulating FSH in premenopausal women [bib_ref] Follicle-stimulating hormone, interleukin-1, and bone density in adult women, Cannon [/bib_ref]. Similarly, the C-C motif chemokine ligand 2 (CCL2/MCP-1) was positively correlated with FSH levels during the early and late menopausal transition [bib_ref] Different circulating levels of monocyte chemoattractant protein-1 and interleukin-8 during the menopausal..., Tani [/bib_ref]. In female rheumatoid arthritis patients, short-term fluctuations of both FSH and LH were positively associated with changes in levels of TNF-α, IL-1β and other pro-inflammatory cytokines [bib_ref] The association of luteinizing hormone and follicle-stimulating hormone with cytokines and markers..., Kass [/bib_ref].
A major contributing factor to the roles of menopausal hormone imbalances and the direct effect of certain cytokines on OC risk and progression in postmenopausal women, is their reproductive history during precedent fertile phase [bib_ref] Oral contraceptive use and reproductive factors and risk of ovarian cancer in..., Tsilidis [/bib_ref]. One or more full-term pregnancies significantly reduces OC risk, while nulliparity increases the risk [bib_ref] Hormonal risk factors and invasive epithelial ovarian cancer risk by parity, Bodelon [/bib_ref]. Since an equivalent extent of OC risk reduction is achieved by progestinbased oral contraceptives, protection could be the result of a reduced number of ovulatory cycles concomitant with increased progesterone (P4) or progestin exposures [bib_ref] Ovarian cancer: etiology, risk factors, and epidemiology, Hunn [/bib_ref]. Thus, uninterrupted ovulation might play a role in OC origin through cumulative DNA damage incurred during the repetitive cycles of wound rupture-repair of the ovarian surface epithelium [bib_ref] Oxidative damage to DNA of ovarian surface epithelial cells affected by ovulation:..., Murdoch [/bib_ref] while the preventive actions of P4 (or progestin) could be mediated by an apoptotic effect [bib_ref] Progesterone-induced apoptosis in immortalized normal and malignant human ovarian surface epithelial cells..., Syed [/bib_ref] or induction of senescence on transformed ovarian surface cells [bib_ref] Progesterone receptors induce FOXO1-dependent senescence in ovarian cancer cells, Diep [/bib_ref]. Whatever the OC pro-or anti-oncogenic mechanisms are, they have to be long-term, i.e. they should develop and persist during cell and tissue aging. were maintained from 3-20 months old in virgin (nulliparous) or multiparous conditions. A smaller third group, composed of young adult 4 months old virgin mice (n=6), was used as reference controls in some assays. Multiparous mice were allowed to breastfeed their pups until 21-days old. Circulating cytokines and hormones were measured once per month in 3-4 distinct, randomly taken mice aged 15-19 months old. Tumor induction was initiated at 16 months old (n=8 per condition) with cytokines and hormones measured similarly. The age scale shown is not proportional. See Methods section for additional details. (B) Chart corresponds to the total number of litters as a function of age. (C) depicts the distribution of number of litters per individual mice over the reproductive period, both data-sets for the entire multiparous group.
With the goal of investigating how parity modulates chronic, age-related systemic inflammation and how that modulation contributes to OC risk at menopause, we herein describe a profile of circulating hormones and cytokines in multiparous versus virgin C57BL/6 female mice subjected to tumor induction at peri-estropausal age. From over 30 cytokines evaluated, seven increased with age regardless of parity status. The same seven cytokines decreased only in multiparous mice as a response to tumor induction with transformed OC syngeneic cells. Both virgin and multiparous mice developed intraperitoneal tumors and ascitic fluid, but multiparous animals showed an extended survival. Our results suggest that parity supports and extends the host immune response against intraperitoneal OC tumor spread during menopause. [fig_ref] Figure 1: Study design and parity records [/fig_ref] summarizes the experimental protocol of this work. Wild type C57BL/6 mice were housed at Bioterio Central of Facultad de Medicina, Universidad de Chile. The animal study, protocol Nº 0536, was approved by the institutionś Comité de Bioetica. Wild type C57BL/6 mice were maintained under 12/12 hours' light/dark schedule with regular food and water ad libitum. Virgin adult female mice, 12 weeks old, were randomly assigned to two experimental groups, virgin and multiparous (n=70 per group). Breeding trios were used for the multiparous mice; the trio included a fertile adult male mouse of proven fertility. Virgin mice were housed three females per cage in the absence of a male. Separate cages with virgin and with multiparous mice were maintained in close proximity, sharing the same rack to preserve estrous cycling in the virgin animals [bib_ref] E-cadherin expression and bromodeoxyuridine incorporation during development of ovarian inclusion cysts in..., Fleming [/bib_ref]. To evaluate reproductive aging, the length of estrous cycles was measured in the virgin group. Estrous cycling was addressed by fresh vaginal cytology at 10:00 AM during 15 consecutive days in random subsets of six virgin mice at 4, 15 and 18 months of age [bib_ref] Assessing reproductive status/stages in mice, Caligioni [/bib_ref]. Pups of multiparous mice were weaned at 21-days old. Diestrous stage blood samples from four randomly chosen mice of each group, were collected monthly by submandibular bleeding [bib_ref] A rapid, simple, and humane method for submandibular bleeding of mice using..., Golde [/bib_ref]. The blood collection period for the intact, untreated mice group was between 15-19 months of age, and for the tumor-induced mice was between 16-19 months of age. Blood was incubated at room temperature for 30 min and centrifuged at 1000g for 5 min. Supernatant sera was carefully pipetted and stored at -20º C in 25 L aliquots for further analysis. Control blood samples were collected and processed similarly from 4 months old female mice at diestrous stage of cycle.
# Materials and methods
## Animals and samples collection
## Tumor induction in a syngeneic mouse model
With the goal of evaluating parity-dependent tumor progression, spontaneously-transformed mouse ovarian surface epithelial (MOSE) cells were injected in the peritoneal cavity of aged mice of both conditions. The IG-10 clonal line of MOSE cells was kindly provided by Dr Katherine Roby (UKMC, Kansas). Cells were cultured in DMEM supplemented with ITS (Sigma-Aldrich, MO) under 5% CO2 atmosphere [bib_ref] Development of a syngeneic mouse model for events related to ovarian cancer, Roby [/bib_ref]. When cultures reached 90% confluency, cells were released with trypsin, centrifuged at low speed and suspended in a minimal volume of HBSS solution (Sigma-Aldrich, MO). An aliquot was diluted and counted with a hemocytometer. A suspension volume containing 5x10 6 cells was injected intraperitoneally with a tuberculin syringe to 16 monthsold multiparous (n=8) and virgin (n=7) mice. One month earlier, males were removed from the multiparous mice cages. A set of control mice (n=4) was injected with HBSS vehicle solution. Animals were monitored once per week after injection. Sera samples were obtained from all animals two weeks after the injection and then monthly until death. Upon initial signs of ascitic fluid accumulation evaluated by abdominal enlargement, mice were supervised daily. Euthanasia by cervical dislocation was conducted according to signs of stress and affliction described by Morton and Griffiths [bib_ref] Guidelines on the recognition of pain, distress and discomfort in experimental animals..., Morton [/bib_ref]. Immediately upon death, blood samples and ascitic fluid were collected and processed for further analysis. Volumes of hemorrhagic ascitic fluid were measured (range 2-9 mL), clarified by centrifugation at 1,000g for 10 min, and stored at -20º C in 0.5 mL aliquots. Intraperitoneal tumor loads were graded and scored as described by Roby et al [bib_ref] Development of a syngeneic mouse model for events related to ovarian cancer, Roby [/bib_ref].
## Circulating pituitary hormones and cytokines
Hormones and cytokines were quantified in serum using multiplex, magnetic bead-based Luminex® xMAP® assays manufactured by Milliplex® (Merck, USA). Specifically, the serum pituitary hormones FSH, LH, TSH and prolactin (PRL) were measured with the MPTMAG-49K panel and cytokines CCL2, IL-10, IL-5, IL-4, TNFa, IL-1b, IL-12p70 and CXCL10 were quantified with the MCYTMAG-70K-PX32 panel. Assay methods were followed as indicated by the supplier. Prior to each assay, test samples were randomized and their identities were blind to the operators. In brief, serum samples were either used directly (10 L; MPTMAG-49K kit) or diluted with an equal volume of assay buffer (25L; MCYTMAG-70K-PX32 kit). Samples were loaded to the plate along with controls and standards provided with the kits. Then, 25 L of the Pre-mixed Beads mixture were added to each well, the plate was sealed, covered with foil and incubated overnight in a shaker at 4°C. The entire content of wells Aging and Disease - Volume 8, Number 5, October 2017 was gently discarded. Plate was washed twice, 25-50 L of Detection Antibodies solution were added and incubated 1 hr at room temperature with shaking. Then, 25-50 L of Streptavidin-Phycoerythrin solution were added and incubated further 30 min at room temperature. The entire content of wells was gently discarded and washed twice. Finally, 100-150 L of Sheath Fluid were added to each well and beads resuspended by shaking for 5 min. Median fluorescence intensity (MFI) of beads was read in a Luminex 200™ instrument (Luminex Corp., USA) operated through the xPONENT 3.1 software. Data processing was conducted with the Milliplex Analyst 3.5.5.0 software (Merck, USA) using the 5-parameter logistic curve.
## Statistics
Hormone and cytokine results are plotted as mean ± standard error of the mean. Since data was not distributed normally, the Kruskal-Wallis test was used to compare non-paired data among young and aged groups. The Dunn's post test was applied for comparisons among these 3 conditions. The two tumor-induced, aged groups were separately compared with a Mann-Whitney U test. Significance was set at p<0.05. Analysis was performed with the GraphPad Prism 5.0 software.
# Results
## Parity records, estrous cycling and pituitary hormone levels
For the parous group composed of 70 mice, a total of 202 litters were recorded. Seven (7) mice never had litters and were thus excluded from the study. This resulted in a mean of 3.2 litters per mouse. As shown in the histogram of [fig_ref] Figure 1: Study design and parity records [/fig_ref] , the maximum reproductive yield was observed at 6 months of age, and 95% of the litters were delivered before the age of 12 months. The number of litters, as a function of age, was consistent with a Gaussian distribution as indicated by three normality tests (not shown). Individual reproductive rates showed that 47.6% of mice had 1-2 litters, while another 46.0% had 3-6 litters [fig_ref] Figure 1: Study design and parity records [/fig_ref]. The mean length of estrous cycles increased from 4.6 days at four months of age, to 9.7 days in 15-18 months-old mice, a result that indicated reproductive senescence in the animals to be studied. The mean serum levels of FSH, LH, TSH and prolactin (PRL) in mice aged 15-19 months are shown in [fig_ref] Figure 2: Circulating pituitary hormones in aged mice [/fig_ref]. FSH, LH and TSH were significantly increased in aged versus young mice, regardless of parity history. PRL levels were unchanged between young and aged virgin mice, but were significantly lower in the aged multiparous group.
## Parity-dependent cytokine profiles
Two predominant patterns of circulating cytokine levels were observed in aged mice: those increased with age regardless of parity -and then specifically altered in response to intraperitoneal tumor induction [fig_ref] Figure 3: Circulating cytokines in control and tumor-induced aged mice [/fig_ref] , and those differing as a sole result of parity (manuscript in preparation). CCL2, IL10, IL5, IL4, TNF-α, IL1-beta and IL12p70 belong to the first pattern. TNF-α, IL1-β and IL4 were significantly increased in aged multiparous mice relative to young mice, and the same tendency was observed in the aged virgin group (TNF-α p=0.057; IL1beta p=0.067; IL4 p=0.024, unpaired t-test with Welch's correction). CXCL10 was distinctive in that no significant increase was observed with age but its parity-dependent tumor response was in the opposite direction with respect to the other cytokines. IL12p70 increased in both groups of aged mice relative to young mice, but their level variations upon tumor response did not reach significance.
## Cytokine profiles and host survival after intraperitoneal tumor induction
Spontaneously-transformed MOSE cells have been widely used as a syngeneic model of OC to study various disease aspects including intraperitoneal tumor dissemination and therapeutic approaches. Here, circulating cytokine levels in both virgin and multiparous mice during tumor progression are shown as dashed bars in [fig_ref] Figure 3: Circulating cytokines in control and tumor-induced aged mice [/fig_ref]. As judged by abdominal enlargement in most cases, ascitic fluid started to accumulate 7-12 days before death, consistent with description provided by Roby et al., who developed this model in young adult mice [bib_ref] Development of a syngeneic mouse model for events related to ovarian cancer, Roby [/bib_ref]. Typically, abundant tumor implants were located in the omental fat band. Implants of smaller size were found in the diaphragm, mesenterium and the pelvic region next to ovaries/oviducts and uterine horns [fig_ref] Figure 4: Tumor spread and survival of host aged mice [/fig_ref]. Median survival times were 87 days (range 72-98) for virgin mice and 98 days (range 87-112) for multiparous mice.
Survival curves were significantly different as determined by two tests (p=0.041, log-rank test; p=0.038, Gehan-Breslow-Wilcoxon Test). [fig_ref] Figure 4: Tumor spread and survival of host aged mice [/fig_ref] shows survival plots. The ascitic fluid volume, tumor load, and percentage of ascitic fluid with respect to body weight at time of death, were not statistically different between conditions.
# Discussion
Natural ovarian decline is associated with hormone and inflammatory changes that overlap with those occurring as a consequence of aging in women. In addition, as suggested from epidemiological data in various populations, periods of suppressed ovulation plus P4 (or progestin) exposure over womanś fertility span are major determinants of OC risk later in menopause [bib_ref] Ovarian cancer: etiology, risk factors, and epidemiology, Hunn [/bib_ref]. We have approached this subject by using a syngeneic immunocompetent OC mouse model maintained under divergent host reproductive conditions (virgin and multiparous) until the peri-estropausal age. Using mouse to study age-associated cancer immunomodulation is supported by recent findings suggesting that the agedependent evolution (adult, mature, old and long lived) of immune functions such as chemotaxis, phagocytosis, natural killer activity and lymphoproliferation is similar between humans and mice of equivalent chronological ages [bib_ref] Immune function parameters as markers of biological age and predictors of longevity, Martinez De Toda [/bib_ref]. Estrous cycle lengthening and increased serum levels of the gonadotropins FSH and LH observed by ≥ 15 months-old in aged mice, were indicative of ovarian senescence and a declining control of the HPG axis. Since gonadotropins in aged animals were higher than in young, but lower than in ovariectomized mice (data not shown), mice of the age range studied in the present work would be equivalent to a human in late menopausal transition or early post-menopause [bib_ref] The menopause and aging, a comparative perspective, Finch [/bib_ref]. Of interest was the significantly reduced PRL level in aged multiparous mice relative to aged virgin mice, a finding consistent with low PRL levels reported in parous women and in those taking oral contraceptives [bib_ref] Circulating prolactin levels and risk of epithelial ovarian cancer, Clendenen [/bib_ref] , factors known to decrease OC risk. PRL exerts pleiotropic metabolic and growth-like effects in various target tissues. In rodents, PRL is synthesized exclusively by the pituitary while in humans it is also produced by several tissues, most interestingly, immune B and T cells. The PRL profile during both the reproductive cycle and pregnancy differs between human and rodents. Besides its function in lactogenesis, PRL plays a luteotropic role in mice to maintain pregnancy [bib_ref] What can we learn from rodents about prolactin in humans?, Ben-Jonathan [/bib_ref]. Importantly, the first pregnancy in women induces a decrease of basal and stimulated PRL levels compared to nulliparous controls [bib_ref] Long-term effect of a first pregnancy on the secretion of prolactin, Musey [/bib_ref]. This effect has been replicated in rats and is linked to a decreased oxidative burst, enhanced phagocytic ability, as well as production of nitric oxide and TNF-α by peritoneal macrophages in multiparous animals [bib_ref] Prior reproductive experience alters prolactin-induced macrophage responses in pregnant rats, Carvalho-Freitas [/bib_ref] [bib_ref] Production of nitric oxide by murine peritoneal macrophages in vitro on treatment..., Tripathi [/bib_ref]. Increasing evidence involves elevated PRL levels in the pathogenesis of certain autoimmune diseases [bib_ref] Prolactin and autoimmunity, Shelly [/bib_ref]. PRL has been recently proposed to impair the tumor-suppressive function of BRCA1 by downregulating expression of the cell cycle inhibitor p21 in cancer cell lines [bib_ref] Prolactin inhibits a major tumor-suppressive function of wild type BRCA1, Chen [/bib_ref]. We also observed higher TSH levels in both aged mice groups regardless of parity history. Though normal serum TSH values have not been clearly defined in the elderly [bib_ref] Diagnosis and Management of Subclinical Hypothyroidism in Elderly Adults: A Review of..., Hennessey [/bib_ref] , TSH levels increase with age in humans [bib_ref] Serum TSH, T(4), and thyroid antibodies in the United States population (1988..., Hollowell [/bib_ref] leading to an apparent sub-clinical hypothyroid condition which has been linked to the risk of atherosclerosis [bib_ref] Even mildly elevated TSH is associated with an atherogenic lipid profile in..., Geng [/bib_ref] , among other chronic diseases.
From a total of 30 circulating cytokines measured in aged female mice in this study, 14 cytokines showed parity-dependent differential serum levels. [fig_ref] Figure 3: Circulating cytokines in control and tumor-induced aged mice [/fig_ref] shows a set of seven cytokines that increased with age, regardless of parity, and decreased in response to tumor induction only in multiparous mice. Notably, this pattern observed in multiparous animals was correlated with a significantly increased survival [fig_ref] Figure 4: Tumor spread and survival of host aged mice [/fig_ref] , suggesting that OC risk reduction associated with parity might involve a particular ability of the senescent immune system of parous mice to react better against the tumor challenge. An exception to this pattern was the chemokine CXCL10, which did not increase significantly with age but exhibited a unique and significant increase in multiparous mice upon tumor induction. A second major pattern included cytokines higher in intact multiparous mice as a sole result of parity (manuscript in preparation). As previously described in young adult mice [bib_ref] Development of a syngeneic mouse model for events related to ovarian cancer, Roby [/bib_ref] , the MOSE cells used in this study induced ascitic fluid accumulation and extensive peritoneal carcinomatosis, mostly in the omental fat band of aged animals [fig_ref] Figure 4: Tumor spread and survival of host aged mice [/fig_ref] , both characteristics frequently observed in human OC [bib_ref] Ovarian cancer microenvironment: implications for cancer dissemination and chemoresistance acquisition, Thibault [/bib_ref].
Among these cytokines, we detected CCL2 (MCP-1) which participates in monocyte recruitment and tissue infiltration, with subsequent differentiation to macrophages. In agreement with the present results in mice, in women CCL2 increased during late menopausal transition showing a positive correlation with FSH [bib_ref] Different circulating levels of monocyte chemoattractant protein-1 and interleukin-8 during the menopausal..., Tani [/bib_ref]. CCL2 expression by luteal cells during corpus luteum (CL) regression enhances macrophage infiltration, a process that seems to be reversed by local P4 and luteotrophic prostaglandin E [bib_ref] Regulated C-C motif ligand 2 (CCL2) in luteal cells contributes to macrophage..., Nio-Kobayashi [/bib_ref]. Only in multiparous aged mice, e.g., animals exposed to P4 during pregnancies early in life, did our results show lower CCL2 levels in response to tumor induction [fig_ref] Figure 3: Circulating cytokines in control and tumor-induced aged mice [/fig_ref]. This tumor invasiondependent decrease of CCL2 at estropause might represent a long-term effect of P4 on infiltration of tumorassociated macrophages (TAMs) during peritoneal tumor spread in this model. In agreement with our results, parity significantly reduced omental monocyte subsets and B1-B lymphocytes in the MOSE model at middle age, with concomitant decreased expression of various chemokines and polarization factors including CCL2 [bib_ref] The parity-associated microenvironmental niche in the omental fat band is refractory to..., Cohen [/bib_ref]. In other tissue contexts, recent reports indicate that progestin suppressed TNF-α induced proliferation and CCL2 secretion of endometrial stromal cells in vitro [bib_ref] Progestin suppressed inflammation and cell viability of tumor necrosis factor-alpha-stimulated endometriotic stromal..., Grandi [/bib_ref] , while P4 prevented macrophage infiltration to brain endothelial cells by blocking CCL2 action after an ischemic stroke [bib_ref] Progesterone protects endothelial cells after cerebrovascular occlusion by decreasing MCP-1-and CXCL1-mediated macrophage..., Remus [/bib_ref]. Importantly, high CCL2 levels have been reported in the ascitic fluid of OC patients [bib_ref] Biochemical composition of malignant ascites determines high aggressiveness of undifferentiated ovarian tumors, Mikula-Pietrasik [/bib_ref].
In the present study, the Th2-class cytokines IL-10, IL-5 and IL4 were also decreased in response to the tumor challenge, but only in multiparous mice. Th1 and Th2 represent two CD4+ T-cells subsets each expressing a distinctive cytokine repertoire. Th1 cells are involved in cell-mediated immunity, whereas Th2 cells participate in the humoral immune response. The normal Th1/Th2 balance becomes altered with aging [bib_ref] Advanced age impairs macrophage polarization, Mahbub [/bib_ref] [bib_ref] Normal aging is associated with an increase in Th2 cells, MCP-1 (CCL1)..., Mansfield [/bib_ref] and during pregnancy [bib_ref] Inflammation and pregnancy, Challis [/bib_ref]. Consistent with our results in aged mice, serum IL-10 and IL-4 increased at menopause [bib_ref] Changes in serum cytokine concentrations during the menopausal transition, Yasui [/bib_ref] [bib_ref] Postmenopausal changes in production of type 1 and type 2 cytokines and..., Deguchi [/bib_ref] , whereas IL-5 has not yet been linked to womenś reproductive aging. Th1 and Th2 responses typically involve macrophage differentiation towards the M1 and M2 polarized phenotypes. The M2 shift observed in a subset of TAMs was characterized by high IL-10 expression leading to immunosuppression and subsequent tumor promotion [bib_ref] Macrophage plasticity and polarization: in vivo veritas, Sica [/bib_ref]. Furthermore, OC stem cells promoted in vitro M2 polarization of raw 264.7 macrophages through the PPARγ/NF-κB pathway [bib_ref] Ovarian cancer stem cells induce the M2 polarization of macrophages through the..., Deng [/bib_ref]. In our model, as the age-induced IL-10 increase was reversed only in multiparous mice exposed to tumorigenic cells, we suggest that parity counteracts tumor immunosuppression by partially repressing M2 polarization of TAMs.
Mediated by pregnancy hormones, the immune system undergoes adaptive changes to tolerate the fetus [bib_ref] Endocrine factors modulating immune responses in pregnancy, Schumacher [/bib_ref] [bib_ref] Progesterone promotes maternal-fetal tolerance by reducing human maternal Tcell polyfunctionality and inducing..., Lissauer [/bib_ref]. Among these, mature B-cells accumulate in bone marrow, in lymph nodes draining the uterus and in the peritoneum of pregnant mice [bib_ref] B cell development undergoes profound modifications and adaptations during pregnancy in mice, Muzzio [/bib_ref]. Both progesterone and estradiol repress IL-10 expression in a subpopulation of splenic B-cells during mouse pregnancy [bib_ref] Progesterone and estradiol exert an inhibitory effect on the production of anti-inflammatory..., Bommer [/bib_ref]. IL-10 is a major effector of B-cell immune regulatory properties [bib_ref] B cell regulation of the anti-tumor response and role in carcinogenesis, Schwartz [/bib_ref] , and has been recently implicated in the weak antitumor immunity observed in OC [bib_ref] Regulatory B cells contribute to the impaired antitumor immunity in ovarian cancer..., Wei [/bib_ref]. In contrast to the effect of IL-10 on B cells, a Th2 shift mediated by transient high IL-4 levels during initial stages of human pregnancy contributes to fetal tolerance and survival [bib_ref] Inflammation and pregnancy, Challis [/bib_ref]. Interestingly, given that IL-4 receptors (IL-4R) are highly expressed in OC and other neoplasia, the IL-4/IL-4R pair has been attempted as a target for OC immunotherapy [bib_ref] Expression and targeting of interleukin-4 receptor for primary and advanced ovarian cancer..., Kioi [/bib_ref]. Because the age-dependent high levels of IL-10, IL-4 and IL-5 were subsequently decreased by tumor induction only in multiparous mice in the present model, we suggest that parity during fertile life might improve antitumor immunity in aged mouse challenged with tumor-inducing cells. In other words, in contrast to aged virgin mice which seem to be unresponsive to OC tumor invasion, the immune system of an aged multiparous mouse would be less tumor tolerant and able to better suppress OC growth. The question then becomes, which of the variables we have noted in multiparous versus virgin aged mice would be responsible for this protection? We have evidence for at least two converging mechanisms that could be in operation: impairment of the M2 macrophage shift and decreased stimulation of regulatory B-cells.
Similar to IL-10, the pro-inflammatory M1-type cytokines TNF-α and IL-1beta have both been shown to increase with age in women thus contributing to the systemic low-grade chronic inflammation status [bib_ref] Effects of menopause and postmenopausal tibolone treatment on plasma TNFalpha, IL-4, IL-10,..., Vural [/bib_ref] [bib_ref] Proinflammatory cytokines, aging, and age-related diseases, Michaud [/bib_ref]. TNF-α is produced mainly by activated macrophages, Aging and Disease - Volume 8, Number 5, October 2017 monocytes and lymphocytes, and participates in multiple normal physiological responses as well as pathogenic processes [bib_ref] TNF-mediated inflammatory disease, Bradley [/bib_ref]. In the present study, the levels of TNF-α, IL1-β and IL12p70 increased in both aged groups irrespective of parity, and decreased as a response to tumor spread only in the parous group, in a similar fashion as observed for the M2-type cytokines [fig_ref] Figure 3: Circulating cytokines in control and tumor-induced aged mice [/fig_ref]. Consistent with this, it was recently shown that a decreased level of TNF-α transcript is associated with parity in epithelial ovarian carcinomas [bib_ref] TNFalpha expression, risk factors, and inflammatory exposures in ovarian cancer: evidence for..., Gupta [/bib_ref]. Indeed, elevated TNF-α levels are associated to increased OC risk [bib_ref] Pre-diagnostic serum levels of inflammation markers and risk of ovarian cancer in..., Trabert [/bib_ref] , while the combination of high TNF-α plus high IL-6 levels in ascitic fluid at primary surgery are predictors of rapid relapse in patients with epithelial OC [bib_ref] Cytokine profiling of ascites at primary surgery identifies an interaction of tumor..., Kolomeyevskaya [/bib_ref]. Furthermore, the constitutive TNF-α expression by OC cells in vitro induces an autocrine network of additional cytokines including CCL2 [bib_ref] The inflammatory cytokine tumor necrosis factor-alpha generates an autocrine tumor-promoting network in..., Kulbe [/bib_ref]. Such links between TNF-α and CCL2 appear early during the fertility cycle in women, where TNF-α expression peaks in the regressing CL and correlates with CCL2 expression, despite the fact that it does not impair steroidogenesis in lutein-granulosa cells [bib_ref] Regulated C-C motif ligand 2 (CCL2) in luteal cells contributes to macrophage..., Nio-Kobayashi [/bib_ref].
Monocytes, macrophages, dendritic cells and brain microglia are the major sources of IL1-β [bib_ref] The interleukin-1 family: back to the future, Garlanda [/bib_ref]. This cytokine plays diverse roles in reproductive ovarian function [bib_ref] The interleukin-1 system and female reproduction, Gerard [/bib_ref] and is found increased at post-menopause [bib_ref] Proinflammatory and anti-inflammatory cytokine changes related to menopause, Malutan [/bib_ref]. IL1-β promotes tumor invasion and angiogenesis while it depresses antitumor immunity [bib_ref] Differential effects of IL-1 alpha and IL-1 beta on tumorigenicity patterns and..., Song [/bib_ref]. As agedependent increased IL1-β levels in the present study were reduced by tumor induction only in multiparous mice, we propose that long-term parity counteracts tumor spread by reducing angiogenesis and relieving immune suppression by the host tissue. Interestingly, FSH levels at pre-menopause were positively correlated with circulating IL-1β, IL-6 and TNF-α levels, while isolated mononuclear cells secreted these three cytokines upon exposure to exogenous FSH [bib_ref] Follicle-stimulating hormone, interleukin-1, and bone density in adult women, Cannon [/bib_ref]. Given that FSH receptor expression has been also detected in osteoclasts [bib_ref] FSH directly regulates bone mass, Sun [/bib_ref] , extragonadal tissue [bib_ref] FSH receptor (FSHR) expression in human extragonadal reproductive tissues and the developing..., Stilley [/bib_ref] and some genitourinary tumors [bib_ref] The folliclestimulating hormone receptor: a novel target in genitourinary malignancies, Gartrell [/bib_ref] , it seems feasible that the crosstalk between systemic FSH and cytokines including IL-1β, IL-6 and TNF-α might contribute to the pro-inflammatory status at postmenopause.
Finally, the pro-inflammatory cytokine IL-12p70 also followed the above described pattern. This is a M1-type heterodimeric cytokine composed of p35 and p40 kDa subunits coded by different genes. IL-12p70 is produced mostly by antigen-presenting cells such as macrophages and dendritic cells. It exerts an immune-stimulatory role on NK cells and promotes differentiation of naive CD4+ T lymphocytes into Th1 cells that produce IFN-γ and TNF-α as part of the innate and adaptive immune responses against endogenous damage [bib_ref] Regulation of Interleukin-12 Production in Antigen-Presenting Cells, Zheng [/bib_ref]. Importantly, IL-12p70 was increased at menopause [bib_ref] Effects of menopause and postmenopausal tibolone treatment on plasma TNFalpha, IL-4, IL-10,..., Vural [/bib_ref] and the level of p40 subunit was positively associated with parity [bib_ref] Factors associated with inflammation markers, a cross-sectional analysis, Clendenen [/bib_ref]. As a potential cancer therapy, IL-12p70 reversed the immunosuppressive phenotype of tumor-associated macrophages in a subcutaneous murine model, with concomitant reduced synthesis of CCL2 and IL-10 [bib_ref] IL-12 rapidly alters the functional profile of tumorassociated and tumor-infiltrating macrophages in..., Watkins [/bib_ref]. In other work, reduction of omental tumors was observed in response to MOSE cells constitutively expressing membrane-bound IL-12p35p40 [bib_ref] Interleukin-12 Immunomodulation Delays the Onset of Lethal Peritoneal Disease of Ovarian Cancer, Cohen [/bib_ref]. In both cases, IL-12p70 expression was effective only in a localized tumor microenvironment since its systemic administration caused toxicity [bib_ref] Gene therapy approaches against cancer using in vivo and ex vivo gene..., Hernandez-Alcoceba [/bib_ref]. IL-12p70 induced IFN-γ production, in turn, induces synthesis of the chemokine CXCL10 (IP-10; 10kD interferon γ-induced protein), which was the only cytokine that increased in multiparous mice in response to tumor induction. Thus, the divergent patterns of IL-12p70 and CXCL10 in our data appeared seemingly inconsistent. CXCL10 is chemotactic for monocytes and T-cells, and has both pro-apoptotic capacity and angiostatic properties against VEGF action, thereby acting as an antitumor agent [bib_ref] The emerging role of CXCL10 in cancer (Review), Liu [/bib_ref].
An important aspect to consider in the present study, is that the levels of cytokines measured somehow reflect the near-senescent state of diverse tissues and organs of aged mice. Among various features, cellular senescence is characterized by secretion of a particular set of cytokines, chemokines, proteases and cell-matrix remodeling factors collectively known as the "senescence-associated secretory phenotype" (SASP) [bib_ref] Cellular senescence: a link between cancer and agerelated degenerative disease?, Campisi [/bib_ref]. However, the relative contributions of each organ/tissue to the pool of circulating cytokines are difficult to estimate. Lastly, a further consequence of the SASP is that senescent tissues become infiltrated with immune cells. As an example, various morphological types of macrophages have been detected in the stroma of the aged mouse ovary in relation to follicle atresia, non-heme iron accumulation, presence of ceroid/lipofuscin and fibrosis [bib_ref] Age-related accumulation of non-heme ferric and ferrous iron in mouse ovarian stroma..., Asano [/bib_ref] [bib_ref] Reproductive ageassociated fibrosis in the stroma of the mammalian ovary, Briley [/bib_ref].
# Conclusions
Elevated serum gonadotropin levels and an extended length of estrous cycle indicated a peri-estropausal state in mice of both study groups >15 months old. PRL levels were lowest in multiparous mice, a finding also reported in multiparous women, and linked to decreased OC risk. Circulating levels of CCL2, IL10, IL5, IL4, TNF-α, IL1b and IL12p70 increased with age regardless of parity history, thus confirming a low-grade chronic inflammatory condition in all peri-estropausal female mice irrespective of parity. Importantly, the levels of these cytokines were significantly reduced in response to tumor induction only in the multiparous animals, suggesting that the drop in these cytokines levels was somehow associated with OC protection. CCL2, IL-10, IL-5 and IL-4 are typically implicated in M2 macrophage polarization, whereas TNF-α, IL1b and IL12p70 participate in M1 polarization. Similar to the human pattern, exogenous syngeneic tumorigenic MOSE cells formed tumor implants spreading across the peritoneal cavity predominantly in the omentum. Survival of multiparous mice was significantly longer than nulliparous ones, suggesting that parity would play a favorable prognostic role. We conclude that, in response to intraperitoneal tumor spread, multiparity partially reverts age-associated systemic inflammation while reducing immunesuppression against OC, i.e, past parity would improve the immune response against the tumor. A decrease of M2 polarization of tumor-associated macrophages, plus a reduced stimulation of regulatory B-cells might be important aspects of this effect, at least in the present mouse model. Further studies are needed to define how the studied cytokines modulate biological responses at the gene expression level in their multiple targets, and in senescent tissues as well as in tumor cells and their associated cellular microenvironments. To our knowledge, this is the first report describing a long-term effect of pregnancy on age-associated chronic inflammation relevant to OC in an animal model.
[fig] Figure 1: Study design and parity records. (A) Two groups of C57BL6 female mice (n=70 per group) [/fig]
[fig] Figure 2: Circulating pituitary hormones in aged mice. Serum levels of the indicated hormones were measured monthly in 4 randomly chosen female mice 15-19 months old. The reference group, was 4 months old young adults (n=6) was. Bars represent mean with SEM corresponding to the abovementioned time period. (*) p<0.05, (**) p<0.01, (***) p<0.005, ( §) 0.05<p<0.10. Further details in Methods. [/fig]
[fig] Figure 3: Circulating cytokines in control and tumor-induced aged mice. Serum levels of the indicated cytokines were measured monthly from 15-19 months old in 3-4 randomly chosen female mice per group. Cytokines in the tumor-induced conditions (dashed bars, n=8 per group) were measured from the time of injection (16 months old) until time of death (19-19.5 months old). The young adult reference group (n=6) was 4 months old. Bars represent the mean with SEM corresponding to the above-mentioned time periods. (*) p<0.05, (**) p<0.01, (***) p<0.005, ( §) 0.05<p<0.10. Further details in Methods. [/fig]
[fig] Figure 4: Tumor spread and survival of host aged mice. (A) Demonstrative image of intraperitoneal tumor implants formed in a 19 months-old virgin C57BL6 female mouse injected with MOSE cells. Tumor implants in the omental fat band are shown pulled-out with clamps. (B) Survival plots of virgin and multiparous aged mice injected with IG-10 MOSE cells; day 0 corresponds to 16.1 ± 0.3 months of age for the two groups. Median survivals were 98 and 87 days for the multiparous and virgin groups, respectively. The p value of log-rank (Mantel-Cox) is shown. Both the log-rank and the Gehan-Breslow-Wilcoxon (p=0.038) tests were performed in GraphPad Prism 5 with 95% CI of 0.7638-1.489 for ratios of survival. [/fig]
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10.1016/j.ijscr.2020.11.146
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CCBY
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7736759
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33310466
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s2orc_pubmed_articles
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Excision of a huge adrenal pheochromocytoma resembling a pancreatic tumor: A case report
# Introduction
Pheochromocytoma is a rare disease. It is known to occur in <0.2% of patients with hypertension. The annual incidence of pheochromocytoma is estimated at 0.8 per 100,000 people. The present case is one of the rarest cases, with large tumors measuring up to 15 cm in diameter.
Since pheochromocytoma cases are based on a state of excess catecholamine production, fluctuations in blood pressure can make surgery dangerous if preparations are not made preoperatively to control blood pressure and circulating blood flow. Therefore, preoperative, intraoperative, and postoperative preparations for blood pressure control are necessary.
If the left adrenal tumor is large, the preoperative preparation and treatment must be fully explained, as reports have described patients who required a combined resection of the distal pancreas or left kidney for tumor resection. Furthermore, if the left adrenal tumor is large, it may be difficult to differentiate from a pancreatic tumor during diagnosis. In the present study, we report a rare case of a large pheochromocytoma wherein the patient was carefully prepared for surgery.
## Presentation of case
A 73-year-old Japanese woman presented to our outpatient clinic with a chief complaint of abdominal pain. She had a history of hypertension. During the initial examination, her vital signs were stable. Her consciousness was clear, and physical examination revealed a flat and soft abdomen without tenderness.
The laboratory data were as follows: white blood cell count, 6730/L; C-reactive protein, 0.03 mg/dL; hemoglobin, 10.2 mg/dL; carcinoembryonic antigen, 2.2 ng/mL; and CA 19-9, 11.4 U/mL. The computed tomography (CT) scan of the abdomen showed an abdominal tumor of 15 cm diameter.
The patient was closely examined with pancreatic tumor in mind, but the adrenal tumors were also considered as a differential disease. The plasma catecholamine fractionation test results showed a high noradrenaline level (828 pg/mL). This led us to strongly suspect a pheochromocytoma. We decided to perform a surgical excision. Because of the excess catecholamine secretion in patients with pheochromocytoma, we anticipated intraoperative blood pressure fluctuations during the excision. Therefore, preoperative blood pressure control with alpha blockers and increased circulating blood volume were important. From 2 weeks before to immediately before the surgery, the patient was given an alpha blocker. The target systolic blood pressure was set at 120 mm Hg. To keep the intravascular volume increased, the patient was admitted to the hospital 1 week before surgery and continued to receive saline at 1000 mL/day, until the day before surgery.
## Operative findings
An upper median incision was made. The tumor was identified by opening the bursa cavity. Owing to the large size of the tumor, the surgical technique was difficult to perform using only a peritoneal approach. Therefore, the Treiz ligament was dis-sected and approached from the transverse mesenteric side to the retroperitoneum and the tumor was safely removed.
First, the left renal artery/vein was identified and then the left adrenal artery/vein branching from each was identified, ligated, and then dissected. Each of the peripheral vessels around the tumor was ligated and dissected. As a result, the tumor was removed. The area in close proximity to the pancreas was safely dissected.
Intraoperatively, grasping the tumor required careful surgical manipulation, as the systolic blood pressure increased quickly to 200 mm Hg when the tumor was grasped. Blood pressure control required the supervision of a skilled anesthesiologist. The surgical operation time was 144 min, and the blood loss was minimal. The histopathological diagnosis was pheochromocytoma of the adrenal gland .
## Postoperative course
The surgery was uneventful, and the patient progressed postoperatively without difficulty in controlling her blood pressure and was discharged 13 days postoperatively.
# Discussion
Left adrenal tumors can be difficult to differentiate from pancreatic tumors if they are large in size. A distal pancreatectomy may also be necessary. Therefore, the operation must be prepared with a pancreatic resection in mind.
In addition, pheochromocytoma is a catecholamine-producing tumor, and preparations before, during, and after surgery are necessary. Specifically, preoperative saline supplementation to maintain the systemic vascular bed, intraoperative administration of antihypertensive alpha blockers and noradrenaline adjustment, and postoperative noradrenaline tapering are necessary.
This case was also difficult to manage intraoperatively because of the pheochromocytoma and the fluctuations in blood pressure due to intraoperative micro-manipulation. Considering the preoperative and postoperative aspects of pheochromocytoma and the possibility of pancreatic tumor resection, this was a case that required more care than the usual surgery.
In addition, adrenal tumors should always be considered as a differentiator of giant pancreatic body/tail tumors. This is because . Resected specimen. The histopathological diagnosis was pheochromocytoma of the adrenal gland. No malignancy was confirmed in this sample. The surgical margin was negative.
surgery to remove a pheochromocytoma can be risky if blood pressure is not controlled in the perioperative period. Adrenal tumors should always be listed as a differential diagnosis for any lesion suspected of being a large pancreatic body tail tumor.
Pheochromocytoma has three main symptoms, but asymptomatic pheochromocytoma has been occasionally reported and should be suspected even in the absence of symptoms. While performing surgery to remove adrenal tumors, the approach is often to remove them from the retroperitoneum. However, in the case of large tumors such as in the present case, the approach is likely through the peritoneal cavity.
To safely and completely resect the large tumor, the pancreas must be identified and the adrenal artery/vein, a feeder vessel, must be ligated and removed. Therefore, we opened the bursa cavity to identify the pancreas and tumor and then dissected the Treiz ligament and approached the transverse mesentery to the retroperitoneum. With the full extent of the massive tumor detailed, we safely removed the tumor. The patient progressed postoperatively without difficulty in controlling her blood pressure and was discharged 13 days postoperatively.
# Conclusion
Careful preparations must be made to resect a giant pheochromocytoma that is difficult to distinguish from a pancreatic tumor. Adrenal tumors should always be considered as a differential diagnosis for any lesion suspected of being a large pancreatic body tail tumor. This case was reported in line with the SCARE guideline.
## Declaration of competing interest
The authors declare that they have no competing interests.
# Funding
None.
# Ethical approval
Not applicable.
## Consent
Informed consent was obtained from the patient for the publication of this case report and accompanying images.
## Registration of research studies
Not applicable.
## Guarantor
On the behalf of all author I am the guarantor. Hideki Kogo.
## Provenance and peer review
Not commissioned, externally peer-reviewed.
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10.1186/s12863-016-0415-0
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CCBY
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4946190
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27421647
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s2orc_pubmed_articles
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Association of single nucleotide polymorphisms in Pre-miR-27a, Pre-miR-196a2, Pre-miR-423, miR-608 and Pre-miR-618 with breast cancer susceptibility in a South American population
Background: MicroRNAs (miRNAs) are a novel class of endogenous, non-coding, single-stranded RNAs capable of regulating gene expression by suppressing translation or degrading mRNAs. Single nucleotide polymorphisms (SNP) can alter miRNA expression, resulting in diverse functional consequences. Previous studies have examined the association of miRNA SNPs with breast cancer (BC) susceptibility. The contribution of miRNA gene variants to BC susceptibility in South American women had been unexplored. Our study evaluated the association of the SNPs rs895819 in pre-miR27a, rs11614913 in pre-miR-196a2, rs6505162 in pre-miR-423, rs4919510 in miR-608, and rs2682818 in pre-mir-618 with familial BC and early-onset non-familial BC in non-carriers of BRCA1/2 mutations from a South American population. Results: We evaluated the association of five SNPs with BC risk in 440 cases and 807 controls. Our data do not support an association of rs11614913:C > T and rs4919510:C > G with BC risk. The rs6505162:C > A was significantly associated with increased risk of familial BC in persons with a strong family history of BC (OR = 1.7 [95 % CI 1.0-2.0] p = 0.05). The rs2682818:C > A genotype C/A is associated with an increased BC risk in non-familial early-onset BC. For the rs895819:A > G polymorphism, the genotype G/G is significantly associated with reduced BC risk in families with a moderate history of BC (OR = 0.3 [95 % CI 0.1-0.8] p = 0.01). Conclusions: The contribution of variant miRNA genes to BC in South American women had been unexplored. Our findings support the following conclusions: a) rs6505162:C > A in pre-miR-423 increases risk of familial BC in families with a strong history of BC; b) the C/A genotype at rs2682818:C > A (pre-miR-618) increases BC risk in non-familial early-onset BC; and c) the G/G genotype at rs895819:A > G (miR-27a) reduces BC risk in families with a moderate history of BC.
# Background
Breast cancer (BC) is the most common cancer among women worldwide. In Chile, BC has the highest mortality rate among cancers (15.8/100,000 women), and its incidence has increased in all age groups analyzed. Genetics factors play an important role in BC development. Currently, there is consensus that mutations in genes BRCA 1 and BRCA 2 are responsible for an average 16 % of the risk for familial BC. It has been proposed that other susceptibility alleles, called moderate or low penetrance, could be responsible for a significant percentage of BC susceptibility. To date, our group has studied the contribution of moderate and low penetrance genes (PALB2 [bib_ref] Association of PALB2 sequence variants with the risk of familial and early-onset..., Leyton [/bib_ref] , BARD1 [bib_ref] The BARD1 Cys557Ser variant and risk of familial breast cancer in a..., Gonzalez-Hormazabal [/bib_ref] , ATM [bib_ref] Association of common ATM variants with familial breast cancer in a South..., Gonzalez-Hormazabal [/bib_ref] , CHEK 2 [bib_ref] Absence of CHEK2 1100delC mutation in familial breast cancer cases from a..., Gonzalez-Hormazabal [/bib_ref] , RAD51 [bib_ref] RAD51 135G > C polymorphism and risk of familial breast cancer in..., Jara [/bib_ref] , FGFR2 [bib_ref] Genetic variants in FGFR2 and MAP3K1 are associated with the risk of..., Jara [/bib_ref] , MAP3K [bib_ref] Genetic variants in FGFR2 and MAP3K1 are associated with the risk of..., Jara [/bib_ref] , TOX 3 [bib_ref] Association of genetic variants at TOX3, 2q35 and 8q24 with the risk..., Elematore [/bib_ref] , 8q24 [bib_ref] Association of genetic variants at TOX3, 2q35 and 8q24 with the risk..., Elematore [/bib_ref] and 2q35 [bib_ref] Association of genetic variants at TOX3, 2q35 and 8q24 with the risk..., Elematore [/bib_ref] to genetic susceptibility for familial BC. Nevertheless, a large part of the genetic component of familial cases remains unidentified [bib_ref] The emerging landscape of breast cancer susceptibility, Stratton [/bib_ref]. Research on known genes continues in order to further understand BC development, with an emerging interest in epigenetics and gene regulation. One of the most surprising advances in understanding the mechanisms of gene regulation has been the discovery of microRNA (miRNA) [bib_ref] Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern..., Wightman [/bib_ref]. miRNAs are single-stranded RNAs of~22 nucleotides that can regulate gene expression by either degrading or blocking translation of target miRNA, mainly by binding to their 3'-UTR [bib_ref] The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity..., Lee [/bib_ref] [bib_ref] MicroRNAs: small RNAs with a big role in gene regulation, He [/bib_ref]. MiRNAs are specific to different mRNAs, and approximately 30 % of all human genes are regulated by miRNA [bib_ref] Conserved seed pairing, often flanked by adenosines, indicates that thousands of human..., Lewis [/bib_ref] [bib_ref] MicroRNAs in development and disease, Erson [/bib_ref]. The discovery of miRNAs has been followed by findings highlighting their important and diverse roles in many molecular pathways and biological processes, including development, apoptosis, differentiation, and cell proliferation [bib_ref] Antisense inhibition of human miRNAs and indications for an involvement of miRNA..., Cheng [/bib_ref] [bib_ref] MicroRNAs and the regulation of cell death, Xu [/bib_ref] , as well as their implication in various human diseases including cancer. Growing evidence indicates that miRNAs can work as oncogenes or tumor suppressors, depending on which gene(s) they modulate [bib_ref] microRNAs as oncogenes and tumor suppressors, Zhang [/bib_ref]. Atypical expression of various miRNAs has been observed in the development and progression of numerous human cancers [bib_ref] MicroRNAs and their target gene networks in breast cancer, O'day [/bib_ref] [bib_ref] MicroRNA expression profiles classify human cancers, Lu [/bib_ref] [bib_ref] Oncomirs -microRNAs with a role in cancer, Esquela-Kerscher [/bib_ref]. Single nucleotide polymorphisms (SNPs) are the most common type of variation in the human genome. SNPs present in the miRNA gene regions can alter expression, lead to maturation to aberrant miRNA, and affect target binding affinity and specificity [bib_ref] Ethnicity modifies the association between functional microRNA polymorphisms and breast cancer risk:..., Chen [/bib_ref]. Many epidemiological studies have examined the association of miRNA SNPs with cancer susceptibility [bib_ref] MicroRNAs and their target gene networks in breast cancer, O'day [/bib_ref]. In BC, several case-control studies and meta-analyses have evaluated associations between miRNA gene polymorphisms and BC risk in European [bib_ref] Evaluation of SNPs in miR-146a, miR196a2 and miR-499 as low-penetrance alleles in..., Catucci [/bib_ref] [bib_ref] The SNP rs895819 in miR-27a is not associated with familial breast cancer..., Catucci [/bib_ref] [bib_ref] Association between hsa-mir-146a genotype and tumor age-of-onset in BRCA1/BRCA2-negative familial breast and..., Pastrello [/bib_ref] [bib_ref] A genetic variant in the pre-miR-27a oncogene is associated with a reduced..., Yang [/bib_ref] [bib_ref] The rs2910164:G > C SNP in the MIR146A gene is not associated..., Garcia [/bib_ref] [bib_ref] Increased risk of breast cancer associated with CC genotype of Has-miR-146a Rs2910164..., Lian [/bib_ref] , Asian [bib_ref] Common genetic variants in pre-microRNAs were associated with increased risk of breast..., Hu [/bib_ref] [bib_ref] Associations of miRNA polymorphisms and female physiological characteristics with breast cancer risk..., Zhang [/bib_ref] , Arab [bib_ref] Differential expression profile and genetic variants of microRNAs sequences in breast cancer..., Alshatwi [/bib_ref] , and Jewish [bib_ref] Single nucleotide polymorphisms in miRNA binding sites and miRNA genes as breast/ovarian..., Kontorovich [/bib_ref] populations. With the exception of one study in a Brazilian population [bib_ref] Evaluation of single nucleotide polymorphisms in microRNAs (hsa-miR-196a2 rs11614913 C/T) from Brazilian..., Linhares [/bib_ref] , the contribution of variant miRNA genes to BC in South American women had been unexplored. In this study, we selected specific SNPs in five miR and evaluated the effects of these SNPs on miR expression and biological function. Recent studies have demonstrated that miR-27a exhibits oncogenic activity by regulating specific transcription factors and the G2-M checkpoint [bib_ref] The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and..., Mertens-Talcott [/bib_ref] [bib_ref] Coordinate regulation of FOXO1 by miR-27a, miR-96, and miR-182 in breast cancer..., Guttilla [/bib_ref] [bib_ref] miR-27a regulates the growth, colony formation and migration of pancreatic cancer cells..., Ma [/bib_ref]. The rs895819:A > G is located at position 40 relative to the first nucleotide of pre-miR-27a [bib_ref] Hsa-mir-27a genetic variant contributes to gastric cancer susceptibility through affecting miR-27a and..., Sun [/bib_ref] , and it has been hypothesized that rs895819 could have an effect on the secondary structure of pre-miR-27a, which subsequently affects the processing and/or maturation of miR-27a. Zhang et al. [bib_ref] A genetic variant in pre-miR-27a is associated with a reduced breast cancer..., Zhang [/bib_ref] showed that miR-27a expression was significantly lower in BC samples with A/G or G/G genotypes as compared to samples with A/A genotypes, indicating that the A-to-G change decreases expression mature miR-27a. The variant rs11614913, located in the mature miR-196a-3p sequence, could lead to less efficient processing of the miRNA precursor to its mature form and diminish its capacity to regulate target genes such as HOXB2, HOXB3, HOXC3, HOXB5, GADD45G, INHBB, and TP63 [bib_ref] The association of miR-146a rs2910164 and miR-196a2 rs11614913 polymorphisms with cancer risk:..., Wang [/bib_ref]. Several studies have shown that miR-423 plays an important role in tumorigenesis [bib_ref] MicroRNA-423 promotes cell growth and regulates G(1)/S transition by targeting p21Cip1/Waf1 in..., Lin [/bib_ref] [bib_ref] microRNA-423-3p promotes tumor progression via modulation of AdipoR2 in laryngeal carcinoma, Guan [/bib_ref] [bib_ref] miRNA423-5p regulates cell proliferation and invasion by targeting trefoil factor 1 in..., Liu [/bib_ref]. In hepatocellular carcinoma, miR-423 promotes cell growth and regulates G(1)/S transition by targeting p21 Cip1/waf1 [bib_ref] MicroRNA-423 promotes cell growth and regulates G(1)/S transition by targeting p21Cip1/Waf1 in..., Lin [/bib_ref]. Zhao et al. [bib_ref] Genetic analysis and preliminary function study of miR-423 in breast cancer, Zhao [/bib_ref] , demonstrated that the SNP rs6505162 in pre-miR-423 affects mature miR expression, and miR-423 plays a potentially oncogenic role in breast tumorigenesis. A few polymorphisms are located in the mature microRNA sequence. Such polymorphisms could directly affect the binding of microRNAs to hundreds of target mRNAs. One of these is rs4919510:C > G, located in mature miR-608. The predicted targets of miR-608 include interleukin-1 alpha (IL1A), growth hormone receptor (GHR), and TP53 [bib_ref] A catalog of polymorphisms falling in microRNA-binding regions of cancer genes, Landi [/bib_ref]. These genes have been reported to be associated with BC [bib_ref] Interleukin-1 and interleukin-6 gene polymorphisms and the risk of breast cancer in..., Hefler [/bib_ref] [bib_ref] Comprehensive analysis of common genetic variation in 61 genes related to steroid..., Canzian [/bib_ref] [bib_ref] Three common TP53 polymorphisms in susceptibility to breast cancer, evidence from meta-analysis, Hu [/bib_ref]. A study by Huang et al. [bib_ref] Polymorphism rs4919510:C > G in mature sequence of human microRNA-608 contributes to..., Huang [/bib_ref] showed that the polymorphism rs4919510:C > G in the mature miR-608 sequence contributes to the risk of HER2+ BC. Deregulation of miR-618 has previously been linked to a number of malignancies, including hepatocellular carcinoma [bib_ref] Promising Candidate Urinary MicroRNA Biomarkers for the Early Detection of Hepatocellular Carcinoma..., Abdalla [/bib_ref] , male BC [bib_ref] MicroRNA expression profiling of male breast cancer, Fassan [/bib_ref] , and Barrett's esophageal cancer [bib_ref] MicroRNA expression profiling in human Barrett's carcinogenesis, Fassan [/bib_ref]. Because SNP rs2682818 is part of the miR-618 precursor's stem-loop sequence, it can affect miR-618 levels. The SNP may alter the secondary stem-loop structure, which in turn influences how pre-miR-618 is processed into its mature form. [bib_ref] Targetome profiling and functional genetics implicate miR-618 in lymphomagenesis, Fu [/bib_ref]. Fu et al. [bib_ref] Targetome profiling and functional genetics implicate miR-618 in lymphomagenesis, Fu [/bib_ref] suggest that the presence of the variant A allele may negatively impact the production of mature miR-618 by interfering with the post-transcriptional miRNA biogenic process. Considering the proceeding information, in this study we evaluated the association of rs895819 in pre-miR27a, rs11614913 in pre-miR-196a2, rs6505162 in pre-miR-423, rs4919510 in miR-608, and rs2682818 in pre-mir-618 with familial BC and early-onset non-familial BC in non-carriers of BRCA1/2 mutations from a South American population.
# Methods
## Families
A total of 440 BC cases (one case per family) belonging to 440 high-risk BRCA1/2-negative Chilean families were selected from the files of the Servicio de Salud del Area Metropolitana de Santiago, Corporación Nacional del Cáncer (CONAC), and other private services in the Metropolitan Area of Santiago. The majority of the cases are from the Metropolitan Region, and all controls are from the Metropolitan Region. All index cases were tested for BRCA1 and BRCA2 mutations as previously described [bib_ref] Spectrum of BRCA1/ 2 point mutations and genomic rearrangements in high-risk breast/ovarian..., Gonzalez-Hormazabal [/bib_ref]. Pedigrees were constructed on the basis of an index case considered to have the highest probability of being a deleterious mutation carrier. None of the families met the strict criteria for other known syndromes involving BC, such as Li-Fraumeni, ataxiatelangiectasia, or Cowden disease. [fig_ref] Table 1: Inclusion criteria for the families studied [/fig_ref] shows the specific characteristics of the families selected according to the inclusion criteria. All families participating in the study were of self-reported Chilean ancestry dating from several generations, confirmed with extensive interviews with several members of each family from different generations. In the selected families; 16 % (70/440) had cases of bilateral BC; 9 % (40/440) had cases of both BC and ovarian cancer (OC); and 1.1 % (5/440) had male BC. In the BC group, the mean age at diagnosis was 42.1 years, and 75.2 % had age of onset <50 years.
This study was approved by the Institutional Review Board of the School of Medicine of the University of Chile. Informed consent was obtained from all of the participants.
## Control population
The sample of healthy Chilean controls (n = 807) was recruited from CONAC files. DNA samples were taken from unrelated individuals with no personal or family history of cancer who consented to anonymous testing. These individuals were interviewed and informed as to the aims of the study. DNA samples were obtained in accordance with all ethical and legal requirements. The control sample was matched by age and socioeconomic strata with respect to the cases.
# Genotyping analysis
Genomic DNA was extracted from peripheral blood lymphocytes of 440 cases belonging to the selected high-risk families and 807 controls. Samples were obtained according to the method described by Chomczynski [bib_ref] A reagent for the single-step simultaneous isolation of RNA, DNA and proteins..., Chomczynski [/bib_ref].
Genotyping of the SNPs rs11614913:C > T, rs6505162:C > A, rs895819:A > G, rs2682818:C > A, and rs4919510:C > G was performed using the commercially-available TaqMan Genotyping Assay (Applied Biosystems, Foster City, CA) (assay IDs C__31185852_10, C__11613678_10, C__305 6952_20, C__286717_10, and C__2826025_10, respectively). The reaction was performed in a 10-uL final volume containing 5 ng of genomic DNA, 1X TaqMan Genotyping Master Mix, and 1X TaqMan SNP Genotyping Assay. The polymerase chain reaction was carried out in a StepOne-Plus Real-Time PCR System (Applied Biosystems, Foster City, CA). The thermal cycles were initiated for 10 min at 95°C, followed by 40 cycles each of 92°C for 15 s and 60°C for 1 min. Each genotyping run contained DNA controls confirmed by sequencing. The alleles were assigned using the StepOne software V2.2 (Applied Biosystems, Foster City, CA). As a quality control, we repeated the genotyping on~10 % of the samples, and all genotype scoring was performed and checked separately by two reviewers unaware of case-control status.
# Statistical analysis
The Hardy-Weinberg equilibrium assumption was assessed in the control sample using a goodness-of-fit chi-square test (HW Chisq function included in the "HardyWeinberg".package v.1.4.1). Fisher's exact test was used to test the association between genotypes and/or alleles for cases and controls. p < 0.05 was used as the criterion of significance. Odds ratios (OR) and 95 % confidence intervals (CI) were calculated to estimate the strength of the associations in cases and controls (odds ratio fisher function included in the EpiTools package v.0.5 − 6).
# Results
Selected characteristics of the 440 BRCA1/2-negative cases are summarized in [fig_ref] Table 1: Inclusion criteria for the families studied [/fig_ref]. For the analysis, the whole case sample was subdivided into two groups: cases with two or more family members with BC and/or OC (n = 269) (subgroup A) and non-familial early-onset BC (B ≤50 years) (n = 171) (subgroup B). The genotype distributions and allele frequencies of the pre-miR-27a rs895819:A > G, pre-miR-196a rs11614913:C > T, pre-miR-423 rs6505162:C > A, miR-608 rs4919510:C > G, and pre-miR-618 rs2682818:C > A polymorphisms in the whole data set and in subgroups A and B with respect to the controls are shown in [fig_ref] Table 2: Genotype and allele frequencies of rs895819, rs11614913, rs6505162, rs4919510 and rs2682818 in... [/fig_ref]. The observed genotype frequencies for four of the five polymorphisms were in Hardy-Weinberg equilibrium in controls (p = 0.12 for rs11614913:C > T, p = 0.7 for rs6505162:C > A, p = 0.3 for rs4919510:C > G, and p = 0.8 for rs2682818:C > A, respectively), while for rs895819:A > G the p-value was 0.02. In the single locus analyses, no significant differences were observed in the genotype and allele distributions for rs11614913:C > T or rs4919510:C > G, either in the whole data set or in subgroups A or B (p > 0.05). With respect to rs6505162:C > A, the genotype and allele distribution was significantly different in the whole sample of BRCA1/2-negative cases and in subgroup A, with respect to the controls (p ≤ 0.05). The minor allele frequency (MAF) (allele A) was higher in subgroup A cases than in controls (0.46 and 0.41, respectively, p = 0.03). Furthermore, in subgroup A, allele A carriers (C/A + A/ A) had a significantly increased BC risk (OR = 1.4 [95 % CI 1.0 − 1.9] p = 0.02) [fig_ref] Table 2: Genotype and allele frequencies of rs895819, rs11614913, rs6505162, rs4919510 and rs2682818 in... [/fig_ref]. We also analyzed the relationship between rs6505162 and BC risk within cases with a history familial BC according to number of BC cases in the family [fig_ref] Table 3: Genotype and allele frequencies of rs895819, rs11614913, rs6505162, rs4919510, and rs2682818 by... [/fig_ref]. No association between rs6505162 and BC risk was found in cases belonging to families with two BC and/or OC cases. However, BC risk was significantly higher in cases with three or more family members affected by BC and/or OC. In these families, the allele A frequency was 0.48 in BC cases versus 0.41 in controls (OR = 1.3 [95 % CI 1.0 − 1.7] p = 0.04), and homozygous A/A were had a significantly increased BC risk (OR = 1.7 [95 % CI 1.0 − 2.0] p = 0.05). No association was found between rs6505162 and non-familial early-onset BC (≤50 years) [fig_ref] Table 2: Genotype and allele frequencies of rs895819, rs11614913, rs6505162, rs4919510 and rs2682818 in... [/fig_ref]. For rs2682818, located in pre-mir-618, in the whole sample, the MAF (allele A) was higher in cases (0.1) than controls (0.07), and the difference was statistically significant (OR = 1.3 [95 % CI 1.0 − 1.8] p = 0.03). This result indicates that allele A is associated with increased BC risk. We also observed increased BC risk for allele A carriers (C/A + A/A) in the whole sample (OR = 1.4 [95 % CI 1.0 − 2.0] p = 0.02) [fig_ref] Table 2: Genotype and allele frequencies of rs895819, rs11614913, rs6505162, rs4919510 and rs2682818 in... [/fig_ref]. When we analyzed the effect of allele A by number of BC cases per family, no association between rs2682818 and BC risk was found. Nevertheless, BC risk increased 1.6-fold in the heterozygous group (OR = 1.6 [95 % CI 1.0 − 2.4] p = 0.04) with non-familial early-onset BC (≤50 years) [fig_ref] Table 3: Genotype and allele frequencies of rs895819, rs11614913, rs6505162, rs4919510, and rs2682818 by... [/fig_ref].
The results for rs895819 showed that the homozygous genotype G/G was marginally associated with a protective effect in the whole sample (OR = 0.6 [CI 0.4 − 1.0] p = 0.08). Nevertheless, in the families with 2 BC and/or OC cases, we observed decreased BC risk associated with
# Discussion
Mutations in BRCA1 and BRCA2 are associated with susceptibility to breast and ovarian cancer. At present, however, those mutations account for only a portion of familial cases, and consequently there is an intensive search for additional targets.
MiRNAs are a class of endogenous, non-coding, single-strand RNAs involved in many molecular pathways and biological processes including apoptosis, differentiation, proliferation, and immune response [bib_ref] MicroRNAs: novel regulators in the hallmarks of human cancer, Ruan [/bib_ref]. SNPs are the most common form of variation present in the human genome. SNPs in miRNA gene regions can affect miRNA function by modulating the transcription of the primary transcript, pri-miRNA and pre-miRNA processing, maturation, or miRNA-mRNA interaction, which could contribute to cancer susceptibility [bib_ref] Genetic variation in microRNA networks: the implications for cancer research, Ryan [/bib_ref]. Recently, many epidemiological studies have examined the association of miRNA SNPs with BC susceptibility, but the results remain inconclusive. Genetic variability is ethnicity-specific, and to date the most miRNA SNP studies have been performed in cases from European, Asian, Arab, and Jewish populations, mainly with sporadic BC. With the exception of one study in a Brazilian population, the role of miRNA variation in BC susceptibility has not been analyzed in a Latin-American population. In the present study, we evaluated the impact of miRNA SNPs on familial and non-familial early-onset BC cases negative for point mutations in BRCA1/2, from a Chilean population. To this end, we studied the association of BC risk with rs895819 in pre-miR27a, rs11614913 in pre-miR-196a2, rs6505162 in pre-miR-423, rs4919510 in miR-608, and rs2682818 in pre-mir-618 in a case-control study.shows the results of association studies between SNPs: rs895819 (mir-27a), rs11614913 (miR196a2), rs6505162 (miR-423), rs4919510 (miR-608), rs2682818 (miR-618) and BC risk in others populations.
Our data do not support an association of rs11614913:C > T and rs4919510:C > G with breast cancer risk. With respect to rs11614913, several case-control studies have been conducted to investigate the association between this SNP with BC susceptibility, but the results have been contradictory. Specifically, case-control studies have shown that rs11614913 SNP is associated with increased BC risk in Han Chinese [bib_ref] Common genetic variants in pre-microRNAs were associated with increased risk of breast..., Hu [/bib_ref] and Saudi Arabian [bib_ref] Ten genes for inherited breast cancer, Walsh [/bib_ref] populations. In contrast, results from studies performed in the United States [bib_ref] microRNA miR-196a-2 and breast cancer: a genetic and epigenetic association study and..., Hoffman [/bib_ref] and China [bib_ref] Comprehensive review of genetic association studies and meta-analyses on miRNA polymorphisms and..., Srivastava [/bib_ref] showed that rs11614913 was associated with decreased BC susceptibility. Other studies in Italian, German, and Australian populations reported that the common SNP rs11614913 was not associated with increased BC risk [bib_ref] Evaluation of SNPs in miR-146a, miR196a2 and miR-499 as low-penetrance alleles in..., Catucci [/bib_ref] [bib_ref] Single nucleotide polymorphism in hsa-mir-196a-2 and breast cancer risk: a case control..., Jedlinski [/bib_ref]. In Brazilian women with BC, the C/C genotype was associated with decreased BC risk, and the presence of the T allele was significantly associated with increased BC risk [bib_ref] Evaluation of single nucleotide polymorphisms in microRNAs (hsa-miR-196a2 rs11614913 C/T) from Brazilian..., Linhares [/bib_ref]. These discrepancies might be explained by different genetic backgrounds. The contemporary Chilean population stems from the admixture of Amerindian peoples with the Spanish settlers in the sixteenth and seventeenth centuries. Later (nineteenth century) migrations of Germans, Italians, Arabs, and Croatians have had only a minor impact on the overall population (not more than 4 % of the total population) and are restricted to the specific locations of the country where they settled [bib_ref] Ethnic origin and evolution of the Chilean population, Cruz-Coke [/bib_ref]. The relationship between ethnicity, Amerindian admixture, genetic markers, and socioeconomic strata has been extensively studied in Chile [bib_ref] Socioeconomic assortative mating in Santiago, Chile: a demonstration using stochastic matrices of..., Valenzuela [/bib_ref] [bib_ref] Sociogenetic gradient in the Chilean population, Valenzuela [/bib_ref]. Thus, it is probable that in the mixed Chilean population, rs11614913 is not a significant contributor to BC, similar to the results described for Caucasian populations. Another SNP found to have no association with BC risk in our study, rs4919510:C > G, is located in mature miR-608. This is important because few polymorphisms are located in the mature microRNA sequence. Moreover, predicted targets of miR-608 include interleukin-1 alpha (IL-1A), growth hormone receptor (GHR), and TP53 [bib_ref] A catalog of polymorphisms falling in microRNA-binding regions of cancer genes, Landi [/bib_ref] , all of which have reported associations with BC. The only case-control study, performed by Huang et al. [bib_ref] Polymorphism rs4919510:C > G in mature sequence of human microRNA-608 contributes to..., Huang [/bib_ref] in Han Chinese women, reported that variant genotypes (C/G + G/G) were specifically associated with increased risk for the HER2-positive subtype in the recessive model, but not for other subtypes. In the Chilean population, we observed no association between this SNP and BC in the whole data set, the familial BC group (subgroup A), or the nonfamilial early-onset BC group (subgroup B). Nevertheless, our results are not comparable with those obtained in the Han Chinese women as our study did not consider pathologic features of the BC. Further studies in different ethnic groups are needed before concluding whether rs4919510:C > G alters BC susceptibility. Several studies have evaluated the association between the SNP rs6505162 in pre-miR-423 and cancer risk in diverse populations and in different cancers, with contradictory outcomes. Nevertheless, there have been scarce association studies on this SNP and BC or OC risk. Kontorovich et al. [bib_ref] Single nucleotide polymorphisms in miRNA binding sites and miRNA genes as breast/ovarian..., Kontorovich [/bib_ref] indicated that rs6505162 was associated with a significantly increased risk of ovarian cancer; on the contrary, Smith [bib_ref] A genetic variant located in miR-423 is associated with reduced breast cancer..., Smith [/bib_ref] showed that it conferred a reduced risk of BC. A meta-analysis published by Chen et al. [bib_ref] Ethnicity modifies the association between functional microRNA polymorphisms and breast cancer risk:..., Chen [/bib_ref] reported no associations between the rs6505162 SNP and BC risk in any genetic model. However, this meta-analysis included only two association studies involving rs6505162 SNP, which is an important limitation to interpreting the results. In our study, we found that the SNP rs6505162:C > A was significantly associated with increased risk of familial BC in the group with a strong family history of BC. In these families, the homozygous genotype A/A was associated with increased BC risk (OR = 1.7 [95 % CI 1.0 − 2.0] p = 0.05). Our results are in accordance with the recent results obtained by Zhao et al. [bib_ref] Genetic analysis and preliminary function study of miR-423 in breast cancer, Zhao [/bib_ref] , who demonstrated that the SNP rs6505162 in pre-miR-423 affects mature miRNA expression and that miR-423 plays a potentially oncogenic role in breast cancer tumorigenesis.
miR-618 deregulation has been related to a number of malignancies, such as hepatocellular carcinoma, [bib_ref] Promising Candidate Urinary MicroRNA Biomarkers for the Early Detection of Hepatocellular Carcinoma..., Abdalla [/bib_ref] , male breast cancer [bib_ref] MicroRNA expression profiling of male breast cancer, Fassan [/bib_ref] , and Barrett's esophageal cancer [bib_ref] MicroRNA expression profiling in human Barrett's carcinogenesis, Fassan [/bib_ref] , suggesting a potential rol of this miRNA as a possible cancer biomarker. Because SNP rs2682818 is part of the miR-618 precursor's stem-loop sequence, it can affect miR-618 levels. The SNP may alter the secondary stem-loop structure, which in turn influences how pre-miR-618 is processed into its mature form [bib_ref] Targetome profiling and functional genetics implicate miR-618 in lymphomagenesis, Fu [/bib_ref]. Recently, Fu et al. [bib_ref] Targetome profiling and functional genetics implicate miR-618 in lymphomagenesis, Fu [/bib_ref] reported that rs2682818:C > A may play a role in susceptibility to follicular lymphoma (OR = 1.65 [95 % CI 1.05-2.50]).; an in vitro analysis indicated that the variant A allele of rs2682818 lowered mature miR-618 levels. This reduction could trigger a deregulation of miR-618-controlled pathways associated with follicular lymphoma. With respect to BC, the only case-control study published to date reported no association between rs2682818 and BC risk in a Chinese population [bib_ref] Associations of miRNA polymorphisms and female physiological characteristics with breast cancer risk..., Zhang [/bib_ref]. Our results showed that the rs2682818 C/A genotype is associated with an increased BC risk both in the whole sample and in the group with non-familial early-onset BC. Our results are the first to contribute to identification of rs6505162 in pre-miR-423 and rs2682818 in pre-miR-618 as polymorphisms associated with increased BC risk in a South American population.
Six studies, including three meta-analyses, have examined the association between the rs895819 polymorphism in miR-27a and BC risk. The studies were conducted in German cases with familial BC, in Italian cases with familial BC, and in Chinese cases with sporadic BC. In the German familial BC cases, the rare (G) allele was shown to have a protective effect limited to cases with age at diagnosis <50 years (OR = 0.83 [95 % CI 0.70 − 0.98] p = 0.0314) and bilateral BC (OR = 0.70 [95 % CI 0.52 − 0.95] p = 0.0238). The results obtained by Catucci et al. [bib_ref] The SNP rs895819 in miR-27a is not associated with familial breast cancer..., Catucci [/bib_ref] in Italian familial BC failed to support the association of rs895819 with BC risk. In a Chinese population, Zhang et al. [bib_ref] A genetic variant in pre-miR-27a is associated with a reduced breast cancer..., Zhang [/bib_ref] showed that in sporadic BC, only younger (<48 years old) allele G (A/G + G/G) carriers showed a significantly reduced BC risk (OR = 0.535 [95 % CI 0.321 − 0.891] p = 0.016). With respect to the meta-analyses, the first, which included 4 studies, concluded that subjects carrying the rs895819 G allele showed reduced BC risk [bib_ref] Pre-miR-27a rs895819A/G polymorphisms in cancer: a meta-analysis, Xu [/bib_ref]. The meta-analysis published by Bai et al. [bib_ref] Association of a pre-miR-27a polymorphism with cancer risk: an updated meta-analysis, Bai [/bib_ref] found a significant association between rs895819 allele G and reduced BC risk in Caucasians, but not in Asians. A protective effect of rs895819 allele G was seen in the younger BC cases and in the subgroup of unilateral BC cases. In addition, the meta-analysis published by Chen et al. [bib_ref] Ethnicity modifies the association between functional microRNA polymorphisms and breast cancer risk:..., Chen [/bib_ref] reported that the miR-27a rs895819 G allele might be a protective factor for BC among Caucasians. Our results in a Chilean mixed population showed that the MAF (allele G) in the controls was low (0.28), similar to the East Asian population . In the whole sample, we observed a marginally protective effect of the genotype G/G, which was likely attributable to SNP frequency and sample sizes. Nevertheless, in the subgroup A, which included families with a moderate BC history, the G/G genotype is significantly associated with reduced BC risk. These results are consistent with the meta-analysis which reported reduced BC risk in Caucasians, as the Chilean population is 60 % Caucasian [bib_ref] Gene geography of Chile: regional distribution of American, European and African genetic..., Fuentes [/bib_ref].
# Conclusions
The contribution of miRNA-gene variants to BC susceptibility in South-American women had been unexplored, with the exception of one study in a Brazilian population. Our findings support the following conclusions: a) rs6505162:C > A in pre-miR-423 increases risk of familial BC in families with a strong history of BC; b) the C/A genotype at rs2682818:C > A (pre-miR-618) increases BC risk in non-familial early-onset BC; and c) the G/G genotype at rs895819:A > G (miR-27a) reduces BC risk in families with a moderate history of BC. Abbreviations miRNA, microRNA; SNP, Single Nucleotide Polymorphism; BR, breast cancer; OC, ovarian cancer; OD, odds ratio; CI, confidence interval
[table] Table 1: Inclusion criteria for the families studied [/table]
[table] Table 2: Genotype and allele frequencies of rs895819, rs11614913, rs6505162, rs4919510 and rs2682818 in BRCA1/2-negative breast cancer cases and controls BC breast cancer, OC ovarian cancer, OR odds ratio, CI confidence interval [/table]
[table] Table 3: Genotype and allele frequencies of rs895819, rs11614913, rs6505162, rs4919510, and rs2682818 by number of BC cases per family, in BRCA1/2-negative breast cancer cases and controls Families with 2 BC and/or OC cases (n = 148) Families with ≥3 BC and/or OC cases (n = 121) BC breast cancer, OC ovarian cancer, OR odds ratio, CI confidence interval [/table]
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10.3390/antiox9040321
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CCBY
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7222398
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32316115
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s2orc_pubmed_articles
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Potential Benefits of Nrf2/Keap1 Targeting in Pancreatic Islet Cell Transplantation
Permanent pancreatic islet cell destruction occurs in type 1 diabetes mellitus (T1DM) through the infiltration of inflammatory cells and cytokines. Loss of β-cell integrity secondary to oxidation leads to an inability to appropriately synthesize and secrete insulin. Allogenic islet cell transplantation (ICT) has risen as a therapeutic option to mitigate problematic hypoglycemia. Nevertheless, during the process of transplantation, islet cells are exposed to oxidatively caustic conditions that severely decrease the islet cell yield. Islet cells are at a baseline disadvantage to sustain themselves during times of metabolic stress as they lack a robust anti-oxidant defense system, glycogen stores, and vascularity. The Nrf2/Keap1 system is a master regulator of antioxidant genes that has garnered attention as pharmacologic activators have shown a protective response and a low side effect profile. Herein, we present the most recently studied Nrf2/Keap1 activators in pancreas for application in ICT: Dh404, dimethyl fumarate (DMF), and epigallocatechin gallate (EGCG). Furthermore, we discuss that Nrf2/Keap1 is a potential target to ameliorate oxidative stress at every step of the Edmonton Protocol.Antioxidants 2020, 9, 321 2 of 12 preparation, and reperfusion-related injuries set the islet cells for engraftment and survival failure. It is no surprise then that the five-year insulin independence rate for allogenic ICTs is 25-50%[13][14][15][16]. Studies have further confirmed that nearly 15-50% of the approximated 1-1.5 million islet cells are lost in the isolation process[17], with transplants often times requiring three to four pancreases to achieve euglycemia[18]. Research efforts have accordingly begun focusing on ways to increase the antioxidant response. Particular attention has been given to the Nrf2/Keap1 pathway, a master regulator of antioxidant genes[19].
# Introduction
Type 1 diabetes mellitus (T1DM) leads to permanent pancreatic islet cell destruction via the infiltration of inflammatory cells and cytokines [bib_ref] Cytokines and their roles in pancreatic islet β-cell destruction and insulin-dependent diabetes..., Rabinovitch [/bib_ref] [bib_ref] A choice of death-the signal-transduction of immune-mediated beta-cell apoptosis, Eizirik [/bib_ref]. T1DM typically occurs in children and adolescents that have a genetic predisposition and have experienced an environmental stressor (i.e., virus or toxin) [bib_ref] Environmental risk factors for type 1 diabetes, Rewers [/bib_ref] [bib_ref] The pain of chronic pancreatitis: A persistent clinical challenge, Goulden [/bib_ref]. Under physiological conditions, β-cells elegantly uptake and convert glucose into ATP, stimulating ion gradients that drive the depolarization-dependent release of insulin. Loss of β-cell integrity leads to an inability to appropriately synthesize and secrete the peptide hormone. This is caustic metabolically, as the tissues are unable to meet their energy demands. In the United States, an estimated 30.3 million people of all ages had diabetes in 2015, with approximately 5% of these having T1DM. Within those 30.3 million, it was estimated that 17,900 were children or adolescents younger than 20 years of age. While the majority of diabetes cases are type 2 diabetes mellitus (T2DM), late-term T2DM is functionally equivalent to T1DM [bib_ref] The epidemiology of pancreatitis and pancreatic cancer, Yadav [/bib_ref] [bib_ref] Pancreatic cancer in chronic pancreatitis; aetiology, incidence, and early detection, Raimondi [/bib_ref]. Allogenic islet cell transplantation (ICT) has thus risen as a therapeutic option to mitigate problematic hypoglycemia [bib_ref] Evidence-informed clinical practice recommendations for treatment of type 1 diabetes complicated by..., Choudhary [/bib_ref]. Following the Edmonton protocol, islet cells are procured from cadaveric donors and perfused into the hepatic portal vein [bib_ref] Is islet transplantation a realistic approach to curing diabetes?, Jin [/bib_ref].
Islet cells are at a baseline disadvantage as they lack a robust anti-oxidant defense system. In fact, relative to other tissues in the rat model, islet cells contain significantly less glutathione peroxidase, superoxide dismutase, and catalase [bib_ref] Two tales of antioxidant enzymes on β cells and diabetes, Lei [/bib_ref] [bib_ref] Low antioxidant enzyme gene expression in pancreatic islets compared with various other..., Lenzen [/bib_ref]. The cells' ability to combat oxidative stress is the key for remaining functional as reactive oxygen species (ROS) directly impact the integrity of the β-cell at the biomolecular level [bib_ref] Islets and their antioxidant defense, Acharya [/bib_ref]. Higher sensitivity to stress along with pancreatic islet during isolation, failure. It is no surprise then that the five-year insulin independence rate for allogenic ICTs is 25-50% [bib_ref] Potent induction immunotherapy promotes long-term insulin independence after islet transplantation in type..., Bellin [/bib_ref] [bib_ref] Beta-cell replacement therapy: Current outcomes and future landscape, Dunn [/bib_ref]. Studies have further confirmed that nearly 15-50% of the approximated 1-1.5 million islet cells are lost in the isolation process [bib_ref] Single-donor islet transplantation and long-term insulin independence in select patients with type..., Al-Adra [/bib_ref] , with transplants often times requiring three to four pancreases to achieve euglycemia [bib_ref] Inhibition of thrombin abrogates the instant blood-mediated inflammatory reaction triggered by isolated..., Özmen [/bib_ref]. Research efforts have accordingly begun focusing on ways to increase the antioxidant response. Particular attention has been given to the Nrf2/Keap1 pathway, a master regulator of antioxidant genes [bib_ref] An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme..., Itoh [/bib_ref].
## Nrf2/keap1 signaling pathway
Nrf2 is a leucine zipper protein that is covalently repressed by its regulator Keap1 in the cytoplasm through ubiquitination [bib_ref] Cloning and characterization of a novel erythroid cell-derived CNC family transcription factor..., Itoh [/bib_ref] [bib_ref] Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding..., Itoh [/bib_ref] [bib_ref] Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase..., Kobayashi [/bib_ref]. Stress induces the separation of these two molecules via modification of the Keap1 cysteine residues, allowing Nrf2 to enter the nucleus and heterodimerize with small Maf Proteins (MafP) [fig_ref] Figure 1: Schematic demonstrating the role of Nrf2/Keap1 signal transduction on oxidative stress regulation [/fig_ref]. The Nrf2/MafP complex binds to genes which promote the transcription of multiple antioxidant enzymes [bib_ref] Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in..., Ishii [/bib_ref] [bib_ref] Embryonic lethality and fetal liver apoptosis in mice lacking all three small..., Yamazaki [/bib_ref] [bib_ref] Nrf2-MafG heterodimers contribute globally to antioxidant and metabolic networks, Hirotsu [/bib_ref] [bib_ref] Measuring reactive species and oxidative damage in vivo and in cell culture:..., Halliwell [/bib_ref] [bib_ref] Cytokines and nitric oxide inhibit the enzyme activity of catalase but not..., Sigfrid [/bib_ref]. have demonstrated that the Nrf2/Keap1 system plays a critical role in the protection of pancreatic β-cells from oxidative damage through repressed apoptosis and enhanced proliferation [bib_ref] Nrf2 protects pancreatic β-cells from oxidative and nitrosative stress in diabetic model..., Yagishita [/bib_ref]. Nrf2/Keap1 thus promises a potential way of reducing the oxidative damage that occurs in ICT.
Dh404, dimethyl fumarate (DMF), and epigallocatechin gallate (EGCG) are the Nrf2 activators that have been most studied in context of pancreatic inflammation and islet cell transplantation.
## Nrf2 activators
Dh404, formally CDDO-9,11-dihydro-trifluoroethyl amide (CDDO-dhTFEA) [bib_ref] The synthetic triterpenoid RTA dh404 (CDDO-dhTFEA) restores Nrf2 activity and attenuates oxidative..., Aminzadeh [/bib_ref] is a synthetic oleanane triterpenoid (SO) plant derivative used in oriental medicine [bib_ref] Pharmacology of oleanolic acid and ursolic acid, Liu [/bib_ref] for anti-inflammatory and anti-tumorigenic purposes [bib_ref] Inhibition of the tumor-promoting action of 12-O-tetradecanoylphorbol-13-acetate by some oleanane-type triterpenoid compounds, Nishino [/bib_ref] [bib_ref] Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic..., Huang [/bib_ref]. Since Yates et al.'s first report of dh404 as a protective agent against aflatoxin-induced tumorigenesis [bib_ref] Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the..., Yates [/bib_ref] , dh404 has been studied in oncology, chronic renal disease, and recently in diseases of the pancreas. As a group, SOs are unique because they are one of the most potent inducers of the Nrf2 pathway with selective induction of phase II detoxifying and antioxidant enzymes [bib_ref] Extremely potent triterpenoid inducers of the phase 2 response: Correlations of protection..., Dinkova-Kostova [/bib_ref].
Dh404 has proven to be protective in the pathogenesis of acute pancreatitis. Dh404 (1 mg/kg)treated rats 24 h before L-arginine (600 mg/100 g)-induced pancreatitis showed reductions in inflammatory cells, acinar structural damage, edema, necrosis (p < 0.001), and rates of apoptosis (p < 0.05). Malondialdehyde (MDA), which is an indicator of lipid peroxidation, was also reduced (p
## Nrf2 activators
Dh404, formally CDDO-9,11-dihydro-trifluoroethyl amide (CDDO-dhTFEA) [bib_ref] The synthetic triterpenoid RTA dh404 (CDDO-dhTFEA) restores Nrf2 activity and attenuates oxidative..., Aminzadeh [/bib_ref] is a synthetic oleanane triterpenoid (SO) plant derivative used in oriental medicine [bib_ref] Pharmacology of oleanolic acid and ursolic acid, Liu [/bib_ref] for anti-inflammatory and anti-tumorigenic purposes [bib_ref] Inhibition of the tumor-promoting action of 12-O-tetradecanoylphorbol-13-acetate by some oleanane-type triterpenoid compounds, Nishino [/bib_ref] [bib_ref] Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic..., Huang [/bib_ref]. Since Yates et al.'s first report of dh404 as a protective agent against aflatoxin-induced tumorigenesis [bib_ref] Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the..., Yates [/bib_ref] , dh404 has been studied in oncology, chronic renal disease, and recently in diseases of the pancreas. As a group, SOs are unique because they are one of the most potent inducers of the Nrf2 pathway with selective induction of phase II detoxifying and antioxidant enzymes [bib_ref] Extremely potent triterpenoid inducers of the phase 2 response: Correlations of protection..., Dinkova-Kostova [/bib_ref]. Dh404 has proven to be protective in the pathogenesis of acute pancreatitis. Dh404 (1 mg/kg)-treated rats 24 h before L-arginine (600 mg/100 g)-induced pancreatitis showed reductions in inflammatory cells, acinar structural damage, edema, necrosis (p < 0.001), and rates of apoptosis (p < 0.05). Malondialdehyde (MDA), which is an indicator of lipid peroxidation, was also reduced (p < 0.05). MDA was further shown to be lower in dh404-cultured cells compared to controls when in 200 µM H 2 O 2 for a 24-h period. Lastly, the effect of dh404 was shown to be temporally dependent, as cells that were incubated with 500 nM dh404 for 1 h had nearly twice the intranuclear Nrf2 concentration as cells incubated for 30 min. When dh404 treatment was prolonged for 24 h, the presence of anti-oxidant enzymes such as Heme Oxygenase-1 (HO-1), superoxide dismutase (SOD), and catalase (CAT) was recorded.
The mechanism of dh404-mediated Nrf2 activation is not yet clear. showed that dh404 is involved in a unique interaction with Cys-151 of Keap1, which under physiological conditions binds Cul3/Rbx1 E3 ligase complex to target Nrf2 ubiquitination and subsequent digestion [bib_ref] Dihydro-CDDO-trifluoroethyl amide (dh404), a novel Nrf2 activator, suppresses oxidative stress in cardiomyocytes, Ichikawa [/bib_ref]. On another hand, Li et al. have shown that dh404-mediated Nrf2-activated pathway involves the autophagy of toxic ubiquitinated proteins driven directly by Nrf2 induction, and not by ROS [bib_ref] Targeting Nrf2 by dihydro-CDDO-trifluoroethyl amide enhances autophagic clearance and viability of β-cells..., Li [/bib_ref]. Because ROS were previously shown [bib_ref] Autophagy as a second level protective process in conferring resistance to environmentally-induced..., Moore [/bib_ref] to endogenously drive the autophagy process as a defense mechanism to inflammation, these findings suggest that dh404 activates Nrf2 to simultaneously carry out two actions that are not mutually exclusive. Whether this response is entirely due to the Nrf2 or supplemented by an additional pathway activated by dh404 necessitates further investigation.
Dimethyl fumarate, otherwise known as BG-12 or Tecfidera, is a fumarate ester that started out as a recognized anti-carcinogen [bib_ref] Effect of dimethyl fumarate on the radiation sensitivity of mammalian cells in..., Held [/bib_ref] , in the 1990s it was licensed in Germany for treatment of psoriasis, and more recently in 2013 has received approval by the US Food and Drug Administration (FDA) for the treatment of relapsing-remitting multiple sclerosis [bib_ref] Dimethyl fumarate, a two-edged drug: Current status and future directions, Saidu [/bib_ref]. Our lab examined the role of DMF as a Nrf2 activator in the setting of pancreatitis [bib_ref] Dimethyl fumarate ameliorates acute pancreatitis in rodent, Robles [/bib_ref] [bib_ref] Dimethyl fumarate protects pancreatic islet cells and non-endocrine tissue in L-arginine-induced chronic..., Robles [/bib_ref]. Pancreata of rats treated with DMF (25 mg/kg) 24 h prior to L-arginine (3 g/kg)-induced acute pancreatitis showed reductions in the severity of inflammatory cell infiltration, acinar damage, perilobar edema, and cell necrosis (p < 0.001) [bib_ref] Dimethyl fumarate ameliorates acute pancreatitis in rodent, Robles [/bib_ref]. Similarly, rats that were orally fed DMF (25 mg/kg) prior to and after L-arginine-induced-chronic pancreatitis resulted in improved glucose tolerance, better-preserved tissue architecture (less atrophy, edema, and fatty infiltration) (p < 0.05), significantly lower levels of inflammatory markers (myeloperoxidase (MPO) and MDA), and significantly higher expression of antioxidants (i.e., HO-1) [bib_ref] Dimethyl fumarate protects pancreatic islet cells and non-endocrine tissue in L-arginine-induced chronic..., Robles [/bib_ref]. Zhang et al. corroborated similar findings and also demonstrated that animals transplanted with DMF-treated-cells had lower blood glucose (p < 0.01) and preserved β-cell function [bib_ref] Effect of dimethyl fumarate on rats with chronic pancreatitis, Zhang [/bib_ref].
Interestingly, and conveniently, DMF has demonstrated to be most efficacious under stressful conditions. In a study performed by Schultheis et al., islet cells from adult mice were cultured for 12-16 h in DMF, and then for 2 or 48 h under control or glucolipotoxic conditions (25 mmol/L glucose and 100 µmol/L palmitate) [bib_ref] Nrf2 Activation Protects Mouse Beta Cells from Glucolipotoxicity by Restoring Mitochondrial Function..., Schultheis [/bib_ref]. Compared to controls, cells in the glucolipotoxic medium had a decrease in oxidized status, superior insulin secretion, and a higher mitochondrial membrane potential (50 vs. 10 µmol/L) at 48 h [bib_ref] Nrf2 Activation Protects Mouse Beta Cells from Glucolipotoxicity by Restoring Mitochondrial Function..., Schultheis [/bib_ref]. While the benefits of DMF in the treatment of inflammatory conditions have been shown to be due to a sundry of anti-inflammatory responses [bib_ref] Dimethylfumarate inhibits TNF-induced nuclear entry of NF-κB/p65 in human endothelial cells, Loewe [/bib_ref] [bib_ref] Dimethylfumarate induces immunosuppression via glutathione depletion and subsequent induction of heme oxygenase..., Lehmann [/bib_ref] [bib_ref] Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the..., Linker [/bib_ref] [bib_ref] Fumarates promote cytoprotection of central nervous system cells against oxidative stress via..., Scannevin [/bib_ref] , the specific mechanism behind Nrf2-activation necessitates further investigation.
Epigallocatechin gallate is a main ingredient of green tea and has been described since the 1990s to have anticarcinogenic, antioxidant, antiangiogenic, antiviral properties, and more recently antidiabetic properties [bib_ref] Inhibition of the infectivity of influenza virus by tea polyphenols, Nakayama [/bib_ref] [bib_ref] Tea antioxidants in cancer chemoprevention, Katiyar [/bib_ref] [bib_ref] Insulin-like biological activity of culinary and medicinal plant aqueous extracts in vitro, Broadhurst [/bib_ref]. It has been shown to act as a neutralizing agent for ROS, and to have anti-inflammatory effects that have reduced liver fibrosis [bib_ref] Tea catechins and polyphenols: Health effects, metabolism, and antioxidant functions, Higdon [/bib_ref] and even contribute to hepatic regeneration [bib_ref] Beneficial effects of green tea catechin on massive hepatectomy model in rats, Saito [/bib_ref].
EGCG has been shown to suppress cytokine-induced pancreatic β-cell damage in vitro. Pretreatment of RINm5f cells with EGCG (0-200 µg/mL) in presence of proinflammatory cytokines resulted in no cell apoptosis compared to the 55% that became apoptotic in the absence of EGCG [bib_ref] Epigallocatechin gallate, a constituent of green tea, suppresses cytokine-induced pancreatic β-cell damage, Han [/bib_ref]. In fact, the response was noted to be concentration-dependent, with 200 µg/mL EGCG nearly fully blocking the cell death response, abrogating the three-fold increase in NO 2 seen in control, Antioxidants 2020, 9, 321 4 of 12 and completely inhibiting the production of inducible NO synthase (iNOS) [bib_ref] Epigallocatechin gallate, a constituent of green tea, suppresses cytokine-induced pancreatic β-cell damage, Han [/bib_ref]. To simulate the inflammatory environment of T1DM in vivo, the authors induced autoimmune diabetes with a 250 mg/kg streptozotocin (STZ) dose in C57BL/KsJ mice for five consecutive days. In the experimental group, EGCG (100 mg/kg) was administered daily with STZ, and alone for the five days thereafter [bib_ref] Epigallocatechin gallate prevents autoimmune diabetes induced by multiple low doses of streptozotocin..., Song [/bib_ref]. Relative to the control group, the EGCG-treated mice had a significantly reduced STZ-induced hyperglycemia, and markedly suppressed iNOS mRNA expression [bib_ref] Epigallocatechin gallate prevents autoimmune diabetes induced by multiple low doses of streptozotocin..., Song [/bib_ref].
Despite current evidence that EGCG is an activator of the Nrf2/Keap1 pathway [bib_ref] Epigallocatechin-3-gallate prevents oxidative stress-induced cellular senescence in human mesenchymal stem cells via..., Shin [/bib_ref] , the exact mechanism of how this occurs has yet to be elucidated. There is evidence that the activation involves the mitogen-activated protein kinase (MAPK) cascade [bib_ref] Involvement of Nrf2, p38, B-Raf, and nuclear factor-κB, but not phosphatidylinositol 3-kinase,..., Andreadi [/bib_ref] , electrophilic interactions with the cysteine residues in Keap1 [bib_ref] Induction of phase I, II and III drug metabolism/transport by xenobiotics, Xu [/bib_ref] , as well as ROS-derived auto-oxidation of EGCG facilitating the release of Nrf2 from its complex [bib_ref] cellular uptake, biotransformation, and efflux of tea polyphenol (−)-epigallocatechin-3-gallate in HT-29 human..., Hong [/bib_ref].
Having introduced the activators with most promise in the setting of pancreatic islet cell transplantation, next we discuss how the Nrf2/Keap1 pathway is a potential target at each step of the transplantation process to protect islet cells from oxidative damage.
## Nrf2 roles in steps of islet transplantation
## Nrf2/keap1: a target for pre-transplant protection of islet cells
## Isolation: metabolic challenges
Ischemia poses a particular challenge to islet cells because of the cells' low antioxidant defenses and lack of glycogen reserves which prevents them from producing ATP (critical for insulin secretion) [bib_ref] Respective effects of oxygen and energy substrate deprivation on beta cell viability, Lablanche [/bib_ref].
The Nrf2 activator dh404 has shown protective benefits when used during the process of islet cell isolation [bib_ref] Pharmacological activation of Nrf2 pathway improves pancreatic islet isolation and transplantation, Li [/bib_ref]. Rats that were fed dh404 (0.6 mg/kg body weight) once daily for three days prior to islet cell isolation had greater islet cell yield (p < 0.05) and β-cell content (p < 0.05) compared to vehicle-treated rats [bib_ref] Pharmacological activation of Nrf2 pathway improves pancreatic islet isolation and transplantation, Li [/bib_ref]. HO-1 upregulation was 10-fold higher and the percentage of apoptotic cells was lower (p < 0.05) in treated cells. The same authors have also highlighted that the capacity of the Nrf2 response is dependent on the stress state of the cell [bib_ref] The effect of Nrf2 pathway activation on human pancreatic islet cells, Masuda [/bib_ref]. When human islets were treated with 500 nM dh404, a significantly higher proportion of β-cell survival was observed in the presence of 200 µM H 2 O 2 when compared to those not treated with dh404 (74% vs. 57%; p < 0.05) [bib_ref] The effect of Nrf2 pathway activation on human pancreatic islet cells, Masuda [/bib_ref]. Within the cells that survived, they also noted higher levels of nuclear Nrf2 and higher mRNA levels of antioxidant genes like NAD(P)H dehydrogenase [quinone] 1(NQO1), HO-1, and Glutamate-cysteine ligase catalytic subunit (GCLC) [bib_ref] The effect of Nrf2 pathway activation on human pancreatic islet cells, Masuda [/bib_ref].
Tetrahydrocurcumin (THC), the final hydrogenated metabolite of curcumin (active ingredient of Curcuma longa L.), has demonstrated very strong antioxidant properties. In an effort to decrease the ROS-mediated islet cell damage prior to transplantation, Kim et al. cultured Balb/c mice islet cells for 24 h in a THC-supplemented medium, and exposed them to various cytokines [bib_ref] Tetrahydrocurcumin enhances islet cell function and attenuates apoptosis in mouse islets, Kim [/bib_ref]. They found that THC-treated cells had 1.3-fold greater glucose sensitivity and produced B-cell lymphoma 2 (BCL2) (antiapoptotic) along with protective caspase proteins [bib_ref] Tetrahydrocurcumin enhances islet cell function and attenuates apoptosis in mouse islets, Kim [/bib_ref]. Although at the time of the study it was established that THC increased glutathione (GSH) (a low molecular weight antioxidant), have recently demonstrated that this occurs specifically through the Nrf2/Keap1 pathway [bib_ref] Tetrahydrocurcumin and octahydrocurcumin, the primary and final hydrogenated metabolites of curcumin, possess..., Luo [/bib_ref]. THC causes Nrf2 to translocate into the nucleus, and induce glutamate cysteine ligase (GCL), which is a key determinant of GSH synthesis [bib_ref] Regulation of glutathione synthesis, Lu [/bib_ref]. Whether THC is a broad antioxidant activator (like dh404) or a more selective one remains to be answered.
## Preservation: dynamic temperature stress on islet cells
Islet cells experience oxidative stress in the freezing and thawing processes that take place during isolation and preparation [fig_ref] Figure 2: Schematic illustration denoting steps of islet cell transplantation [/fig_ref] [bib_ref] Improved insulin secretion of cryopreserved human islets by antioxidant treatment, Janjic [/bib_ref]. have demonstrated the protective effect of curcumin against extreme temperatures [bib_ref] Curcumin treatment enhances islet recovery by induction of heat shock response proteins,..., Kanitkar [/bib_ref]. Isolated mice islet cells were cryopreserved using a standard protocol. These cells were stored in liquid nitrogen for seven days, and thawed rapidly from -196 - C to 37 - C [bib_ref] Curcumin treatment enhances islet recovery by induction of heat shock response proteins,..., Kanitkar [/bib_ref]. Experimental groups included islets treated with 10 µM curcumin during cryopreservation, during the 24 h post-thaw incubation period, and during both [bib_ref] Curcumin treatment enhances islet recovery by induction of heat shock response proteins,..., Kanitkar [/bib_ref]. Inclusion of curcumin at both steps demonstrated increased yield, better morphology and integrity, enhanced basal and stimulated insulin secretion, as well as induction of Hsp70 and HO-1 [bib_ref] Curcumin treatment enhances islet recovery by induction of heat shock response proteins,..., Kanitkar [/bib_ref]. While the study did not directly discuss the activation of Nrf2/Keap1, we infer its role given that curcumin is metabolized into THC, a confirmed activator [bib_ref] Tetrahydrocurcumin and octahydrocurcumin, the primary and final hydrogenated metabolites of curcumin, possess..., Luo [/bib_ref].
The antioxidant response during cold-storage appears to be more protective than oxygenation alone by means of perfluorohexyloctane (F6H8) (a low specific density, oxygen carrier) [bib_ref] Quality of isolated pig islets is improved using perfluorohexyloctane for pancreas storage..., Brandhorst [/bib_ref]. Brandhorst et al. may have indirectly shown the involvement of Nrf2/Keap1 in their study where 5 mmol/L L-glutamine was administered to pig islet cells exposed to warm (30 min) and cold (3 h) storage [bib_ref] Pancreatic L-glutamine administration protects pig islets from cold ischemic injury and increases..., Brandhorst [/bib_ref]. Their hypothesis was based upon previous evidence that glutamine infusion led to formation of intra-islet GSH [bib_ref] Intra-ductal glutamine administration reduces oxidative injury during human pancreatic islet isolation, Avila [/bib_ref] [bib_ref] Improvement of pancreatic islet isolation outcomes using glutamine perfusion during isolation procedure, Avila [/bib_ref]. This was anticipated, as glutamate (deaminated glutamine) is a building block of GSH [bib_ref] as a precursor of glutathione, and oxidative stress, Amores-Sánchez [/bib_ref]. While the authors noted a protective effect, they could not explain why glutamine leads to GSH formation specifically at a time of stress. Recently, Sayin et al. have shown that tumor cells hyperactivate the Nrf2 system in order to maintain oxidative homeostasis. In fact, they discovered that tumor cells depend heavily on exogenous glutamine for GSH synthesis. In Brandhorst et al.'s study, because glutamine ameliorated the temperature-induced oxidative damage, it is possible that stress upregulated the Nrf2/Keap1 system, which, in effect, increased the demand for the supplied glutamine to synthesize GSH.
## Digestion and isolation: chemical and mechanical stress on islet cells
Pancreata digestion is required to extract the islet cells from the donor organ. It is a harsh process that involves chemical and mechanical exposures. The islet cells undergo collagenase digestion, agitation in marble glass, and centrifugal forces all while experiencing ischemia and temperature The antioxidant response during cold-storage appears to be more protective than oxygenation alone by means of perfluorohexyloctane (F6H8) (a low specific density, oxygen carrier) [bib_ref] Quality of isolated pig islets is improved using perfluorohexyloctane for pancreas storage..., Brandhorst [/bib_ref]. Brandhorst et al. may have indirectly shown the involvement of Nrf2/Keap1 in their study where 5 mmol/L L-glutamine was administered to pig islet cells exposed to warm (30 min) and cold (3 h) storage [bib_ref] Pancreatic L-glutamine administration protects pig islets from cold ischemic injury and increases..., Brandhorst [/bib_ref]. Their hypothesis was based upon previous evidence that glutamine infusion led to formation of intra-islet GSH [bib_ref] Intra-ductal glutamine administration reduces oxidative injury during human pancreatic islet isolation, Avila [/bib_ref] [bib_ref] Improvement of pancreatic islet isolation outcomes using glutamine perfusion during isolation procedure, Avila [/bib_ref]. This was anticipated, as glutamate (deaminated glutamine) is a building block of GSH [bib_ref] as a precursor of glutathione, and oxidative stress, Amores-Sánchez [/bib_ref]. While the authors noted a protective effect, they could not explain why glutamine leads to GSH formation specifically at a time of stress. Recently, Sayin et al. have shown that tumor cells hyperactivate the Nrf2 system in order to maintain oxidative homeostasis. In fact, they discovered that tumor cells depend heavily on exogenous glutamine for GSH synthesis. In Brandhorst et al.'s study, because glutamine ameliorated the temperature-induced oxidative damage, it is possible that stress upregulated the Nrf2/Keap1 system, which, in effect, increased the demand for the supplied glutamine to synthesize GSH.
## Digestion and isolation: chemical and mechanical stress on islet cells
Pancreata digestion is required to extract the islet cells from the donor organ. It is a harsh process that involves chemical and mechanical exposures. The islet cells undergo collagenase digestion, agitation in marble glass, and centrifugal forces all while experiencing ischemia and temperature changes [fig_ref] Figure 2: Schematic illustration denoting steps of islet cell transplantation [/fig_ref] [bib_ref] Improved quality and yield of islets isolated from human pancreata using a..., Kenmochi [/bib_ref]. Although there are no published studies on the direct effect of Nrf2 during pancreatic digestion, one study by Ito et al. indicates the likelihood of its involvement [bib_ref] Improvement of canine islet yield by donor pancreas infusion with a p38MAPK..., Ito [/bib_ref]. Pancreata removed from beagle dogs were treated with p38MAPK inhibitor (P38IH) prior to preservation, and were assessed after isolation [bib_ref] Improvement of canine islet yield by donor pancreas infusion with a p38MAPK..., Ito [/bib_ref]. The intraductal infusion of P38IH was found to reduce TNF-alpha expression, reduce B-cell apoptosis, and significantly improve the islet cell yield (76). Naidu et al.'s publication one year later elucidated that inhibition of p38MAPK upregulates HO-1 expression via activation of the Nrf2 pathway [bib_ref] Inhibition and genetic deficiency of p38 MAPK up-regulates heme oxygenase-1 gene expression..., Naidu [/bib_ref].
## Nrf2/keap1: a target for post-transplant protection of islet grafts
After isolation, the islet cells are injected into the portal vein and distribute heterogeneously in the liver's peripheral branches [bib_ref] Evidence for instant blood-mediated inflammatory reaction in clinical autologous islet transplantation, Naziruddin [/bib_ref]. It takes approximately 10 days [bib_ref] Angiogenesis and vascularization of murine pancreatic islet isografts, Vajkoczy [/bib_ref] before islet cells establish revascularization, hence the liver is a desirable site due to the constant blood flow which the diffusion-limited islets heavily depend on. While hypoxia is certainly a limiting factor in the survival capacity of the cells once infused, Muthyala et al. showed that even when groups of islet cells were spaced in alginate microcapsules to effectively increase the surface area available for diffusion, neither metabolic activity nor insulin secretion differed significantly from those that were infused as free cells. On the contrary, several groups have shown in both mouse and human models that tissue factor (TF) (expressed on the islet cell membrane) is a major player in the activation and release of a downstream inflammatory cascade that has been coined the immediate blood mediated inflammatory response (IBMIR). IBMIR consists of leukocyte infiltration, complement activation, and thrombosis which has been seen on MRI and positron-emission tomography monitoring to lead to a dramatic reduction of islet cells in the very early peri-transplant period [bib_ref] Visualization of early engraftment in clinical islet transplantation by positron-emission tomography, Eich [/bib_ref] [bib_ref] Positron emission tomography: A real-time tool to quantify early islet engraftment in..., Eich [/bib_ref] [bib_ref] Magnetic resonance imaging of pancreatic islets transplanted into the liver in humans, Saudek [/bib_ref] [bib_ref] Monitoring the survival of islet transplants by MRI using a novel technique..., Jirak [/bib_ref]. Utilization of Kosinova et al.'s model for in vivo monitoring of liver ischemia in relation to pancreatic islet cell transplants shows promise as a system capable of quantifying the alleviating extent of inflammation [bib_ref] A novel model for in vivo quantification of immediate liver perfusion impairment..., Kosinova [/bib_ref] with Nrf2/Keap1 activation.
Wada et al. have for the first time shown that EGCG treatment of mouse islets in vivo improves viability and function through the activation the Nrf2 system [bib_ref] The protective effect of epigallocatechin 3-gallate on mouse pancreatic islets via the..., Wada [/bib_ref]. To test the response of EGCG in context of cell transplantation, the 100 µM EGCG group (concentration found to promote significantly higher cell viability) and a control group were transplanted under the kidney capsule of a 200 mg/kg STZ-induced diabetic mouse [bib_ref] The protective effect of epigallocatechin 3-gallate on mouse pancreatic islets via the..., Wada [/bib_ref]. The cytoprotective effect of EGCG was evident as the ROS production was lower (p < 0.01), and the HO-1 mRNA expression of the nuclear-translocated Nrf2-positive cells was higher (p < 0.05) in treated cells [bib_ref] The protective effect of epigallocatechin 3-gallate on mouse pancreatic islets via the..., Wada [/bib_ref]. Of note, while 100 µM was established as the optimal concentration for cell viability and stimulation index, higher concentrations actually proved harmful [bib_ref] The protective effect of epigallocatechin 3-gallate on mouse pancreatic islets via the..., Wada [/bib_ref]. The viability and function tests of islets treated with 500 µM EGCG were significantly lower than those treated with 100 µM [bib_ref] The protective effect of epigallocatechin 3-gallate on mouse pancreatic islets via the..., Wada [/bib_ref].
While a majority of the studies on Nrf2 activators have revolved around their protective role in acute and chronic pancreatitis, there are currently no published studies demonstrating their antioxidant effect in context of the immediate reperfusion period after islet cell transplantation. Nevertheless, liver and renal tissue damage secondary to ischemia/reperfusion has been studied in the presence of Nrf2 activator pretreatment, and the results are promising. Our group looked at the effect of DMF pretreatment in rats that were subjected to ischemia for 1 h and reperfusion for 2 h [bib_ref] Treatment with dimethyl fumarate ameliorates liver ischemia/reperfusion injury, Takasu [/bib_ref]. Rats that received orally-administered DMF (25 mg/kg, 2x/day) pretreatment had a significant decrease in levels of MDA (p = 0.0009), increased expression of CAT (p = 0.03) and Glutamate-Cysteine Ligase Modifier Subunit (GCLM) (p = 0.04), decreased neutrophils and markers of inflammation, superior endothelial function and histopathological integrity (p = 0.02), and increased ATP levels (apoptosis consumes NAD + ) (p = 0.02) [bib_ref] Treatment with dimethyl fumarate ameliorates liver ischemia/reperfusion injury, Takasu [/bib_ref].
Yoon et. al. looked at the effect of sulforaphane pretreatment in Hexokinase 2 (HK2) renal tubular epithelial cells incubated in anaerobic jars at 37 - C, and observed a concentration-dependent benefit [bib_ref] Sulforaphane protects kidneys against ischemia-reperfusion injury through induction of the Nrf2-dependent phase..., Yoon [/bib_ref]. Sulforaphane pretreatment at 20 µM improved survival to nearly 93% (vs. 57% in control), and increased HO-1, NQO1, Glucocorticoid receptor (GR), and glutathione peroxidase (GPx) mRNA [bib_ref] Sulforaphane protects kidneys against ischemia-reperfusion injury through induction of the Nrf2-dependent phase..., Yoon [/bib_ref]. In vivo pretreatment resulted in significantly depressed (p < 0.01) serum creatinine and less macroscopic histological evidence of tubular damage by a factor of 10 relative to untreated controls [bib_ref] Sulforaphane protects kidneys against ischemia-reperfusion injury through induction of the Nrf2-dependent phase..., Yoon [/bib_ref].
The potential application of Nrf2 activators extends beyond the peri-transplant period given evidence of Nrf2 s ability to prevent the side-effects caused by immunosuppressants [bib_ref] Treatment with dimethyl fumarate attenuates calcineurin inhibitor-induced nephrotoxicity, Takasu [/bib_ref]. Our group has previously published that DMF treatment confers renal protection against cyclosporine A nephrotoxicity [bib_ref] Treatment with dimethyl fumarate attenuates calcineurin inhibitor-induced nephrotoxicity, Takasu [/bib_ref]. Given that transplant patients require long-term immunosuppression with calcineurin inhibitors, we postulate that Nrf2/Keap1 pathway activation plays a critical role in the long-term wellbeing of our patients as well. Future studies are needed to determine whether certain activators will act and be tolerated better as adjuncts to immunosuppressive therapy than others.
# Discussion
Activation of the Nrf2/Keap1 pathway has been demonstrated to confer antioxidant protection in every step of the islet cell transplantation. This is not surprising as the cell is exposed to taxing conditions every step of the way. While dh404, DMF, and EGCG have all been described to have good safety profiles, it is important to note that such is the case when the appropriate dose is used. Many studies have proven the dose-dependent effects of Nrf2 activators. Thus far, dh404 has proven to have an optimal concentration in vivo at 500 nM, DMF at 0.25-5 µM, and EGCG at 100 µM. When the concentrations are too low or too high relative to their optimum, oxidative stress eventually leads to cell death [bib_ref] The protective effect of epigallocatechin 3-gallate on mouse pancreatic islets via the..., Wada [/bib_ref] [bib_ref] Dimethyl fumarate controls the NRF2/DJ-1 axis in cancer cells: Therapeutic applications, Saidu [/bib_ref] [bib_ref] Dimethyl fumarate is highly cytotoxic in KRAS mutated cancer cells but spares..., Saidu [/bib_ref]. Thus, further confirmatory testing at optimal dose of those medications in different applications are necessary prior to human experimentation. Furthermore, the context of application for these Nrf2 activators must be determined as it has been described that they are most effective under stress. Whether the recipient may benefit from Nrf2 activators prophylactically to achieve antioxidant protection prior to transplant would also be worth investigation.
While dh404, DMF, and EGCG are the Nrf2 activators that have been most studied in context of pancreatic inflammation and islet cell transplantation, it is important to note that there are more Nrf2 activators that have been described [bib_ref] Activators and inhibitors of NRF2: A review of their potential for clinical..., Robledinos-Antón [/bib_ref] [bib_ref] The emerging role of redox-sensitive Nrf2-Keap1 pathway in diabetes, Bhakkiyalakshmi [/bib_ref]. Establishing a profile index of which activator is most effective and safe would be prudent. Since different activators appear to have unique mechanisms of Nrf2 activation, the simultaneous use of multiple types of activators might result in a summative response. Once the appropriate Nrf2 activator(s) and their optimal dose(s) are confirmed, the next step will be to utilize such activators during recognized oxidative-heavy steps of the peri-transplant process.
# Conclusions
The antioxidant response promulgated by the activation of the Nrf2/Keap1 pathway offers a potential protective mechanism during pancreatic islet cell transplantation. By reducing the amount of oxidative damage islet cells experience in the preservation, isolation, and transplantation process, Nrf2 activators promise a means to enhance viability and henceforth prolong the longevity of transplanted islet cells in Type 1 diabetic recipients.
[fig] Figure 1: Schematic demonstrating the role of Nrf2/Keap1 signal transduction on oxidative stress regulation. [/fig]
[fig] Figure 2: Schematic illustration denoting steps of islet cell transplantation. Nrf2/Keap1 pathway activation at each of these steps has the potential to ameliorate oxidative damage that occurs as a result of temperature (1), isolation (2-4), reperfusion(5), and immunosuppression side-effectmediated insults. [/fig]
[fig] Author: Contributions: H.I. and A.J.L. conceived and designed the study; A.J.L. and H.L. drafted the manuscript; S.L. and H.I. edited and revised the manuscript. All authors have read and agreed to the published version of the manuscript. [/fig]
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10.3390/s22114216
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s2orc_pubmed_articles
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A Novel Lightweight Anonymous Proxy Traffic Detection Method Based on Spatio-Temporal Features
# Introduction
In recent years, anonymous proxy services, e.g., Shadowsocks [bib_ref] Security analysis of shadowsocks(r) protocol, Ji [/bib_ref] , VPN (Virtual Private Network) [bib_ref] Evaluating performances of VPN tunneling protocols based on application service requirements, Akter [/bib_ref] , and V2ray [3], have been used by increasingly more Internet users. On one hand, they can help users to access restricted resources by circumventing Internet censorship. On the other hand, they have become an important means for criminals to engage in illegal network activities, e.g., data theft, darknet transactions, cyber-attacks, and pornographic propagation [bib_ref] Flow context and host behavior based shadowsocks's traffic identification, Zeng [/bib_ref]. Thus, anonymous proxy traffic detection is of great significance for network security.
Anonymous proxy traffic detection methods can be categorized into traditional machine learning methods and deep learning-based methods. Traditional machine learning methods require manually crafting and selecting features based on professional experience following a trial-and-error paradigm. This paradigm is labor-intensive and time-consuming. In recent years, the detection based on deep learning has become a hot research topic, since deep learning algorithms can automatically extract and select traffic features.
Currently, most deep learning-based methods convert the network traffic into images, for the purpose of making (heterogeneous) network traffic adapt to the homogeneous input of typical deep learning algorithms. However, these methods have a common drawback, i.e., large-sized converted images. For example, many methods (e.g., [bib_ref] MATEC: A lightweight neural network for online encrypted traffic classification, Cheng [/bib_ref] [bib_ref] Darknetsec: A novel self-attentive deep learning method for darknet traffic classification and..., Lan [/bib_ref] convert the payloads of the first few packets of a flow into an image. They connect the payloads of the first few packets of a flow into a byte stream, and then convert a byte into an integer (0 to 255). As the byte stream comprises a lot of bytes, the converted images are large, e.g., 784 bytes and 1521 bytes. The method in [bib_ref] Flowpic: A generic representation for encrypted traffic classification and applications identification, Shapira [/bib_ref] converts the sequences of packet sizes and packet arrival time of a flow into a two-dimensional square histogram. The method extracts the size and arrival time of each packet in the flow as a record pair, and then plots the record pairs by defining the X-axis as the packet arrival time and the Y-axis as the packet size. As the MTU (Maximum Transmission Unit) is 1500 bytes, the Y-axis is set between 1 and 1500. The size of converted images is 2250 KB (1500 × 1500 pixels). Large-sized images result in very large storage and computational resource overhead.
To address the problems above, a novel method for anonymous proxy traffic detection is proposed. The method is one of the solutions to reduce storage and computational resource overhead. It converts the sequences of the size and inter-arrival time of the first N packets of a flow into an image, and then categorizes the converted images using the one-dimensional convolutional neural network (1D-CNN). As the method converts the two-way and one-way spatio-temporal features of a flow into an image, the method can comprehensively capture the flow differences. The method achieves comparable detection performance to the state-of-the-art methods. Meanwhile, since the method uses only a small amount of data regarding the size and inter-arrival time (rather than data including packet headers and payloads), the converted images of the method are much smaller than that of existing image-based deep learning methods. Thus, the method is very lightweight and efficient. Compared with existing image-based deep learning methods, the method can significantly reduce storage and computational resource overhead. Due to its high efficiency and low storage requirements, the method can be applied to traffic analysis tasks in large-scale networks.
To the best of our knowledge, we make the first effort towards building a lightweight image representation of encrypted network traffic, with applications to anonymous proxy traffic detection. The approach is not only lightweight, but also incorporates spatiotemporal features, thereby achieving both high efficiency and accuracy. The main contributions of this paper can be summarized below.
## -
A novel lightweight anonymous proxy traffic detection method is proposed. The method can convert the two-way and one-way spatio-temporal features of the flow into an image. The converted images of the method are at least 90% smaller than that of existing image-based deep learning methods, hence drastically lowering space and time computational complexity. - Besides smaller image sizes, the proposed method achieves comparable detection performance to the state-of-the-art methods, i.e., F1 scores up to 98.51% in Shadowsocks traffic detection and 99.8% in VPN traffic detection. - Since the proposed approach features a compact image-based representation of encrypted traffic, it could be incorporated into existing traffic analysis systems that take a mirrored copy of network traffic as input as needed. The analysis results of the systems can be further transmitted to IDS/IPS systems deployed on the network border, which then generate network management policies to allow or drop traffic.
The rest of this paper is structured as follows. Section 2 surveys the literature. Section 3 details our method, and Section 4 presents the experiments. Section 5 presents the performance comparison. We finally discuss limitations in Section 6 and conclude in Section 7.
# Related work
In this section, anonymous proxy traffic detection methods and application traffic identification methods are outlined. These methods can be divided into traditional machine learning methods and deep learning-based methods.
Traditional machine learning methods: These methods utilize handcrafted features, statistical features, and traditional machine learning algorithms to identify anonymous proxy traffic or application traffic.
Miller et al. [bib_ref] Multilayer perceptron neural network for detection of encrypted VPN network traffic, Miller [/bib_ref] extract flow statistics including time-based statistics and other metrics, and then compile them into a dataset. They determine strongest features using Pearson's Correlation Coefficient algorithm. They detect VPN web traffic using multi-layered per-ceptron neural network. Parchekani et al. [bib_ref] Differentiation of sliding rescaled ranges: New approach to encrypted and VPN traffic..., Nigmatullin [/bib_ref] utilize the time-related traffic features (e.g., the forward inter-arrival time and the flow bytes per second) and the random forest algorithm to detect VPN traffic. Deng et al. [bib_ref] The random forest based detection of shadowsock's traffic, Deng [/bib_ref] propose several features, e.g., the fraction of outcoming packets, the average burst length, and the time of the whole transmission. They detect Shadowsocks traffic using the Random Forest algorithm.
Zeng et al. [bib_ref] Flow context and host behavior based shadowsocks's traffic identification, Zeng [/bib_ref] propose 12 features (e.g., the number of flow bursts and the sum of all flow burst lengths) from three aspects: the hosts' flow behavior, the relationship between flows, and the hosts' DNS behavior. They detect Shadowsocks traffic using the Random Forest algorithm. Cheng et al. [bib_ref] ACER: detecting shadowsocks server based on active probe technology, Cheng [/bib_ref] propose an active method for Shadowsocks servers detection. They collect the IP and port of the server as a dataset, and then classify servers of the Shadowsocks using machine learning algorithm XGBoost.
Shim et al. [bib_ref] Application traffic classification using payload size sequence signature, Shim [/bib_ref] use the packet order, direction, and payload size of the first N packets of a flow to generate unique payload size sequence (PSS) signatures for each application. They use the unique PSS signatures to identify application traffic (e.g., Skype, Outlook, and GomTV). Hajjar et al. [bib_ref] Network traffic application identification based on message size analysis, Hajjar [/bib_ref] propose an identification model for network traffic application (e.g., SMTP, FTP, and MSN) identification. The identification model is based on the features (i.e., the size, the direction, and the position) of the first application-layer messages of the flow.
Deep learning-based methods: Compared with traditional machine learning methods, deep learning-based methods can automatically learn nonlinear relationships between the input and the output.
Wang et al. [bib_ref] The applications of deep learning on traffic identification, Wang [/bib_ref] propose a method for protocol traffic identification and anomalous protocol traffic detection. They first propose to convert network traffic into images. They connect the payload bytes of a TCP flow, and then a byte is transformed into an integer (0 to 255). They classify the converted images using Artificial Neural Network (ANN) and Stacked Auto-Encoder (SAE). Many methods improve the classification performance on the basis of the method in [bib_ref] The applications of deep learning on traffic identification, Wang [/bib_ref]. Tang et al. [bib_ref] Caps-lstm: A novel hierarchical encrypted VPN network traffic identification using capsnet and..., Tang [/bib_ref] propose a novel deep neural network for encrypted VPN network traffic identification. The deep neural network consists of CapsNet and Long Short-Term Memory (LSTM) network. Guo et al.propose two deep learningbased models for VPN traffic detection and VPN traffic classification, i.e., convolutional auto-encoding (CAE) and convolutional neural network (CNN). Cheng et al. [bib_ref] MATEC: A lightweight neural network for online encrypted traffic classification, Cheng [/bib_ref] design a lightweight model for online encrypted traffic classification. The number of parameters and training time of the model are significantly reduced.
Lan et al. [bib_ref] Darknetsec: A novel self-attentive deep learning method for darknet traffic classification and..., Lan [/bib_ref] propose a self-attentive deep learning method for application identification and darknet traffic classification. They use a 1D-CNN and a bidirectional LSTM network to capture local spatial-temporal features from the payload content of packets. They also extract side-channel features (e.g., Number of packets/bytes per second) from payload statistics to improve the classification performance. Lotfollahi et al. [bib_ref] Deep packet: a novel approach for encrypted traffic classification using deep learning, Lotfollahi [/bib_ref] propose to categorize network traffic by classifying packets. They convert a packet into an image, and then classify the converted images using the stacked autoencoder (SAE) and the convolution neural network (CNN). Wang et al. [bib_ref] End-to-end encrypted traffic classification with one-dimensional convolution neural networks, Wang [/bib_ref] [bib_ref] Malware traffic classification using convolutional neural network for representation learning, Wang [/bib_ref] show that the performance of 1D-CNN is better than 2D-CNN in encrypted traffic classification. Hu et al. [bib_ref] Cld-net: A network combining CNN and LSTM for internet encrypted traffic classification, Hu [/bib_ref] propose a network for encrypted traffic classification. The network consists of CNN and LSTM.
Johnson et al. [bib_ref] Application of deep learning on the characterization of tor traffic using time..., Johnson [/bib_ref] use various traditional machine learning algorithms (e.g., Random Forest, Decision Tree, k-Nearest Neighbor) and Deep Neural Networks and time-based statistical features to detect tor traffic. They demonstrate that time-based features are effective for Tor traffic detection.
# Method
In this section, a framework is used to introduce the implementation process of the method. This framework consists of three stages, i.e., raw traffic preprocessing stage, image conversion stage, and CNN model training and detection stage. [fig_ref] Figure 1: The framework of the method [/fig_ref] presents the details of the framework.
# Feature analysis
The first few packets of the flow are the key negotiation stage of the application. The negotiation process of the stage is based on predefined rules by the application. The key negotiation stage is different for different applications. Therefore, the size sequence of the first few packets of the flow can be used to identify application traffic [bib_ref] Application traffic classification using payload size sequence signature, Shim [/bib_ref] [bib_ref] High performance traffic classification based on message size sequence and distribution, Lu [/bib_ref].
Many anonymous proxies (e.g., Shadowsocks, V2ray, and VPN) have a similar operational mechanism. They consist of the client (Proxy-client) and the remote server (Proxy-server). The Proxy-client is generally deployed on a local machine, router, or other machines on the local network. The Proxy-server is deployed outside the firewall. The operational mechanism of anonymous proxies is as follows: the user client sends request data to the Proxy-client. The Proxy-client encrypts the request data and forwards them to the Proxy-server. The Proxy-server decrypts the request data and forwards them to the target server. The response data from the target server are returned to the original user client in the same pattern [bib_ref] Flow context and host behavior based shadowsocks's traffic identification, Zeng [/bib_ref] [bib_ref] The random forest based detection of shadowsock's traffic, Deng [/bib_ref]. However, in a regular network environment, the user client sends the request data directly to the target server. The target server sends the response data directly to the user client. This comparison shows that using anonymous proxies increases the time overhead of data transmission. The packet inter-arrival time of anonymous proxy traffic is longer than that of regular traffic. Therefore, the packet inter-arrival time sequence of a flow can be used as a distinguishable feature to detect anonymous proxy traffic.
# Image conversion method
The sequences of sizes and inter-arrival time of the first N packets of a flow are considered as a grayscale image. The size of the packet is transformed into a binary integer. Most packet inter-arrival time of regular traffic and anonymous proxy traffic is less than 1 s. Therefore, the packet inter-arrival time is converted into an integer by Equation (1), and then the integer is transformed into a binary integer. The binary integers of each feature are connected into a binary stream. The binary stream is divided into bytes (8 bits), and if there is binary data that less than a byte, 0 is added at the end of it to complement to a byte. After that, a byte is converted into an integer (0 to 255), which corresponds to a pixel value of an image.
[formula] Integer = round(Time × Value),(1) [/formula]
where the Time is the packet inter-arrival time. The Value is the integer conversion value of the packet inter-arrival time (the value that transforms the packet inter-arrival time into an integer is called the integer conversion value). CNN is used to detect anonymous proxy traffic. The images fed into the CNN must have a unified size. In this work, the size of the converted images is set based on N (the first N packets of a flow) and the MTU of the network. Specifically, to reduce data loss, the size of the converted images is set according to the special case that the size of the first N packets of a flow is MTU. For Shadowsocks traffic detection, the size of converted images of the method is 49 bytes (7 × 7 pixels). For VPN traffic detection, the size of converted images of the method is 16 bytes (4 × 4 pixels).
Taking the converted images of 49 bytes as an example, the construction process of the converted image is introduced. To reduce the interference between different features, the pixel value sequences of the features are unified to the same length (i.e., 7 or 14 bytes). If the sequence of pixel values is less than 7 bytes, 0 is appended to the end of it to complement to 7 bytes. If the sequence of pixel values is more than 7 bytes and less than 14 bytes, 0 is appended to the end of it to complement to 14 bytes. The pixel value sequences of all the features are connected into a sequence. If the connected sequence is less than 49 bytes, 0 is appended at the end of it to complement to 49 bytes. If the connected sequence is more than 49 bytes, it is truncated to 49 bytes. Finally, the pixel value files and the label files are transformed into IDX format files.
The process of converting the inter-arrival time of packets into pixel values is similar to the process of converting the size of the packet into pixel values. Thus, taking the process of converting the size of the packet into pixel values of an image as an example, the image conversion process of the method is introduced. The image conversion process of the method is shown in [fig_ref] Figure 2: The image conversion process of the method [/fig_ref]. In this figure, the + and − stand for the forward direction (client to server) and the backward direction (server to client) of the flow, respectively.
## Cnn model
CNNs have been widely used in the field of computer vision, such as image recognition [bib_ref] Gesture recognition using dual-stream CNN based on fusion of semg energy kernel..., Xu [/bib_ref] [bib_ref] SAR target recognition using only simulated data for training by hierarchically combining..., Zhang [/bib_ref] [bib_ref] Image scene geometry recognition using low-level features fusion at multi-layer deep CNN, Khan [/bib_ref] and video analysis [bib_ref] Application research of key frames extraction technology combined with optimized faster R-CNN..., Jiang [/bib_ref].
The 1D-CNN model consists of 7 layers, i.e., an input layer, two convolutional layers, two pooling layers, a fully connected layer, and an output layer. The convolutional layer extracts different features of the input image by the convolution operation. The convolution operation is defined as:
[formula] S(i, j) = (K * I)(i, j) = ∑ m ∑ n I(i − m, j − n)K(m, n),(2) [/formula]
where I is the input, and K is a kernel function. The pooling layer selects important features, which can reduce the parameters of the model. In this model, max-pooling is used. The activation function introduces the nonlinearity into the network, which improves the expressive ability of the model. In this model, the Rectified Linear Units (ReLU) activation function is used. The ReLU activation function is defined as:
[formula] ReLU(x) = max(0, x),(3) [/formula]
As mentioned before, the input of the model is grayscale images of size 16 bytes (4 × 4 pixels) and 49 bytes (7 × 7 pixels). Taking the input images of 49 bytes as an example, the parameter of the model is introduced. The size of the filter of the 1-dimensional convolutional layers is 1 × 25. The size of the filter of the max-pooling layer is 1 × 3. The output of the first convolutional layer is 32 feature maps of size 1 × 49. The output of the first max-pooling layer is 32 feature maps of size 1 × 17. The output of the second convolutional layer is 64 feature maps of size 1 × 17. The output of the second max-pooling layer is 64 feature maps of size 1 × 6. The output of the fully connected layer is 1024. Moreover, the Dropout layer is used to improve the generalization ability of the model. The details of the 1D-CNN model are presented in [fig_ref] Table 1: The parameters of 1D-CNN model [/fig_ref].
## Experiment
In this section, the datasets and the process of traffic preprocessing are presented. After that, metrics for evaluating the method are introduced. The parameters of the method are analyzed. The performance of the method on different versions of Shadowsocks traffic detection tasks is analyzed.
## Datasets
Both the self-collected dataset Shadowsocks-Regular and the public dataset ISCX VPN-nonVPN [bib_ref] Characterization of encrypted and VPN traffic using time-related features, Draper-Gil [/bib_ref] are used to validate the proposed approach. The self-collected dataset Shadowsocks-Regular was built by Wireshark [bib_ref] Performance of packet analysis between observer and wireshark, Kim [/bib_ref]. When collecting the Shadowsocks traffic, the Shadowsocks client is set to global mode. The Shadowsocks-Regular dataset consists of regular network traffic and the network traffic via Shadowsocks. Both the regular traffic and Shadowsocks traffic consist of data of multiple popular applications.
The public ISCX VPN-nonVPN dataset consists of 14 types of network traffic: 7 types of regular network traffic (i.e., P2P, VoIP, Browsing, Streaming, Chat, File transfer, and Email) and 7 types of network traffic via VPN (i.e., VPN-P2P, VPN-VoIP, VPN-Browsing, VPN-Streaming, VPN-Chat, VPN-File transfer, and VPN-Email). Each type of traffic comprises the data of multiple popular applications. For example, the Chat traffic comprises the data of ICQ, AIM, Facebook, Hangouts, and Skype. The details of these two datasets are presented in [fig_ref] Table 2: Shadowsocks-Regular dataset [/fig_ref] and 3.
## Data preprocessing
The flow is a traffic unit based on the same 5-tuple (source IP, destination IP, source port, destination port, and transport-level protocol) [bib_ref] Darknetsec: A novel self-attentive deep learning method for darknet traffic classification and..., Lan [/bib_ref] [bib_ref] Accurate decentralized application identification via encrypted traffic analysis using graph neural networks, Shen [/bib_ref]. The packets in a flow are sorted by the arrival time. The raw traffic is divided into multiple flows, each of which is saved as a file. During Shadowsocks traffic collection, some regular traffic still was captured even though the Shadowsocks client was set to global mode. Thus, the regular traffic was filtered out based on the port number of the Shadowsocks server. The FIN and RST are the end marker of TCP flows. If there is no packet including the FIN or RST in the TCP flow, the end of the flow file is the termination of the flow.
Some network traffic that is useless to us was removed, such as the Domain Name System (DNS) traffic. Flows without a payload were removed. Moreover, for the ISCX VPN-nonVPN dataset, incomplete TCP flows and very small flows were removed. An incomplete TCP flow has no connection establishment phase (Three-way handshake). Flows with less than two packets with a payload are very small flows.
## Evaluation metrics
Four metrics are used to assess the proposed approach, i.e., Accuracy (Acc), Precision (Pre), Recall (Rec), and F1 Score (F1). These four metrics are defined as follows:
[formula] Acc = TP + TN TP + FP + FN + TN (4) Pre = TP TP + FP (5) Rec = TP TP + FN (6) F1 = 2 * Pre * Rec Pre + Rec(7) [/formula]
where FP, FN, TP, and TN stand for false positives, false negatives, true positives, and true negatives, respectively.
## Experimental evaluation under different parameter settings
The validity of the features is validated. After that, the N (the first N packets of a flow) value and the integer conversion value of the packet inter-arrival time that enable the method to achieve better performance are analyzed. The detection performance of the method using the payload size sequence of the flow is analyzed. The effect of zero-padding between different features on the performance of the method is analyzed.
## The features
We evaluate the effectiveness of the features (i.e., the two-way and one-way packet size sequences of a flow and the two-way and one-way packet inter-arrival time sequences of a flow). The integer conversion value of the packet inter-arrival time and the N (the first N packets of the flow) values that enable the method to achieve better performance are analyzed. The performance of the method using temporal features of the flow on VoIP traffic and VPN-VoIP traffic classification is not good. Therefore, 12 types of network traffic of the ISCX VPN-nonVPN dataset except VoIP and VPN-VoIP traffic are used to validate the temporal features of the flow. The analysis results are presented in . In these figures, the Two-way stands for the two-way packet size sequence of the flow or the two-way packet inter-arrival time sequence of the flow. The One-way stands for the one-way packet size sequences of the flow or the one-way packet inter-arrival time sequences of the flow. The 5 packets stands for the first 5 packets of a flow.
As shown in [fig_ref] Figure 4: When the integer conversion value of the packet inter-arrival time is 1000,... [/fig_ref] and 7, the two-way and one-way packet size sequences of the flow and the two-way and one-way packet inter-arrival time sequences of the flow are effective in Shadowsocks and VPN traffic detection. Moreover, these features have optimal N values that enable the method to achieve the best performance. For the time features, the two-way and one-way packet inter-arrival time sequences of the flow have optimal integer conversion values that enable the method to attain the best performance.
As shown in [fig_ref] Figure 4: When the integer conversion value of the packet inter-arrival time is 1000,... [/fig_ref] , for Shadowsocks traffic detection, converting the twoway size sequence of the first N packets of a flow into an image, the approach obtains the best performance when N is 10. Converting the one-way size sequences of the first N packets of a flow into an image, when N is 10, the approach achieves the best performance. Converting the two-way inter-arrival time sequence of the first N packets of a flow into an image, the approach attains the best performance when N is 10. Converting the one-way inter-arrival sequences of the first N packets of a flow into an image, when N is 10, the approach attains the best performance. As shown in [fig_ref] Figure 5: e i n t e g e r c o n v... [/fig_ref] , the approach achieves the best performance when the integer conversion value of the packet inter-arrival time is 1000/1500.
As shown in [fig_ref] Figure 7: When the integer conversion value of the packet inter-arrival time is 1250,... [/fig_ref] , for VPN traffic detection, converting the two-way size sequence of the first N packets of a flow into an image, the approach obtains the best performance when N is 10/15. Converting the one-way size sequences of the first N packets of a flow into an image, when N is 10, the approach achieves the best performance. Converting the two-way inter-arrival time sequence of the first N packets of a flow into an image, the approach achieves the best performance when N is 5. Converting the one-way inter-arrival sequences of the first N packets of a flow into an image, when N is 5, the approach attains the best performance. As shown in , the approach achieves the best performance when the integer conversion value of the packet inter-arrival time is 1250.
Through the above analysis, for the two-way and one-way packet size sequences of the flow and the two-way and one-way packet inter-arrival time sequences of the flow, the uniform N value is used. For Shadowsocks traffic detection, 1000/1500 is used as the integer conversion value of the packet inter-arrival time. As the performance of the method using temporal features of the flow on VoIP traffic and VPN-VoIP traffic classification is not good, the temporal features of the flow are not used to detect VPN traffic. T w o -w a y O n e -w a y . The VPN traffic detection performance of the method using the two-way and one-way packet size sequences of the flow, respectively.
## N (the first n packets of a flow)
We evaluate the parameter N that enables the approach to achieve better performance. The evaluation results are shown in [fig_ref] Figure 1: The framework of the method [/fig_ref]. As shown in these two figures, for Shadowsocks and VPN traffic detections, when the first 10 packets of the flow are used, the method achieves the best detection performance. In comparison to [fig_ref] Figure 1: The framework of the method [/fig_ref] , for VPN traffic detection, the best performance of the method using the two-way and one-way packet size sequences of the flow is the same as that using only the two-way packet size sequence of the flow. Therefore, for VPN traffic detection, only the two-way packet size sequence of the flow is used.
These experimental results show that the first N packets (the key negotiation stage) of the flow have unique features of an anonymous proxy. The sequences of the size and inter-arrival time of the first N packets of the flow can be used as distinguishable features to detect anonymous proxy traffic. The two-way and one-way spatio-temporal features of the flow have different distinguishable features, they make different contributions to anonymous proxy traffic detection.
## Payload size sequence
We analyze the detection performance of the method using the payload size sequence of the first N packets of a flow. The analysis results are shown in [fig_ref] Table 4: The detection performance of the method using the payload size sequence of... [/fig_ref]. In this table, the N stands for the first N packets of a flow. The Payload stands for the payload size sequence of the flow. The Packet stands for the packet size sequence of the flow.
As shown in [fig_ref] Table 4: The detection performance of the method using the payload size sequence of... [/fig_ref] , in Shadowsocks and VPN traffic detections, the performance of the method using the packet size sequence of the flow is better than that using the payload size sequence of the flow. For Shadowsocks traffic detection, the F1 score of the method using the packet size sequence of the flow is 1.83% higher than that using the payload size sequence of the flow. For VPN traffic detection, the F1 score of the method using the packet size sequence of the flow is 19.71% higher than that using the payload size sequence of the flow. These experimental results show that the acknowledgment packet at the Key negotiation stage can also make contributions to Shadowsocks and VPN traffic detections.
## Zero padding
We analyze the effect of zero-padding between different features on the performance of the method. Since only one feature is used in VPN traffic detection, the effect of zeropadding on VPN traffic detection is no longer analyzed. The analysis results on Shadowsocks traffic detection are shown in [fig_ref] Table 5: The Shadowsocks traffic detection performance of the method using zero-padding and non-padding,... [/fig_ref]. In this table, the Non-padding means that the pixel value sequences of all features are directly connected into a sequence, without zero padding. Zero-padding means that the pixel value sequences of the features are unified to the same length, if the sequence is less than the uniform length, then zero-padding. Zero-padding (More) refers to padding more zeros between different features.
As shown in [fig_ref] Table 5: The Shadowsocks traffic detection performance of the method using zero-padding and non-padding,... [/fig_ref] , in Shadowsocks traffic detection, the F1 score of the method using zero-padding is 1.36% higher than that without zero-padding. The F1 score of the method setting the size of converted images to 49 bytes is 0.04% higher than that setting the size of converted images to 81 bytes. It can be seen that the performance of the method using zero-padding is better than that without zero-padding. Padding more zeros between different features does not improve the detection performance.
## Different versions of shadowsocks traffic
In this section, the performance of the method on different versions of Shadowsocks (i.e., Shadowsocks and ShadowsocksR) traffic detections is analyzed. Shadowsocks traffic of different versions was collected in the same pattern. They consists of the traffic of the same applications (i.e., Youtube, Spotify, Twitter, Instagram, some posts, and some blogs). The analysis results are showed in [fig_ref] Table 6: The performance of the method on different versions of Shadowsocks traffic detections [/fig_ref]. In this table, the Size represents the two-way and one-way packet size sequences of the flow. The Time represents the two-way and one-way packet inter-arrival time sequences of the flow. The Value is the integer conversion value of the packet inter-arrival time.
As shown in [fig_ref] Table 6: The performance of the method on different versions of Shadowsocks traffic detections [/fig_ref] , the method achieves comparable performance in different versions of Shadowsocks traffic detection tasks. Moreover, the parameter settings of the method in different versions of Shadowsocks traffic detection tasks are almost the same. It can be seen that the method can be applied to different versions of Shadowsocks traffic detection tasks. The method has wide applicability and robustness.
## Performance comparison
We compare the detection performance of our approach against the state-of-the-art methods. Specifically, the proposed method is compared with the methods in [bib_ref] Flowpic: A generic representation for encrypted traffic classification and applications identification, Shapira [/bib_ref] from two aspects: accuracy and the size of the converted images (Imgsize). Both methods in [bib_ref] Flowpic: A generic representation for encrypted traffic classification and applications identification, Shapira [/bib_ref] are image-based deep learning methods, but the ways they convert network traffic into images are different. Both our method and the methods in [bib_ref] Flowpic: A generic representation for encrypted traffic classification and applications identification, Shapira [/bib_ref] employ the public ISCX VPN-nonVPN dataset to validate the performance of the method. Therefore, we copied the experimental results from [bib_ref] Flowpic: A generic representation for encrypted traffic classification and applications identification, Shapira [/bib_ref].
As shown in [fig_ref] Table 7: Performance comparison of VPN traffic detection [/fig_ref] , for VPN traffic detection, the accuracy of our method is 0.15% higher than that of the method in [bib_ref] Flowpic: A generic representation for encrypted traffic classification and applications identification, Shapira [/bib_ref]. The accuracy of our method is 0.02% lower than that of the method in. The converted images of our method are much smaller than that of the methods in [bib_ref] Flowpic: A generic representation for encrypted traffic classification and applications identification, Shapira [/bib_ref]. Thus, our method is more lightweight and efficient than the methods in [bib_ref] Flowpic: A generic representation for encrypted traffic classification and applications identification, Shapira [/bib_ref]. In the same case (e.g., the same hardware devices), our method can save a lot of resource overhead, e.g., storage resource overhead, computational resource overhead, and model training and execution time overhead. The proposed method is efficient and has low storage requirements. Thus, it can work in a large-scale network environment. [bib_ref] Flowpic: A generic representation for encrypted traffic classification and applications identification, Shapira [/bib_ref] 2250 KB 99.7 ---Guo et al.1521 B 99.87 ---
# Discussion
The proposed method has several limitations that may affect its applicability in certain scenarios. First, the integer conversion value of the packet inter-arrival time and the parameter N (the first N packets of a flow) are obtained empirically. Although an optimal setting can be found through experiments, empirically setting parameters is not userfriendly. Second, it is possible to convert the traffic data into a non-image form other than an image. Researchers converting network traffic into images may want to exploit this advantage of CNN, since it is well known that CNN achieves better performance in image classification, image recognition, and natural language processing. We leave these issues as open questions for future study.
# Conclusions
A novel anonymous proxy traffic detection method is proposed. Benefiting from converting the two-way and one-way spatio-temporal features of a flow into an image, the method is effective and attains comparable detection performance to the state-of-the-art methods. Moreover, the method is lightweight. Compared with existing image-based deep learning methods, the size of the converted images of the method are reduced at least 90%. Thus, the method can reduce storage and computational resource overhead. Since it is based on CNN, the method can automatically extract and select features, omitting the works of manually crafting and selecting features. Experiments have shown that our approach can detect anonymous proxy traffic effectively, with the capability of detecting different versions of Shadowsocks traffic and VPN traffic. Due to its high efficiency resulting from compact image-based traffic representation, the method can be applied to traffic analysis tasks in large-scale networks.
[fig] Figure 1: The framework of the method. [/fig]
[fig] Figure 2: The image conversion process of the method. [/fig]
[fig] Figure 4: When the integer conversion value of the packet inter-arrival time is 1000, the Shadowsocks traffic detection performance of the method using the two-way and one-way packet inter-arrival time sequences of the flow, respectively. [/fig]
[fig] Figure 5: e i n t e g e r c o n v e r s i o n v a l u e o f t h e p a c k e t i n t e r -a r r i v a l t i The Shadowsocks traffic detection performance of the method using different integer conversion values of packet inter-arrival time. [/fig]
[fig] Figure 7: When the integer conversion value of the packet inter-arrival time is 1250, the VPN traffic detection performance of the method using the two-way and one-way packet inter-arrival time sequences of the flow, respectively. [/fig]
[fig] 5 p a c k e t s 1 0 p a c k e t s, Figure 8: e i n t e g e r c o n v e r s i o n v a l u e o f t h e p a c k e t i n t e r -a r r i v a l t i m e The VPN traffic detection performance of the method using different integer conversion values of packet inter-arrival time. [/fig]
[fig] Author: Contributions: Conceptualization, methodology, software, data curation, writing-original draft preparation, writing-reviewing and editing, Y.H.; supervision, writing-review and editing, funding acquisition, W.L. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This work was supported by the National Natural Science Foundation of China (Grant No. 61672026). [/fig]
[table] Table 1: The parameters of 1D-CNN model. [/table]
[table] Table 2: Shadowsocks-Regular dataset. [/table]
[table] Table 3: ISCX VPN-nonVPN dataset. [/table]
[table] Table 4: The detection performance of the method using the payload size sequence of the flow (%). [/table]
[table] Table 5: The Shadowsocks traffic detection performance of the method using zero-padding and non-padding, respectively (%). [/table]
[table] Table 6: The performance of the method on different versions of Shadowsocks traffic detections (%). [/table]
[table] Table 7: Performance comparison of VPN traffic detection (%). Shapira et al. [/table]
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Aminoacyl-tRNA synthetases in medicine and disease
# Introduction
Aminoacyl-tRNA synthetases (ARSs) comprise an ancient ubiquitous family of enzymes in all cells from three major kingdoms of life. They catalyse the esterification reactions that link amino acids with cognate tRNAs bearing the correct anticodon triplet to ensure the accurate transfer of information directed by the genetic code [bib_ref] Aminoacyl tRNA synthetases: general scheme of structurefunction relationships in the polypeptides and..., Schimmel [/bib_ref]. Generally, the aminoacylation reaction is performed by a two-step process in which amino acids are first activated by ATP, forming an intermediate aminoacyl adenylate, and then transferred to the 3 0 -end of tRNA to form the aminoacyl-tRNA end-product [bib_ref] Aminoacyl-tRNA synthesis, Ibba [/bib_ref]. All ARSs contain catalytic and anticodon recognition domains to catalyse the aminoacylation reactions specific for their cognate amino acids. To ensure translational fidelity and maintain normal cellular function, several ARSs have developed editing activities to hydrolyse misactivated amino acids or mischarged tRNAs and prevent insertion of incorrect amino acids during protein synthesis [bib_ref] Development of tRNA synthetases and connection to genetic code and disease, Schimmel [/bib_ref].
The canonical functions of ARSs, including aminoacylation and editing, are highly conserved throughout the three kingdoms. However, during evolution from prokaryotes to vertebrates, including mammals, certain ARSs acquired appended domains with unique structural characteristics that are neither a part of the enzymatic core nor present in bacterial homologues. These newly evolved domains, generally affixed to the amino or carboxy terminus, are not essential for tRNA charging, but instead are responsible for noncanonical activities unrelated to aminoacylation [bib_ref] New functions of aminoacyl-tRNA synthetases beyond translation, Guo [/bib_ref] , including translation control, transcription regulation, signal transduction, cell migration, angiogenesis, inflammation, and tumourigenesis. Emerging evidence suggests that defects in either canonical or noncanonical ARS functions can cause or contribute to human diseases. Potential associations of ARSs with several types of cancer through aberrant expression and interactions have been described [bib_ref] Cancer association study of aminoacyl-tRNA synthetase signaling network in glioblastoma, Kim [/bib_ref]. This review will focus on the most recent and illustrative discoveries about genetic mutations of ARSs in pathology, on ARSs regulating disease-related processes, and the potential use of ARSs as pharmacological targets and therapeutic reagents.
In human cells, two distinct sets of ARSs encoded by separate genes, can be distinguished by their cytoplasmic or mitochondrial localization. According to the standard nomenclature for human ARS genes and proteins, the cytoplasmic forms are abbreviated as the single-letter amino acid code followed by 'ARS'. For mitochondrial ARS genes, a '2' is appended. Human cells contain 17 cytoplasmic ARS polypeptides (including the bifunctional glutamyl-prolyl-tRNA synthetase, EPRS, responsible for aminoacylation of Glu and Pro; note the exception to the 'ARS' nomenclature), 18 mitochondrial ARSs, and 2 duallocalized ARSs present in both cytoplasm and mitochondria . In human and other mammalian cells, a large tRNA multi-synthetase complex (MSC) organizes 9 cytoplasmic ARSs and 3 non-enzyme factors, namely MSC p43, p38 and p18 (alternatively known as aminoacyl-tRNA synthetase-interacting multifunctional proteins 1, 2 and 3, or AIMP1, AIMP2 and AIMP3, respectively).
Charcot-Marie-Tooth disease: An inheritable human disease caused by mutations in cytoplasmic ARSs
Remarkably, to this date all known disease-associated mutations in cytoplasmic ARSs are associated with Charcot-Marie-Tooth (CMT) and related neuropathies [fig_ref] Figure 1: Genetic mutations in human cytosolic ARSs cause CMT disease [/fig_ref]. In contrast, mutations in mitochondrial ARSs are associated with a wider variety of syndromes and diseases [fig_ref] Table 1: Compilation of mitochondrial ARS-derived genetic mutations and their connections to human diseases [/fig_ref]. In 2003, the first human disease-related genetic mutation in an ARS was reported in GARS (glycyl-tRNA synthetase) [bib_ref] Glycyl tRNA synthetase mutations in Charcot-Marie-Tooth disease type 2D and distal spinal..., Antonellis [/bib_ref]. A mutation in the GARS coding region was associated with CMT disease type 2D and distal spinal muscular atrophy type V. CMT disease comprises a genetically and clinically heterogeneous group of autosomal-dominant peripheral neuropathies characterized by progressive degeneration of distal motor and sensory neuron function. Additional dominantly inherited missense mutations in GARS have been implicated in CMT disease [bib_ref] Mutant glycyl-tRNA synthetase (Gars) ameliorates SOD1(G93A) motor neuron degeneration phenotype but has..., Banks [/bib_ref] [bib_ref] Charcot-Marie-Tooth disease type 2D with a novel glycyl-tRNA synthetase gene (GARS) mutation, Hamaguchi [/bib_ref] [bib_ref] Severe childhood SMA and axonal CMT due to anticodon binding domain mutations..., James [/bib_ref] [bib_ref] GARS axonopathy: not every neuron's cup of tRNA, Motley [/bib_ref]. In this section we focus on mutations in cytoplasmic ARSs associated with CMT disease and their potential underlying mechanisms.
## Yars mutations in dominant-intermediate cmt disease
Dominant-intermediate CMT (DI-CMT) is characterized by slow progressive neuropathy, intermediate nerve conduction velo-cities, axonal degeneration, and demyelination of peripheral motor and sensory neurons. Three dominant mutations (G41R, E196K, V153_V156del) in YARS (tyrosyl-tRNA synthetase) are associated with DI-CMT type C (DI-CMTC) [bib_ref] Disrupted function and axonal distribution of mutant tyrosyl-tRNA synthetase in dominant intermediate..., Jordanova [/bib_ref]. A Drosophila model of DI-CMTC was developed in which over-expression of each of the three mutant YARS genes, but not the wild-type gene, promotes axon atrophy and impaired motor function, major hallmarks of the human disease [bib_ref] Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of..., Storkebaum [/bib_ref]. The loss of tRNA charging activity is not a generally observed feature of DI-CMTC-associated YARS mutant proteins, and is neither necessary nor sufficient to cause the disease phenotype [bib_ref] Dominant Intermediate Charcot-Marie-Tooth disorder is not due to a catalytic defect in..., Froelich [/bib_ref]. Thus, the DI-CMTC phenotype is not due to haploinsufficiency of the canonical synthetase activity, but more likely is related to a gain-offunction of mutant YARS or possibly to a loss-of-function of an as-yet unknown secondary activity of wild-type YARS.
KARS and AARS mutations in CMT disease KARS (lysyl-tRNA synthetase) is the third ARS gene associated with CMT disease. Compound heterozygous mutations in the KARS gene were identified in a patient with severe neurological symptoms including peripheral neuropathy [bib_ref] Compound heterozygosity for lossof-function lysyl-tRNA synthetase mutations in a patient with peripheral..., Mclaughlin [/bib_ref]. Three variants (p.L133H, p.Y173SfsX7 and p.I302M) were found in KARS, with the former two containing loss-offunction mutations that severely inhibit enzymatic activity. An editing-defective, recessive sticky missense mutation (p.A734E) in AARS (alanyl-tRNA synthetase) in a mouse model of ataxia was reported in 2006 [bib_ref] Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration, Lee [/bib_ref]. The mutant enzyme mischarges tRNA Ala with Gly or Ser, leads to amino acid misincorporation and protein misfolding, and causes cerebellar Purkinje cell loss and ataxia, but not peripheral axon degeneration. Surprisingly, mutations affecting editing functions have not been observed in patients. Recently, a single family affected by the axonal form of CMT (CMT2) was investigated [bib_ref] A major determinant for binding and aminoacylation of tRNA(Ala) in cytoplasmic Alanyl-tRNA..., Latour [/bib_ref]. The subjects exhibited sensorymotor distal degeneration secondary to predominant axonal neuropathy and mild demyelination. Sequencing of candidate genes identified a novel mutation in AARS, p.R329H. The mutant AARS exhibits reduced aminoacylation activity and might engender reduced translation and consequent neurodegeneration. Moreover, three new pathological mutations were identified in AARS: p.N71Y, p.E778A and p.D893N V 1 5 3 -V 1 5 6 d e l E 1 9 6 K E 7 1 G P 9 8 L L 1 2 9 P C 1 5 7 R P 2 3 4 K Y G 2 4 0 R P 2 4 4 L I 2 8 0 F H 4 1 8 R D 5 0 0 N G 5 2 6 R S 5 8 1 L G 5 9 8 A G 6 5 2 A [bib_ref] Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy, Zhao [/bib_ref]. The N71Y mutant also exhibited reduced aminoacylation activity; however, the E778A mutant maintained full activity thus providing a second example of a mutated ARS in which a defect in the primary function is unlikely to be responsible for the consequent CMT pathology.
Towards a unifying mechanism of CMT pathology An expanding ensemble of genetic mutations in GARS, YARS, KARS and AARS associated with CMT and related disorders have been discovered and investigated [fig_ref] Figure 1: Genetic mutations in human cytosolic ARSs cause CMT disease [/fig_ref]. However, the molecular and cellular mechanisms linking ARS mutations and the consequent pathology remain unclear. Elucidation of a unifying mechanism underlying CMT caused by mutations at multiple sites within four ARSs remains a major intellectual and experimental challenge. Of the four mutated ARSs, GARS is the most extensively studied, leading to several important mechanistic insights. Fifteen mutations in GARS have been identified as responsible for clinical phenotypes ranging from CMT neuropathy to a severe infantile form of spinal muscular atrophy. Multiple plausible mechanisms have been proposed [bib_ref] The role of aminoacyl-tRNA synthetases in genetic diseases, Antonellis [/bib_ref] [bib_ref] GARS axonopathy: not every neuron's cup of tRNA, Motley [/bib_ref] [fig_ref] Figure 2: Potential mechanisms underlying CMT caused by ARS mutations [/fig_ref] , and some have been tested using mouse models [bib_ref] Charcot-Marie-Tooth-linked mutant GARS is toxic to peripheral neurons independent of wild-type GARS..., Motley [/bib_ref] [bib_ref] An assessment of mechanisms underlying peripheral axonal degeneration caused by aminoacyl-tRNA synthetase..., Stum [/bib_ref]. Mutations in human cytoplasmic ARSs are primarily located in the catalytic domains responsible for aminoacylation activity. In these cases, a plausible cause is reduced aminoacylation due to mutated synthetic active sites or altered dimerization [fig_ref] Figure 2: Potential mechanisms underlying CMT caused by ARS mutations [/fig_ref] mechanism a,e), and consequent defective global protein synthesis. However, multiple findings argue against this mechanism. For example, the Pro to Lys-Tyr mutation (P278KY) in the Gars Nsf249/þ mouse model of CMT2D did not exhibit reduced synthetase activity, and furthermore, heterozygous mice with a single null loss-of-function GARS allele exhibited reduced synthetase activity but none of the symptoms of CMT [bib_ref] An active dominant mutation of glycyl-tRNA synthetase causes neuropathy in a Charcot-Marie-Tooth..., Seburn [/bib_ref]. As an example, studies of two dominant mouse models of CMT2D (Gars Nmf249/þ and Gars C201R/þ ) showed that increased dosage of disease-causing alleles led to more severe neurological phenotypes due to gainof-function effects that could not be rescued by over-expression of wild-type GARS, arguing against a loss-of-function defect [bib_ref] Charcot-Marie-Tooth-linked mutant GARS is toxic to peripheral neurons independent of wild-type GARS..., Motley [/bib_ref]. These observations suggest, at best, a weak relationship between pathological phenotype in CMT and compromised protein synthesis caused by GARS mutations. Moreover, several YARS, KARS and AARS mutants associated with CMT retain full catalytic activity, thus providing additional evidence against this mechanism. Reduced aminoacylation activities of some mutant ARSs could intensify the symptoms but might not be sufficient or required. A distinct catalysisrelated mechanism could contribute to CMT pathology, namely, mischarging of cognate tRNA and subsequent global incorporation of erroneous amino acids due to compromised editing activity [fig_ref] Figure 2: Potential mechanisms underlying CMT caused by ARS mutations [/fig_ref]. However, a linkage between defective editing and CMT phenotypes has not been described, in fact, no human disease has been associated with an editing defect. Also, neither misfolding and consequent aggregation nor protein destabilization has been observed for any of the mutant GARS associated with CMT pathology [fig_ref] Figure 2: Potential mechanisms underlying CMT caused by ARS mutations [/fig_ref] mechanism c,d).
Mutant ARSs might contribute to CMT pathology by at least two alternative mechanisms, namely, altered cellular localization or dysfunctional noncanonical activity. Arguing against the former mechanism, abnormal intracellular distribution of GARS was not detected in mouse central nervous system (CNS) or in peripheral nerves of GARS mutant mice (C57BL/6J-GarsNmf249/J, Gars Nmf249/þ mice) [bib_ref] An assessment of mechanisms underlying peripheral axonal degeneration caused by aminoacyl-tRNA synthetase..., Stum [/bib_ref]. However, given the differences between human and rodent nervous systems, a change in localization of mutant GARS in the CNS or peripheral neurons of human CMT patients, e.g., in nuclei, mitochondria, or cytosolic granules, cannot be formally excluded [fig_ref] Figure 2: Potential mechanisms underlying CMT caused by ARS mutations [/fig_ref]. Supporting this possibility is the observation that mast cell KARS undergoes conditiondependent nuclear import and transcriptional regulation of gene expression [bib_ref] LysRS serves as a key signaling molecule in the immune response by..., Yannay-Cohen [/bib_ref] , as well as the presence of both GARS and KARS in multiple intracellular compartments including cytoplasm and mitochondria. Furthermore, there is experimental evidence for local protein translation in distal regions of axons supporting the concept that the peripheral motor axon is translationally active [bib_ref] Axonal protein synthesis provides a mechanism for localized regulation at an intermediate..., Brittis [/bib_ref] [bib_ref] Local RNA translation at the synapse and in disease, Liu-Yesucevitz [/bib_ref] [bib_ref] RNA transport and localized protein synthesis in neurological disorders and neural repair, Wang [/bib_ref]. Consistent with this observation, GARS protein is detectable in sciatic axons and Schwann cells in mice. However, the axonic localization is indistinguishable between mutant Gars Nmf249/þ mice and controls [bib_ref] An assessment of mechanisms underlying peripheral axonal degeneration caused by aminoacyl-tRNA synthetase..., Stum [/bib_ref]. Notwithstanding these observations in mice, we cannot exclude the possibility that defective axon transport of mutant GARS could compromise local translation and cause axonal degeneration [fig_ref] Figure 2: Potential mechanisms underlying CMT caused by ARS mutations [/fig_ref].
GARS secreted from human macrophages exhibits antitumourigenic activity . This finding raises the intriguing possibility that GARS may have a noncanonical, axonspecific function unrelated to tRNA charging, and it is a defect in this activity that is a determinant of pathology [fig_ref] Figure 2: Potential mechanisms underlying CMT caused by ARS mutations [/fig_ref]. Alternatively, a mutation may influence pathology via an injurious gain-of-function unrelated to the canonical activity, for example by acquisition of a new interaction with protein or RNA [fig_ref] Figure 2: Potential mechanisms underlying CMT caused by ARS mutations [/fig_ref]. A recent structural study of CMT-related GARS mutants suggested that multiple spatially dispersed mutations induced a common conformational change that released inhibition by the a-helical WHEP domain, and exposed a potential gain-of-function interaction surface [bib_ref] Dispersed disease-causing neomorphic mutations on a single protein promote the same localized..., He [/bib_ref]. Despite the above-described mechanisms, each supported by limited evidence, generalized etiologic mechanisms for mutant GARS-directed CMT diseases remain largely unknown.
## Defective mitochondrial ars aminoacylation activity and disorders of mitochondrial metabolism
Mutations in two mitochondrial ARSs, namely, DARS2 (mitochondrial aspartyl-tRNA synthetase) and RARS2 (mitochondrial arginyl-tRNA synthetase), have been implicated in leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) [bib_ref] Clinically asymptomatic adult patient with extensive LBSL MRI pattern and DARS2 mutations, Labauge [/bib_ref] [bib_ref] Leukoencephalopathy with brainstem and spinal cord involvement and normal lactate: a new..., Lin [/bib_ref] [bib_ref] Mitochondrial aspartyl-tRNA synthetase deficiency causes leukoencephalopathy with brain stem and spinal cord..., Scheper [/bib_ref] and infantile encephalopathy [bib_ref] Deleterious mutation in the mitochondrial arginyltransfer RNA synthetase gene is associated with..., Edvardson [/bib_ref] , respectively. Similarly, several genetic mutations in EARS2 (mitochondrial glutamyl-tRNA synthetase), MARS2 (mitochondrial methionyl-tRNA synthetase) and FARS2 (mitochondrial phenylalanyl-tRNA synthetase) cause leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) [bib_ref] Leukoencephalopathy with thalamus and brainstem involvement and high lactate 'LTBL' caused by..., Steenweg [/bib_ref] , spastic ataxia with leukoencephalopathy [bib_ref] Mutations in the mitochondrial methionyl-tRNA synthetase cause a neurodegenerative phenotype in flies..., Bayat [/bib_ref] and Alpers encephalopathy [bib_ref] Mitochondrial phenylalanyl-tRNA synthetase mutations underlie fatal infantile Alpers encephalopathy, Elo [/bib_ref] , respectively. An expanding ensemble of genetic mutations in mitochondrial ARSs has been identified in a variety of pathological contexts [fig_ref] Table 1: Compilation of mitochondrial ARS-derived genetic mutations and their connections to human diseases [/fig_ref].
Mutation of YARS2 causes myopathy, lactic acidosis and sideroblastic anemia (MLASA) syndrome Mitochondrial respiratory chain disorders are heterogeneous and among the most common inborn defects of metabolism, with an incidence of more than 1 in 8000 births. MLASA is a mitochondrial respiratory chain disorder characterized by progressive exercise intolerance and sideroblastic anemia. A pathogenic, recessive mutation (p.F52L) in the YARS2 gene (mitochondrial tyrosyl-tRNA synthetase) has been identified [bib_ref] Mutation of the mitochondrial tyrosyl-tRNA synthetase gene, YARS2, causes myopathy, lactic acidosis,..., Riley [/bib_ref]. Importantly, recombinant mutant YARS2 protein exhibited 9-fold lower tyrosylation activity in vitro compared to wild-type enzyme. Reduced aminoacylation activity of the mutant enzyme could cause decreased mitochondrial protein synthesis and consequent mitochondrial respiratory chain dysfunction.
HARS2 mutations cause ovarian dysgenesis and sensorineural hearing loss in Perrault syndrome Perrault syndrome is a rare, genetically heterogeneous, recessive disorder characterized by ovarian dysgenesis and sensorineural hearing loss, or other neurological manifestations. In a nonconsanguineous family with five affected siblings, compound heterozygosity for mutations in HARS2 (mitochondrial histidyl-tRNA synthetase) at two highly conserved amino acids (p.L200V and p.V368L), and an alternative splice-derived deletion of 12 inframe codons, were shown to be causative [bib_ref] Mutations in mitochondrial histidyl tRNA synthetase HARS2 cause ovarian dysgenesis and sensorineural..., Pierce [/bib_ref]. Point-mutant HARS2 V368L protein exhibited reduced aminoacylation activity, and the deletion mutant lost essentially all activity. RNAi-mediated knockdown of hars-1 in Caenorhabditis elegans caused severe gonadal defects, e.g., the absence of oocytes or fertilized eggs, resulting in compromised fertility. Possibly, Perrault syndrome is related to aberrant mitochondrial translation and gonadal dysgenesis.
SARS2 mutations cause hyperuricemia, pulmonary hypertension, renal failure in infancy and alkalosis (HUPRA) syndrome A new multisystemic mitochondrial cytopathy was diagnosed in three infants from consanguineous Palestinian kindred. The key clinical findings were tubulopathy, pulmonary hypertension and progressive renal failure in infancy. The patients experienced severe feeding difficulties and developmental delay and died of multi-organ failure, respiratory insufficiency, or refractory pulmonary hypertension. The enzymatic activities of the respiratory chain complexes I-IV were reduced in mitochondria from patients' muscle specimens. A pathogenic, recessive mutation (p.D390G) in the enzymatic active site of SARS2 (mitochondrial seryl-tRNA synthetase) was identified [bib_ref] Mutations in the mitochondrial seryl-tRNA synthetase cause hyperuricemia, pulmonary hypertension, renal failure..., Belostotsky [/bib_ref]. In peripheral lymphocytes obtained from patients, the D390G mutation exhibited lower serylation activity for the isoacceptor tRNA Ser AGY but not for tRNA Ser UCN , and the uncharged tRNA is destabilized and degraded. This mutation is the first example of a defective ARS differentially recognizing two tRNA isoacceptors leading to translational inefficiency.
AARS2 mutations in mitochondrial infantile cardiomyopathies Infantile mitochondrial cardiomyopathies (CMPs) are fatal disorders occurring during the neonatal period or first year after birth. The exome of a hypertrophic mitochondrial CMP infant with cardiac respiratory chain deficiency, who died at 10 months of age, was sequenced. A homozygous missense mutation (p.R592W) was identified in AARS2 (mitochondrial alanyl-tRNA synthetase) [bib_ref] Exome sequencing identifies mitochondrial alanyl-tRNA synthetase mutations in infantile mitochondrial cardiomyopathy, Gotz [/bib_ref]. Two siblings from an unrelated family, both of whom died perinatally of hypertrophic CMP, harboured the same mutation, compound heterozygous with a second missense mutation (p.L155R). The mutations in AARS2 cause perinatal or infantile CMP with near-total deficiency of mitochondrial respiratory chain complexes I and IV in the heart. Computational modelling of AARS2 structure suggested that these mutations might affect aminoacylation or editing functions.
To date, all mitochondrial ARSs bearing genetic mutations causing human diseases exhibit compromised aminoacylation activity, and effects on noncanonical functions unrelated to translation have not been demonstrated. Thus, the etiology underlying human mitochondrial disorders might be restricted to defective enzymatic activities. These observations raise an important question: Given that genetic mutations from mitochondrial ARSs affect fundamental mitochondrial functions, for example, translational repression of mitochondrial proteins and consequent respiratory chain defects, why do they cause a diversity of disease phenotypes in distinct affected tissues? Possibly, the threshold amount of an ARS required for optimal enzymatic activity differs for each individual ARS and tissue combination. Thus, a change in the activity of a mutant ARS might be tolerated in some tissues but not in others. Alternatively, there might exist tissue-specific proteins that bind the mutant ARS, leading to disease phenotypes by inhibiting the enzymatic activity, or by diminishing potential noncanonical functions.
Human mitochondrial DNA encodes 22 mitochondrial tRNAs [bib_ref] Mitochondrial tRNA mutations and disease, Yarham [/bib_ref]. Inheritable mutations in mitochondrial tDNA (mtT-DNA) are hot spots of pathology causing a wide spectrum of clinical phenotypes including neuromuscular disorders. Intriguingly, genetic mutations in either member of at least five cognate ARS-tRNA pairs (AARS2-mtT-DNA Ala , EARS2-mtT-DNA Glu , HARS2-mtT-DNA His , RARS2-mtT-DNA Arg and YARS2-mtT-DNA Tyr ) cause the same or similar pathological phenotypes, possibly due to a common loss-of-activity mechanism [fig_ref] Table 1: Compilation of mitochondrial ARS-derived genetic mutations and their connections to human diseases [/fig_ref]. However, the pathological outcomes of mutations in other pairs do not appear to overlap (i.e. DARS2-mtT-DNA Asp , FARS2-mtT-DNA Phe , MARS2-mtT-DNA Met and SARS2-mtT-DNA Ser ). Mutations at distinct sites in the same mtT-DNA gene sometimes lead to multiple disease types, possibly due to diverse effects on tRNA synthesis, maturation, accepting activity, or stability. In contrast, mutations in mitochondrial ARSs impact aminoacylation activity without affecting production, processing or turnover of cognate tRNAs, possibly accounting for the phenotype mismatch between mutations of ARS-tRNA pairs.
## Arss as pharmacological targets against pathogenic microorganisms and in autoimmune disease
Mupirocin targets aminoacylation site of bacterial IARS Antibiotic resistance of pathogenic bacteria is a major threat to public health and provides a rationale for screening natural products to seek new antibiotic therapies. Clinical effectiveness and low side-effects of ARS inhibitors demand extremely high selectivity for bacterial versus human ARS targets. ARSs exhibit multiple characteristics potentially advantageous for targets, including (i) high evolutionary divergence of prokaryotic and eukaryotic ARSs; (ii) the full complement of bacterial ARSs provides 20 distinct targets; (iii) a wealth of structural information on both bacterial and eukaryotic ARSs and (iv) an abundance of known natural inhibitors that can be used for starting points for drug development. Mupirocin (pseudomonic acid) is currently the only ARS inhibitor commercially used as an antibiotic. It is a natural product isolated from Pseudomonas fluorescens that inhibits eubacterial and archaeal IARS catalytic activity, and is topically effective against grampositive pathogens [bib_ref] Insights into editing from an ile-tRNA synthetase structure with tRNAile and mupirocin, Silvian [/bib_ref]. The antibiotic inhibits the enzymatic activity of IARS by blocking the synthetic binding site of the intermediate Ile-AMP [fig_ref] Figure 3: ARSs as drug targets [/fig_ref] [bib_ref] Structural basis for the recognition of isoleucyl-adenylate and an antibiotic, mupirocin, by..., Nakama [/bib_ref].
Most antibiotics under development to target ARS active sites are natural or synthetic compounds mimicking the aminoacyl-AMP moieties to repress aminoacylation activity of the corresponding ARSs.
AN2690 targets editing site of fungal LARS AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole) is a broad-spectrum, anti-fungal therapeutic agent specifically developed for topical treatment of onychomycosis [bib_ref] Discovery of a new boron-containing antifungal agent, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), for the potential..., Baker [/bib_ref]. Onychomycosis, commonly referred to as 'nail fungus', is primarily caused by dermatophytes, fungi that infect the skin or nails. AN2690 inhibits the essential fungal enzyme, LARS, and was designed with three distinguishing characteristics, namely, enhanced nail penetration, absence of systemic side effects, and potent mechanism-based antifungal activity [bib_ref] An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the..., Rock [/bib_ref]. The inhibition of protein synthesis by AN2690 leads to specific repression of fungal cell growth [bib_ref] Unique residues crucial for optimal editing in yeast cytoplasmic leucyl-tRNA synthetase are..., Yao [/bib_ref] , thereby eliminating the infection. Mechanistically, AN2690 inhibits synthesis of leucyl-tRNA Leu by formation of a stable boronmediated tRNA Leu -AN2690 adduct in the editing site of LARS and consequently blocking protein synthesis [fig_ref] Figure 3: ARSs as drug targets [/fig_ref]. Importantly, these findings have established the editing site as a bona fide target for ARS inhibitors. AN2690 did not produce systematic side-effects during phase I and phase II clinical trials (www. anacor.com). The high specificity of AN2690 for the fungal enzyme is possibly due to inefficient penetration of human cell plasma membranes or inaccessibility of LARS in the MSC.
## Prs-dead eprs
## Halofuginone targets human eprs, activates amino acid response (aar) pathway and inhibits t h 17-cell differentiation
Febrifugine is a bioactive natural product extracted from roots of the hydrangea Dichroa febrifuga Lour and used in traditional Chinese medicine. Febrifugine derivatives have been used to treat malaria, cancer, fibrosis and inflammatory disease. T helper 17 (T h 17) cells, a T helper cell sub-class characterized by robust interleukin 17 (IL-17) production, have a key role in autoimmune diseases [bib_ref] Th17 cells: biology, pathogenesis of autoimmune and inflammatory diseases, and therapeutic strategies, Maddur [/bib_ref]. Halofuginone (HF), a low-toxicity, halogenated febrifugine derivative, activates the amino acid response (AAR) pathway and inhibits the development and progression of T h 17-driven autoimmunity in a mouse model of multiple sclerosis [fig_ref] Figure 3: ARSs as drug targets [/fig_ref] [bib_ref] Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase, Keller [/bib_ref]. HF is in clinical trials as a therapeutic against cancer and fibrotic disease. The HF target in human cells is human EPRS. HF acts as an ATPdependent, prolyl adenylate-mimetic that specifically inhibits PRS aminoacylation activity. At an appropriate dose that does not cause global translational arrest, HF triggers the AAR pathway, inhibits T h 17 differentiation, and induces antifibrotic activities in fibroblasts. The AAR pathway is triggered by amino acid starvation, and results in an increase of transcription factor ATF4, which in turn limits or increases the production of other downstream target proteins. The novel pharmacological mechanism suggests that the AAR pathway might be an important target for drugs accelerating the resolution of inflammation. HF has long been applied as an anti-parasitic agent, and the recent realization of its activity as an antiautoimmune response inhibitor expands its potential clinical utility.
It remains an open question whether other ARS-targeting antibiotics with low efficiency for human enzymes can be developed into therapeutic agents targeting human disease.
## Noncanonical functions of arss in regulating gene expression and cellular functions related to disease
The noncanonical functions of ARSs beyond global translation have been reviewed elsewhere [bib_ref] New functions of aminoacyl-tRNA synthetases beyond translation, Guo [/bib_ref] [bib_ref] Aminoacyl tRNA synthetases and their connections to disease, Park [/bib_ref]. In mammalian cells, three non-enzyme factors that reside with nine of the ARSs in the MSC, i.e. MSC p43, p38 and p18, also exhibit cellular activities beyond translation outside of the MSC. The physiological and pathological implications of these non-synthetase components of the MSC have been reviewed elsewhere [bib_ref] Functional expansion of aminoacyl-tRNA synthetases and their interacting factors: new perspectives on..., Park [/bib_ref] [bib_ref] Aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMPs): a triad for cellular homeostasis, Park [/bib_ref]. Here, we focus on recent advances in our understanding of noncanonical functions of human cytoplasmic ARSs [fig_ref] Figure 4: Noncanonical functions of ARSs in regulating cell functions [/fig_ref].
Phosphorylated EPRS directs gene-specific translational silencing of inflammation-related mRNAs Macrophages function in innate immunity in vertebrates by phagocytosing pathogens and cellular debris, and by releasing cytotoxic proteins as a defence against infection. Overaccumulation of these toxic agents can be detrimental to host tissues and organisms, and contribute to chronic inflammatory diseases. The EPRS-bearing GAIT (gamma-interferon-activated inhibitor of translation) complex suppresses translation of a 'regulon' of inflammation-related mRNAs, including ceruloplasmin (Cp), vascular endothelial growth factor-A (VEGF-A), and several chemokine receptors and ligands among others [bib_ref] DAPK-ZIPK-L13a axis constitutes a negative-feedback module regulating inflammatory gene expression, Mukhopadhyay [/bib_ref] [bib_ref] A stressresponsive RNA switch regulates VEGFA expression, Ray [/bib_ref] [bib_ref] Genome-wide polysome profiling reveals an inflammationresponsive posttranscriptional operon in gamma interferon-activated monocytes, Vyas [/bib_ref]. The complex might participate in the 'resolution of inflammation' by restricting production of injurious proteins [bib_ref] Noncanonical function of glutamyl-prolyl-tRNA synthetase: gene-specific silencing of translation, Sampath [/bib_ref]. EPRS is the unique GAIT constituent that binds the GAIT RNA element in the 3 0 -UTR of target mRNAs. In human myeloid cells, interferon-g induces sequential phosphorylation of Ser 886 and Ser 999 of EPRS in the noncatalytic linker connecting the synthetase cores [bib_ref] Two-site phosphorylation of EPRS coordinates multimodal regulation of noncanonical translational control activity, Arif [/bib_ref]. Ser 886 phosphorylation is required for binding NSAP1 (NS1-associated protein 1) to form an inactive pre-GAIT complex. Ser 999 phosphorylation directs the formation of the active GAIT complex that binds initiation factor eIF4G and blocks recruitment of the preinitiation complex. Unexpectedly, a C-terminus truncated form of EPRS, termed EPRS N1 , was found to bind GAIT target mRNAs and prevent binding of the functional GAIT complex, thereby acting as a dominant-negative inhibitor of GAIT activity. This imposes a 'translational trickle' of target protein expression at basal levels required for tissue well-being [bib_ref] Coding region polyadenylation generates a truncated tRNA synthetase that counters translation repression, Yao [/bib_ref]. Genetic or condition-dependent defects in EPRS have not been reported; however, pathological dysregulation of another GAIT constituent protein, ribosomal protein L13a, by oxidized lipoproteins disrupts the GAIT system, potentially contributing to progression of chronic inflammatory disorders [bib_ref] Protection of extraribosomal RPL13a by GAPDH and dysregulation by S-nitrosylation, Jia [/bib_ref].
## Mars regulates global translation and tumour suppressor activity of msc p18
Human MARS has an especially critical role in translation initiation by transferring Met to initiator tRNA i Met . MARS is a component of the MSC and provides a docking site for MSC p18 in the cytoplasm [bib_ref] Dual role of methionyl-tRNA synthetase in the regulation of translation and tumor..., Kwon [/bib_ref]. MSC p18 is a potent tumour suppressor that translocates to the nucleus for DNA repair upon DNA damage [bib_ref] The haploinsufficient tumor suppressor p18 upregulates p53 via interactions with ATM/ATR, Park [/bib_ref] , and also regulates translational initiation by mediating the delivery of charged tRNA i Met to the initiation complex . UV irradiation induces phosphorylation of MARS at Ser 662 by GCN2 (general control nonrepressed-2), releasing tumour suppressor MSC p18 for nuclear re-localization. Phosphorylated MARS exhibits significantly reduced catalytic activity due to diminished tRNA i Met binding, and a consequent repression of global translation. Interestingly, UV-stress-induced MARS phosphorylation, and subsequent global translational repression, mirrors IFN-g-induced EPRS phosphorylation and transcriptselective translational silencing program, revealing a noncanonical theme common to these and possibly other synthetases.
## Sars mediates transcriptional repression during vascular development
In a genetic screen in zebrafish, mutations in the sars gene were found that affect vascular development and maintenance, including aortic arch vessel dilatation, aberrant hindbrain capillary patterning, abnormal intersomitic vessels, or disorganized vessels with abnormal branching of established intersegmental vessels [bib_ref] Noncanonical activity of seryl-tRNA synthetase is involved in vascular development, Fukui [/bib_ref] [bib_ref] Genetic evidence for a noncanonical function of seryl-tRNA synthetase in vascular development, Herzog [/bib_ref]. Phenotypic rescue by over-expression of an enzymatically inactive form of sars T429A and knockdown of vegf receptor or its ligand vegf-a, suggest that a noncanonical function of SARS in repression of VEGF-A expression contributes to vascular development. Injections of highly homologous human SARS mRNA rescued the vascular branching phenotype in sars mutant-bearing zebrafish suggesting that the regulatory function of SARS in vascular development might be conserved between zebrafish and human. The UNE-S (Unique-S) domain, appended to SARS at the C-termini and containing nuclear localization signal (NLS), was identified as an essential domain for shuttling the enzyme into nucleus and repressing VEGF-A mRNA transcription, thereby permitting normal vascular development . It remains an open question whether SARS directly interacts with VEGF-A gene promoter and modulates its transcription, or acts by an indirect mechanism.
WARS mediates crosstalk between IFN-g and p53 signalling pathways WARS exhibits a noncanonical regulatory function for activation of p53 [bib_ref] Trp-tRNA synthetase bridges DNA-PKcs to PARP-1 to link IFN-gamma and p53 signaling, Sajish [/bib_ref]. IFN-g upregulates WARS and facilitates formation of a nuclear trimeric complex with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and poly(ADP-ribose) polymerase 1 (PARP-1). WARS bridges the N-terminal domain of PARP-1 with the kinase domain of DNA-PKcs. Within the complex, PARP-1 catalyses poly(ADP-ribosyl)ation of DNA-PKcs, thereby activating the kinase which in turn activates the tumour suppressor p53 by phosphorylation at Ser 15 . Over-expression of WARS by itself activates p53 indicating that other IFN-g signalling pathways are not required. These results suggest that the antiproliferative and antiangiogenic responses induced by IFN-g-mediated activation of p53 are triggered in part by upregulation and nuclear localization of WARS.
## Lars is a leucine sensor in mtorc1 signalling
The protein kinase mTOR (mammalian target of rapamycin) controls various critical cellular processes including cell growth, autophagy, protein synthesis and metabolism. Activation of the mTOR signalling pathway contributes to multiple human pathologies including cancer and tissue hypertrophy. Pharmacological inhibitors of mTOR are used clinically to prevent graft rejection and restenosis after angioplasty. mTOR complex 1 (mTORC1) is activated by metabolic inputs such as elevated Leu and other amino acids [bib_ref] signaling: what we still don't know, Wang [/bib_ref]. Recently, human LARS was identified as an important intracellular sensor for mTORC1 signalling through its Leu activation activity, independent of tRNA Leu . In the presence of Leu, LARS translocates to the lysosome, interacts with mTORC1, Raptor (regulatory associated protein of mTOR) and the GTPbound form of RagD, and promotes GTP hydrolysis and mTORC1 activation. Knockdown of LARS reduces cell size and increases autophagy. The interaction between RagD and LARS is mediated by the non-catalytic C-termini of both proteins. Consistent with these findings, yeast LARS was identified as a sensor for TORC1 activation [bib_ref] Leucyl-tRNA synthetase controls TORC1 via the EGO complex, Bonfils [/bib_ref] , suggesting evolutionary conservation of this important noncanonical function. It remains to be determined whether elevated LARS in cancer cells causes hyperactivation of mTORC1 signalling with pathological consequences such as tumourigenesis [bib_ref] Implication of leucyl-tRNA synthetase 1 (LARS1) over-expression in growth and migration of..., Shin [/bib_ref].
Membrane complex of KARS and laminin receptor promotes cell migration Human KARS exhibits regulatable cellular localization and multiple functions beyond translation including transcription control upon nuclear import [bib_ref] LysRS serves as a key signaling molecule in the immune response by..., Yannay-Cohen [/bib_ref] and cytokine signalling after release into the extracellular space [bib_ref] Human lysyl-tRNA synthetase is secreted to trigger proinflammatory response, Park [/bib_ref]. Recently, a new membrane translocation event for KARS was reported [bib_ref] Interaction of two translational components, lysyl-tRNA synthetase and p40/37LRP, in plasma membrane..., Kim [/bib_ref]. Upon laminin stimulation, KARS is phosphorylated at Thr 52 residue by p38 MAP kinase, dissociates from the MSC, and translocates to the plasma membrane where it assembles into a complex with the 67 kDa laminin receptor (67LR). KARS inhibits ubiquitin-dependent degradation of 67LR, thereby promoting laminin-induced cell migration. This pro-migratory function of KARS suggests a possible role in tumour cell metastasis.
## Gars as an endogenous anti-tumourigenesis reagent
Human GARS is released from macrophages in response to tumour-derived Fas ligand . GARS binds ERKactivated tumour cells through cadherin (CDH)6 and induces release of phosphatase 2A (PP2A) from CDH6. Activated PP2A inhibits ERK signalling through ERK dephosphorylation, and induces apoptosis. In vivo administration of GARS strongly suppresses tumour growth in a mouse model, accompanied by CDH6 induction and ERK activation. Thus, GARS has potential applicability in anti-tumour therapy.
## Concluding remarks
ARSs have a triple-faceted relationship with human health and disease. First, the loss-of-function of an ARS resulting from genetic mutations can cause a broad spectrum of diseases. Human LARS can correct mitochondrial dysfunctions caused by tRNA Leu UUR A3243G mutation-related neurodegenerative disorder, MELAS syndrome [bib_ref] Human mitochondrial leucyl-tRNA synthetase corrects mitochondrial dysfunctions due to the tRNALeu(UUR) A3243G..., Li [/bib_ref]. This finding provides a step toward therapeutic interventions for human disorders by introducing exogenous ARSs. Second, antibiotic-directed inhibition of ARS activities from lower species, including bacteria and fungi, can treat infectious diseases. Interestingly, targeting specific functions of human ARSs can have beneficial clinical output rather than cytotoxic effect, for example, for treatment of autoimmune diseases. Third, elucidation of emerging noncanonical functions of human ARSs will provide unique opportunities for therapeutic intervention. Because many therapeutic reagents with high potency also exhibit adverse side effects, e.g., elevated risk of coronary heart disease by nonsteroidal anti-inflammatory drugs, and disturbance of blood vessel maintenance by anti-VEGF-A inhibitors, the application or stimulation of natural secretory or endogenous ARSs (e.g., GARS and EPRS), might provide a new set of physiologic extracellular or intracellular pathways as basis for developing novel therapeutics with minimal side-effects.
[fig] Figure 1: Genetic mutations in human cytosolic ARSs cause CMT disease. The domain organization and sites of genetic mutations in human KARS, AARS, YARS and GARS are shown. [/fig]
[fig] Figure 2: Potential mechanisms underlying CMT caused by ARS mutations. Possible etiologic mechanisms of CMT are shown. (a) Defective aminoacylation activity; (b) defective editing activity; (c) destabilization or degradation; (d) aggregate formation; (e) defective dimerization; (f) abnormal nuclear import; (g) abnormal mitochondrial import; (h) abnormal localization in cytosolic granules; (i) gain-of-function by generation of new protein interactions; (j) loss of noncanonical function and (k) defective axonal transport. Genetic mutations in ARSs are indicated (red stars). [/fig]
[fig] 2013: The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. [/fig]
[fig] Figure 3: ARSs as drug targets. A. The anti-bacterial reagent mupirocin targets the bacterial IARS synthetic active site by blocking Ile-AMP binding. B. The anti-fungal reagent AN2690 targets the yeast LARS editing active site by boron-mediated trapping of tRNA Leu . C. The anti-fibrotic reagent halofuginone targets synthetic active site of PRS in human EPRS, triggering the AAR pathway and inhibiting T h 17-cell differentiation. [/fig]
[fig] Figure 4: Noncanonical functions of ARSs in regulating cell functions. Recent discoveries of noncanonical functions of human ARSs and their underlying mechanisms. IFN-g-induced release of EPRS from the MSC and assembly of the GAIT complex that mediates translational silencing of inflammation-related mRNAs. UV-triggered phosphorylation of MARS inhibits global translation and activates tumour suppressor MSC p18. SARS directs transcriptional repression of VEGFA during vertebrate vascular development. WARS mediates crosstalk between IFN-g and p53 signalling pathway to activate p53. LARS acts as a leucine sensor in mTORC1 signalling to regulate cell size and autophagy. Laminin-stimulated membrane localization of KARS promotes cell migration. Human macrophages secret GARS to suppress tumour growth. See details in the text. [/fig]
[table] Table 1: Compilation of mitochondrial ARS-derived genetic mutations and their connections to human diseases [/table]
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s2orc_pubmed_articles
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Verbal Memory Impairments in Children after Cerebellar Tumor Resection
This study was designed to investigate cerebellar lobular contributions to specific cognitive deficits observed after cerebellar tumor resection. Verbal working memory (VWM) tasks were administered to children following surgical resection of cerebellar pilocytic astrocytomas and age-matched controls. Anatomical MRI scans were used to quantify the extent of cerebellar lobular damage from each patient's resection. Patients exhibited significantly reduced digit span for auditory but not visual stimuli, relative to controls, and damage to left hemispheral lobule VIII was significantly correlated with this deficit. Patients also showed reduced effects of articulatory suppression and this was correlated with damage to the vermis and hemispheral lobule IV/V bilaterally. Phonological similarity and recency effects did not differ overall between patients and controls, but outlier patients with abnormal phonological similarity effects to either auditory or visual stimuli were found to have damage to hemispheral lobule VIII/VIIB on the left and right, respectively. We postulate that damage to left hemispheral lobule VIII may interfere with encoding of auditory stimuli into the phonological store. These data corroborate neuroimaging studies showing focal cerebellar activation during VWM paradigms, and thereby allow us to predict with greater accuracy which specific neurocognitive processes will be affected by a cerebellar tumor resection.
# Introduction
As early as 1809, anatomists and physiologists such as Rolando and Flourens concluded, based on ablation experiments in animals, that the cerebellum is "the organ controlling locomotion". The notion that the cerebellum is primarily or exclusively engaged in activities of motor control and balance has been widely ac-cepted in clinical neurology for decades. Recent findings from neuroimaging and patient studies, however, have suggested that the cerebellum also plays a role in higher-order, non-motor processes such as learning, memory and emotion. A growing body of evidence suggests that children can suffer intellectual disabilities after cerebellar tumor resection [bib_ref] Cognitive and adaptive outcome in low-grade pediatric cerebellar astrocytomas: evidence of diminished..., Beebe [/bib_ref] [bib_ref] Surgery of tumors of the cerebellum and prefrontal cortex, and sensory memory..., Castro-Sierra [/bib_ref] [bib_ref] Late effects of treatment on the intelligence of children with posterior fossa..., Duffner [/bib_ref] [bib_ref] Critical risk factors for intellectual impairment in children with posterior fossa tumors:..., Grill [/bib_ref] [bib_ref] Relevance of the cerebellar hemispheres for executive functions, Karatekin [/bib_ref] [bib_ref] Preoperative and postoperative analysis of visual and auditory memory in children with..., Lazareff [/bib_ref] [bib_ref] Neuropsychological consequences of cerebellar tumour resection in children: cerebellar cognitive affective syndrome..., Levisohn [/bib_ref] [bib_ref] Cerebellum and procedural learning: evidence from focal cerebellar lesions, Molinari [/bib_ref] [bib_ref] Muteness of cerebellar origin, Rekate [/bib_ref] [bib_ref] The cerebellum contributes to higher functions during development: evidence from a series..., Riva [/bib_ref] [bib_ref] Lateralized cognitive deficits in children following cerebellar lesions, Scott [/bib_ref] [bib_ref] Neuropsychological long-term sequelae after posterior fossa tumour resection during childhood, Steinlin [/bib_ref] [bib_ref] Cognitive impairments in patients with congenital nonprogressive cerebellar ataxia, Steinlin [/bib_ref]. Due to the heterogeneity of tumor pathology, tumor location and treatment modalities (e.g., irradiation and chemotherapy) used in these studies, it has been difficult to make inferences as to the role of the cerebellum in specific cognitive processes. In addition, most of the studies exploring cognition in cerebellar damaged patients are limited in their abili-ty to determine which specific cerebellar regions (i.e. lobules) are responsible for specific cognitive impairments.
Much of our current knowledge of cerebellar function is derived from studies of adult patients with cerebellar pathology. For example, studies of patients with isolated cerebellar infarcts have implicated a role for the right posterolateral cerebellum in verb generation [bib_ref] Role of the posterolateral cerebellum in language, Gebhart [/bib_ref] , linguistic production [bib_ref] The cerebellum contributes to linguistic production: a case of agrammatic speech following..., Silveri [/bib_ref] , and spatial cognition (e.g. hemineglect) [bib_ref] Right side neglect in right cerebellar lesion, Silveri [/bib_ref] , whereas the left posterior cerebellum as been associated with the cognitive affective syndrome [bib_ref] Pure post-stroke cerebellar cognitive affective syndrome: a case report, Paulus [/bib_ref]. In a comparison of patients with isolated posterior inferior cerebellar artery (PICA) and superior cerebellar artery (SCA) territory infarcts, Exner and colleagues showed that patients with inferior and posterior cerebellar damage (including lobules VIIb, VII-Ia, VIIIb, IX and Crus II bilaterally) performed worse on tests of verbal episodic long term memory, short term memory and attention than patients with anterior and superior lesions [bib_ref] Cerebellar lesions in the PICA but not SCA territory impair cognition, Exner [/bib_ref]. In a similar study Neau et al. demonstrated no statistical differences between patients with isolated infarcts in the PICA and SCA regions on tests of verbal cognition [bib_ref] Neuropsychological disturbances in cerebellar infarcts, Neau [/bib_ref]. However, patients with SCA territory infarcts showed decreased performance on a task of inhibition (i.e. the Stroop naming task with and without interference). Qualitatively, five patients with PICA territory infarcts showed impairments on tests of learning and interference,memory (recall and recognition), and verbal fluency.
The present study was designed to assess the role of specific cerebellar regions for human cognition. Verbal working memory (VWM), the process by which finite units of information are maintained in memory for a brief period of time, served as an assay for cognition in these experiments. Baddeley proposed a framework for VWM, called the phonological loop, which consists of two sub-components, a phonological shortterm store, which can hold speech-related information for 1-2 seconds, and an articulatory control system, which serves to sub-vocally refresh the contents of the phonological store [bib_ref] Working memory, Baddeley [/bib_ref] [bib_ref] Working memory and executive control, Baddeley [/bib_ref]. Using functional magnetic resonance imaging (fMRI) activations elicited from a VWM task and known cerebro-cerebellar projections [bib_ref] The cerebrocerebellar system, Schmahmann [/bib_ref] , Desmond and colleagues proposed an extension to Baddeley's VWM framework, in which both the superior and inferior cerebellar hemispheres provide supportive processing to enhance efficiency of neocortical functions through a feed-forward network [bib_ref] Lobular patterns of cerebellar activation in verbal working-memory and finger-tapping tasks as..., Desmond [/bib_ref].
It is well known that VWM continues to develop throughout childhood and adolescence [bib_ref] Relationships among processing speed, working memory, and fluid intelligence in children, Fry [/bib_ref] [bib_ref] The structure of working memory from 4 to 15 years of age, Gathercole [/bib_ref]. Although the majority of studies investigating VWM through psychophysical tasks and neuroimaging paradigms have focused on adult populations, a few studies have examined VWM and its neural correlates during various stages of development [bib_ref] Functional developmental similarities and differences in the neural correlates of verbal and..., Brahmbhatt [/bib_ref] [bib_ref] Putative biomarker of working memory systems development during childhood and adolescence, Keage [/bib_ref] [bib_ref] Combined analysis of DTI and fMRI data reveals a joint maturation of..., Olesen [/bib_ref]. In one of the earliest studies of functional neuroimaging with children, Casey showed activation of inferior and middle frontal gyri during performance of a VWM task which correlated to areas visualized in adults by Cohen et al. [bib_ref] Activation of the prefrontal cortex in a nonspatial working memory task with..., Cohen [/bib_ref]. Diffusion tensor and functional MR imaging results from another study of children demonstrated activation of superior frontal and inferior parietal regions [bib_ref] Combined analysis of DTI and fMRI data reveals a joint maturation of..., Olesen [/bib_ref] , both classic centers underlying VWM processing in adults. Brahmbhatt and colleagues [bib_ref] Functional developmental similarities and differences in the neural correlates of verbal and..., Brahmbhatt [/bib_ref] further demonstrated activation of the known functional network subserving VWM, including the right cerebellum, and found that certain regions within these networks, specifically the superior parietal cortex, show age-related differences between adolescents and adults. Similarities in functional neuroanatomy between adults and children have also been demonstrated in several studies for spatial working memory [bib_ref] Increased brain activity in frontal and parietal cortex underlies the development of..., Klingberg [/bib_ref] [bib_ref] Neural basis of protracted developmental changes in visuo-spatial working memory, Kwon [/bib_ref] [bib_ref] Functional neuroanatomy of spatial working memory in children, Nelson [/bib_ref] [bib_ref] A developmental functional MRI study of spatial working memory, Thomas [/bib_ref].
Currently, patients with cerebellar pathology are not routinely evaluated for cognitive deficits. Even if they are administered a standard neuropsychological assessment, comprehensive tests of phonological storage and articulatory rehearsal, the two sub-components of VWM, are not typically included. Therefore, to assess for the necessity of the cerebellum's contribution to the phonological loop, we applied a series of computerized psychophysical tasks to systematically probe the two sub-components of VWM. In addition, detailed anatomical analyses were performed to determine precisely which cerebellar lobules were compromised for each patient. Based on patterns of fMRI activation during verbal working memory [bib_ref] Lobular patterns of cerebellar activation in verbal working-memory and finger-tapping tasks as..., Desmond [/bib_ref] , we hypothesized that articulatory rehearsal deficits would be correlated with damage to superior cerebellar hemispheres and portions of the vermis. Deficits in phonological storage, on the other hand, should be correlated with damage to inferior cerebellum. It is conceivable that if the cerebellum does play an important role in the process of VWM, many of the higher-order cognitive deficits described in cerebellar damaged patients could potentially be explained by this involvement.
# Subjects and methods
## Subjects
Subjects were 12 patients recruited through the Pediatric Neuro-Oncology Clinic at Lucile Packard Chil- [fig_ref] Table 2: Distribution of cerebellar lobular damage [/fig_ref]. Images are presented in radiological convention. dren's Hospital who had undergone complete surgical resection for a cerebellar pilocytic astrocytoma, without any adjuvant radiotherapy or chemotherapy, and an equal number of gender and age-matched healthy controls. Representative coronal sections illustrating the damage to the cerebellum are depicted in [fig_ref] Figure 1: T1-weighted coronal MRI images showing the cerebellum of all 12 patients after... [/fig_ref]. Patients ranged from 6-19 years old (mean = 12.5 years, S.D. = 4.1, distribution = 13.9, 6.5, and were an average of 5.5 ± 3.1 (SD) years post surgical resection. Control subjects were selected to match age and education level of the patients, and statistical tests confirmed the lack of any significant differences in either age (t(11) = 0.31, p = 0.764) or level of education (t(11) = −0.56, p = 0.586) between patients and controls. Informed consent was obtained from each individual subject prior to their participation, which was approved by the Institutional Review Board at Stanford University. All subjects received limited financial compensation for their participation.
All subjects spoke English fluently and all but one patient were right handed. All patients had a normal neurological exam without any gross cerebellar deficits at the time of testing and none reported a history of head injury with loss of consciousness or diagnosis of a psychological disorder before or after their tumor resection. In addition, none of the patients or controls reported being diagnosed with a learning disability or an attention deficit disorder, or being enrolled in special education courses. All healthy controls also reported no history of a head injury with loss of consciousness, neurological or psychological disorders, or major illnesses or surgeries.
## Neuropsychological assessment
Subjects completed the following battery of neuropsychological tests to assess general intellectual functioning, specific components of the VWM circuitry (e.g. phonological processing), and fine motor coordination:
Wechsler Abbreviated Scale of Intelligence (WASI TM ). The WASI has been shown to be a reliable measure of intelligence. The two subtests, vocabulary and matrix reasoning, were used.
Conner's Continuous Performance Test-Second Edition (CPT-II). The CPT-II measures attention and simple reaction time.
Comprehensive Test of Phonological Processing (CTOPP). The CTOPP measures phonological processing abilities.
Controlled Oral Word Association (COWA). The COWA test measures both phonemic and semantic verbal fluency.
Purdue Pegboard Test. The Purdue Pegboard measures finger and hand dexterity for fine motor co-ordination. This test has been used to localize cerebral lesions and deficits [bib_ref] Purdue Pegboard: test-retest estimates, Reddon [/bib_ref].
## Behavioral vwm tasks
The most fundamental VWM task requires subjects to recall serial sequences of digits of increasing length. Pre-recorded sequences of one, two, three, four, five, six or seven digits (0-9) were presented either aurally through audio speakers or visually on a computer monitor. Subjects received 5 trials at each sequence length, beginning with one item. If a subject failed to correctly recall at least 2 sequences at a given length the following longer sequence was not administered. Memory span for each condition was calculated by summing the accuracy for each of the seven sequence lengths [bib_ref] Verbal short-term store-rehearsal system and the cerebellum. Evidence from a patient with..., Silveri [/bib_ref] [bib_ref] The phonological short-term store-rehearsal system: patterns of impairment and neural correlates, Vallar [/bib_ref]. Memory span was measured using two modes of response. For the first, subjects verbally repeated the sequence; for the second, a 2 × 5 array of letters, randomly arranged, were presented on the computer screen and subjects pointed to the items in sequence.
The immediate serial recall of phonologically dissimilar items is generally more accurate than recall for phonologically similar items, a phenomenon that has been referred to as the phonological similarity effect (PSE) [bib_ref] Short-term memory for word sequences as a function of acoustic, semantic and..., Baddeley [/bib_ref] [bib_ref] Information, acoustic confusion and memory span, Conrad [/bib_ref]. This phenomenon is thought to arise because of conflicts in the phonological store. Thus, impairments in the phonological store should result in an attenuation of the PSE and similar performance for both phonologically similar and dissimilar stimuli (i.e. a negative PSE). Subjects were instructed to remember a list of six consonants taken from a set of either phonologically similar (B, C, D, G, P, T, V) or phonologically dissimilar (F, K, Q, R, X, W, Z) letters presented at a rate of 2 items per second either aurally or visually through a computer system. After subjects rehearsed these letters sub-vocally during a 5000 ms retention interval, they responded "yes" or "no" via a button press to indicate if a probe item, presented for the initial 1500 ms of a 2000 ms response interval, matched a remembered letter in the preceding list. The probe and response period was followed by an inter-trial-interval of 3000 ms. A fixation cross, presented for 750 ms (followed by a 250 ms delay) indicated the start of each trial. Subjects completed 48 trials of the PSE task in each presentation modality.
The continuous uttering of irrelevant speech (e.g., repeating the words "the" or "blah") during a VWM task disrupts the articulatory rehearsal system and leads to impairments in performance [bib_ref] Exploring the articulatory loop, Baddeley [/bib_ref]. To test this articulatory suppression effect, subjects were instructed to remember a list of six randomly generated consonant letters presented at a rate of 2 per second either aurally or visually. During half of the trials, subjects were instructed to start uttering the syllable "blah" before the presentation of the stimuli, and to stop after the 5000 ms sub-vocal rehearsal period when the probe item was presented. Subjects again responded via a button press to the probe and completed 48 trials of the task in both visual and auditory modalities. The inter-trial-interval for this task was 3000 ms, and trials also began with a 750 ms fixation cross followed by a 250 ms delay.
Another way to assess the integrity of VWM circuitry is to measure subjects' performance when they freely recall sequences of stimuli that exceed the length of their immediate memory span. Normal subjects show greater recall of the final items in the list (recency effect), compared to the preceding stimuli [bib_ref] Two storage mechanisms in free recall, Glanzer [/bib_ref] [bib_ref] The phonological short-term store-rehearsal system: patterns of impairment and neural correlates, Vallar [/bib_ref] [bib_ref] Phonological short-term store and the nature of the recency effect: evidence from..., Vallar [/bib_ref]. In immediate auditory free recall, the phonological short term store provides a greater contribution to the recency effect than the articulatory control system [bib_ref] Phonological short-term store and the nature of the recency effect: evidence from..., Vallar [/bib_ref] [bib_ref] The anatomical localization of selective impairment of auditory verbal shortterm memory, Warrington [/bib_ref]. Thus, patients with an impaired phonological store should show an attenuation of the recency effect. On the other hand, patients with a reduced memory span and a normal recency effect, the deficit could be reasonably attributed to the articulatory rehearsal process [bib_ref] The phonological short-term store-rehearsal system: patterns of impairment and neural correlates, Vallar [/bib_ref].
Twenty lists, composed of 12 common nouns, chosen at random and without replacement from a pool of high frequency nouns [bib_ref] Theta and gamma oscillations during encoding predict subsequent recall, Sederberg [/bib_ref] , were visually presented to the subjects at a rate of 1 item per second. Immediately after the presentation of the list, subjects were instructed to verbally recall as many words as they could remember in any order. The words were manually recorded by the experimenter in the order they were recalled (an audio tape recording was used to verify recalled words). Subjects indicated they were ready to proceed to the next list by pushing a button. A cross, presented for 1 second, indicated the start of a new trial.
## Anatomical analysis of cerebellar tumors
In order to determine the effect of a particular cerebellar resection on behavioral performance,coronal T1weighted multi-echo multi-planar post-surgical MRI brain images were acquired for each subject from clinical scans. These scans consisted of 24-30 coronal sections 5 mm thick with 1 mm gap and an inplane resolution of 0.43 mm-0.94 mm. Repetition time (TR) ranged from 417-800 ms, and echo time (TE) was 20 ms. These slices were assembled into a 3D volumetric data setand aligned into the Montreal Neurological Institute (MNI) coordinate system. Using custom-made software previously described [bib_ref] On-and offline Talairach registration for structural and functional MRI studies, Desmond [/bib_ref] , this brain volume was then realigned such that the brainstem was oriented vertically as visualized on a midline sagittal image. The x, y, and z extent of the cerebellum in this new orientation was measured manually for each patient using a utility built in to the software, thereby allowing the volume to be adjusted for potential age-related differences in cerebellar dimensions and scaled to match the cerebellar dimensions of the MNI template. The net result was a 9-paramter (translation, rotation, and scaling adjustments in the x, y, z dimensions) normalization for each subject to the MNI cerebellum. Eleven coronal sections were then resliced parallel to the brainstem, and the perimeter of the surgical resection was manually outlined from these sections using a closed polygon utility that captured the x, y, z coordinates of all the voxels contained within the polygon. From these captured coordinates a lesion "volume of interest" could be created in the same volume coordinate system. The lobular distribution of each lesion was determined by overlaying the lesion volume of interest on a separate volume containing anatomical codes from the Automated Anatomical Labeling (AAL) map [bib_ref] Automated anatomical labeling of activations in SPM using a macroscopic anatomical parcellation..., Tzourio-Mazoyer [/bib_ref] distinguishing the individual cerebellar lobules. Deep (dentate/interposed) nucleus regions of interest were created using probabilistic X, Y, Z dimensional boundaries for the MNI brain published by Dimitrova et al.. Values corresponding to 51-60% of subject overlap were used (see their [fig_ref] Table 1: Neuropsychological assessment scores All measures are represented in T scores [/fig_ref] in the ROI creation. The per-centage of voxels occupied by the lesion in each lobule or deep nuclear ROI was computed and recorded. All patients were confirmed to have isolated pilocytic cerebellar astrocytomas. A complete set of neuroimaging scans, including T2-weighted scans and contrast studies, were inspected to ascertain that no other tumors or lesions were present.
## Statistical analyses 2.5.1. analysis of main behavioral variables
The effects of cerebellar damage on verbal digit span were assessed with a 2 × 2 repeated measures analysis of variance (ANOVA) with one between subject factor of group (patient vs. control) and one within subject factor of modality of presentation (auditory vs. visual). The effects of cerebellar damage on articulatory control were assessed by comparing performance (i.e., accuracy, defined as the proportion of the total number of trials in which a correct response was made) with and without articulatory suppression, and was evaluated using a 2 × 2 × 2 repeated measures ANOVA with within subject factors of modality (auditory vs. visual) and rehearsal type (with vs. without suppression), and between subject factor of group. The effects of cerebellar damage on phonological storage were assessed with two analyses. First, the phonological similarity effect was evaluated by comparing performance (i.e., accuracy, as defined above for articulatory suppression) with phonologically similar vs. dissimilar letters. A 2 × 2 × 2 repeated measures ANOVA was therefore used for analysis with within subject factors of modality (auditory vs. visual) and phonological similarity (similar vs. dissimilar), and between subject factor of group. Second, the recency effect of freely recalled items was assessed by examining accuracy of recall as a function of serial position of presentation. Statistical analysis of this effect was conducted using a 2 × 12 repeated measures ANOVA with within subject factor of serial position and between subject factor of group (this test was conducted only with visual presentation so there is no factor of modality). Because two neuropsychological test measures (FSIQ and Matrix measures, see below) were significantly different between the groups, ANOVAs that indicated significant group effects were repeated using each of these measures as a covariate to insure that significant group effects of interest remained significant.
## Mapping behavioral performance to lesion
locations For behavioral measures in which patient performance was found to be significantly worse than that of controls, regression analyses were conducted to determine if the extent of lobular damage was correlated with the degree of behavioral impairment. If the behavioral measure was found to be correlated with the age of the patient, both lobular damage and age were regressed on the behavioral measure and partial correlations were computed to determine if lobular damage significantly predicted performance above and beyond the contribution of age. To correct for multiple comparisons (24 lobular/deep nuclear regions of interest) a Bonferroni correction was applied to achieve an overall family wise error rate of 0.05, taking into account for the fact that cerebellar region of interest measures were correlated with each other [bib_ref] Some comments on frequently used multiple endpoint adjustment methods in clinical trials, Sankoh [/bib_ref]. The mean correlation of the regions of interest was determined by averaging all 276 pairwise combinations of correlations from the 24 regions of interest, and this average was 0.325. From these values, a Bonferroni corrected p value of 0.00585 was required. [fig_ref] Table 1: Neuropsychological assessment scores All measures are represented in T scores [/fig_ref] displays results of the neuropsychological assessment measures from patients and their age-matched controls. Results from the WASI (vocabulary and matrix subtests), COWA, CPT-II and Purdue Pegboard are presented as T-scores with a mean of 50 and standard deviation of 10. Results from the WASI (full scale IQ) and CTOPP are presented as standard scores with a mean of 100 and a standard deviation of 15. Although all of the tumor patients except for one had IQ scores in the average or above average range, overall, the patients scored significantly lower than their respective controls (t(22) = −2.336, p = 0.029). This effect was carried primarily by the matrix subtest of the WASI which was significantly higher for controls (t(11) = −2.29, p = 0.032). There was no significant difference between patients and controls on tests of verbal fluency (FAS), animal naming, attention (CPT-II), phonological processing (CTOPP), or fine motor control (pegboard, preferred hand).
# Results
## Neuropsychological measures
As described above, one cerebellar patient was left handed. The specific neuropsychological scores for this patient were as follows: 2. For the behavioral tests described above, the main abnormality displayed for this patient was an abnormal phonological similarity effect to visually presented stimuli. The lesion for this subject was located in the vermis, right inferior cerebellum, and right deep nuclei. [fig_ref] Figure 2: Anatomical distribution of cerebellar damage averaged over the 12 patients [/fig_ref] illustrates the anatomical distribution of cerebellar damage averaged over the 12 patients. The X axis depicts the names of the lobules and the Y axis depicts the mean percent damage to the lobule. Table 2 summarizes the subject by subject distribution of lobular damage. These analyses indicate that the vermis suffered the greatest amount of damage in these patients.
## Anatomical analyses
## Behavioral vwm tasks
Analysis of variance of verbal digit span revealed a significant group x modality interaction (F(1,22) = 7.69, p = 0.011, [fig_ref] Figure 3: Digit span performance for patients and controls in auditory and visual modalities [/fig_ref] , which remained significant when FSIQ and Matrix scores were entered as covariates (group x modality interaction p = 0.007 and p = 0.001, respectively). Analysis of simple main effects indicated that patients performed worse than controls in the auditory modality (t(11) = 2.65, p = 0.023, paired t-test of patients with age-matched controls), but not in the visual modality. Analysis of performance when patients pointed rather than verbally responded indicated no difference in the two modes of responding. Patients did not perform significantly different for auditory and visual modalities (t(11) = 0.532, p = 0.605). Controls on the other hand, performed significantly better in the auditory modality (t(11) = 4.29, p < 0.01). In addition, the ANOVA indicated a significant main effect of group (F(1,22) = 5.65, p = 0.027).
Analyses of structure/behavior correlation were confined to the auditory modality because patient and control digit span differed only in this modality. Auditory digit span performance was found to be correlated
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−less than 5% of lobule resected; +5-50% of lobule resected; ++more than 50 % of lobule resected.
with patient age (Pearson r = 0.88, p < 0.0001), so both lobular damage and age were regressed on patient digit span performance, and partial correlations were performed to determine if lobular damage significantly increased the proportion of accountable variance. This analysis resulted in only one significant lobule, the left hemispheral lobule VIII (lobule beta test: t(9) = 3.65, p = 0.0054; multiple-R for lobule and age = 0.955, F(2,9) = 46.73, p < 0.00001). Analysis of articulatory suppression revealed a significant rehearsal type x group interaction, indicating that patients were significantly less affected by articulatory suppression than controls (F(1,22) = 5.65, p = 0.027,. This effect remained significant when the ANOVA was repeated with either FSIQ or Matrix scores entered as covariates (rehearsal type x group interaction p = 0.046 and p = 0.014, respectively). There was also a significant main effect of rehearsal type (F(1,22) = 45.16, p < 0.0001). The modality x group and rehearsal type x modality interactions both approached significance (p = 0.10 and p = 0.12, respectively). The articulatory suppression measurement (i.e. no suppression -suppression accuracy) for visual, but not auditory, stimuli was correlated with age (r = 0.61, p = 0.035) so both lobular damage and age were regressed on patient articulatory suppression measures for visual stimuli. This analysis indicated that damage to vermian lobule VII (lobule beta t(9) = 3.95, p = 0.0034, multiple R = 0.878, p = 0.0013) and vermian lobule VIII (lobule beta t(9) = 3.97, p = 0.0033, multiple R = 0.879, p = 0.0013) was significantly correlated with impairment in the articulatory suppression measurement for visual stimuli (i.e., as lobule damage increased, the non-suppressed minus suppressed accuracy decreased), with p-values below the Bonferroni threshold. In addition, the following lobules exhibited significant correlations at standard p-value thresholds but did not survive with the Bonferroni correction: left hemispheral lobule IV/V (lobule beta t(9) = 2.54, p = 0.032, multiple R = 0.797, p = 0.011), vermian lobule IV/V (lobule beta t(9) = 2.80, p = 0.0074, multiple R = 0.815, p = 0.0074), vermian lobule VI (lobule beta t(9) = 2.84, p = 0.019, multiple R = 0.818, p = 0.0069), and vermian lobule IX (lobule beta t(9) = 2.82, p = 0.02, multiple R = 0.816, p = 0.0071). In addition the right hemispheral lobule IV/V approached significance (lobule beta t(9) = 1.744, p = 0.11, multiple R = 0.729, p = 0.033). As the auditory articulatory suppression measure was not correlated with age, lobular damage was simply correlated with behavioral measures. In contrast to the visual modality, no lobules were correlated with the auditory articulatory suppression measure.
Analysis of the phonological similarity accuracy measures revealed a main effect of phonological similarity (F(1,22) = 14.92, p = 0.0008), indicating that subjects in general responded with greater accuracy for phonologically dissimilar stimuli (78.9% accuracy) than for similar stimuli (71.7% accuracy). However, there was no significant subject type x phonological similarity interaction or subject type x modality x phonological similarity interaction, indicating that both patients and controls exhibited a standard phonological similarity effect.
Although the ANOVA did not indicate overall group differences in the phonological similarity effect between patients and controls, we noted that in 3 cases for the auditory modality (subjects 01, 02 and 08) and 3 different cases for the visual modality (subjects 03, 06, and 07) behavioral performance was worse than controls by at least 3 standard errors of the mean performance for control subjects. In these cases the performance on the phonologically similar condition was actually slightly better than that for the dissimilar condition. In order to assess anatomical damage that was common to these subjects, a conjunction of the lesions for the 3 subjects in each modality was performed. The results, illustrated in [fig_ref] Figure 5: = 51 [/fig_ref] , indicate that damage to hemispheral lobule VIII/VIIB was common to all subjects that showed abnormal performance, and that the modality affected was correlated with the lateralization of the damage such that left cerebellar damage was associated with abnormal performance in the auditory modality and right cerebellar damage was associated with abnormal visual modality performance.
Analysis of free recall data revealed a significant main effect of serial position [fig_ref] Figure 1: T1-weighted coronal MRI images showing the cerebellum of all 12 patients after... [/fig_ref] serial position x subject type interaction were not significant. [fig_ref] Figure 6: Free recall performance as a function of serial position of remembered items... [/fig_ref] shows that patients and controls had a similar U-shaped function signifying normal recency effects for both groups. In contrast to the phonological similarity experiment, we did not find individual outliers among the cerebellar patients in serial position performance.
# Discussion
The findings from this study confirm and extend our current knowledge of the role of the cerebellum in cognition and VWM in particular. This is the first systematic study of the neurocognitive sequelae of cerebellar tumor resection in children that focuses on the contributions of individual cerebellar lobules to behavioral impairments. Lobular based analyses enable us to compare these results with activation maps from neuroimaging studies and with predictions from current models of cerebro-cerebellar VWM circuitry.
Our results indicated that cerebellar patients had a significantly reduced memory span for aurallypresented digits relative to control subjects. In contrast to the patient data reported by Silveri and colleagues, memory span did not improve when patients were allowed to point to items rather than speak [bib_ref] Verbal short-term store-rehearsal system and the cerebellum. Evidence from a patient with..., Silveri [/bib_ref]. While digit span performance for control subjects was superior when digits were presented aurally relative to visual presentation, patients did not exhibit modality differences. These data confirm and extend two previous reports of impaired auditory short-term memory performance in children with cerebellar tumors [bib_ref] Surgery of tumors of the cerebellum and prefrontal cortex, and sensory memory..., Castro-Sierra [/bib_ref] [bib_ref] Preoperative and postoperative analysis of visual and auditory memory in children with..., Lazareff [/bib_ref]. Although the first of these reports could not distinguish behavioral performance between patients with midline tumors and those with hemispheric lesions [bib_ref] Preoperative and postoperative analysis of visual and auditory memory in children with..., Lazareff [/bib_ref] , a subsequent study by the same researchers concluded that lesions of the midline portions of the cerebellum do not appear to cause cognitive deficits and that short term memory impairments could be explained by hemispheric damage alone [bib_ref] Surgery of tumors of the cerebellum and prefrontal cortex, and sensory memory..., Castro-Sierra [/bib_ref]. They observed that three of the patients with midline tumors from their initial study who showed memory deficits had lesions which also affected the adjacent cerebellar hemisphere. The lobular analyses from the present study are relevant to these earlier findings and suggest that damage to the left inferior hemispheral lobule VIII, which lies in relatively close proximity to the midline, may be associated with impaired auditory digit span performance. Studying adult cerebellar stroke and tumor patients, Ravizza and colleagues similarly reported impaired digit span performance for aurally-presented stimuli [bib_ref] Cerebellar damage produces selective deficits in verbal working memory, Ravizza [/bib_ref]. They further reported greater impairment of digit span for backward relative to forward recall, and no impairment relative to controls for spatial working memory span. Consistent with the present study, they found that digit span performance was correlated with inferior cerebellar damage, although they did not find a laterality effect.
Relative to control subjects, patients' verbal working memory performance in the present study was significantly less affected by articulatory suppression. A lack of an articulatory suppression effect is typically found in patients with articulatory control system (phonological output buffer) deficits [bib_ref] The phonological short-term store-rehearsal system: patterns of impairment and neural correlates, Vallar [/bib_ref]. Although the lack of an articulatory suppression effect in the group of cerebellar patients appears to be more pronounced in the auditory modality, statistical analyses found significant effects for rehearsal type x group interaction but not for the rehearsal type x modality x group interaction.
Ravizza et al. noted that verbal working memory impairment was correlated with dysarthria severity, consistent with a cerebellar role in articulatory rehearsal, but in contrast to the present study, did not observe significant articulatory suppression effects [bib_ref] Cerebellar damage produces selective deficits in verbal working memory, Ravizza [/bib_ref]. Likely sources for the discrepancy between these studies are the ages of the patients and the distributions of the lesions. They did note, however, that patients with more superior and anterior cerebellar lesions were more dysarthric and were more affected by the increased demands on rehearsal that occurred from an extended delay period. Our lobular analyses of the articulatory suppression effect indicated that damage to the superior cerebellar hemispheres (lobule IV/V) and large portions of the vermis were particularly correlated with the suppression effect. These results are consistent with the observations of Ravizza et al. as well as other neuroimaging and patient investigations linking superior cerebellum to articulation [bib_ref] Speech deficits in ischaemic cerebellar lesions, Ackermann [/bib_ref] [bib_ref] Does the cerebellum contribute to cognitive aspects of speech production? A functional..., Ackermann [/bib_ref] [bib_ref] A functional study of auditory verbal imagery, Shergill [/bib_ref] [bib_ref] Cerebellar speech representation: lesion topography in dysarthria as derived from cerebellar ischemia..., Urban [/bib_ref]. Event-related neuroimaging investigations have found that superior cerebellar activations tend to be strongly related to the encoding phase of the verbal working memory task [bib_ref] Dissociation of verbal working memory system components using a delayed serial recall..., Chein [/bib_ref] [bib_ref] Temporal dynamics of cerebrocerebellar network recruitment during a cognitive task, Chen [/bib_ref] and verbal working memory performance can be impaired by transcranial magnetic stimulation administered just after the encoding period [bib_ref] Cerebellar transcranial magnetic stimulation impairs verbal working memory, Desmond [/bib_ref] , suggesting that the superior cerebellar role in articulation may be to assist in orthographic to phonological conversion of visual information and/or to set up an initial motor articulatory trajectory.
Statistical analyses of patients and control subjects in the present investigation revealed overall normal phonological similarity and recency effects. However, inspection of individual patient performance did reveal outlier performance in the phonological similarity experiment for 3 patients in the auditory modality and 3 patients in the visual modality. Conjunction of the lesion data for these subjects suggested that left hemispheral lobule VIII damage was associated with an abnormal phonological similarity effect with aurally presented stimuli, while damage to the same region on the right side was associated with an abnormal phonological similarity effect in the visual modality. These results are consistent with the findings of Justus and colleagues who found that patients with lesions which included the inferior cerebellum, either unilaterally or bilaterally, disrupted the phonological store and yielded a negative phonological similarity effect [bib_ref] Reduced phonological similarity effects in patients with damage to the cerebellum, Justus [/bib_ref]. Similarly, Chiricozzi et al. [bib_ref] Phonological short-term store impairment after cerebellar lesion: a single case study, Chiricozzi [/bib_ref] reported a case study of a patient with cerebellar damage to left hemispheral lobule VIII and right hemispheral lobule V that showed impaired phonological similarity effects as well as other signs of impaired phonological storage. It can be further noted that our group results may have underestimated disruption of phonological similarity effects by using a recognition rather than recall procedure. Similarly, non-disruption of the recency effect in patients was observed using visually presented letters, whereas impairment might have been more readily detected with auditory presentation.
With respect to the concordance of the present results with previous findings from functional neuroimaging, we have consistently found with visually presented letters in a Sternberg verbal working memory task that right HVIII/HVIIB is activated mostly during the maintenance phase of the memory task, but not during motoric rehearsal conditions in which phonological storage is not required. On the basis of this observation as well as evidence from neuroanatomy regarding cerebro-cerebellar connectivity we have suggested that this inferior cerebellar region is linked with temporal/parietal neocortical regions that have been implicated in phonological storage [bib_ref] Temporal dynamics of cerebrocerebellar network recruitment during a cognitive task, Chen [/bib_ref] [bib_ref] Cerebro-cerebellar networks during articulatory rehearsal and verbal working memory tasks, Chen [/bib_ref] [bib_ref] Lobular patterns of cerebellar activation in verbal working-memory and finger-tapping tasks as..., Desmond [/bib_ref]. As described below, the emphasis from neuroimaging on right inferior cerebellum may have been biased from the use of visually-presented letters. Recent data from our lab indicates that with aurally-presented letters, activation of left inferior cerebellar regions (as well as right) is much more apparent. The presence of overall phonological similarity and recency effects in the present study (albeit a null result) are not inconsistent with the neuroimaging data because lobular damage to HVIII and HVIIB was small in most patients. However, the disruptive effect of inferior cerebellar damage on the phonological similarity effect in a subset of our patients is consistent with previous neuroimaging findings.
Functional neuroimaging has also revealed activation in superior cerebellar hemispheres (right HVI, and left Crus I) as well as in the vermis (VI and VII) when contrasting high vs. low load conditions for both working memory and motoric rehearsal (non-memory) tasks, suggesting that these regions might be involved in common articulatory or encoding operations for both types of tasks [bib_ref] Cerebro-cerebellar networks during articulatory rehearsal and verbal working memory tasks, Chen [/bib_ref] [bib_ref] Lobular patterns of cerebellar activation in verbal working-memory and finger-tapping tasks as..., Desmond [/bib_ref]. We found that patients were significantly less disrupted by articulatory suppression than controls, indicating a possible deficit in articulatory control. Although damage to right HVI and left Crus I was also very modest in our patients (mean of 9.7 and 6.2 percent, respectively), damage to the vermis was extensive and the significant correlation of damage to vermian lobule VI and VII with articulatory suppression measures is consistent with the functional neuroimaging evidence.
In summary, damage to left hemispheral lobule VIII in the present study was associated with reduced digit span to auditory stimuli. There is some indication from our data that damage to this lobule may affect phonological storage. The abnormal articulatory suppression effects as well as the selective disruption of auditory digit span are patterns similar to those observed in patients with lesions in premotor, Rolandic, and insular regions who are diagnosed with primary impairment in the articulatory control system [bib_ref] The phonological short-term store-rehearsal system: patterns of impairment and neural correlates, Vallar [/bib_ref]. While these similarities suggest that cerebellar lesions in the present study caused impairment of the articulatory control sys-tem, two observations should be considered. The first is that no improvement in auditory digit span was observed when subjects were allowed to point rather than to respond verbally. If articulatory control was the primary deficit for the cerebellar patients, one would expect that bypassing the articulatory system through pointing would have improved performance. A memory advantage from the pointing procedure has been viewed as evidence of an impaired articulatory control system, and such an advantage from pointing has been observed in a case study of verbal working memory impairment in a cerebellar patient [bib_ref] Verbal short-term store-rehearsal system and the cerebellum. Evidence from a patient with..., Silveri [/bib_ref]. The second observation is that the patients showing the most severe visual articulatory control deficits were not the same patients that showed the most disruption of the phonological similarity effect. In patients with articulatory control deficits due to left frontal lesions, phonological similarity effects are typically preserved for the auditory modality, but impaired for visual stimuli, due to the need for the articulatory system in translating the visual letters into a phonological code [bib_ref] The phonological short-term store-rehearsal system: patterns of impairment and neural correlates, Vallar [/bib_ref]. We also note that while damage to lobule HVIII was highly correlated with reduced auditory digit span performance, this lobule was not correlated with behavioral suppression effects -rather, such effects were correlated with superior cerebellar and vermis damage. Although the articulatory suppression data suggests impairment of the articulatory control system, from the overall pattern of the data we hypothesize that damage to left lobule HVIII may interfere with normal encoding of auditory stimuli into phonological storage, possibly affecting item sequencing [bib_ref] The contribution of the cerebellum to speech production and speech perception: clinical..., Ackermann [/bib_ref] [bib_ref] Cognitive sequencing impairment in patients with focal or atrophic cerebellar damage, Leggio [/bib_ref] [bib_ref] Discrimination of temporal information at the cerebellum: functional magnetic resonance imaging of..., Mathiak [/bib_ref] or item associations [bib_ref] Enhancement of phonological memory following Transcranial Magnetic Stimulation (TMS), Kirschen [/bib_ref] that may normally contribute to phonological loop execution. Such an effect would be generally consistent with a hypothesized role of the cerebellum in sensory acquisition [bib_ref] Control of sensory data acquisition, Bower [/bib_ref] [bib_ref] Lateral cerebellar hemispheres actively support sensory acquisition and discrimination rather than motor..., Parsons [/bib_ref]. In support of this hypothesis, functional MRI observations from our laboratory, directly contrasting activation in a Sternberg verbal working memory task under conditions of aurally vs. visually presented stimuli, have shown that while right lateral HVIII (HVIIIA) and HVIIB is activated for both auditory and visual presentation of letters, only the auditory condition activates more medial inferior cerebellar regions including bilateral medial HVIII (HVIIIB) [bib_ref] Modality specific cerebellar activation during verbal working memory: a fMRI study, Kirschen [/bib_ref]. The concordance of these functional neuroimaging data, in which the modality of the letters presented was the only difference in conditions, with the results of the present study suggest that the medial inferior hemispheres may be a critical entry point for auditory information in the cerebellum, and that damage to this region can impair working memory performance in the auditory modality.
[fig] Figure 1: T1-weighted coronal MRI images showing the cerebellum of all 12 patients after tumor resection. Image numbers correspond to subject numbers in [/fig]
[fig] Figure 2: Anatomical distribution of cerebellar damage averaged over the 12 patients. Mean percent damage to the lobule is plotted against the cerebellar lobules. Yellow bars = superior cerebellar regions; Blue = inferior cerebellar regions; Gray = cerebellar tonsils; Green = cerebellar vermis, Maroon = deep cerebellar nuclei. [/fig]
[fig] Figure 3: Digit span performance for patients and controls in auditory and visual modalities. Average digit span performance for 12 patients and 12 controls is depicted and error bars represent standard error of the mean. [/fig]
[fig] Figure 4: (A) Articulatory suppression performance (expressed as the difference between non-suppressed and suppressed performance) for patients and controls in auditory and visual modalities. Average accuracy (i.e., proportion of total number of trials answered correctly) for the 12 patients and 12 controls is presented and error bars represent standard error of the mean. (B) Articulatory suppression results with breakdown of non-suppressed and suppressed performance. [/fig]
[fig] Figure 5: = 51.4, p < 0.0001), but the main effect of subject type and the Conjunction of lesions of subjects exhibiting abnormal phonological similarity effects. The figure represents conjunctions for 3 subjects with abnormal performance for aurally presented stimuli and 3 different subjects who showed abnormal performance for visually presented stimuli. The conjunction region in both cases extends into posterior portions of hemispheral lobules VIII and VIIB. [/fig]
[fig] Figure 6: Free recall performance as a function of serial position of remembered items for patients and controls. Error bars represent standard error of the mean. [/fig]
[table] Table 1: Neuropsychological assessment scores All measures are represented in T scores (mean = 50 and SD = 10) unless otherwise stated. 2 Standard scores (mean = 100 and SD = 15). 3 Only 10 out of 12 patients performed this test. * Significant at p < 0.05. [/table]
[table] Table 2: Distribution of cerebellar lobular damage [/table]
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10.3390/ijms23105472
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CCBY
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9143493
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35628279
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s2orc_pubmed_articles
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Reduced Environmental Dose Rates Are Responsible for the Increased Susceptibility to Radiation-Induced DNA Damage in Larval Neuroblasts of Drosophila Grown inside the LNGS Underground Laboratory
Citation: Porrazzo, A.; Esposito, G.; Grifoni, D.; Cenci, G.; Morciano, P.; Tabocchini, M.A. Reduced Environmental Dose Rates Are Responsible for the Increased Susceptibility to Radiation-Induced DNA Damage in Larval Neuroblasts of Drosophila Grown inside the LNGS Underground Laboratory. Int. J. Mol. Sci. 2022, 23, 5472. https://doi.
# Introduction
Natural ionizing radiation (IR) has been regarded as a crucial factor in the evolution of life forms on earth since the first cell came into being about 4 billion years ago. It has been postulated that the ability of present-day organisms to cope with radiation-induced DNA damage could in part result from the adaptation of their ancestors, who experienced many fluctuations of environmental radiation exposure (i.e., very high radiation exposure) during primeval times. The natural radiation, whose dose rate currently varies in the range of 10 −7 -10 −5 Gy/h, results from a combination of cosmic, terrestrial, and internal sources of different forms of energy and electrically charged particles, generally referred to as background radiation. It is noteworthy that background radiation also shows large geographical variations. Since the exposure of organisms, including humans, to background radiation is unavoidable, understanding its role is important to address basic questions about life's evolution on earth and the health effects of low-dose ionizing radiation exposure, a relevant issue in radiation protection. In this respect, controlled experiments with model organisms, conducted in parallel in underground laboratories where the radiation background is largely reduced, and in reference conditions at a natural background radiation, could provide useful insight into the overall role of natural radiation. Among the various locations that hosted experiments in conditions of deprived background radiation, the Deep Underground Laboratories (DULs) stand out for their unique shielding characteristics that make it possible to reduce the total radiation background. These research infrastructures are built under a rock overburden greater than about 1000 m of water equivalent (m.w.e), originally created to host particle, astroparticle, and/or nuclear physics experiments (i.e., the search for neutrino interaction, proton decay, matter particles), which would require stringent experimental conditions of strongly reduced cosmic ray particle interference [bib_ref] History, advancements, and perspective of biological research in deep-underground laboratories: A brief..., Liu [/bib_ref] [bib_ref] Fruit Flies Provide New Insights in Low-Radiation Background Biology at the INFN..., Morciano [/bib_ref]. Being among the most efficient places for experimental isolation from radiation, some DULs have become multidisciplinary sites hosting important studies in fields such as geology, geophysics, the climate, environmental, and space sciences, technology/instrumentation development, and radiobiology. So far, all biological studies carried out in DULs strongly indicate that the deprivation of natural background radiation affects, although to varied degrees, the growth and transcription profiles of bacteria as well as of unicellular and multicellular eukaryotes [bib_ref] Deinococcus radiodurans UWO298 Dependence on Background Radiation for Optimal Growth, Castillo [/bib_ref] [bib_ref] Chinese hamster V79 cells' dependence on background ionizing radiation for optimal growth, Castillo [/bib_ref] [bib_ref] First transcriptome profiling of D. melanogaster after development in a deep underground..., Zarubin [/bib_ref]. Moreover, several experiments indicate that cell cultures kept in this strongly reduced background show higher susceptibility to subsequent radiationinduced DNA damage than parallel cultures kept in external radiation background [bib_ref] The Cosmic Silence experiment: On the putative adaptive role of environmental ionizing..., Carbone [/bib_ref] [bib_ref] Low-radiation environment affects the development of protection mechanisms in V79 cells, Fratini [/bib_ref] [bib_ref] Influence of a low background radiation environment on biochemical and biological responses..., Satta [/bib_ref]. These observations challenge the Linear No-Threshold (LNT) model, which posits that all radiation exposure is always considered harmful with no threshold point and that risks increases linearly with the dose [bib_ref] The linear No-Threshold (LNT) dose response model: A comprehensive assessment of its..., Calabrese [/bib_ref].
The Gran Sasso National Laboratory (LNGS) at Assergi (L'Aquila, Italy) is one of the largest DULs in the world, hosting the largest number of biological experiments performed to date. Due to the 1400-m coverage of dolomitic rocks poor in uranium and thorium, at LNGS the flux of cosmic rays is considered negligible as it is reduced by approximately 6 orders of magnitude, while the neutron flux is 1000 times reduced with respect to the external environment [bib_ref] Effects of reduced natural background radiation on Drosophila melanogaster growth and development..., Morciano [/bib_ref]. The setting up of two dedicated facilities, namely, PULEX and COSMIC SILENCE for cell culture and animal housing, respectively, allowed researchers to gather several pieces of evidence on the influence of the deprivation of a natural level of environmental radiation in different model systems. In particular, a few years ago, we launched the FLYINGLOW project that aimed to determine whether the LNGS's underground environment could affect the growth and development of the well-established model organism, Drosophila melanogaster. Our results indicated that the reduced background radiation affects the lifespan and fertility of adult flies. Providing the first evidence of the influence of radiation background in a complex organism [bib_ref] Fruit Flies Provide New Insights in Low-Radiation Background Biology at the INFN..., Morciano [/bib_ref]. Since then, other organisms such as fishes [bib_ref] A novel specialized tissue culture incubator designed and engineered for radiobiology experiments..., Pirkkanen [/bib_ref] and nematodeshave been demonstrated to promptly modify their physiological responses following changes in the radiation background. Collectively, these observations suggest that the stimulation of defense mechanisms against stress triggered by the natural background radiation is an evolutionarily conserved phenomenon. However, to date, no direct evidence has been provided about the involvement of environmental radiation in determining these different responses. It should be considered that. not only underground but also in an external environment, a small number of cells within an organism interact with radiation every week [bib_ref] Spatial and temporal distribution of energy, Goodhead [/bib_ref]. Nevertheless, even if few cells are directly affected by radiation, cell-cell communication mechanisms may be able to propagate signals to other (neighboring) cells and thus amplify the total number of perturbed cells [bib_ref] The complex interactions between radiation induced nontargeted effects and cancer, Campa [/bib_ref]. Moreover, the low-and high-LET components of the environmental background could have a different weight. Thus, dedicated experiments are needed to clarify this issue.
In different DULs, various components contribute to the radiation field. Generally, whereas both the high-LET (e.g., neutrons) and low-LET (e.g., muons) component of cosmic rays can be reduced by several orders of magnitude, the low-LET terrestrial gamma rays and the products deriving from 222 Rn decay contribute significantly to the overall dose/dose rate. The characterization of the radiation field remains a crucial point for the interpretation of the biological effects, and dosimetric measurements should be constantly carried out not only underground but also in the reference laboratory. Furthermore, the modulation of the radiation field inside the DULs through the implementation of devices that could modulate some of these components could provide insights into the mechanisms underpinning the observed biological effects [bib_ref] Stress induction in the bacteria Shewanella oneidensis and Deinococcus radiodurans in response..., Castillo [/bib_ref].
Here, we show for the first time that the reduced natural background radiation (herein Low Radiation Environment, LRE) at LNGS affects the response to radiation-induced DNA damage in the complex eukaryotic system Drosophila melanogaster. In particular, we show that neuroblasts from wild-type flies grown for one and five generations at the LNGS LRE exhibit a frequency of chromosome breaks (CBs) induced by acute exposure to ionizing radiation higher than that observed in cells from flies kept at the external (herein Reference Radiation Environment, RRE) LNGS facility. Moreover, we show that increasing the low-LET component by means of a Marinelli beaker designed ad hoc and filled with tuff, a natural gamma emitter building material, rescues the IR sensitivity in cells from flies kept at LRE. This finding provides the first evidence that the modulation of the DNA response observed in Drosophila melanogaster neuroblasts is indeed radiation-dose-rate-dependent and that a minimum dose rate level (comparable to the environmental one) is required to trigger efficient DNA damage response mechanisms.
# Results and discussion
In the present paper. we set out to investigate if reduced background radiation affects the response to radiation-induced DNA damage, expressed as CBs/cell, induced by an acute dose of gamma rays in Drosophila melanogaster wild-type mitotic cells. To this purpose, we raised Oregon-R (wild-type) flies at LRE and in parallel at RRE for one and five generations. Third instar larvae were collected from generation 1 and generation 5 and irradiated with an acute challenging gamma dose of 10 Gy; four hours later, larval neuroblasts were analyzed for CB frequency. The average number of total CBs/cell was calculated by measuring the ratio of the total number of chromatid deletions (CDs, scored as a single event; see also Figure 1b) and isochromosome breaks (ISOs or chromosome deletions, scored as two events; see also [fig_ref] Figure 1: IR-induced CBs and TFs in DAPI-stained larval neuroblasts [/fig_ref] to the total number of metaphases [fig_ref] Figure 1: IR-induced CBs and TFs in DAPI-stained larval neuroblasts [/fig_ref]. Whereas the CB frequency induced by the challenging dose in the RRE larvae was~0.6, larvae from LRE flies exhibited a~2-fold increase in the CB frequency [fig_ref] Figure 2: Effects of radiation background on IR-induced CBs [/fig_ref]. This finding suggests that the reduced natural background affects the response to radiation-induced chromosome damage. Interestingly, we found that the CB frequency from the generation 5 LRE was indistinguishable from that of the generation 1 LRE, indicating that the Drosophila cell sensitivity is not caused by the occurrence of spontaneous dominant mutations but rather represents an early response to the switch from a normal to reduced radiation background. Similar effects were also previously described in flies kept at LRE for several generations, which showed reduced fertility [bib_ref] Effects of reduced natural background radiation on Drosophila melanogaster growth and development..., Morciano [/bib_ref]. It is also interesting to note that the deprivation of the radiation background at LRE did not render the DNA repair and telomere capping mutant flies [bib_ref] DNA Repair in Drosophila: Mutagens, Models, and Missing Genes, Sekelsky [/bib_ref] [bib_ref] Silence at the End: How Drosophila Regulates Expression and Transposition of Telomeric..., Cacchione [/bib_ref] [bib_ref] Terminin: A protein complex that mediates epigenetic maintenance of Drosophila telomeres, Raffa [/bib_ref] [bib_ref] Effete, a Drosophila chromatinassociated ubiquitin-conjugating enzyme that affects telomeric and heterochromatic position..., Cipressa [/bib_ref] more sensitive to the chromatin burden induced by endogenous genome instability compared to RRE [fig_ref] Figure 3: Effects of the deprivation of the radiation background on DDR and telomere... [/fig_ref]. This confirms the idea that extremely low doses, such as the environmental ones, increase the ability of cells and organisms to cope with exogenous damage through a stress response, not a damage response. The mechanisms involved in these phenomena need to be further investigated, although we can envisage that epigenetic mechanisms could play a relevant role. Since the beginning of our underground biology activity, we applied an experimental approach to minimize all the possible differences between the underground and reference environments except radiation, and collected convincing indications that the different behavior depends on the different radiation field in LRE and RRE [bib_ref] Low-radiation environment affects the development of protection mechanisms in V79 cells, Fratini [/bib_ref] [bib_ref] Effects of reduced natural background radiation on Drosophila melanogaster growth and development..., Morciano [/bib_ref] [bib_ref] Low environmental radiation background impairs biological defence of the yeast Saccharomyces cerevisiae..., Satta [/bib_ref]. It is clear that the percentage of cells in the biological system that interacts with radiation at RRE and LRE is very low, and therefore we must assume a strong involvement of cellular communication effects. These effects have been clearly demonstrated to occur at low doses above ground [bib_ref] Review of Quantitative Mechanistic Models of Radiation-Induced Non-Targeted Effects (Nte), Shuryak [/bib_ref]. In principle, the observed difference could also be linked to some other factors difficult to control (i.e., differences in air quality, vibrations related to nearby installations, the presence of sunlight at RRE and not at LRE, etc.) as proposed by Zarubin et al. [bib_ref] First transcriptome profiling of D. melanogaster after development in a deep underground..., Zarubin [/bib_ref]. We used incubators to avoid at least some of the proposed confounding factors and tried to get more insight on the involvement of the environmental radiation on the modulation of the radiation-induced DNA damage, directly investigating the role of specific components of the environmental radiation field on the response of Drosophila larval neuroblasts. In line with the aims of our recently launched RENOIR (Radiation Environment Triggers Biological Responses in Flies) program at LNGS, we sought to elucidate the role of a specific component of the environmental radiation field on the LRE-dependent sensitivity to IR-induced DNA damage responses. Since it is not possible to restore the radiation field present at RRE in LRE, as a first approximation, we decided to increase the dose rate value present at LRE regardless of the type of radiation that generates it. The idea was to place an irradiator in LRE to increase the dose rate value of the gamma component. To this end, we took advantage of especially designed Marinelli beakers consisting of aluminum hollow cylinders filled with tuff (a natural gamma emitter building material) and sealed to avoid any radon exposure due to the tuff decay products. The low-LET component measurements carried out in LRE using TLD-700H placed inside the cylindrical hole of Marinelli filled with tuff (herein irradiator) revealed a dose rate value Since the beginning of our underground biology activity, we applied an experimental approach to minimize all the possible differences between the underground and reference environments except radiation, and collected convincing indications that the different behavior depends on the different radiation field in LRE and RRE [bib_ref] Low-radiation environment affects the development of protection mechanisms in V79 cells, Fratini [/bib_ref] [bib_ref] Effects of reduced natural background radiation on Drosophila melanogaster growth and development..., Morciano [/bib_ref] [bib_ref] Low environmental radiation background impairs biological defence of the yeast Saccharomyces cerevisiae..., Satta [/bib_ref]. It is clear that the percentage of cells in the biological system that interacts with radiation at RRE and LRE is very low, and therefore we must assume a strong involvement of cellular communication effects. These effects have been clearly demonstrated to occur at low doses above ground [bib_ref] Review of Quantitative Mechanistic Models of Radiation-Induced Non-Targeted Effects (Nte), Shuryak [/bib_ref]. In principle, the observed difference could also be linked to some other factors difficult to control (i.e., differences in air quality, vibrations related to nearby installations, the presence of sunlight at RRE and not at LRE, etc.) as proposed by Zarubin et al. [bib_ref] First transcriptome profiling of D. melanogaster after development in a deep underground..., Zarubin [/bib_ref]. We used incubators to avoid at least some of the proposed confounding factors and tried to get more insight on the involvement of the environmental radiation on the modulation of the radiation-induced DNA damage, directly investigating the role of specific components of the environmental radiation field on the response of Drosophila larval neuroblasts. In line with the aims of our recently launched RENOIR (Radiation Environment Triggers Biological Responses in Flies) program at LNGS, we sought to elucidate the role of a specific component of the environmental radiation field on the LREdependent sensitivity to IR-induced DNA damage responses. Since it is not possible to restore the radiation field present at RRE in LRE, as a first approximation, we decided to increase the dose rate value present at LRE regardless of the type of radiation that generates it. The idea was to place an irradiator in LRE to increase the dose rate value of the gamma component. To this end, we took advantage of especially designed Marinelli beakers consisting of aluminum hollow cylinders filled with tuff (a natural gamma emitter building material) and sealed to avoid any radon exposure due to the tuff decay products. The low-LET component measurements carried out in LRE using TLD-700H placed inside the cylindrical hole of Marinelli filled with tuff (herein irradiator) revealed a dose rate value slightly higher than that of RRE (~100 nGy/h vs.~66 nGy/h).
We have previously shown that the Marinelli beaker without tuff (phantom) does not affect the growth and fertility of flies grown inside the cylinders at RRE, indicating that this device represents an appropriate tool to test the flies' responses in both LRE and RRE environments. We measured the frequency of IR-induced CBs in larval neuroblasts from wild-type line grown at both LRE and RRE inside the irradiator and the phantom using the challenging dose approach described above. Interestingly, we found that the CB frequency in larvae raised for a complete developmental cycle from embryo to third instar larvae (7 days) inside the irradiator at LRE and then exposed to an acute dose of 10 Gy of gamma rays was~40% lower than the CBs found in larvae maintained either inside or outside the phantom at LRE [fig_ref] Figure 2: Effects of radiation background on IR-induced CBs [/fig_ref]. This result indicates that the partial restoring of low-LET flux (specifically of gamma rays) at LRE is able to rescue the IR sensitivity of flies maintained in a reduced radiation background and is direct evidence of the involvement of the radiation field in the different behavior observed in our biological model system at LRE and RRE. It is reasonable to speculate that there is a threshold value of the dose rate between 20 nGy/h and 66 nGy/h (the reference external value) that switches the response of our biological system, making it pass from one state to another. What we know is that a further increase in the dose rate, obtained by keeping the larvae in Marinelli with tuff at RRE, does not significantly change the radiosensitivity in terms of the CBs induced by 10 Gy (data not shown).
Our results are in agreement with the pioneering work by [bib_ref] Influence on cell proliferation of background radiation or exposure to very low,..., Planel [/bib_ref] on Paramecium tetraurelia, showing that the ciliate growth rate decreases inside the Pyrenees Mountain underground laboratory but is restored when cultures are exposed to very low doses of gamma radiation in the same underground environment [bib_ref] Influence on cell proliferation of background radiation or exposure to very low,..., Planel [/bib_ref].
More recently, carried out an analysis of the effects on the cell number and viability and gene expression of Chinese hamster V79 cells under two background-radiation-deprived conditions (below background and in the presence of KCl source) with respect to the external natural background. Their results indicate that cells growing below background show lower viability than that of KCl-amended underground controls or surface controls. This effect appears after 5 days of incubation and lasts, intermittently, for up to 21 days. The data suggest that the sole emission of γ-rays from K-40 in KCl-amended controls is able to only partially rescue the V79's viability, and although there is a clear differentiation between the underground and the surface controls, the authors argue that it could depend on differences among the radiation spectrum in the two conditions, although the experimental set up doesn't allow for the exclusion of the potential influence of other environmental factors such as air pressure or gas composition [bib_ref] Chinese hamster V79 cells' dependence on background ionizing radiation for optimal growth, Castillo [/bib_ref].
# Materials and methods
## Drosophila strains
The Drosophila Oregon R (OR-R) line was used as a wild-type strain. The Drosophila mutant alleles mre 11 , nbs 1 , and rad50 ∆5.1 in genes encoding for DDR factors and cav 1 , moi 1 , and eff tre1 in telomere capping encoding genes were previously described [bib_ref] The Drosophila Mre11/Rad50 complex is required to prevent both telomeric fusion and..., Ciapponi [/bib_ref] [bib_ref] The Drosophila Nbs protein functions in multiple pathways for the maintenance of..., Ciapponi [/bib_ref] [bib_ref] UbcD1, a Drosophila ubiquitin-conjugating enzyme required for proper telomere behavior, Cenci [/bib_ref] [bib_ref] The Drosophila HOAP protein is required for telomere capping, Cenci [/bib_ref] [bib_ref] The Drosophila modigliani (moi) gene encodes a HOAP-interacting protein required for telomere..., Raffa [/bib_ref]. The detailed information on these stocks as well as images of adult and larval wild-type developmental stages is available on Flybase (http://flybase.org (accessed on 11 April 2022)). Flies were maintained in Drosophila medium (Nutri-FlyrGF; Genesee Scientific, San Diego, CA, USA) treated with propionic acid at controlled parameters of temperature (22 - C), relative humidity (about 55%rH), and 12 h light-dark alternation in identical cooled incubators (BioloG-Lux140 Cooled Incubator; F.lli Galli G.& P., Fizzonasco, Italy) placed at LRE and RRE. As the Gran Sasso underground laboratory is a tunnel with horizontal access, the difference in atmospheric pressure between the two environments of RRE (approx. 903 mbar) and LRE (approx. 906 mbar) is negligible. To create generation 1, an RRE population of young adults was divided into two groups. One group was kept in RRE and the other transferred to LRE. Both in RRE and LRE, adults were left to lay embryos for 3-4 days and then discarded. Deposited embryos represent generation 0. Young adults from generation 0 were transferred to a new vial for 3-4 days (and then discarded) to lay embryos for generation 1. Young adults from a generation were used as parents for the next generation and so on for the following generations [bib_ref] Fruit Flies Provide New Insights in Low-Radiation Background Biology at the INFN..., Morciano [/bib_ref] [bib_ref] Deinococcus radiodurans UWO298 Dependence on Background Radiation for Optimal Growth, Castillo [/bib_ref] [bib_ref] Chinese hamster V79 cells' dependence on background ionizing radiation for optimal growth, Castillo [/bib_ref]. The generations 1 and 5 were analyzed to measure the frequency of CBs.
## Dosimetry
Radiation dosimetry at RRE and at LRE of the different components of the radiation field was performed with measurements and evaluations from literature data, as previously described [bib_ref] Low-radiation environment affects the development of protection mechanisms in V79 cells, Fratini [/bib_ref]
## Irradiation treatments
Third instar larvae were exposed to an acute dose of 10 Gy of gamma rays from a 137 Cs source at a dose rate of about 0.7 Gy/min using the Gammacell Exactor 40 (Nordion) of the Istituto Superiore di Sanità (ISS), Italy.
To increase the low-LET component of the underground radiation spectrum, we developed an irradiator consisting of an especially designed Marinelli beaker consisting of aluminum hollow cylinders filled with tuff and sealed to avoid any radon exposure due to tuff decay products (see. For the Marinelli irradiator experiment, 10 young males and 10 young females were crossed overnight and then discarded. Laid embryos were kept inside the Marinelli irradiator (with tuff) or in the Marinelli phantom (without tuff) for 7 days in both LRE and RRE.
## Chromosome cytology and microscopy
Metaphases of colchicine-treated neuroblasts from Drosophila third instar larval brains were obtained as previously described [bib_ref] A role for Separase in telomere protection, Cipressa [/bib_ref] [bib_ref] an essential Drosophila melanogaster gene, encodes a protein required for chromatin organization, Messina [/bib_ref]. For CB scoring, larval brains were dissected 3 h after irradiation and incubated for one additional hour with 10 −6 M colchicine before preparing the slides. The CBs and TFs were analyzed using the inverted fluorescence microscope Nikon TE 2000 (Nikon Instruments Inc., Melville, NY, USA) equipped with a Charged-Coupled Device (CCD camera; Photometrics CoolSnap HQ). At least 100 metaphases (for mutant strains) and 400 (for Oregon-R) for each experimental condition were counted for the statistical analysis.
# Statistical analysis
For experiments with Oregon-R, we compared the means of two data sets each obtained from at least three independent experiments. For each experiment, the mean number of CBs per cell was determined (>400 cells for each condition). Error bars show the standard deviation. Having two independent groups of samples (collected independently of one another) that follow normal distributions, we used a parametric test (unpaired Student's t-test) to determine statistical differences between the means obtained from these two different groups. Values of p < 0.05 were considered as statistically significant.
For the mutants' analysis, no statistical differences were observed between LRE and RRE (t-test). At least 100 metaphases for each mutant were counted for the statistical analysis.
# Conclusions
Despite the difficulties in eliminating all confounding factors when comparing the underground to the reference environment, our results show that the natural background radiation dose rate is indeed capable of modulating the radiation-induced DNA damage response in the complex multicellular organism Drosophila melanogaster, probably through the involvement of stress defense mechanisms. However, additional experiments using different endpoints and/or other biological systems should be carried out to substantiate and possibly extend our conclusions. Furthermore, although our results points to the gamma rays as a relevant component in triggering defense mechanisms, we cannot rule out the possibility that other components (e.g., neutrons) of the environmental radiation spectrum may play a similar role. Given that the modulation of neutrons in underground facilities is not an easy task to accomplish, their contribution to the cellular response to radiation-induced DNA damage remains unclear at the present.
[fig] Figure 1: IR-induced CBs and TFs in DAPI-stained larval neuroblasts. (a) Examples of IR CBs and TFs in third instar larvae neuroblasts scored for this study: (a) wild-type Drosophi gaster female metaphase, (b-d) examples of metaphases showing (b) autosomal chromatid (arrow), (c) autosomal isochromosome deletion (arrows), and (d) TFs giving rise to a ring some involving the chromosome 3 (asterisk), a chromosome 2-chromosome 3 dicentric somes, and a X-X dicentric chromosome. Bright DAPI-stained chromosome regions refer t tromeric and pericentromeric portions of the two major autosomes[23]. [/fig]
[fig] Figure 2: Effects of radiation background on IR-induced CBs. (a) Average number of CB induced by gamma rays in neuroblasts from Oregon-R third instar larval brains analyzed post-irradiation with the acute dose of 10 Gy. Net data are reported, after subtraction of th values. CB frequency was analyzed in Oregon-R larvae from generations 1 and 5. (b). Aver ber of CBs per cell induced by gamma rays in larvae kept for 7 days in the Marinelli bea tuff (irradiator) or without tuff (phantom) at both LRE and RRE and then exposed to the a of 10 Gy. At least three independent experiments were performed for each condition. The e represent the standard error of the mean. Values of p < 0.05 (*) were considered as statist nificant (t-test). In our experiments, we observed a frequency of CBs in the control sam irradiated with the acute dose) ranging from 0.03 to 0.15 CBs per cell. CBs: Chromosom LRE: Low Radiation Environment; RRE: Reference Radiation Environment. [/fig]
[fig] Figure 3: Effects of the deprivation of the radiation background on DDR and telomere capping mutants. (a) Analysis of Chromosome Breaks (CBs) in DNA damage response (DDR) mutants. [/fig]
[fig] Author: Contributions: Conceptualization, M.A.T. and G.E.; methodology, P.M. and G.C.; formal analysis, G.E. and G.C.; investigation, A.P., P.M. and G.E.; writing-original draft preparation, G.C. and P.M.; writing-review and editing, A.P., G.E., D.G., G.C., P.M. and M.A.T.; visualization, A.P., G.E. and P.M.; project administration, G.E. and P.M.; funding acquisition, G.C. and M.A.T. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This work has been supported by grant number: "Anna Tramontano 2018"; funder: Fondazione Cenci Bolognetti/Istituto Pasteur Italia, Rome, Italy to G.C., Programmes Transversaux de Recherche (PTR), Pasteur Institute (France) to G.C., by the "Operative Collaboration for R&D Activities in the Field of on Radiobiology" (Assegno di ricerca n. 20573/2018 to P.M.) in the framework of the General INFN-ISS Agreement, and by 2020-2022 INFN-CSN5 RENOIR experiment to M.A.T. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data generated or analyzed during this study are included in this published article. [/fig]
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Young People's Views on Accelerometer Use in Physical Activity Research: Findings from a User Involvement Investigation
The use of accelerometers to objectively measure physical activity is important in understanding young people's behaviours, as physical activity plays a key part in obesity prevention and treatment. A user-involvement qualitative study with young people aged 7-18 years (n = 35) was carried out to investigate views on accelerometer use to inform an obesity treatment research study. First impressions were often negative, with issues related to size and comfort reported. Unwanted attention from wearing an accelerometer and bullying risk were also noted. Other disadvantages included feeling embarrassed and not being able to wear the device for certain activities. Positive aspects included feeling "special" and having increased attention from friends. Views on the best time to wear accelerometers were mixed. Advice was offered on how to make accelerometers more appealing, including presenting them in a positive way, using a clip rather than elastic belt to attach, personalising the device, and having feedback on activity levels. Judgements over the way in which accelerometers are used should be made at the study development stage and based on the individual population. In particular, introducing accelerometers in a clear and positive way is important. Including a trial wearing period, considering practical issues, and providing incentives may help increase compliance.
# Introduction
The benefits of physical activity among young people are well documented. Evidence shows physical inactivity during childhood to be associated with higher risk of childhood obesity, as well as being a risk factor for cardiovascular disease, cancer, and osteoporosis in later life. Physical activity has an important role in body weight regulation, both in preventing weight gain and maintaining weight loss over time [bib_ref] Physical activity plays an important role in body weight regulation, Chaput [/bib_ref]. As such, understanding young people's physical activity behaviours is important in promoting participation among this group, particularly when activity patterns may vary between lean and obese children [bib_ref] How children move: activity pattern characteristics in lean and obese Chinese children, Mcmanus [/bib_ref].
In order to establish physical activity participation levels, accurate measurement is required. Objective measures are being increasingly used to quantify the amount and intensity of physical activity, as well as levels of sedentary behaviour, with accelerometers currently being the favoured objective measuring device [bib_ref] Objective measurement of physical activity and sedentary behaviour: review with new data, Reilly [/bib_ref]. Accelerometers have been used in studies to measure general physical activity levels among young people [bib_ref] Objective measurement of levels and patterns of physical activity, Riddoch [/bib_ref] [bib_ref] Physical activity and dietary behaviour in a population-based sample of British 10-year..., Van Sluijs [/bib_ref] , as well as in conjunction with measurement of body composition [bib_ref] Objectively measured physical activity and obesity prevention in children, adolescents and adults:..., Wilks [/bib_ref] or alongside a health intervention [bib_ref] Utility of accelerometers to measure physical activity in children attending an obesity..., Robertson [/bib_ref]. Although publications relating to accelerometer data analyses are common [bib_ref] Accelerometer assessment of physical activity in children: an update, Rowlands [/bib_ref] [bib_ref] Actigraph accelerometer cutpoints for youth, but...:a systematic review, Kim [/bib_ref] , those reporting findings related to practical issues in data collection are less so [bib_ref] Feasibility of using the Tritrac motion sensor over a 7-day trial with..., Crocker [/bib_ref] [bib_ref] Adolescent perspectives on wearing accelerometers to measure physical activity in population-based trials, Audrey [/bib_ref].
The acceptability to young people of wearing accelerometers has previously been assessed as good, based on low study dropout rates from children not wanting to wear the monitor [bib_ref] Feasibility of using accelerometers to measure physical activity in young adolescents, Van Coevering [/bib_ref]. However, previous studies found some parents reporting that their children were unwilling to wear accelerometers at school and during sports due to the risk of stigma and bullying [bib_ref] Utility of accelerometers to measure physical activity in children attending an obesity..., Robertson [/bib_ref]. There is limited qualitative research on how young people themselves feel about wearing accelerometers for physical activity research. Investigations of adults' views on wearing accelerometers have shown noncompliance to be related to occupational factors or discomfort during sitting or driving [bib_ref] Utility of the RT3 triaxial accelerometer in free living: an investigation of..., Perry [/bib_ref]. However, these are adult-specific issues, and different reasons for noncompliance are likely to be relevant to children. One recent study [bib_ref] Adolescent perspectives on wearing accelerometers to measure physical activity in population-based trials, Audrey [/bib_ref] investigated the use of accelerometers among adolescents in relation to recruitment, retention, and adherence to the study protocol. The authors reported that adherence may be improved by following a two-part reward system, whereby part one is for returns and part two is for adherence. The use of personal activity graphs and less obtrusive belts and monitors was also suggested. A review on the use of accelerometers in field-based research suggests that researchers need to devise a plan to promote compliance with the monitoring protocol and minimise the possibility of acquiring incomplete data [bib_ref] Conducting accelerometer-based activity assessments in field-based research, Trost [/bib_ref]. The authors suggest that further information is required to identify the psychological, sociocultural, and environmental factors that are associated with or predict compliance among individuals wearing accelerometers. The importance of young people's participation at the onset of the research process is well recognised and advocated by professional bodies. User involvement in research has been interpreted as a shift away from assumptions that "experts" are the best judges [bib_ref] Setting agendas for relevant research: a participatory approach, Tighe [/bib_ref]. High quality research and good retention rates depend on listening to the voices of children and young people themselves and taking account of their experiences, priorities, and perspectives. This paper presents findings from a user involvement study with young people, aged 7-18 years. The data form part of a larger user involvement study, aimed at informing a proposed obesity interventional randomised control trial. This paper reports data sought about accelerometer use. It presents the practicalities of using accelerometers within the research setting from young people's perspective and, based on these findings, goes on to offer practical advice to researchers using accelerometers with this group.
# Methods
A total of five focus group discussions took place; involving 35 young people aged 7-18 years and facilitated by two user involvement coordinators from the West Midlands Medicines for Children Research Network (MCRN) (CC, CT). Participants comprised members of two MCRN young person's advisory groups (YPAG) (groups 1-2) and pupils (groups 3-5) from two local primary schools. Group 2 was made up predominantly of young people who were overweight. MCRN young person's advisory groups have previously been shown to be effective in providing a forum for young people to learn about and comment on various aspects of the research cycle. Focus groups with young people allow for differences between participants to be revealed and are considered particularly useful in attitudinal research and when interaction between participants can help to illuminate a research issue. The MCRN young person's advisory group had previously taken part in a weight-related trial which meant that they formed a particularly appropriate group to give advice on the use of accelerometers. The two schools were selected from locations where the obesity interventions were to take place, and in liaison with school staff, pupils were selected to take part in a focus group discussion. Participants included both boys and girls and a range of ethnicities and weight status. A broad range of ages were included, to identify differences in views across the age span. Written consent was obtained from parents in advance of the research. Verbal consent was attained from all participating young people. Prior to participation in focus group discussions, some participants (n = 18) were given the opportunity to wear an accelerometer (GT1M Actigraph, Fort Walton, FL) for a given time in order to inform the discussion. The accelerometer was worn around the waist using an elastic belt. Details of the groups and main focus group discussion topic are shown in [fig_ref] Table 1: Details of focus group discussion participants/topic [/fig_ref].
The researchers took notes throughout the FGDs or wrote participants' thoughts on a flipchart. These were later analysed using thematic analysis [bib_ref] Using thematic analysis in psychology, Braun [/bib_ref] ; based on what themes emerged and how often these themes were discussed. Ethical approval was obtained from the NRES Committee West Midlands-Coventry and Warwickshire, REC (Reference 11/WM/0290).
# Results
Five broad themes were drawn out from the discussions. Although it is acknowledged that there is overlap across the themes, for the purposes of data presentation, these are reported as follows.
## First impressions.
The majority of first impressions on accelerometers were negative. For younger schoolchildren (7-11 years) the main concern was a feeling that the accelerometer was "watching" them, believing it to have a camera or microphone contained within it. This was present even after the researcher had assured them this was not the case. Across the full age range (7-18 years) many expressed a general dislike in the physical appearance of accelerometers, mainly around the size and appearance, describing them as being too big and bulky. Words used to describe the devices included "unattractive," "too thick," and "not nice." The elastic waistband onto which the accelerometer was attached was also described as being uncomfortable.
## How it feels to
Wear an Accelerometer. Some positive aspects of wearing an accelerometer were noted, for example, feeling "different" or "special" in some way. Several young people stated that they were constantly asked questions about the accelerometers by their peers, who also wanted to be involved or try them on.
"I wanted my friends to be jealous that I had something different." (girl, aged 10; wore accelerometer 1 week)
"After a while I got used to it [accelerometer], so it did not annoy me. Only one child asked me about it but was very interested and understanding about why I needed to wear it." (girl, aged 10; wore accelerometer 1 week) Some reported that it took them a little while to get used to wearing the device, either taking it on and off several times Among children (7-11 years) who wore the accelerometer for one school day (n = 12), half felt constantly reminded they had them on, while the other half forgot. Girls appeared to be more self-conscious and aware of wearing them. Some reported finding them distracting to wear and constantly wanted to touch the accelerometer. In general, all age groups expressed a preference to wearing accelerometers under rather than over their clothes, although three pupils (all girls) did not mind and one (boy) felt strongly that he should wear it under his clothes as he did not want anyone to see it. Some felt it would be a good idea to wear them over one layer of clothes so that it would feel more comfortable while still being hidden.
## Best time for
Wearing an Accelerometer. There were mixed views as to when the best time to wear accelerometers might be. Some felt that holiday time would be better in order to reduce questioning from peers. However, young people understood that, from a research perspective, wearing the device during school time provided a more accurate account of their activity levels. Pupils from one primary school showed preferences for wearing during term time, whereas those from the other primary school had no preference, highlighting that there are varied views on this subject. One boy aged 9 reported not wanting to wear the accelerometer at school.
## Disadvantages of accelerometers.
For those who had had the opportunity to wear an accelerometer prior to the FGD, concerns were raised as to the way it felt and how it might be perceived by their peers. The device was described by some as being "uncomfortable" or "irritating," especially the elastic belt onto which it was attached. Some also described being embarrassed about having to wear it or felt that it gave other pupils a reason for picking on them, especially if they had weight-related issues.
"It gave people an excuse to be more horrible to me." (girl, aged 10 with weightrelated issues, wore accelerometer 1 week)
The risk of bullying was therefore heightened among those wearing the accelerometer. A participant with weightrelated issues suggested that they would feel being asked to wear an accelerometer as "patronising" and therefore suggested that care should be taken by researchers when introducing the device.
A practical disadvantage of the accelerometer was that children were not able to wear the device for certain activities, such as swimming. Indeed, questionnaire data (not presented in this paper) showed swimming to be among one of the most popular activities amongst the young people in this study. One young person described how he was not able to wear his accelerometer during rugby because of the danger in a contact sport. The device used in this study (GT1M) had a marker button which would allow marking the start of an event, such as a run. The young people reported that they were constantly pressing the button and wondering whether doing so might affect readings. They suggested it would be better to have a device without buttons, to reduce temptation to press. Activity diaries are commonly used alongside accelerometers to record what activities the participant is taking part in. However, when questioned, younger boys (aged 7-11 years) reported that they would not use the diary. Girls were more positive about the thought of completing an activity diary, but they admitted that they would be likely to forget and that it would largely depend on what the activity diary looked like.
## How accelerometers can
Be Made More Appealing/Incentives for Use. Young people made several suggestions as to how accelerometers could be made more appealing or how incentives might be used to improve compliance. All age groups felt that a clip mechanism which fitted onto a child's own belt or clothes would be preferable to an elastic belt. Other suggestions included incorporating the accelerometer as part of a watch, sweatband, or bracelet. The way in which the device looked, in particular its colour, was an important consideration. The option of choosing from a variety of colours was thought to be a good way of making it more appealing. Participants also suggested that being able to personalise the device would encourage its use, for example, using stickers or having the young person's name or school on it. A "clip-on" cover, similar to those available for mobile phones, was suggested as a way of personalising. Offering tangible rewards, such as money, was also mentioned as a way to encourage young people to wear accelerometers.
"I would be happy to wear [accelerometer] if paid." (boy, aged 15; handled accelerometer at start of focus group) Across the age groups a major incentive put forward for wearing the accelerometer was being able to have feedback on their own activity levels, as this would provide worth and encouragement. However, it was also acknowledged that this might encourage "cheating" or asking friends to wear the accelerometer. Young people felt that in order to address this, it would be better to provide accelerometers to the whole class rather than just certain individuals. Doing this may also prevent an individual from feeling singled out.
Younger children felt that it would be good for researchers or teachers to discuss with the whole class the reasons for wearing an accelerometer if a child in that class was going to wear one. This would reduce curiosity from other children. However, it was also acknowledged that this might bring unwanted attention to the child wearing the accelerometer and even encourage bullying or make the child feel different. All young people said they would feel comfortable discussing their accelerometer in front of a class of peers but embarrassed in front of older children in the school. Giving parents an accelerometer to wear alongside their children was also suggested, making accelerometer wearing a whole family activity and hence encouraging children to wear one. Having role models (such as sports people) wearing accelerometers might help further encourage its use. Young people recommended that wearing the accelerometer should be presented by the researcher in a positive way. For example, how it may benefit the child, explaining how it could encourage them and other children to stay healthy, and fit. Introducing the importance of the accelerometer as a device for seeing how fit, healthy and active a person is was suggested. It could be emphasised how those young people wearing the accelerometers are helping research and potentially helping other young people. Young people also felt they would feel more comfortable about wearing an accelerometer if they knew that others were also wearing one.
# Discussion
To our knowledge, there has been little focus on young people's views on wearing accelerometers in physical activity research [bib_ref] Conducting accelerometer-based activity assessments in field-based research, Trost [/bib_ref]. Of the research available, it has been suggested that compliance may be increased with the use of rewards, personal activity graphs, and less obtrusive belts and monitors [bib_ref] Adolescent perspectives on wearing accelerometers to measure physical activity in population-based trials, Audrey [/bib_ref]. This small scale user-involvement study adds to initial data in this area, confirming some already highlighted issues, as well as introducing further areas for consideration. This paper provides practical issues for researchers to consider when embarking on accelerometer research among young people. Based on the current study findings, we make a number of suggestions to researchers at the design stage of a study involving young people and accelerometers. These suggestions are focussed around introducing the accelerometer, practical issues, individual factors associated with wearing an accelerometer, and incentives for compliance .
Young people's reports of their first impressions of the accelerometer highlight the need for introducing the accelerometers in a clear and informative way. Furthermore, this study begins to reveal that individual factors such as gender, weight status, or current physical activity behaviours may affect an individual's likelihood of wearing the device. It highlights the need to make judgements on an individual participant basis. As suggested in , a trial wearing of the device (e.g., 24 hours) may highlight issues that can then be addressed during this time and potentially increase the likelihood of compliance during the study period. Practicalities of a "trial period" will of course depend on the study size and duration and number of participants. Previous studies have suggested that accelerometer measures tend to be higher on the first day of recording [bib_ref] Use of accelerometers in a large field-based study of children: protocols, design..., Mattocks [/bib_ref]. Some young people in the current study did report that it took them a little to get used to wearing the device. As such, a period of habituation once a participant has started to wear an accelerometer for data collection is advised.
The current study begins to reveal that individuals with weight-related issues may have certain opinions about the use of an accelerometer that others may not, for example, being at an increased risk of bullying. As current literature on young people's views on accelerometer use is limited, this study highlights an even greater need for understanding the needs and concerns of overweight/obese children in this context.
Previous findings have demonstrated how young people's physical activity patterns can vary between school days and weekends [bib_ref] Eating and activity habits of overweight children on ISRN Obesity 7 weekdays..., Hart [/bib_ref] and time of year [bib_ref] Seasonal variation in accelerometer-determined sedentary behaviour and physical activity in children: a..., Rich [/bib_ref]. The time at which the device is worn is, therefore, likely to have an impact. There were mixed findings as to whether young people preferred wearing accelerometers during school term or school holidays. For research studies which are measuring change in physical activity over time, it is important that longitudinal measurements are carried out either in school term or school holidays, if permitted by the study protocol, in order to minimise intraindividual variation. Giving children the choice of school term or school holidays may help compliance.
Previous research on adolescent perspectives on accelerometer use has been conducted in large-scale population-based school studies [bib_ref] Adolescent perspectives on wearing accelerometers to measure physical activity in population-based trials, Audrey [/bib_ref]. While this is a common setting for accelerometer based research [bib_ref] Physical activity and dietary behaviour in a population-based sample of British 10-year..., Van Sluijs [/bib_ref] , studies involving community-based interventions (e.g., family-based obesity treatment interventions) are likely to involve one-to-one : Practical advice for researchers using accelerometers for physical activity research with young people.
Introducing the accelerometer (i) For school-based studies, introduce the research to the whole class. This may help reduce curiosity of others and reduce the risk or bullying (ii) Providing alternative "placebo" devices (e.g., pedometer) to other pupils may help in involving all pupils and reduce stigma (iii) Explain to participants that the accelerometer is not "watching" them, that is, there is no camera or microphone, and only the researcher will see the results. This is particularly important for younger children. Showing participants an example of what these data look like (e.g., a graph) may help them to understand what is being recorded (iv) Consider the use of "role models" to promote the wearing of an accelerometer
(v) Present the accelerometer in a positive way, for example, by wearing it, the participant is helping us to understand how they and other children can live healthier, happier lives (vi) Consider a trial wearing of the device (e.g., 24 hours)/period of habituation prior to data collection Practical issues (i) Use a clip rather than elastic band to attach the accelerometer (ii) Where possible, choose a small accelerometer without extra buttons * (iii) Certain popular activities, such as swimming or rugby, cannot be recorded due to practicalities of wearing the accelerometer at these times. This highlights the need for an activity diary, not only to record data on the accelerometer use, but also at times when it cannot be used. Diary use compliance is however low, and therefore alternative ways of gaining these data should be explored Individual factors associated with wearing an accelerometer (i) Judgements over the way in which an accelerometer is used should be made on an individual participant basis (ii) Discuss with the child what would make them feel more comfortable wearing the device (e.g., under or over clothes, timing, e.g., term time or holiday time) and administer accordingly where study protocol permits Incentives for compliance (i) Provide small stickers to personalise the accelerometers. These could be supplied at the same time as the accelerometer for the children is uses and removed once the child returns the device (ii) Unlike the pedometer, participants are not able to instantly view their activity levels. Therefore, provide an incentive to children for wearing the accelerometer for the whole study period. This could be in the form of feedback about their activity levels, as well as a certificate for wearing it for the full time (iii) Provide the parent with an accelerometer at the same time and ask them to wear it at the same time as their child, in order to make it a family activity and potentially increase compliance * The new Actigraph model GT3X is smaller than the one used in this study (GT1M) and is free of buttons.
communication with participants, perhaps within their home or a community venue. As such, different issues may be present compared with population-based research. For example, the overweight child is being singled out to wear the accelerometer and is likely to be the only one in their class. This may present specific challenges to the researcher to minimise the risk of bullying. Strategies could include offering other children in the child's class the opportunity to have their physical activity measured and support to the child to help them explain the monitor to their classmates. The allocation of accelerometers to children is more likely to be on a one-to-one level between the researcher and participant and therefore offers the potential for more time to be invested in individual concerns. Providing activity graphs [bib_ref] Adolescent perspectives on wearing accelerometers to measure physical activity in population-based trials, Audrey [/bib_ref] may therefore be easier in this context and was certainly an incentive which participants in the current study supported.
Although not within the control of the researcher, accelerometer manufacturers may want to consider the aesthetics of devices, with the suggestion from children that clip-on covers, such as those used on mobile phones, may be a valuable option for increasing its appeal and ultimately compliance. These are extra costs for the researcher but may improve the quality of the data being collected.
This is a small-scale study within a specific group of young people in the West Midlands, England. As such, the study findings are limited to a small group of individuals which are not necessarily representative of young people within different communities. However, initial findings are revealing and certainly warrant further exploration. These findings are to be used in a current randomised controlled trial of the "Families for Health" programme, an intervention for overweight and obese children and their families in Coventry, Warwickshire, and Wolverhampton.
# Conclusions
In conclusion, this user-involvement study provides initial data on young people's views on accelerometer use in physical activity research. Children report both positive and negative aspects of wearing accelerometers. While positive aspects relate to feeling "special" and having increased attention from friends, negative aspects include the size and comfort of the device as well as an increased risk of bullying. Young people offered advice on how to make wearing accelerometers more appealing, including presenting the device in a positive way, using a clip rather than elastic belt to attach it, personalising the device, and having feedback on activity levels. Judgements over the way in which accelerometers are used should be made at the study development stage and based on the individual population. Further research in this area is suggested, in particular exploring the views of specific populations, such as overweight/obese children or those attending a community health intervention.
## Disclaimer
The views and opinions expressed therein are those of the authors and do not necessarily reflect those of the HTA programme, NIHR, NHS, or the Department of Health.
[table] Table 1: Details of focus group discussion participants/topic. when initially worn, or simply taking some time to forget they had it on."It took me a while to get used to having it on, so the first day I took it on and off several times." (girl aged 10; wore accelerometer 1 week) [/table]
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Thyroid Hormone Activates Brown Adipose Tissue and Increases Non-Shivering Thermogenesis - A Cohort Study in a Group of Thyroid Carcinoma Patients
Background/ObjectivesThyroid hormone receptors are present on brown adipose tissue (BAT), indicating a role for thyroid hormone in the regulation of BAT activation. The objective of this study was to examine the effect of thyroid hormone withdrawal followed by thyroid hormone in TSH-suppressive dosages, on energy expenditure and brown adipose tissue activity.Subjects/MethodsThis study was a longitudinal study in an academic center, with a follow-up period of 6 months. Ten patients with well-differentiated thyroid carcinoma eligible for surgical treatment and subsequent radioactive iodine ablation therapy were studied in a hypothyroid state after thyroidectomy and in a subclinical hyperthyroid state (TSH-suppression according to treatment protocol). Paired two-tailed t-tests and linear regression analyses were used.ResultsBasal metabolic rate (BMR) was significantly higher after treatment with synthetic thyroid hormone (levothyroxine) than in the hypothyroid state (BMR 3.8 ± 0.5 kJ/min versus 4.4 ± 0.6 kJ/min, P = 0.012), and non-shivering thermogenesis (NST) significantly increased from 15 ± 10% to 25 ± 6% (P = 0.009). Mean BAT activity was significantly higher in the subclinical hyperthyroid state than in the hypothyroid state (BAT standard uptake value (SUV Mean ) 4.0 ± 2.9 versus 2.4 ± 1.8, P = 0.039).
After complete resection of the thyroid gland, thyroid carcinoma patients will develop a hypothyroid state in order to be able to effectively eradicate possible thyroid remnants with radioactive iodine ablation therapy. The high levels of TSH will then maximally stimulate the thyroid remnant to incorporate iodine and thus increase the effect of the administered radioactive iodine dose. After administering the I 131 -therapy, thyroid hormone suppletion with levothyroxin is started in TSH-suppressive doses to decrease plasma TSH to levels < 0.1 mU/L. Since large changes in thyroid hormone levels are routinely observed in these patients this patient group offers the unique opportunity to observe the effect of hypothyroidism on physiological and metabolic parameters like blood pressure, skin temperature, BMR, NST (non-shivering thermogenesis) and BAT activity both in a hypothyroid and in the subsequent subclinical hyperthyroid state within the same subject. Therefore, we investigated the combined the effect of well-differentiated thyroid carcinoma treatment on several physiological and metabolic parameters, including BAT activity. So far, no studies have directly investigated the proposed synergism between thyroid hormone and cold-induced regulation of BAT activity.
# Materials and methods
The study was reviewed and approved of by the medical ethics committee of the Maastricht University Medical Centre (METC 11-3-081). This study was registered on ClinicalTrials.gov after enrollment of the subjects and in the Dutch CCMO register before enrollment of the subjects. The authors confirm that all ongoing and related trials for this drug/intervention are registered. Written informed consent was obtained from twelve subjects with well-differentiated thyroid carcinoma, two male and 10 female, who were treated and included in the study at Maastricht University Medical Center between June 2012 and July 2014. Unfortunately, there were two female dropouts during the study. One subject could not withstand the cold and one subject started using a beta-blocker between the first and second set of measurements [fig_ref] Fig 1: Consort 2010 flow chart [/fig_ref]. These subjects were excluded from the analyses.
To avoid interference of the menstrual cycle with the measurements, all female subjects were postmenopausal or used a specific oral contraceptive pill (ethinylestradiol/levonorgestrel 20 μg/100 μg) throughout the study. Subjects were screened before participation and all reported to have stable physical activity for at least six months (no participation in vigorous exercise program in the last six months, exercise maximum 2 times a week and maximum 3 hours a week). For compliance reasons, subjects who were psychologically unstable and subjects with mental retardation or severe behavior disorders were excluded from the study. Other exclusion criteria were pregnancy, the use of beta-blockers, and participation in an intensive weight-loss program during the last year before the start of the study, drugs and/or alcohol abuse and insulin-dependent type 2 diabetes or diabetes-related complications. All subjects were their own control, as they were all measured during the two conditions; hypothyroid state six to eight weeks after surgical removal of the thyroid gland and subclinical hyperthyroid state after administration of levothyroxine for four to six months.
## Study protocol
The first set of measurements took place between June 2012 and November 2013, on average 6.8 ± 2.8 weeks after surgery, when plasma free T4-levels were at the minimum. Subjects were measured in the morning after overnight fasting and asked to refrain from heavy exercise 24 hours before the measurements. During the measurements subjects were allowed to wear light standardized clothes (socks 0.02 clo, shirt with long sleeves 0.20 clo, sweatpants 0.28 clo, underwear 0.04 clo, total clo factor 0.54 clo). Clo units refer to clothing insulation [bib_ref] Glossary of terms for thermal physiology, Bligh [/bib_ref]. First, the subjects' body composition was determined using dual x-ray absorptiometry (DXA, Hologic, type Discovery A, Bedford, MA). For the determination of body core temperature and heart rate, a telemetric pill (CoreTemp, HQ Inc., Palmetto, FL USA) was orally ingested by the study subjects. This measurement failed in three subjects. Skin temperature was determined by applying wireless iButtons on 14 ISO-defined sites of the body [bib_ref] Evaluation of wireless determination of skin temperature using iButtons, Van Marken Lichtenbelt [/bib_ref]. The second set of measurements were done four to six months after radioactive iodine ablation therapy, when the subjects were in the subclinical hyperthyroid state, on levothyroxine therapy, with low TSH and high fT4 levels [fig_ref] Fig 2: Schematic representation of study measurements after total thyroidectomy in the Maastricht University... [/fig_ref]. For detailed description see S1 Text-Study Protocol.
## Personal cooling protocol and 18 f-fdg-pet-ct
Subjects were placed inside a specially equipped air permeable climate tent (Colorade Altitude Training, Louisville, CO). Inside this tent the air temperature was regulated by an air Blue arrows indicate moment of study measurements. FT4 indicates free thyroxine, TSH indicates thyroid-stimulating hormone, levothyroxine treatment indicates pharmacological levothyroxine suppletion that suppresses endogenous TSH. I 131 indicates radioactive iodine, used for radioactive ablation therapy of thyroid gland remnants after thyroid gland resection for well-differentiated thyroid carcinoma. I 124 indicates a proton-rich isotope of iodine used as a radiochemical for determination of thyroid gland remnants after thyroid gland resection for well-differentiated thyroid carcinoma. conditioner to maintain the air temperature within the tent with an accuracy of 1°C [bib_ref] Increased systolic blood pressure after mild cold and rewarming: relation to cold-induced..., Kingma [/bib_ref]. To guarantee a comfortable position during the measurements, subjects were placed in a semisupine position in a nephrodialysis chair. Subjects underwent a personalized cooling protocol. The seat of the nephrodialysis chair in which the subjects were placed was covered with a water-perfused matrass (Blankett role, Cincinatti sub zero 2000, USA), allowing for extra cooling options for the dorsal side of the body. To guarantee maximum non-shivering thermogenesis, we used a personalized cooling protocol described earlier [bib_ref] Cold acclimation recruits human brown fat and increases nonshivering thermogenesis, Van Der Lans [/bib_ref]. NST was defined as percentage increase in EE after cold exposure.
Energy expenditure was measured by indirect calorimetry during the three hours of the measurements. During the first hour, measurements were performed in thermoneutrality (room temperatures 23.7 ± 0.85°C in hypothyroid state, 23.8 ± 0.26°C in TSH-suppressed state), followed by a gradual step-wise decrease of the temperature of the air and cooling mattress during the second hour. This was continued until the onset of shivering. Shivering was detected visually and subjects were asked to report shivering on a visual analog scale every ten minutes, as described in earlier studies by our group [bib_ref] Systemic beta-adrenergic stimulation of thermogenesis is not accompanied by brown adipose tissue..., Vosselman [/bib_ref]. At the first signs of shivering, air and water temperatures were increased by steps of 1°C until shivering just stopped and temperatures were then kept stable at these points. After the first hour of cold exposure 75 MBq of 18 -Fluoro-Deoxy-Glucose ( 18 F-FDG) was injected through a intravenous catheter. After injection, cold-exposure was maintained for another hour, in which subjects were instructed to lay still to prevent uptake of 18 F-FDG in muscle tissue. Blood was withdrawn from the cubital vein catheter once during thermoneutrality (55 minutes after the start of the protocol and right before the onset of cooling) and once during cold exposure (115 minutes after the start of the protocol and right before the 18 F-FDG injection).
After the third hour, subjects were moved to the positron emission and computed tomography scanner (PET-CT-scanner) (Gemini TF PET-CT, Philips, The Netherlands) for quantification of metabolically active BAT. The scanning protocol and data interpretation methods were identical to those used in earlier studies by our group [bib_ref] Cold-activated brown adipose tissue in healthy men, Van Marken Lichtenbelt [/bib_ref]. For detailed description see S1 Text-Study Protocol.
## Follow-up
All patients underwent their thyroidectomy and radioactive iodine ablation therapy, according to treatment protocol, without any complications. The second set of measurements took place between October 2012 and July 2014, four to six months after the initial measurements, after subjects were stable on a daily dose of synthetic thyroid hormone (levothyroxine, fT4 levels 23.1 ± 3.9 pmol/L, TSH 0.5 ± 0.6 mU/L levothyroxine dose 137.75 ± 23.75 μg/day), the above described measurement protocol was repeated.
## Blood analyses
Blood was collected from the antecubital vein for analyses of several blood variables. Plasma concentrations of free fatty acids (NEFA-HR set; Wako Chemicals), free glycerol (Glycerol kit; R-Biopharm), total glycerol (ABX Triglyceriden CP; Horiba ABX), and glucose (ABX Glucose HK CP; Horiba ABX) were measured on a COBAS PENTRA centrifugal spectrophotometer (Horiba ABX). Plasma triglyceride concentrations were calculated by subtracting free glycerol from the total glycerol concentrations, and serum insulin was analyzed on a Gamma Counter (2470 Automatic Gamma Counter Wizard2 Wallac; Perkin-Elmer) with a Human Insulin specific RIA kit (Millipore). Plasma norepinephrine and adrenaline were analyzed by using reagents from Recipe Chemicals and Instruments with HPLC through electrochemical detection. Serum TSH was measured by an Electrochemiluminescence Immunoassay Kit on a COBAS 6000 system (Roche Diagnostica), and free thyroxine (FT4) was analyzed by a solidphase time-resolved fluoroimmunoassay kit on an AutoDELFIA system (PerkinElmer). Plasma inflammatory marker C-reactive protein (CRP) was measured with a particle-enhanced immunoturbidimetric assay on a COBAS PENTRA system (Horiba ABX). For detailed description see S1 Text-Study Protocol.
# Statistical analysis
Statistical analysis was performed by PASW Statistics version 20.0 for MacBook Pro.
Unfortunately, no studies have been performed with thyroid hormone in which our most important outcome parameters (BAT activity) is studied. Still, we decided to base our power calculation on the SUV of BAT. A previous study from our group showed an increase of BAT activity in lean subjects upon 10 days of cold acclimation from 2.4 standard uptake value (SUV) mean to 2.8 SUV mean, with a standard deviation of 0.5 [bib_ref] Increased sympathetic tone in forearm subcutaneous tissue in primary hypothyroidism, Vagn Nielsen [/bib_ref]. In the current study, in which slightly older subjects are included, we expect BAT values to be lower.
We expect a slightly bigger increase in BAT activity as compared to 10 days of mild cold exposure. Therefore, we expect BAT activity values to be 1.4 SUV mean in the hypothyroid state and 1.85 SUV mean in the subclinical hyperthyroid state, with a standard deviation of 0.5. With this information, a two-sided alpha of 0.05, and a power of 0.80, our study group should consist of 10 participants (calculated using G Ã Power 3.1 software, Faul, Erdfelder, Land and Buchner, University of Trier). Accounting for a possible dropout rate of 15%, a total of (10/ 0.85 =) 12 subjects were included. In the end, two subjects dropped out.
Reported data is expressed as means of ±SD. Total BAT activity was expressed in standard uptake values (SUV; as calculated by uptake (kilobequerels per milliliter) per injected dose (kilobequerels) per patient weight (grams)). BAT activity of each region was determined by the average SUV (SUV mean) times the volume of the region (cubic centimeter), expressed as SUV total. Paired two-tailed t-tests were used in order to compare data before and after cold exposure and data before and after treatment. Linear regression analyses were used to identify correlations between variables. For detailed description see S1 Text-Study Protocol.
## Subject characteristics
This study included eight female and two male patients with well-differentiated thyroid carcinoma that were selected for total thyroidectomy according to national and local oncology guidelines as defined in the Dutch oncology guidelines for thyroid carcinoma . Subject characteristics are shown in [fig_ref] Table 1: Subject characteristics [/fig_ref]. Surgical removal of the thyroid gland was on average 6.8 ± 2.8 weeks before the first set of measurements and subjects did not have any adjustments to their levothyroxine medication (last adjustment > one month before the second set of measurements). The first set of measurements took place on the same day as the radioactive iodine ablation therapy, after which levothyroxine therapy was started. On average, subjects were remeasured 4.2 ± 1.4 months after the first measurement, and average dosage of levothyroxine at the time of the second set of measurements was 137.75 ± 19.75 μg/day.
# Results
## Energy expenditure
Basal metabolic rate significantly increased in the subclinical hyperthyroid state versus the hypothyroid state (4.4 ± 0.6 kJ/min versus 3.8 ± 0.5 kJ/min, P = 0.012, [fig_ref] Fig 2: Schematic representation of study measurements after total thyroidectomy in the Maastricht University... [/fig_ref]. Furthermore, EE during cold exposure significantly increased after thyroid substitution therapy (4.3 ± 0.7 J/min versus 5.4 ± 0.5 J/min, P = 0.006). NST, also increased significantly in the presence of thyroid hormone (15 ± 10% versus 25 ± 6%, P = 0.009, [fig_ref] Fig 3: BAT activity, BMR and NST before and after levothyroxine therapy [/fig_ref].
## Bat activity
All subjects (N = 10) had active cold-stimulated BAT, both in the hypothyroid and subclinical hyperthyroid state. Representative images of FDG-uptake on PET-CT in the studied group are shown in [fig_ref] Fig 3: BAT activity, BMR and NST before and after levothyroxine therapy [/fig_ref] The mean SUV was higher in the presence of thyroid hormone in 9 of the 10 subjects. This increase was significant (BAT SUV mean; 4.0 ± 2.9 versus 2.4 ± 1.8, P = 0.039, [fig_ref] Fig 3: BAT activity, BMR and NST before and after levothyroxine therapy [/fig_ref]. No correlations were found between changes in fT4 levels and changes in BAT activity (BAT SUVmean: r = -0.100, P = 0.783). Also, changes in TSH levels did not correlate with changes in BAT activity (BAT SUV mean: r = -0.122, P = 0.754). Changes in fT4 levels (delta fT4) were defined as: fT4 level in hypothyroid state minus fT4 level in subclinical hyperthyroid state. Changes in TSH levels and BAT activity were defined in a similar manner.
## Core and skin temperature
Body core temperature in thermoneutral and cold conditions did not significantly change in the presence of thyroid hormone (core temperature thermoneutral 37.1 ± 0.5°C versus 37.3 ± 0.6°C, P = 0.633; core temperature cold 37.4 ± 0.4°C versus 37.2 ± 0.3°C, P = 0.518, [fig_ref] Table 2: Skin perfusion, blood pressure, and body temperature under thermoneutral conditions and during... [/fig_ref]. Mean skin temperature in thermoneutral and cold conditions was significantly lower in the hypothyroid state (mean skin temperature thermoneutral 32.2 ± 0.3°C versus 33.3 ± 0.4°C, P < 0.001; mean skin temperature cold 29.5 ± 0.6°C versus 30.9 ± 0.7°C P = 0.001, [fig_ref] Table 2: Skin perfusion, blood pressure, and body temperature under thermoneutral conditions and during... [/fig_ref]. No correlations were found between mean skin temperature during cold exposure and BAT activity in both the hypothyroid and subclinical hyperthyroid state (r = -0.161, P = 0.656 and r = 0.035, P = 0.925 respectively).
## Skin perfusion and blood pressure
Hand skin blood flow was significantly reduced during cold exposure (hand; -47% ± 48%, P = 0.048, [fig_ref] Table 2: Skin perfusion, blood pressure, and body temperature under thermoneutral conditions and during... [/fig_ref]. There were no differences in the extent of skin blood flow reduction between the hypothyroid and subclinical hyperthyroid situation. Diastolic blood pressure in the thermoneutral condition was significantly higher in the hypothyroid state (93 ± 18 versus 82 ± 15, P = 0.004, [fig_ref] Table 2: Skin perfusion, blood pressure, and body temperature under thermoneutral conditions and during... [/fig_ref]. Also, mean arterial pressure (MAP) in both thermoneutral and mild cold conditions was significantly higher in the hypothyroid state (105 ± 20 versus 96 ± 18, P = 0.013 and 113 ± 18 versus 100 ± 18, P = 0.009 respectively, [fig_ref] Table 2: Skin perfusion, blood pressure, and body temperature under thermoneutral conditions and during... [/fig_ref].
## Blood values
Baseline free fatty acids and total glycerol were significantly lower in the subclinical hyperthyroid state than in the hypothyroid state (P = 0.042 and P = 0.001 respectively, [fig_ref] Table 2: Skin perfusion, blood pressure, and body temperature under thermoneutral conditions and during... [/fig_ref] , whereas baseline CRP and glucose levels were significantly higher in the clinical hyperthyroid state (P = 0.021 and P = 0.024 respectively, [fig_ref] Table 2: Skin perfusion, blood pressure, and body temperature under thermoneutral conditions and during... [/fig_ref]. During cold exposure, both noradrenaline and adrenaline levels were significantly lower in the subclinical hyperthyroid state than in the hypothyroid situation (P = 0.001 and P = 0.007 respectively, [fig_ref] Table 2: Skin perfusion, blood pressure, and body temperature under thermoneutral conditions and during... [/fig_ref]. In our study, changes in TSH levels (deltaTSH) did not correlate with changes in total glycerol (r = 0.429, P = 0.250) or changes in free fatty acid levels (r = -0.039, P = 0.921). For full study data see S1 Dataset.
# Discussion and conclusion
In this study we showed that BAT activity significantly increased in the subclinical hyperthyroid state compared to the hypothyroid state after thyroid carcinoma treatment. The data also demonstrated the presence of functional BAT in the hypothyroid group. Furthermore, we showed that the increase in BAT activity was accompanied by a 26% increase in NST.
## Effects on bmr, nst and bat activity
It has long been accepted that one of the main functions of thyroid hormone is homeostasis of overall EE and BMR to the benefit of growth and development [bib_ref] The role of thyroid hormones in the control of energy expenditure, Danforth [/bib_ref]. Thyroidectomized rats have 20-30% reduction in BMR, which is immediately restored upon T4 injection [bib_ref] Metabolism of tissues excised from thyroxine-injected rats, Barker [/bib_ref]. Here, we showed that BMR significantly increased in the presence of thyroid hormone, when compared to the hypothyroid state.
It is well known that BAT thermogenesis is activated by the hypothalamus via the sympathetic nervous system (SNS) [bib_ref] UCP1: the only protein able to mediate adaptive non-shivering thermogenesis and metabolic..., Nedergaard [/bib_ref]. Here we showed a significant cold-stimulated BAT activation in hypothyroid patients, which is in line with a recent case report, which showed active BAT in a case of severe hypothyroidism [bib_ref] Presence of brown adipose tissue in an adolescent with severe primary hypothyroidism, Kim [/bib_ref]. Furthermore, a significant increase in BAT activity accompanied by an increased NST was detected after thyroid hormone substitution therapy. Changes in T4 and TSH values were not correlated with changes in BAT activity. The positive effect of high-dose levothyroxine treatment on BAT activity after treatment of thyroid carcinoma with total thyroidectomy and subsequent radioactive iodine ablation therapy was previously shown in a case report. However, without levothyroxine no BAT activity was detected in that patient [bib_ref] Thyroid hormone induced brown adipose tissue and amelioration of diabetes in a..., Skarulis [/bib_ref]. The additive effect of thyroid hormone and cold-exposure on BAT activity found in this study could possibly be attributed to the up-regulation of betaadrenergic receptors in response to thyroid hormone [bib_ref] The influence of hyperthyroidism and hypothyroidism on alpha-and beta-adrenergic receptor systems and..., Bilezikian [/bib_ref] , leading to increased sensitivity to catecholamines.
## Body composition and temperatures
In our study, we found that in the hypothyroid state, average skin temperature at thermoneutrality was significantly lower than in the subclinical hyperthyroid state. Hypothyroidism patients often report cold extremities and cold intolerance 37, personal observations . The increase in mean skin temperature upon thyroid hormone replacement is probably accounted for by thyroid-induced modulation of metabolic rate. Also, skin temperatures during cold exposure were significantly higher in the clinical hyperthyroid state than in the hypothyroid state, although no correlations between average skin temperature during cold exposure and BAT activity were found. The reduction of thermogenesis seen in hypothyroidism has been known to be partially compensated by cutaneous vasoconstriction [bib_ref] Quantitation of thyroid hormone effect on skin perfusion by laser Doppler flowmetry, Weiss [/bib_ref] [bib_ref] Increased sympathetic tone in forearm subcutaneous tissue in primary hypothyroidism, Vagn Nielsen [/bib_ref]. The decreased skin temperature seen in the hypothyroid situation could be this thyroid-driven vasoconstriction, for the benefit of internal heat preservation. However, in the absence of thyroid hormone, EE still increased and functionally active BAT was still observed upon cold exposure, as was expected from animal studies [bib_ref] Disruption of thyroid hormone activation in type 2 deiodinase knockout mice causes..., Castillo [/bib_ref]. Interestingly, hypothyroid noradrenalin and adrenalin levels during mild cold exposure where significantly higher than those in the subclinical hyperthyroid state, which could indicate compensatory adrenergic activation. Systemic hyperthyroidism or central administration of thyroid hormone to rats has been shown to cause a decrease in hypothalamic AMP-activated protein kinase, leading to sympathetic activation and BAT induction. BAT can also be activated in the case of hypothyroidism. Exposing D2-deficient mice to a cold environment still leads to thermogenesis via activation of the sympathetic nervous system, despite the limited availability of active plasma thyroid hormone. However, when these animals are ot exposed to cold, they become glucose intolerant and develop non-alcoholic fatty liver disease and diet-induced obesity [bib_ref] Disruption of thyroid hormone activation in type 2 deiodinase knockout mice causes..., Castillo [/bib_ref] , suggesting the synergism between the sympathetic nervous system and thyroid axis in the control of BAT-mediated thermogenesis. The additive effect of thyroid hormone and cold-exposure on BAT activity could also be attributed to the up-regulation of beta-adrenergic receptors in response to thyroid hormone [bib_ref] The influence of hyperthyroidism and hypothyroidism on alpha-and beta-adrenergic receptor systems and..., Bilezikian [/bib_ref] , leading to increased sensitivity to catecholamines. In hypothyroidism, responses to adrenergic stimulation are relatively low or blunted, leading to cold intolerance and limited metabolic response to cold exposure [bib_ref] Thyroid-adrenergic interactions: physiological and clinical implications, Silva [/bib_ref]. Possibly the significant cold-induced increase in catecholamines in the hypothyroid state is a feedback mechanism to the benefit of heat production.
## Metabolic health effects
In this study, during the hypothyroid state, blood pressure was significantly increased. Hypothyroidism has been known to have profound effects on multiple organs and organ systems. Lately, increased risk for cardiovascular disease due to hypothyroidism has received strong scientific attention. In this study we found that thyroid hormone substitution therapy has a positive effect on serum free fatty acids and total glycerol, alongside an improvement of systolic and diastolic blood pressure. A positive association between TSH levels and systolic and diastolic blood pressure has been described earlier [bib_ref] The association between subclinical hyperthyroidism and blood pressure in a population-based study, Volzke [/bib_ref]. In another publication by Liu et al. the prevalence of hypertension (defined as systolic blood pressure ! 140 mmHg and/or diastolic blood pressure ! 90 mmHg) was significant higher in a group with subclinical hypothyroidism (TSH > 4.8 mIU -1 ) than in the euthyroid group (TSH 0.3-4.8 mIU -1 ) [bib_ref] A cross-sectional survey of relationship between serum TSH level and blood pressure, Liu [/bib_ref]. Another possible explanation for the elevated blood pressure in hypothyroidism observed is the compensatory adrenergic activation (see above).
Recently thyroid-stimulating hormone has been identified as an inducer for lipolysis [bib_ref] PKC activation is required for TSH-mediated lipolysis via perilipin activation, Thrush [/bib_ref]. In our study we found significantly higher serum free fatty acids in the hypothyroid state (with high TSH, [fig_ref] Table 2: Skin perfusion, blood pressure, and body temperature under thermoneutral conditions and during... [/fig_ref] , alongside increased serum total glycerol [fig_ref] Table 2: Skin perfusion, blood pressure, and body temperature under thermoneutral conditions and during... [/fig_ref] , suggesting a role for TSH in metabolic health. This finding is in accordance with earlier studies, in which recombinant TSH was administered to 19 patients with differentiated thyroid carcinoma, who had previously undergone surgical thyroidectomy with consecutive radioactive iodine ablation therapy. In this study a 42% increase in FFAs was found [bib_ref] Thyroid-stimulating hormone stimulates lipolysis in adipocytes in culture and raises serum free..., Gagnon [/bib_ref]. In our study, no correlations were found between TSH, total glycerol and FFA levels. The higher levels of total glycerol observed in the hypothyroid state could result from lower BAT activation in this state, since BAT is known to control triglyceride clearance [bib_ref] Brown adipose tissue activity controls triglyceride clearance, Bartelt [/bib_ref].
The major limitations in this study are the small number of subjects enrolled. However, the used protocol, which allowed us to study the same subject in the hypothyroid as well as the subclinical hyperthyroid state, increased the power of the study. Taken together, in the present study we showed that higher levels of thyroid hormone positively affect BAT activity, BMR and NST. This is in line with the postulated thyroid hormone and sympatho-adrenal synergy. This suggests that thyroid hormone is an effective activator for brown adipose tissue and coldinduced thermogenesis. If safe, BAT-targeted thyroid thermogenesis could increase energy expenditure [bib_ref] Thyroid hormones: igniting brown fat via the brain, Cannon [/bib_ref]. However, since this study consisted of measurements in a group of cancer patients, more studies regarding the effects of thyroid hormone on BAT activity and energy metabolism in healthy subjects are warranted and detailed data on possible side effects is needed.
Supporting Information S1 Checklist. TREND Checklist. (PDF) S1 Dataset. (PDF) S1 Text. Study Protocol. (PDF)
[fig] Fig 1: Consort 2010 flow chart. doi:10.1371/journal.pone.0145049.g001 Thyroid Hormone and Brown Adipose Tissue PLOS ONE | DOI:10.1371/journal.pone.0145049 January 19, 2016 [/fig]
[fig] Fig 2: Schematic representation of study measurements after total thyroidectomy in the Maastricht University Medical Centre. [/fig]
[fig] Fig 3: BAT activity, BMR and NST before and after levothyroxine therapy. Brown adipose tissue (BAT) activity before and after levothyroxine substitution therapy. (A) Basal metabolic rate (BMR) in joules per minute. (B) Non-shivering thermogenesis (NST) before and after levothyroxine replacement therapy. (C) BAT activity in Mean Standard Uptake Values (SUV mean) before and after levothyroxine therapy. Subject indicated with X is also depicted in Fig 2D and 2E. (D) Transversal CT (top) and PET/CT fusion (bottom) slice of the supraclavicular region demonstrating 18 F-FDG-uptake in BAT locations (white arrows) after cold exposure in hypothyroid state. (E) Transversal CT (top) and PET/CT fusion (bottom) slice of the supraclavicular region demonstrating 18 F-FDG-uptake in BAT locations (white arrows) after cold exposure in subclinical hyperthyroid state. doi:10.1371/journal.pone.0145049.g003 [/fig]
[table] Table 1: Subject characteristics (N = 10). [/table]
[table] Table 2: Skin perfusion, blood pressure, and body temperature under thermoneutral conditions and during mild cold exposure, before and after levothyroxine substitution (N = 10.) [/table]
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10.1038/s41419-021-03874-7
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s2orc_pubmed_articles
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G-MDSCs promote aging-related cardiac fibrosis by activating myofibroblasts and preventing senescence
Aging is one of the most prominent risk factors for heart failure. Myeloid-derived suppressor cells (MDSCs) accumulate in aged tissue and have been confirmed to be associated with various aging-related diseases. However, the role of MDSCs in the aging heart remains unknown. Through RNA-seq and biochemical approaches, we found that granulocytic MDSCs (G-MDSCs) accumulated significantly in the aging heart compared with monocytic MDSCs (M-MDSCs). Therefore, we explored the effects of G-MDSCs on the aging heart. We found that the adoptive transfer of G-MDSCs of aging mice to young hearts resulted in cardiac diastolic dysfunction by inducing cardiac fibrosis, similar to that in aging hearts. S100A8/A9 derived from G-MDSCs induced inflammatory phenotypes and increased the osteopontin (OPN) level in fibroblasts. The upregulation of fibroblast growth factor 2 (FGF2) expression in fibroblasts mediated by G-MDSCs promoted antisenescence and antiapoptotic phenotypes of fibroblasts. SOX9 is the downstream gene of FGF2 and is required for FGF2-mediated and G-MDSC-mediated profibrotic effects. Interestingly, both FGF2 levels and SOX9 levels were upregulated in fibroblasts but not in G-MDSCs and were independent of S100A8/9. Therefore, a novel FGF2-SOX9 signaling axis that regulates fibroblast self-renewal and antiapoptotic phenotypes was identified. Our study revealed the mechanism by which G-MDSCs promote cardiac fibrosis via the secretion of S100A8/A9 and the regulation of FGF2-SOX9 signaling in fibroblasts during aging.
# Introduction
Aging is known to be one of the most important cardiovascular risk factors [bib_ref] Differences between men and women in mortality and the health dimensions of..., Crimmins [/bib_ref]. The proportion of individuals with diastolic dysfunction or even heart failure significantly increases with advancing age 2 . Aging induces cardiac biological changes, resulting in hypertrophy, a decline in diastolic function, or even heart failure [bib_ref] Ageing, metabolism and cardiovascular disease, Costantino [/bib_ref] [bib_ref] Cardiovascular aging and heart failure: JACC review topic of the week, Triposkiadis [/bib_ref].
Functionally, aging-related heart disease is characterized by heart failure with preserved ejection fraction (HFpEF) caused by cardiac fibrosis, which results from changes in the cardiac microenvironment, including senescence, collagen deposition, inflammation and cell death, and has been shown to have clinical importance [bib_ref] Heart failure with preserved ejection fraction: molecular pathways of the aging myocardium, Loffredo [/bib_ref]. Thus, the mechanisms of aging-related cardiac fibrosis deserve further exploration.
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress innate and adaptive immunity [bib_ref] Myeloid-derived suppressor cells, Gabrilovich [/bib_ref] [bib_ref] The terminology issue for myeloid-derived suppressor cells, Gabrilovich [/bib_ref]. MDSCs consist of two large groups of cells: granulocytic MDSCs (G-MDSCs) are phenotypically and morphologically similar to neutrophils, and monocytic MDSCs (M-MDSCs) are similar to monocytes [bib_ref] Coordinated regulation of myeloid cells by tumours, Gabrilovich [/bib_ref]. Previous studies have demonstrated that MDSCs are most commonly identified by LOX or CD33 expression in humans, whereas in mice, MDSCs were found to be myeloid-derived cells (Gr1 + CD11b + ) [bib_ref] Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells..., Condamine [/bib_ref] [bib_ref] Myeloid-derived suppressor cells coming of age, Veglia [/bib_ref]. Initially, MDSCs were identified as a major obstacle for natural antitumor immunities and were found in many other abnormal conditions, such as autoimmunity, infection, and diabetes [bib_ref] Myeloid-derived suppressor cells coming of age, Veglia [/bib_ref]. MDSCs act as a double-edged sword in different disorders. Although a series of studies have verified the expansion of MDSCs with age [bib_ref] Targeting macrophages rescues age-related immune deficiencies in C57BL/6J geriatric mice, Jackaman [/bib_ref] [bib_ref] Blood CD33( + )HLA-DR(-) myeloid-derived suppressor cells are increased with age and..., Verschoor [/bib_ref] , the effects of these cells on aging hearts are still unclear. In this study, we sought to explore the role of MDSCs in the aging heart and the mechanism of the G-MDSC profibrotic function.
# Results
The G-MDSC levels increase with advancing age
To explore the dynamic transcriptome of myeloidderived cells with age, we obtained RNA-Seq data of 135 human whole blood transcriptomes from the GEO database (GSE123698) and performed correlation analysis. The total coexpressed genes were divided into 10 modules by weighted gene coexpression network analysis (WGCNA), and the analysis showed that module 0 (ME0) was closely related to age (correlation coefficient = 0.15) but not to sex (correlation coefficient = 0.043) . Then, hypergeometric tests were performed on 913 genes in ME0 according to immunologic gene sets. The bubble plot showed that a series of myeloid-derived cell-related gene sets passed the filtering criteria. Notably, G-MDSC-related genes were significantly linked to ME0 . Next, the correlation between the M-MDSC/G-MDSC signature and age was detected by a deconvolution method. Compared with the M-MDSC signature, the G-MDSC signature was associated with age in both male and female individuals, indicating that the correlation between the G-MDSC signature and age was more significant than that of the M-MDSC signature . To further verify these findings, through single-cell RNA-Seq (scRNA-Seq) data (GSE145477) of myeloid-derived cells in mice, we calculated the M-MDSC/G-MDSC signature and found that the G-MDSC signature increased in the mice at 16 months compared with that in the mice at 3 months, while the M-MDSC signature remained almost unchanged . We therefore examined the changes in G-MDSCs/M-MDSCs in aging mice. Flow cytometric analysis revealed that the level of CD11b + GR1 + Ly6G+ cells (G-MDSCs) was significantly elevated in the hearts of aging mice, while there was no obvious change in CD11b + GR1 + Ly6C+ cells (M-MDSCs) , similar results were observed in the spleen, blood, and bone marrow (Supplement . To determine whether these granulocyte-like cells exert an immunosuppressive effect, we cocultured granulocyte-like cells purified from hearts and spleens with CD4 + and CD8 + T cells at a ratio of 2:1. As expected, these granulocytelike cells strongly inhibited the proliferation of CD4 + T cells and CD8 + T cells and Supplement . Overall, our results demonstrated that G-MDSCs are enhanced in human and mouse hearts with aging.
The G-MDSCs from aging mice induce cardiac fibrosis
To investigate the effect of the G-MDSCs from aging mice on the heart, we performed adoptive transfer of G-MDSCs (1 × 10 7 ) extracted from aging mice (20 months) to young mice (6 weeks) in vivo. We found that the transferred G-MDSCs did not survive for more than 48 h in mice heart (Supplement ; thus, the transfer of G-MDSCs was performed through tail vein injection every 5 days for 40 days. Compared with that of the young mice, the diastolic function of the aging mice was substantially reduced, as indicated by obvious decreases in dP/dT max and dP/dT min. Notably, transfer of the G-MDSCs also reduced the diastolic function in the young mice . Cardiomyocyte hypertrophy and fibrosis are typical pathological features of cardiac diastolic dysfunction. However, WGA staining showed no obvious difference in cardiomyocyte size between the young mice with or without G-MDSCs . By Masson staining, we observed a substantial increase in fibrotic areas in the cardiac sections of the aging mice, and similar phenomena were also observed in the mice with transfer of G-MDSCs , indicating that G-MDSCs can induce cardiac fibrosis. Through scRNA-Seq data (E-MTAB7869), we observed the enhancement of fibroblasts in aging mouse hearts (Supplement . Hence, we detected the difference in cardiac fibroblast phenotypes between the mice with or without transfer of G-MDSCs. Through flow cytometry, we found that transfer of G-MDSCs significantly increased the number of myofibroblasts (collagen I + α-SMA + ) in the hearts of the young mice . Moreover, transfer of G-MDSCs induced strong upregulation of the mRNA expression of fibrotic markers (Col3a1, Postn, Acta2), fibrosis-related factors (MMP9, Lox, and Lgals3), and inflammatory cytokines (IL6 and IL10) . Interestingly, the change in the Tgf-β level was unremarkable in the aging mice and the young mice with transfer of G-MDSCs . Furthermore, we cocultured fibroblasts with granulocyte-like cells from young and aging mice and found that the G-MDSCs from aging mice increased the levels of fibrotic markers (Acta2, Spp1, Fgf2), while the granulocyte-like cells from young mice had no obvious effects (Supplement , revealing that the role of G-MDSCs is due to a functional change and not accumulation. Overall, our in vivo experiments suggested that aging-related G-MDSCs can impair cardiac diastolic function by inducing cardiac fibrosis. Levels of MDSCs in the heart are positively associated with age. A WGCNA of the coexpressed genes from 135 human whole blood transcriptomes from the GEO database (GSE123698). ME0 was related to age (correlation coefficient = 0.15) but not to sex (correlation coefficient = 0.043). Red indicates a positive correlation, and blue indicates a negative correlation. B Hypergeometric test of the genes in ME0 based on the immunologic gene set from GSE123698. The size of the dot represents the number of genes. A darker color indicates a lower correlation. C The bubble plots show the correlation between the G-MDSC/M-MDSC signature and age in males and females based on GSE123698. D The ratio (left) and signature value (right) of the G-MDSCs and M-MDSCs in 3month-old and 16-month-old mice determined by scRNA-Seq analysis (GSE145477). E Representative flow cytometric profile showing the quantitative analysis of Cd11b + Gr1 + Ly6G+ cells and Cd11b + Gr1 + Ly6C+ cells in the hearts of young and aging mice; n = 5 per group. F CFSE-labeled CD4 + and CD8 + T cells and G-MDSCs separated from hearts were cocultured at a ratio of 2:1 for 24 h. Representative images and quantitative analysis of the proliferation of CD4 + or CD8 + T cells analyzed by flow cytometry; n = 6 per group. The data are presented as the means ± SDs. Differences were determined by Student's t test. *P < 0.05. G-MDSCs induce inflammatory phenotypes and increase the level of OPN in fibroblasts by secreting S100A8/9
To help elucidate the mechanism of aging-related G-MDSC-mediated cardiac fibrosis, we explored the phenotypes of cardiac fibroblasts in aging mice from the scRNA-Seq database (E-MTAB7869). Through cell type recognition, we found that the proportion of fibroblasts significantly increased in aging hearts compared with young hearts . Fibrotic markers (Acta2, Fn1, and Col1a1) were significantly increased in the aging hearts (Supplement . G-MDSCs promoted the proliferation of fibroblasts and enhanced the levels of fibrotic markers (Col3a1, Postn, and Acta2) . Interestingly, we found that G-MDSCs increased the levels of cytokines, including IL10, Tnf, and IL6 . Previous studies have reported that OPN is required for fibroblast activation and promotes aging-related cardiac fibrosis [bib_ref] Visceral adipose tissue drives cardiac aging through modulation of fibroblast senescence by..., Sawaki [/bib_ref]. We therefore tested the levels of OPN and α-SMA in fibroblasts. By immunofluorescence staining, we observed that the levels of α-SMA and OPN were increased in the fibroblasts cocultured with G-MDSCs . These results demonstrated that G-MDSCs could promote cardiac fibrosis. S100A8/A9, biomarkers of MDSCs, were reported to upregulated proinflammatory genes in the inflammatory response [bib_ref] S100A8/A9 in inflammation, Wang [/bib_ref]. The single-cell RNA-seq (GSE145477) analysis suggested that S100A8/9 expression levels were higher in G-MDSCs (Supplement [fig_ref] Figure 7: SOX9 knockdown alleviates the fibrotic phenotypes induced by G-MDSCs in vivo [/fig_ref] than that in M-MDSCs (Supplement [fig_ref] Figure 7: SOX9 knockdown alleviates the fibrotic phenotypes induced by G-MDSCs in vivo [/fig_ref] , which is consistent with our qPCR results (Supplement [fig_ref] Figure 7: SOX9 knockdown alleviates the fibrotic phenotypes induced by G-MDSCs in vivo [/fig_ref] and the study by Veglia, F et al. [bib_ref] Myeloid-derived suppressor cells coming of age, Veglia [/bib_ref]. In addition, we also found that the level of S100A8/9 in granulocyte-like cells (CD11b + Gr1 + Ly6G + ) was higher in aging mice than that in young mice (Supplement . Compared with S100A8, the level of S100A9 increased more significantly in granulocyte-like cells (Supplement .
Neutralizing S100A8 and S100A9 decreased the levels of proinflammatory cytokines (Tnf and IL6) . By flow cytometry, we found that G-MDSCs increased reactive oxygen species (ROS) generation and that neutralizing S100A8/A9 reduced the level of intracellular ROS . Interestingly, immunofluorescence revealed that neutralizing S100A8 and S100A9 decreased the OPN level in fibroblasts but had no obvious effect on the level of α-SMA , F). Altogether, our results indicated that G-MDSCs can promote inflammatory phenotypes and increase the level of OPN in fibroblasts by releasing S100A8/A9.
## Aging-related g-mdscs inhibit the cellular senescence and apoptosis of fibroblasts
Through scRNA-Seq data (E-MTAB7869), we found that the levels of inflammatory cytokines (IL6, IL1b, IL10, and Tnf) and senescence markers (Ccl2, Cdkn2a, Cdkn2b, and Mki67) increased in the cardiac fibroblasts of aging mice, confirming cell senescence (Supplement . Immunofluorescence staining indicated that the level of OPN, a marker of cell senescence that is involved in fibrosis, increased in the fibroblasts from young mice cultured with G-MDSCs . Therefore, further explorations were conducted on the effect of G-MDSCs on fibroblast senescence. Interestingly, G-MDSCs decreased the level of β-galactosidase in fibroblasts , suggesting that G-MDSCs may blunt fibroblast senescence. We next induced cellular senescence of fibroblasts by ultraviolet (UV) treatment in vitro. The β-galactosidase staining results showed that G-MDSCs decreased fibroblast senescence induced by UV treatment . As demonstrated by cell cycle assays, the fibroblasts cocultured with G-MDSCs showed significant cell cycle arrest at S phase compared with the fibroblasts cultured alone, regardless of UV treatment . Similarly, G-MDSCs suppressed UV-induced mRNA upregulation of the senescence markers Cdkn2a and Cdkn2b. . Furthermore, flow cytometry revealed that G-MDSCs inhibited fibroblast apoptosis induced by UV treatment . Overall, our data indicated that aging-related G-MDSCs could inhibit fibroblast senescence and apoptosis by inducing cell cycle arrest.
(see figure on previous page) G-MDSCs from aging mice induce cardiac fibrosis. Adoptive transfer of G-MDSCs (1 × 10 7 ) was performed in young mice (6 weeks) every 5 days for 40 days. Echocardiography, pathological examination, and qPCR were performed in young mice, old mice (20 months), and young mice that were administered G-MDSCs. A-D Left ventricular ejection fraction (LVEF; A), LV end-systolic diameter (LVDs; B), LV dp/dTmax (C), and LV dp/ dTmin (D) of the young mice, aging mice, and young mice with G-MDSCs; n = 5 per group. E WGA staining revealed the cardiomyocyte crosssectional area of the young mice, aging mice, and young mice with G-MDSCs. Representative cytograms are shown on the left, and statistical data are shown on the right; n = 4 per group. Scale bars, 50 μm. F Masson staining revealed fibrotic tissue in the mouse hearts. Representative cytograms are shown on the left, and statistical data are shown on the right; n = 4 per group. Scale bars, 50 μm. G The ratio of α-SMA + fibroblasts detected by flow cytometry of the young mice, aging mice, and young mice with G-MDSCs. Representative cytograms are shown on the left, and statistical data are shown on the right; n = 4 per group. H-J The mRNA levels of fibrosis markers (Col1a1, Col3a1, Postn, and Acta2; H), fibrosis-related factors (Mmp2, Mmp9, Timp1, Lox, Lgals3, and Tcf21; I), and cytokines (IL6, IL1b, IL10, and Tgfβ1; J) in the hearts of the young mice, aging mice and young mice with G-MDSCs; n = 4 per group. The data are presented as the means ± SDs. Differences were determined by one-way ANOVA (for more than 2 groups), and Tukey's HSD post hoc test was performed. *P < 0.05.
## G-mdscs have antisenescence and antiapoptotic effects on fibroblasts by upregulating fgf2 levels in fibroblasts
We used RNA-Seq to investigate the mechanism underlying the effect of G-MDSCs on cardiac fibroblasts. GSEA indicated that several signaling pathways were enhanced in the fibroblasts cocultured with G-MDSCs. Among these pathways, FGFR signaling is believed to be involved in fibrosis . As shown in the heatmap, the levels of FGF family members strongly increased in the mice treated with G-MDSCs . Among these factors, FGF2 was selected because of its enrichment in cardiac tissue. To explore the origin of FGF2, we examined the mRNA levels of FGF2 in fibroblasts and G-MDSCs. Interestingly, the level of FGF2 was lower in G-MDSCs and higher in fibroblasts, especially when they were cocultured with G-MDSCs. . Our data revealed that G-MDSCs can upregulate the level of FGF2 in fibroblasts. Next, we performed RNA-Seq on fibroblasts cocultured with G-MDSCs or 100 μg/mL S100A8/9 for 48 h to explore whether S100A8/9 affects the FGF2 level. The heatmap indicates that the S100A8/9 treatment significantly increased the levels of Spp1 (OPN) of fibroblasts but downregulated Fgf2 expression (Supplement , which is consistent with our qPCR results (Supplement . Thus, the above results suggested that the level of FGF2 in fibroblasts was independent of S100A8/9. To further investigate whether FGF2 inhibits fibroblast senescence and apoptosis, we performed adoptive transfer of G-MDSCs with/without BGJ398 (FGF2 inhibitor) treatment in young mice. Echocardiography revealed that neutralization of FGF2 partly alleviated the diastolic dysfunction induced by the transfer of G-MDSCs, as indicated by the increased dP/dT max and dP/dT min values . Flow cytometric analysis of β-galactosidase showed that neutralization of FGF2 partly relieved the G-MDSC-mediated antisenescence effects . Neutralization of FGF2 increased the mRNA levels of the senescence markers Cdkn2a and Cdkn2b but had little effect on the mRNA levels of inflammatory cytokines . Then, we examined the senescence and apoptotic phenotypes of fibroblasts in vitro. β-Galactosidase staining of senescent fibroblasts showed that neutralization of FGF2 induced an increase in senescent cells . Similarly, flow cytometry showed that neutralization of FGF2 increased the ratio of apoptotic fibroblasts . Thus, our data revealed that G-MDSCs mediate antisenescence and antiapoptotic functions by enhancing the release of FGF2 in fibroblasts.
## Sox9 is required for fgf2-mediated antisenescence and antiapoptotic functions
To explore the downstream targets of FGF2, we performed further experiments. GSEA of transcriptome data showed that SOX9, which has a profibrotic role, was enriched in the mice administered G-MDSCs . To investigate whether FGF2-induced fibrosis was dependent on SOX9, we transfected fibroblasts with a luciferase reporter plasmid (containing 12 repeats of a 48bp COL2A1 intron 1 enhancer element, which is known to be activated by SOX9). As expected, FGF2 increased the luciferase activity driven by SOX9 . Western blotting results also showed that FGF2 treatment increased the level of SOX9 in fibroblast nuclei . The RNA-seq analysis showed that the Sox9-related gene set was significantly enriched with the G-MDSC treatment, but showed no significance between the S100A8/9 treatment and control group (Supplement , suggesting that SOX9 expression was independent of S100A8/9. Then, we examined the senescence and apoptotic phenotypes of fibroblasts in vitro to verify the link between FGF2 and SOX9. β-Galactosidase staining showed that SOX9 knockdown enhanced the number of senescent cells . SOX9 knockdown increased the mRNA levels of the senescence markers Cdkn2a and Cdkn2b . As shown by the cell cycle assay, SOX9 knockdown alleviated the arrest at S phase induced by FGF2 . Moreover, SOX9 knockdown increased the number of apoptotic fibroblasts compared with those of the fibroblasts cultured with FGF2 alone . In conclusion, our data revealed that SOX9 is a downstream gene of FGF2. B CCK-8 analysis shows the proliferative potential of the fibroblasts cultured alone or cocultured with G-MDSCs at 12 h and 24 h; n = 5 per groups. C, D The mRNA levels of fibrosis markers (Col3a1, Postn, Acta2; C) and inflammatory cytokines (Tnf, IL6, IL10; D) in the fibroblasts cultured alone or cocultured with G-MDSCs for 24 h analyzed by qPCR; n = 5 per group. E-H Fibroblasts were cocultured with G-MDSCs for 24 h, and anti-S100A8/anti-S100A9 (50 µg) was added to the culture to neutralize S100A8/A9 secreted by G-MDSCs. Immunofluorescence staining revealed the levels of OPN (E) and α-SMA (F) in fibroblasts. Scale bars, 20 μm. G The mRNA levels of Tnf and IL6 in the fibroblasts of each group were analyzed by qPCR; n = 5 per group. H Intracellular ROS generation was analyzed by flow cytometry through DCFH-FA staining. Representative cytograms are shown on the top, and statistical data are shown on the bottom; n = 6 per group. The data are presented as the means ± SDs. Differences were determined by Student's t test (for 2 groups) or one-way ANOVA (for more than 2 groups), and Tukey's HSD post hoc test was performed. Repeated measures ANOVA was performed for time series data, and correlations were evaluated by Pearson's test. *P < 0.05.
## Sox9 is required for g-mdsc-mediated cardiac fibrosis
Next, we knocked down SOX9 expression in the G-MDSC-transferred mouse hearts by injecting adenoassociated virus (AAV) harboring the SOX9-KD plasmid in the mice, and the results were confirmed by Western blot analysis [fig_ref] Figure 7: SOX9 knockdown alleviates the fibrotic phenotypes induced by G-MDSCs in vivo [/fig_ref]. Then, we analyzed the effect of SOX9 knockdown on cardiac fibrosis. Immunofluorescence analysis revealed that the level of α-SMA in the SOX9_KD hearts returned to a level similar to that of the control group, indicating that fibroblast activation mediated by G-MDSCs was suppressed [fig_ref] Figure 7: SOX9 knockdown alleviates the fibrotic phenotypes induced by G-MDSCs in vivo [/fig_ref]. Similarly, the mRNA levels of fibrosis markers (Col3a1, Postn, Acta2) decreased in the SOX9_KD mouse hearts [fig_ref] Figure 7: SOX9 knockdown alleviates the fibrotic phenotypes induced by G-MDSCs in vivo [/fig_ref]. Moreover, SOX9 knockdown induced elevated levels of Cdkn2a, Cdkn2b [fig_ref] Figure 7: SOX9 knockdown alleviates the fibrotic phenotypes induced by G-MDSCs in vivo [/fig_ref] , and β-galactosidase [fig_ref] Figure 7: SOX9 knockdown alleviates the fibrotic phenotypes induced by G-MDSCs in vivo [/fig_ref] in fibroblasts, indicating that SOX9 is involved in G-MDSCmediated suppression of cellular senescence. These results demonstrated that SOX9 is required for the G-MDSC-mediated profibrotic functions.
# Discussion
In our study, we revealed the role of G-MDSCs as an important profibrotic factor in the aging heart. We found that G-MDSCs can promote fibroblast proliferation and increase the levels of fibrosis markers. S100A8 and S100A9 released by G-MDSCs mediated the inflammatory phenotypes and increased the OPN level in fibroblasts. In addition, we demonstrated that G-MDSCs can suppress fibroblast senescence and apoptosis via FGF2-SOX9 signaling. Since aging-related fibrosis is an important pathological factor of HFpEF, G-MDSCs could be a new antifibrogenic therapeutic target for HFpEF.
MDSCs develop from common myeloid progenitor cells during the myelopoietic process and possess a powerful array of immune-suppressive mechanisms [bib_ref] Immunosenescence: the potential role of myeloid-derived suppressor cells (MDSC) in age-related immune..., Salminen [/bib_ref]. These cells are recruited to inflammatory tissue to suppress acute inflammation by inhibiting the functions of innate and adaptive immunity. Accumulating evidence has indicated that the numbers of MDSCs increase in the spleen, bone marrow, peripheral lymph nodes, and peripheral blood with aging [bib_ref] A role for immature myeloid cells in immune senescence, Enioutina [/bib_ref] [bib_ref] Age-related increase of tumor susceptibility is associated with myeloid-derived suppressor cell mediated..., Grizzle [/bib_ref] [bib_ref] Myeloid-derived suppressor cells promote age-related increase of lung cancer growth via B7-H1, Chen [/bib_ref] [bib_ref] Conditions that diminish myeloid-derived suppressor cell activities stimulate cross-protective immunity, Heithoff [/bib_ref] [bib_ref] Expansion of myeloid-derived suppressor cells with aging in the bone marrow of..., Flores [/bib_ref]. In our study, we confirmed that G-MDSCs, rather than M-MDSCs, accumulated in aging hearts and verified their immunosuppressive function through their suppressive effect on T-cell proliferation. The accumulation of G-MDSCs in aging hearts may be associated with aging-associated chronic low-grade inflammation, which is always accompanied by aging progression [bib_ref] Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from..., Franceschi [/bib_ref]. Low levels of persistent inflammation have been verified to be related to pathophysiological cardiovascular diseases (CVDs), such as hypertension and arteriosclerosis [bib_ref] Inflammageing: chronic inflammation in ageing, cardiovascular disease, and frailty, Ferrucci [/bib_ref] [bib_ref] Inflammation and cardiovascular disease mechanisms, Libby [/bib_ref]. In detail, the secretome of the senescence-associated secretory phenotype (SASP) and the high levels of proinflammatory cytokines in circulation, the most typical inducers of MDSCs, can activate the proliferation and recruitment of MDSCs in inflammatory tissue [bib_ref] CXCR2-expressing myeloid-derived suppressor cells are essential to promote colitis-associated tumorigenesis, Katoh [/bib_ref] [bib_ref] Myeloid-derived suppressor cells (MDSC): an important partner in cellular/tissue senescence, Salminen [/bib_ref]. As G-MDSCs accumulate in aging hearts, their role in senile CVDs remains unclear. By transferring G-MDSCs, we found that these cells can substantially decrease cardiac diastolic function by inducing cardiac fibrosis, which is similar to the pathological changes in aging hearts. As relevant studies have shown that MDSCs are closely related to pulmonary fibrosis 26 , we next explored the relationship between G-MDSCs and fibroblast phenotypes. As expected, we demonstrated that G-MDSCs can activate the proliferation of fibroblasts. In addition, G-MDSCs increased the levels of the fibrosis markers Col3a1, Postn, and Acta2 in fibroblasts, indicating that G-MDSCs play a crucial role in cardiac fibrosis. S100A8 and S100A9, also known as migration inhibitory factor-related proteins 8 (MRP8) and 14 (MRP14), are biomarkers of MDSCs 10 . Extracellular S100A8/A9 promote the production of inflammatory cytokines by binding to two pattern recognition receptors: Toll-like receptor 4 (TLR4) and receptor of advanced glycation endproducts (RAGE) [bib_ref] Development and evaluation of a non-peptidic ligand for the molecular imaging of..., Faust [/bib_ref] [bib_ref] Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal,..., Vogl [/bib_ref] [bib_ref] S100A8 and S100A9 mediate endotoxin-induced cardiomyocyte dysfunction via the receptor for advanced..., Boyd [/bib_ref]. Notably, S100A8/A9 can induce the accumulation and migration of MDSCs through multiple mechanisms [bib_ref] Myeloid-derived suppressor cells as regulators of the immune system, Gabrilovich [/bib_ref] [bib_ref] Proinflammatory S100 proteins regulate the accumulation of myeloid-derived suppressor cells, Sinha [/bib_ref]. Here, our data revealed that release of S100A8/A9 by G-MDSCs (see figure on previous page) Aging-related G-MDSCs suppress fibroblast senescence and apoptosis. A, B Young fibroblasts were cultured with aging-related G-MDSCs for 24 h to determine the changes induced by aging-related G-MDSCs. A Immunofluorescence results revealed the level of OPN in fibroblasts. Scale bars, 50 μm. B The levels of β-galactosidase in fibroblasts analyzed by flow cytometry through C12FDG staining. Representative cytograms are shown on the top, and statistical data are shown on the bottom; n = 7 per group. C-F The senescent fibroblast model was induced by UV treatment. Then, we investigated the influence of G-MDSCs on fibroblast senescence and apoptosis. C Representative images of senescent fibroblasts detected by Xgal staining are shown on the top, and statistical data are shown on the bottom. Red arrows represent X-Gal + fibroblasts; n = 8 per group. Scale bars, 50 μm. D Cell cycle distribution and the percentage of fibroblasts in S phase were detected by flow cytometry. Representative cytograms are shown on the top, and statistical data are shown on the bottom; n = 8 per group. E The mRNA levels of the senescence markers Cdkn2a and Cdkn2b in the fibroblasts with or without UV treatment analyzed by qPCR; n = 8 per group. F The percentage of apoptotic fibroblasts was detected by flow cytometry using Annexin V-FITC and PI staining. Representative cytograms are shown on the top, and statistical data are shown on the bottom; n = 8 per group. The data are presented as the means ± SDs. Differences were determined by an unpaired t test, one-way ANOVA (more than 2 groups) or two-way ANOVA (more than 2 factors), and Tukey's HSD post hoc test (for one-way ANOVA) or a Sidak HSD post hoc test (for two-way ANOVA) was performed. *P < 0.05.
promoted the secretion of proinflammatory cytokines and elevated OPN levels in fibroblasts. Accumulation of MDSCs in aging tissue is believed to prevent excessive inflammation caused by aging [bib_ref] Immunosenescence: the potential role of myeloid-derived suppressor cells (MDSC) in age-related immune..., Salminen [/bib_ref]. In contrast, recent studies have shown that MDSCs can aggravate chronic inflammation by inhibiting both innate and adaptive immunity in chronic inflammatory tissue or aging tissue [bib_ref] Myeloid-derived suppressor cells: the dark knight or the joker in viral infections?, Goh [/bib_ref] [bib_ref] Myeloid-derived suppressor cells in bacterial infections, Ost [/bib_ref] [bib_ref] Obesity-driven inflammation and cancer risk: role of myeloid derived suppressor cells and..., Okwan-Duodu [/bib_ref] [bib_ref] Cardiac fibrosis in the ageing heart: contributors and mechanisms, Lu [/bib_ref]. We observed that G-MDSCs increased the levels of proinflammatory cytokines, such as IL-6, IL-10, and TNF-α in fibroblasts. The aging process was shown to be associated with a balance between proinflammatory and anti-inflammatory responses, and MDSCs play an important role in this process [bib_ref] Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from..., Franceschi [/bib_ref] [bib_ref] The role of myeloid-derived suppressor cells (MDSC) in the inflammaging process, Salminen [/bib_ref] [bib_ref] The pleiotropic association between IL-10 levels and CVD prognosis: evidence from a..., Ni [/bib_ref]. A previous study indicated that activated MDSCs suppress the functions of invading inflammatory cells, limiting the excessive inflammatory response but promoting the presence of low-grade chronic inflammation [bib_ref] Myeloid-derived suppressor cells (MDSC): an important partner in cellular/tissue senescence, Salminen [/bib_ref]. Thus, our results suggested that G-MDSCs promote the inflammatory phenotypes of fibroblasts, indicating that the role of G-MDSCs in the immune microenvironment of aging tissue deserves further exploration. OPN is a proinflammatory cytokine and a crucial matricellular protein of the extracellular matrix (ECM). As documented by several studies, OPN is essential for fibroblast activation [bib_ref] Novel role for osteopontin in cardiac fibrosis, Zahradka [/bib_ref] [bib_ref] Targeting osteopontin, the silent partner of Na + /H + exchanger isoform..., Mohamed [/bib_ref] [bib_ref] Osteopontin: role in extracellular matrix deposition and myocardial remodeling post-MI, Singh [/bib_ref]. Daigo Sawaki et al. confirmed that the accumulation of OPN, derived from visceral adipose tissue, in aging hearts can promote cardiac fibrosis 13 , identifying the origin of OPN. In addition, increased cardiac stiffness induced by a high level of OPN represents an increased risk of cardiac diastolic dysfunction, which is an important pathological characteristic of HFpEF [bib_ref] Cellular and molecular differences between HFpEF and HFrEF: a step ahead in..., Simmonds [/bib_ref] [bib_ref] Syndecan-4 is a key determinant of collagen cross-linking and passive myocardial stiffness..., Herum [/bib_ref]. In summary, our data suggested that extracellular S100A8 and S100A9 are involved in G-MDSC-mediated profibrotic progression.
Another interesting finding is that G-MDSCs trigger fibroblast cell cycle self-renewal and antiapoptotic effects in aging hearts. With increasing age, fibroblasts also exhibit aging phenotypes, such as galactosidase enhancement and cell cycle arrest. Our results confirmed that G-MDSCs can suppress fibroblast senescence and apoptosis. According to current studies, fibroblast senescence is beneficial in attenuating cardiac fibrosis 13 , suggesting that the induction of fibroblast senescence may be a strategy to inhibit tissue fibrosis. Recent studies have revealed that TGF-β expression was downregulated in fibrotic tissue of aging mice, which is consistent with our results. Therefore, further elucidation of the mechanism of the G-MDSC-mediated profibrotic function is needed. Our data identified a novel FGF2-SOX9 signaling axis in fibroblasts [fig_ref] Figure 7: SOX9 knockdown alleviates the fibrotic phenotypes induced by G-MDSCs in vivo [/fig_ref]. FGF2 is an endogenous heparin-binding multifunctional growth factor expressed and secreted predominantly by cardiac nonmyocytes (fibroblasts) in the heart and has long been known to stimulate the proliferation of fibroblasts 43 . SOX9 plays an essential role during mammalian development, in which it crucially regulates chondrogenesis and sex differentiation [bib_ref] SOX E genes: SOX9 and SOX8 in mammalian testis development, Barrionuevo [/bib_ref] [bib_ref] Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription..., Lefebvre [/bib_ref]. SOX9 was shown to be an important transcription factor in cardiac fibrosis and is mainly active in fibroblasts 46 . Through regulation by G-MDSCs, FGF2 secretion is increased in fibroblasts, where it upregulates the SOX9 gene, resulting in cell cycle self-renewal and antiapoptotic effects in fibroblasts. However, the mechanism by which G-MDSCs act on fibroblasts to release FGF2 remains unclear and deserves further exploration. In summary, our data revealed that G-MDSCs can suppress fibroblast senescence and apoptosis by regulating fibroblast FGF2-SOX9 signaling.
Notably, Zhou, L. et al. have reported that MDSCs (CD11b + Gr1 + ) show a cardioprotective effect in heart failure models through their antihypertrophic and antiinflammatory effects [bib_ref] Cardioprotective role of myeloid-derived suppressor cells in heart failure, Zhou [/bib_ref]. The differences between the two studies may be explained by the following points. First, Zhou, L. et al. performed the experiments on mice with acute or chronic pressure overload-induced heart failure, which was characterized by excessive activation of the inflammation process [bib_ref] Inflammation and fibrosis in murine models of heart failure, Bacmeister [/bib_ref]. Due to their immunosuppression, MDSCs may alleviate cardiac injury by reducing the levels of pro-inflammatory cytokines. Second, Zhou, L. et al. found that MDSCs showed an antihypertrophic effect on cardiomyocytes. However, our study found that G-MDSC aggravated stiffness of cardiac fibroblasts, which (see figure on previous page) FGF2 secreted by fibroblasts is involved in G-MDSC-mediated suppression of senescence and apoptosis. A KEGG pathway enrichment showing signaling pathways related to G-MDSCs. B Heatmap showing the expression levels of genes involved in signaling by the FGFR pathway in the MDSC-treated mice and the control mice. Red indicates a positive correlation, and blue indicates a negative correlation. C The mRNA levels of FGF2 in the fibroblasts and G-MDSCs cultured alone or cocultured with each other for 24 h analyzed by qPCR; n = 6 per group. D LVEF, LVDs, LV dp/ dTmax, and LV dp/dTmin of the mice administered G-MDSCs with or without BGJ398 (5 µm); n = 5 per group. E-H Senescent fibroblasts were cocultured with G-MDSCs for 24 h, and BGJ398 (5 µm) was used to neutralize FGF2. E The levels of β-galactosidase in fibroblasts analyzed by flow cytometry through C12FDG staining. Representative cytograms are shown on the left, and statistical data are shown on the right; n = 7 per group. F The mRNA levels of the senescence markers Cdkn2a and Cdkn2b in fibroblasts; n = 8 per group. G Representative images and statistical data of senescent fibroblasts using X-gal staining. Red arrows represent X-Gal + fibroblasts; n = 7 per group. Scale bars, 50 μm. H Representative flow cytometric profile and statistical data of fibroblast apoptosis through Annexin V-FITC and PI staining; n = 7 per group. The data are presented as the means ± SDs. The data are presented as the means ± SDs. Differences were determined by one-way ANOVA (more than 2 groups), and Tukey's HSD post hoc test was performed. *P < 0.05. The profibrotic factor SOX9, induced by FGF2 elevation, is required for G-MDSC-mediated suppression of senescence and apoptosis. A GSEA showing the SOX9-related gene set enriched in the fibroblasts treated with FGF2. B Luciferase activity driven by the SOX9 motif in the fibroblasts treated with FGF2. An asterisk indicates a significant difference vs. the pShuttle promoter; n = 5 per group. C-G The fibroblasts with SOX9 knockdown in vitro were cultured with medium supplemented with FGF2 (4 ng/mL) for 24 h. C Western blotting analysis showing the expression level of SOX9 in senescent fibroblasts. D Representative X-gal staining images (top) and statistical data (bottom) on fibroblast senescence; n = 6 per group. Scale bars, 50 μm. E The mRNA levels of the senescence markers Cdkn2a and Cdkn2b in senescent fibroblasts; n = 8 per group. F Cell cycle distribution and the percentage of fibroblasts in S phase were detected by flow cytometry through PI staining; n = 6 per group. G Representative flow cytometric images and statistical data of apoptosis rates of senescent fibroblasts determined by Annexin V-FITC and PI staining; n = 8 per group. The data are presented as the means ± SDs. Differences were determined by one-way ANOVA (more than 2 groups), and Tukey's HSD post hoc test was performed. *P < 0.05. C The mRNA levels of fibrosis markers (Col3a1, Postn, and Acta2) in mouse hearts analyzed by qPCR; n = 5-6 per group. D The mRNA levels of the senescence markers Cdkn2a and Cdkn2b in mouse hearts analyzed by qPCR; n = 5-6 per group. E The levels of β-galactosidase in fibroblasts analyzed by flow cytometry through C12FDG staining. Representative cytograms are shown on the left, and statistical data are shown on the right; n = 5-6 per group. F Diagram of the mechanism by which G-MDSCs act on fibroblasts. The data are presented as the means ± SDs. Differences were determined by an unpaired t-test or two-way ANOVA (more than 2 factors), and a Sidak HSD post hoc test was performed. *P < 0.05. is a salient feature of age-related cardiac dysfunction [bib_ref] Cardiac fibrosis in the ageing heart: contributors and mechanisms, Lu [/bib_ref]. Third, Zhou, L. et al. suggested that MDSC exerted cardioprotective effects through nitric oxide, which is synthesized more in M-MDSCs than in G-MDSCs [bib_ref] Myeloid-derived suppressor cells coming of age, Veglia [/bib_ref]. Taken together, these discrepancies may partly explain the differences between the two studies.
Although our data suggested that the transferred G-MDSCs in heart cannot survive for more than 48 h, we still cannot completely rule out the effect of excessive cell accumulation. Nevertheless, our results provide new insight into the pathology of HFpEF and identify the role of G-MDSCs in inducing aging-related cardiac fibrosis through release of S100A8/A9 and regulation of FGF2-SOX9 signaling.
# Methods
## Cell isolation and culture
Murine MDSCs were isolated from the spleens of aging C57BL/6 mice (20 months). The spleens were filtered by a cell strainer with 40 μm nylon. Then, the red blood cells were eliminated by red blood cell lysis. G-MDSCs were defined as CD11b + Ly6Clow cells and obtained using fluorescence-activated cell sorting; this method provides nearly 90% CD11b + Gr1 + Ly6G+ cell purity assessed by flow cytometry. The immunosuppression of G-MDSCs was detected by a T-cell proliferation assay.
Mouse fibroblasts were isolated from 5-week-old C57BL/6 mice using enzymatic digestion according to our previous protocol [bib_ref] Arctigenin alleviates myocardial infarction injury through inhibition of the NFAT5-related inflammatory phenotype..., Ni [/bib_ref]. Mouse heart pieces were enzymatically digested with stirring for 10 min, and the supernatant was collected. The different cell types were isolated by Percoll gradient separation (top Percoll layer: 55%; bottom Percoll layer: 65%). We collected the intermediate layer of cardiomyocytes and the upper layer of cardiac fibroblasts.
The cells were cultured in medium containing 10% fetal bovine serum and 1% streptomycin/penicillin and incubated at 37°C with 5% CO 2 .
## Animal experiments and adoptive transfer
Young (6 weeks) and aging (20 months) male C57BL/6 mice [obtained from the Experimental Animal Center of Guangzhou University of Chinese Medicine, China, certification no. SCXK (Yue) 2016-0168] weighing 20 ± 1 g were housed in an individual ventilated cage system. The mice were maintained according to the Guidelines for the Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of China, and all experimental procedures were approved by the Ethics Committee of Guangzhou University of Chinese Medicine. All animals were housed at an ambient temperature of 21°C under a 12/12 h light-dark schedule and maintained on food formulated according to the American Institute of Nutrition for Rodent Diets, with ad libitum access to water. Randomization was used to assign samples to the experimental groups for all in vivo studies.
For adoptive transfer, G-MDSCs (CD11b + Ly6Clow) were isolated from the spleens of aging C57BL/6 mice (20 months), this method provides nearly 90% CD11b + Gr1 + Ly6G+ cell purity assessed by flow cytometry. G-MDSCs (1 × 10 7 ) were injected through the tail vein of recipient mice (more than 5 young mice per group; randomly assigned, not a blinded method) every 5 days during the experiments. At the end of the experiments, the recipient mice were euthanized.
# Rna-seq and analysis
Total mRNA was extracted from the mouse hearts using an RNA extraction kit (Qiagen K.K., Tokyo, Japan). RNA-Seq was performed using an Ion Proton system for next-generation sequencing, according to the manufacturer's instructions. Sequenced reads were mapped to the mm9 genome using the Ion Torrent TMAP aligner with the 'map4' option. The RNA-Seq reads that were aligned against the exon regions of genes were quantified with HTSeq-Count in the RefSeq mm9 annotation. Gene set enrichment analysis (GSEA) was performed on mRNA expression datasets using our gene set data. Gene signatures were considered enriched if the false discovery rate (FDR) q-values and the familywise error rate (FWER) p-values showed significant differences.
# Flow cytometric analysis
## Beta-galactosidase activity assay
Cells cocultured in vitro were fixed with 0.5% paraformaldehyde and stained with 1 mg/mL 5-bromo-4chloro-3-indoyl β-D-galactopyranoside (X-gal) for 24 h. The stained cells (blue) were imaged and quantified by differential interference contrast brightfield microscopy.
For the beta-galactosidase activity of cardiac fibroblasts in vivo, 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C 12 FDG) was used according to a previous protocol [bib_ref] Arctigenin alleviates myocardial infarction injury through inhibition of the NFAT5-related inflammatory phenotype..., Ni [/bib_ref]. Briefly, cardiac fibroblasts were isolated using enzymatic digestion and Percoll gradient separation. Then, the cells were treatment with 50 μM C 12 FDG for 2 h. Flow cytometric analysis was used to estimate the relative β-galactosidase activity based on the green fluorescence intensity.
## Echocardiography
Echocardiography was performed using a Vevo 2100 Imaging System (VisualSonics, Inc., Toronto, Canada). All parameters were automatically obtained using the LV Trace measurement tool in the Cardiac Package and the Short Axis (SAX) module in Vevo 2100 analysis software. The measurements were performed using a single-blinded method.
## Western blotting
The protein content was quantified by the BCA method, and 100 μg per sample was loaded onto 12% SDS polyacrylamide gels. After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% BSA and probed with primary antibodies (Affinity, 1:1000, SOX9: Cat: AF6330; Lamin-B: Cat: DF7356). After the PVDF membranes were washed, they were incubated with a secondary antibody (Affinity, 1:2000, Cat. No. S0001) for 2 h. Finally, the blots on the membranes were scanned with a charge-coupled device (CCD) system (ImageStation 2000 MM, Kodak, USA).
## Real-time fluorescence quantitative pcr
The TRIzol method was used to extract total RNA from cells (Life Technologies, USA). A cDNA Synthesis Kit (TaKaRa, Japan) was used to synthesize cDNA for realtime fluorescence quantitative PCR (qPCR) assays. PCR amplification was carried out on a thermal cycler in a reaction volume of 20 µL containing SYBR Green (TaKaRa, Japan) using a MyiQ real-time PCR detection system (Bio-Rad). The data were analyzed using the 2 -ΔΔCt method, and the gene primers are listed in .
## Wga staining and immunofluorescence
Hearts were harvested and fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into sections. The sections were dewaxed and incubated in water at 80°C for 20 min. After cooling, the sections were washed, stained with wheat germ agglutinin (WGA), and subsequently incubated at 37°C for 10 min. After the section were washed, they were sealed with glycerol and finally observed and imaged.
For immunofluorescence, the heart tissue was fixed, embedded, and sliced as described above. After the tissue sections were washed, they were incubated with anticollagen I (1:200, Affinity, Cat: AF0134), anti-α-SMA (1:200, Affinity, Cat: BF9212), and anti-osteopontin (OPN) (1:200, Affinity, Cat: BF0002) primary antibodies and a fluorescently labeled secondary antibody before being observed and imaged.
## Luciferase reporter assay
We assayed the transcriptional activity of SOX9 by a luciferase reporter assay. The pGL3-SOX9-Luc reporter separately contained 12 repeats of a 48-bp Col2a1 intron 1 enhancer element, which is known to be activated by SOX9, and the pRL-TK reporter was used as a control reporter. HEK-293T cells were cultured in 24-well plates and then transfected with 600 ng of pGL3-SOX9-Luc and 30 ng of pRL-TK. After 48 h, the cells were lysed, and the luciferase activity was measured with a Cytation 5 Cell Imaging Multi-Mode Reader.
## Lentiviral transfection
Lentiviruses were synthesized and purchased from GenePharma (Shanghai, China). The viral stocks were titrated by transduction of cells to 1 × 10 7 transduction units (TU)/mL. For cell transfection, cells were transfected with siRNA lentivirus or a control lentivirus in 5 μg/mL polybrene. After transfection, the cells were used for intravenous injection or in vitro experiments.
# Statistical analysis
Bioinformatics data were analyzed using the R program (3.6.2, Austria) with the Bioconductor packages described above. GraphPad Prism 7.0 (CA, USA) was used for statistical analysis. The data are presented as the mean ± SD (for in vivo experiments) and were analyzed by t-tests, one-way ANOVA (for more than 2 groups) or two-way ANOVA (for more than 2 factors). A post hoc Tukey's HSD test (for one-way ANOVA) or a Sidak HSD post hoc test (for two-way ANOVA) was performed when the ANOVA results showed significant interactions between variables. Repeated measures ANOVA was performed for time series data, and correlations were evaluated by Pearson's test. A P-value < 0.05 was considered to indicate statistical significance.
## Data availability
Data from GEO database (GSE145477 and GSE145477), and Array express (E-MTAB7869) were used for analysis in this study.
# Ethics statement
All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Guangzhou University of Chinese Medicine (20190329008).
[fig] Figure 7: SOX9 knockdown alleviates the fibrotic phenotypes induced by G-MDSCs in vivo. Induction of SOX9 knockdown in MDSC-treated mice through injection of AAV harboring the SOX9-KD plasmid. A Western blotting analysis showing the expression levels of SOX9 in the WT and SOX9_KD hearts. B Representative immunofluorescence images of the level of α-SMA in the control, G-MDSC, and SOX9_KD hearts. Scale bars, 50 μm. [/fig]
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Machine Learning of Protein Interactions in Fungal Secretory Pathways
In this paper we apply machine learning methods for predicting protein interactions in fungal secretion pathways. We assume an inter-species transfer setting, where training data is obtained from a single species and the objective is to predict protein interactions in other, related species. In our methodology, we combine several state of the art machine learning approaches, namely, multiple kernel learning (MKL), pairwise kernels and kernelized structured output prediction in the supervised graph inference framework. For MKL, we apply recently proposed centered kernel alignment and p-norm path following approaches to integrate several feature sets describing the proteins, demonstrating improved performance. For graph inference, we apply input-output kernel regression (IOKR) in supervised and semi-supervised modes as well as output kernel trees (OK3). In our experiments simulating increasing genetic distance, Input-Output Kernel Regression proved to be the most robust prediction approach. We also show that the MKL approaches improve the predictions compared to uniform combination of the kernels. We evaluate the methods on the task of predicting protein-protein-interactions in the secretion pathways in fungi, S.cerevisiae, baker's yeast, being the source, T. reesei being the target of the inter-species transfer learning. We identify completely novel candidate secretion proteins conserved in filamentous fungi. These proteins could contribute to their unique secretion capabilities.
# Introduction
Protein secretion is a fundamental cellular process that is required for transporting proteins into cellular compartments, the cell surface and the external space of the cell as well as for covalent modification i.e. disulphide bond formation and glycosylation of proteins. As can be expected from its central role, the protein secretion machinery is conserved in eukaryotes. Fundamental research to unravel its functioning has been carried out in the fungus Saccharomyces cerevisiae [bib_ref] Charting the secretory pathway in a simple eukaryote, Schekman [/bib_ref]. However, the baker's yeast S. cerevisiae of the subphylum Saccharomycotina does not naturally secrete large amounts of proteins unlike the filamentous fungi of the subphylum Pezizomycotina. For example the Pezizomycotina Trichoderma reesei (Hypocrea jecorina) is able to secrete its native cellulase proteins with yields of over 100 g/l in industrial problem, to predict, whether a pair of proteins interact or not. Thus, any general model for classification learning is applicable in this setting, including ensemble learners [bib_ref] Libd3c: ensemble classifiers with a clustering and dynamic selection strategy, Lin [/bib_ref] [bib_ref] Binmempredict: a web server and software for predicting membrane protein types, Zou [/bib_ref] [bib_ref] An empirical study of features fusion techniques for protein-protein interaction prediction, Zeng [/bib_ref] , Naive Bayes, and support vector machines (SVM). SVM models rely on so called pairwise kernels, where the similarities of protein pairs are compared to each other. Another class of PPI learning methods aim to predict interaction patterns by learning similarities between proteins in the protein interaction network. Output kernel trees [bib_ref] Inferring biological networks with output kernel trees, Geurts [/bib_ref] and input-output kernel regression [bib_ref] Semi-supervised penalized output kernel regression for link prediction, Brouard [/bib_ref] are recent examples of this kind of methods.
The above approaches have not been explicitly applied to cross-species transfer learning, perhaps due to the limited amount of verified PPIs in a majority of species. Beyond basic sequence comparisons, more advanced computational methods have been applied in the crossspecies setting only sparingly. In [bib_ref] Cross-species cluster co-conservation: a new method for generating protein interaction networks, Karimpour-Fard [/bib_ref] , a cross-species cluster co-conservation method is proposed, that exploits phylogenetic profiles for predicting protein interaction networks. In [bib_ref] Simultaneous inference of biological networks of multiple species from genome-wide data and..., Kashima [/bib_ref] , a link propagation approach was proposed relying on gene expression and sequence similarity, applied to cross-species metabolic network reconstruction.
There is a dire need for novel function and interaction prediction methods that would be locally available, able to cross large sequence similarity distances and not require the solving of orthology-paralogy relationships to cope with the rising amount of genomes. In this paper, we introduce a framework of machine learning methods that can be used for predicting physical or functional protein-protein interaction or more specific biological networks i.e. metabolic pathways depending on what type of training labels are used. Our method uses as features various sequence similarity and protein family analysis derived from the CoReCo pipeline [bib_ref] Comparative genome-scale reconstruction of gapless metabolic networks for present and ancestral species, Pitkänen [/bib_ref]. Although our method relies partly on sequence similarity, it is, through a combination of methods, still able to predict for proteins that do not belong into any known protein family. Hence our method can give clues for PPIs of previously unknown proteins. Our method introduces recently proposed multiple kernel learning (MKL) methods [bib_ref] Algorithms for learning kernels based on centered alignment, Cortes [/bib_ref] to supervised network inference, thus boosting the performance of the latter method family and making full use of the wide array of sequence-derived features.
We focus in predicting the secretion machinery in industrially relevant fungi, in particular, T.reesei. Our focus is in predicting functional protein-protein interactions (PPI) in the secretory pathway. As there are no verified protein interaction data available for these organisms, we assume the cross-species transfer learning setting, where the training data comes from S. cerevisiae, and prediction targets is T.reesei.
# Materials and methods
## Data and preprocessing
Sequence data. In this paper the models are based on features that can be computational derived from protein sequence data. The sequence data for the two studied organisms were downloaded from SGD database (http://www.yeastgenome.org) for S. cerevisiae and from JGI Mycocosm database (http://genome.jgi.doe.gov/Trire2/Trire2.home.html) for T. reesei.
Protein-protein interaction data. The machine learning methods require a set of known PPIs to be used as ground truth for the model output, used for training and testing the model. We obtained our PPI data from the recently published genome-scale model of the yeast secretory machinery [bib_ref] Genome-scale modeling of the protein secretory machinery in yeast, Feizi [/bib_ref] that gathers knowledge of 50 years of research on secretion in S.cerevisiae. The authors identified 162 proteins to be involved in secretion that are assigned to 16 subsystems such as translocation, ER glycosylation, COP, Golgi processing etc. These protein complexes give 2200 undirected interactions between the 162 secretion proteins which are used as training labels.
Feature extraction. For our models we use several types of features to characterize the similarity of proteins as well as the similarity of protein pairs. For all protein sequences of the 2 organisms we computed the following features using the CoReCo pipeline [bib_ref] Comparative genome-scale reconstruction of gapless metabolic networks for present and ancestral species, Pitkänen [/bib_ref] : sequence alignment with BLAST against the UniProt database as well as Global Trace Graph (GTG) [bib_ref] The global trace graph, a novel paradigm for searching protein sequence databases, Heger [/bib_ref] , protein domains and functional sites gathered by InterProScan [bib_ref] Interproscan 5: genome-scale protein function classification, Jones [/bib_ref] from its member databases: Pfam [bib_ref] The pfam protein families database, Punta [/bib_ref] , Panther [bib_ref] Panther version 10: expanded protein families and functions, and analysis tools, Mi [/bib_ref] , Gene3D [bib_ref] Gene3d: comprehensive structural and functional annotation of genomes, Yeats [/bib_ref] , PRINTS, Prosite, PIRSF [bib_ref] Pirsf family classification system for protein functional and evolutionary analysis, Nikolskaya [/bib_ref] , SMART [bib_ref] Smart: recent updates, new developments and status in 2015, Letunic [/bib_ref] , and SUPERFAMILY [bib_ref] Superfamily 1.75 including a domain-centric gene ontology method, De Lima Morais [/bib_ref] (See S1 Table for details on these data sources).
Artificial sequences. We used artificial data to test if the different biological network inference algorithm that have been developed for intra-species prediction also work for interspecies prediction with low sequence similarity. They are well below commonly used amino acid sequence identity cut-off values. For obtaining artificial sequences with varying levels of sequence similarity we altered the sequences of the 162 secretion proteins of S. cerevisiae based on Blosum matrices [bib_ref] Amino acid substitution matrices from protein blocks, Henikoff [/bib_ref]. These matrices represent the substitution probabilities from an amino acid to an other amino acid in natural sequence data sets. Hence, they allow approximation of natural sequence evolution. We created four different data sets where we deleted and mutated 70%, 60%, 38% and 20% of the amino acids according to the Blosum30, Blosum40, Blosum62 and Blosum80 respectively. The Blosum matrices were downloaded from NCBI Blast site (ftp://ftp.ncbi.nih.gov/blast/matrices/). Each different Blosum matrix has been made by combining proteins that are no more similar than a given percentage (30%, 40%, 62% and 80%) to one single sequence and then comparing only those sequences [bib_ref] Amino acid substitution matrices from protein blocks, Henikoff [/bib_ref]. In [fig_ref] Fig 1: Frequency distribution of percentage of amino acid sequence identity between natural S [/fig_ref] the percentage of amino acid sequence identity between the artificially mutated protein sequences of S. cerevisiae and T. reesei based on the Smith-Waterman alignment is shown. Based on visual comparison, the generated Blosum30 data set has a similar level of sequence similarity to S. cerevisiae as T. reesei. In the experiments, the artificially perturbed sequences were coupled with the labels of the corresponding labels of the original sequences.
Transcriptomic data analysis for biological network validation. The transcriptomic data for the validation of T. reesei PPI network was composed by eight publicly available data sets taken from Gene expression omnibus [bib_ref] Gene expression omnibus: Ncbi gene expression and hybridization array data repository, Edgar [/bib_ref] plus eight in-house data sets. The public data sets contained 76 samples all together and the in-house data sets 499 samples. Once combined the final data set contained 575 samples and 9078 genes. Each data set was normalized separately using quantile normalizationand normalized again after they were combined using COM-BAT normalization [bib_ref] Adjusting batch effects in microarray expression data using empirical bayes methods, Johnson [/bib_ref].
## Problem formalization
Supervised graph inference has been introduced a decade ago in [bib_ref] Supervised graph inference, Vert [/bib_ref] and has been widely used for biological network reconstruction subsequently. Given a set of nodes V = v 1 , .. ,v m a biological network can be defined as an undirected graph G = (V, E) where E & V × V are the edges between the m vertices. The graph can be represented by a symmetric adjacency matrix Y = (y ij ) of size m × m where y ij = y ji = 1 if the nodes v i and v j are connected and y ij = y ji = 0 otherwise. We will also use the shorthand yðv i Þ ¼ ðy ij Þ m j¼1 to denote the connectivity pattern of protein v i in the network. In addition, we assume that each node has assigned features x(v i ) 2 χ, for some input space χ.
The learning task is then defined as follows: given partial knowledge of the graph G = (V, E) and the feature representation of the nodes, determine a function f: V × V ! {0, 1} that best approximates the unknown edges of the graph.
Note that the main difficulty for solving this problem is that the features are assigned to individual nodes and the labels to pairs of nodes [bib_ref] On protocols and measures for the validation of supervised methods for the..., Schrynemackers [/bib_ref]. To transform the task into a standard classification problem, we use a global approach that tries to find a feature representation for pairs of nodes. Another issue inherent to biological network inference is the substantial class imbalance since the number of positive interactions is small compared to the number of all possible interactions. Thus special care is needed for setting up the evaluation experiments, see e.g. [bib_ref] ndna-prot: identification of dna-binding proteins based on unbalanced classification, Song [/bib_ref]. First of all, the evaluation metrics should be chosen such that the class imbalance does not lead to incorrect conclusions (e.g AUPR metric explained below). Secondly, methods that predict for each protein an interaction profile (see OK3 and IOKR below), represented as a multilabel, a binary vector containing interaction labels for all other proteins, are able to mitigate the class imbalance, since in general the set of multilabels are diverse with no very frequent multilabel. In [bib_ref] On protocols and measures for the validation of supervised methods for the..., Schrynemackers [/bib_ref] it is recommended to perform cross validation on the nodes as cross validation on pairs tends to give too optimistic results. A schematic representation of the duality between the biological network and the adjacency matrix and the cross validation on nodes is given in [fig_ref] Fig 2: Schematic representation of the duality between [/fig_ref] Finally, for performing inter-species biological network inference we use the protein sequences and their interactions from one species as training set and the protein sequences from the second species as testing set. Note that in this setting the training-testing interactions are not of interest and that the feature representation needs to be the same for training and testing proteins.
## Inference algorithms
In this section we present three different approaches for supervised network inference that we have applied to inter-species PPI network prediction. Additionally, we present different approaches for learning kernels that account for the relevance of a data source for the learning task.
Output kernel trees (OK3). have been proposed by [bib_ref] Inferring biological networks with output kernel trees, Geurts [/bib_ref] and are based on the kernel embedding of the graph where the kernel function is defined as
[formula] k Y : V × V ! < with k Y (v, v 0 ) = hψ(v), ψ(v 0 )i. The kernel k Y (v, v 0 ) [/formula]
is defined such that adjacent vertices have higher values of k Y than non-adjacent ones. To achieve this, the diffusion kernel is commonly used K Y = exp(−βL) where L is the Laplacian matrix of the graph L = D − Y with D being the degree matrix and Y the adjacency matrix. Additionally, β > 0 is a user defined parameter that controls the diffusion degree.
The OK3 algorithm relies on the top-down induction algorithm widely used to learning decision trees (e.g. CART. The methods start with a tree represented by a single leaf and then recursively partition (or split) the input data S until the data is homogeneous enough (in our case: the proteins in S have similar connectivity patterns). The data arriving to leaf L of the decision tree is split into two parts S l and S r , using a binary test T t (x) 2 {0, 1} based on a value of a single input feature of x (e.g. does protein have a given motif or not). The two sets S l = {x 2 S|T(x) = 0} and S r = {x 2 S|T(x) = 1} will be recursively used to grow subtrees which then will be attached as the children of L.
For learning the decision trees on the input vectors
[formula] x i = x(v i ), i = 1. [/formula]
.m the following score is maximized to select a test T to be inserted in the decision tree leaf given the set of inputs S routed to the current decision tree leaf:
[formula] ScoreðT; SÞ ¼ varfcðvÞ j Sg À N l N varfcðvÞ j S l g À N r N varfcðvÞ j S r gð1Þ [/formula]
where ψ(v) is the output feature vector, N, N l and N r are the sizes of the training sample S and its left and right split, S l and S r , respectively. The variance of the output feature vectors in the set S can be easily computed using the kernel trick:
[formula] varfcðvÞ j Sg ¼ 1 N X N i¼1 k Y ðv i ; v i Þ À 1 N 2 X N i;j¼1 k Y ðv i ; v j Þ [/formula]
One main advantage of the OK3 approach is that the decision tree on the input features results in a ranking of relevant features for the learning task.
Then for prediction each leaf L is labeled with a predictionĉ L ¼ 1 N l P N L i¼1 cðv i Þ analog to standard regression trees where N L are the number of samples that reach the leaf. Finally, the kernel value between two vertices v and v 0 where x(v) reaches leaf L 1 and x(v 0 ) leaf L 2 respectively can be approximated by thresholdinĝ
[formula] k Y ðv; v 0 Þ ¼ 1 N L 1 N L 2 X N L 1 i¼1 X N L 2 j¼1 k Y ðv 1 i ; v 2 j Þ [/formula]
where v k i ; i ¼ 1; . . . ; N L k enumerate the vertices routed to leaf L k . For improving the accuracy of the method an ensemble of decision trees also known as a random forest is used. In our experiments we used the C code provided by the authors [bib_ref] Inferring biological networks with output kernel trees, Geurts [/bib_ref].
Kernels on protein pairs. The main idea of the biological network reconstruction methods presented inis to reformulate the task as a pattern recognition problem: given a training set τ = {(u 1 , t 1 ),(u 2 , t 2 ), .. ,(u N , t N )} of patterns u i 2 < q with a binary label t i 2 {−1, 1} infer a function f: < q ! {−1, 1} for any new pattern u. The main hindrance in doing so is that in network reconstruction the labels are defined on pairs of vertices and the input features or patterns on individual vertices. Thus in a first step a so called linear kernel on pairs of vertices induced by their input features is defined by their inner product k X (v, v 0 ) = x(v) T x(v 0 ). These kernels k X represent the similarity of any pair of protein sequences that are then used to compute kernels on pairs of protein pairs as follows
[formula] 1. Direct product kernel: k DRCT ((a, b), (c, d)) = k X (a, c) Ã k X (b, d) 2. Tensor product pairwise kernel: k TPPK ((a, b), (c, d)) = k X (a, c) Ã k X (b, d) + k X (a, d) Ã k X (b, c) 3. Metric learning pairwise kernel: k MLPK ((a, b), (c, d)) = (k X (a, c) − k X (a, d) − k X (b, c) + k X (b, d)) 2 [/formula]
Now a standard support vector machine (SVM) can be used to solve the binary classification task. Since PPI networks are undirected the tensor product kernel k TPPK and the metric learning pairwise kernel k MLPK are best suited for modelling the similarity between protein pairs.
Despite the method's good predictive performance it has a major drawback: the kernels between pairs of proteins can become quickly very large even for a reasonable amount of protein sequences. The space complexity for storing the kernel matrix turns out to be O(m 4 ) where m is the number of proteins in the biological network which leads to serious scalability problems and usage of computational resources [bib_ref] Simultaneous inference of biological networks of multiple species from genome-wide data and..., Kashima [/bib_ref].
Input-Output Kernel Regression (IOKR). This method combines elements of the two previous algorithms that circumvent their respective disadvantages-on the input side it uses the simple kernels on protein pairs and on the output side it uses the diffusion kernel built from the adjacency matrix of the output graph. But the classification problem is addressed by solving a kernel learning problem using regularized regression [bib_ref] Semi-supervised penalized output kernel regression for link prediction, Brouard [/bib_ref]. The method comes in two flavors: the supervised version learns only the kernel ridge regression model and the semi supervised one adds a smoothness constraint using the inputs of labeled data and auxiliary data, called unlabeled data.
As the OK3 method, IOKR proposes to solve the link prediction problem by learning an output kernel k Y : V Â V ! R, that encodes the similarities between the proteins in the interaction network. After learning this kernel, positive interactions can be predicted for the kernel values that are higher than some threshold θ:
[formula] f y ðv; v 0 Þ ¼ sgnðk Y ðv; v 0 Þ À yÞ [/formula]
As k Y is a kernel, its values can be written as:
[formula] k Y (v, v 0 ) = hψ(v), ψ(v 0 )i, where ψ is called the output feature map. [/formula]
The IOKR method approximates the output feature map ψ with a function h and then build an approximation of the output kernel k Y by taking the inner product between the values of this function:k Y ðv; v 0 Þ ¼ hhðvÞ; hðv 0 Þi :
Thus learning f θ reduces to learn the single variable function h. Then given models of the general form h M (v) = Mϕ(v) and assuming a regularized square loss function the parameters of the supervised IOKR model can be estimated based on l training samples as follows:
[formula] argmin M X l 1 k h M ðv i Þ À cðv i Þk 2 þ l 1 k M k 2 F [/formula]
where λ 1 > 0 is a regularization parameter that is tuned with cross validation for the experiments.
The method has also been extended to the semi-supervised setting where the input of unlabeled data is taken into account. The new cost function that has to be minimized is:
[formula] argmin M X l 1 k h M ðv i Þ À cðv i Þk 2 þ l 1 k M k 2 F þl 2 traceðh M L X n h T M Þ [/formula]
where L X n = exp(−β(D n − K X n )) denotes the diffusion kernel associated to input kernel matrix on labeled and unlabeled data. The last term constrains proteins that are similar to each other in input to be similar in the predicted interaction network. λ 1 > 0 and λ 2 > 0 are two regularization parameters that are tuned with cross validation for the experiments. Both minimisation problems lead to a closed form solution that can be found in Propositions 4 and 6 of [bib_ref] Semi-supervised penalized output kernel regression for link prediction, Brouard [/bib_ref].
Multiple Kernel Learning (MKL). The heterogeneous set of features that we extracted from the protein sequences is expected not to uniformly contribute information to the learned model which makes the uniform combination of the kernels over the different data sources suboptimal. Therefore we apply Multiple Kernel Learning (MKL) to take the feature's relevance into account. We focus on linear mixtures of kernels,
[formula] K μ ¼ X r q¼1 m q K q [/formula]
where the weights μ q are typically restricted to be non-negative to ensure the PSD property of the resulting mixture. Note that setting μ q = 1 for all kernels yields the uniform kernel combination. A major step forward in the MKL field was learning kernels based on centered kerneltarget alignment [bib_ref] Algorithms for learning kernels based on centered alignment, Cortes [/bib_ref]
[formula] r ðK; K Y Þ ¼ hK c ; K Y i F k K c k F k K Y k F [/formula]
where h.i F is the Frobenius product, k.k F the Frobenius norm, K Y is a target kernel and K c denotes a centered version of the input kernel K, achieved by the centering operation
[formula] K c ¼ I À 11 T m K I À 11 T m [/formula]
where 1 denotes the vector of ones and I is the identity matrix. This gives a simple improvement over the uniform combination of kernels be directly using the kernel-target alignment scoresrðK q ; K Y Þ as a mixture weights:
[formula] K μ / X p k¼1r ðK k ; K Y ÞK k [/formula]
This MKL method is called ALIGN. In [bib_ref] Algorithms for learning kernels based on centered alignment, Cortes [/bib_ref] it is claimed that the kernel centering is critical for the kernel alignment score to correlate well with performance.
The previously presented independent kernel alignment neglects the correlation between the base kernels which can be overcome by jointly maximization the alignment between the convex combination kernel with the target kernel and is also referred to as ALIGNF:
[formula] max μ hK μ ; K Y i F k K μ k F [/formula]
With the constraints that kμk 2 = 1 and μ ! 0 the alignment maximization problem can be rewritten as:
[formula] μ Ã ¼ argmax μ μ T aa T μ μ T Mμ [/formula]
where a = (hK 1c , K Y i F , . . ., hK rc , K Y i F ) T records the kernel-target alignments of the input kernels and M = (M ql ) ql with M ql = hK qc ,K lc i F contains the pairwise kernel alignments between the input kernels. The problem can be solved by quadratic programming [bib_ref] Algorithms for learning kernels based on centered alignment, Cortes [/bib_ref]. Another approach for optimizing the kernel target alignment has been proposed in [bib_ref] On p-norm path following in multiple kernel learning for non-linear feature selection, Jawanpuria [/bib_ref]. The method aims at sparse combinations of kernels by regularizing the kernel weights by ℓ pnorm, where 1 ! p is simultaneously optimized. The proposed generalized ℓ p -norm kernel target alignment formulation is as follows:
[formula] min μ!0 l 1 k μ À μ 0 k 2 2 þl 2 X r i¼1 m p i À X r i¼1 m i a [/formula]
The squared Euclidean distance in the first term is an instantiation of Bregman divergence [bib_ref] On p-norm path following in multiple kernel learning for non-linear feature selection, Jawanpuria [/bib_ref] B F ðμÞ ¼ FðμÞ À ðμ À μ 0 Þ T rFðμ 0 Þ for F(μ) = hμ, μi, and μ 0 is a fixed point in the domain of F (Following [bib_ref] On p-norm path following in multiple kernel learning for non-linear feature selection, Jawanpuria [/bib_ref] we used μ 0 = 0 in our experiments.). Additionally, λ 1 0 and λ 2 0 are the regularization parameters. For implementing the sparsity inducing l p regularizer p is systematically reduced towards unity till a sufficient level of sparsity is obtained. The solution of the path following is computed with a Predictor-Corrector algorithm [bib_ref] On p-norm path following in multiple kernel learning for non-linear feature selection, Jawanpuria [/bib_ref].
## Evaluation metrics for binary predictions
Binary classification problems are typically evaluated with the accuracy measure which is computed as the number of correctly predicted pairs divided by the total number of pairs. For highly imbalanced problems like network inference accuracy is not an appropriate measure because it favours the majority class and thus the non-interactions. In the following Receiver-Operator-Characteristic (ROC) and Precision-Recall (PR) curves are presented which are better suited for evaluating network inference predictions [bib_ref] The relationship between precision-recall and roc curves, Davis [/bib_ref]. Both measures are based on a so called confusion matrix which is 2 x 2 for binary classification with the columns and rows representing the predicted and the actual classes respectively. Denoting interactions as positive and non-interactions as negative the confusion matrix is given in.
From this matrix several measures for model evaluation can be derived:
- True positive rate (TPR): also known as sensitivity or recall, is the number of true positives divided the number of the actual positives TP/P All of these measures need to be combined in order to give a reliable performance measure of an algorithm e.g. specificity and sensitivity or precision and recall. Note as well that a threshold needs to be defined if predictions are confidence scores. For evaluating algorithms with varying confidence thresholds ROC and PR curves can be used.
ROC curves. plot the TPR over the FPR for varying confidence thresholds. More specifically, each threshold corresponds to a different confusion matrix and thus a different pair of values for TPR and FPR and a point on the ROC curve. The end points are always (0, 0) and (1, 1) and a perfect classifier would pass through the point (0, 1), while a random classifier would be a diagonal connecting (0, 0) and (1, 1). A common summary statistic of the ROC curve is the area under the ROC curve (AUROC). AUROC is one for a perfect classifier and 0.5 for a random one. For the highly imbalanced network prediction tasks even moderate FPR can lead to more FP predictions than TP predictions and hence a very low precision.
PR curves. plot the precision over the recall for varying confidence thresholds. The curve starts at a pseudo point (0, 1) and ends at (1, P/(P + N)) which corresponds to to predicting all pairs as positive. An optimal classifier would pass as well through (1, 1). The area under the PR curve (AUPR) is also a common summary statistic. As for AUROC one assumes that the higher the AUPR the better the performance of the method. One advantage of PR curves over ROC curves is that they allow to measure early precision where recall is low and thus gives a tool to evaluate the quality of the top ranks of the result list.
# Results
We report here on three sets of experiments. First, we evaluate how the prediction methods perform under simulated sequence data, representing differing amount of genetic distance between the source and target species. Second, we check how well the methods separate the secretory pathway from the rest of the genome. Third, we evaluate the PPI prediction in the cross-species transfer learning from S. cerevisiae to T. reesei.
## Network reconstruction for evolutionary distant sequences
Here we compare the performance of the network inference methods Output Kernel Trees (OK3), Tensor kernel SVM on protein pairs (PP), and supervised and semi-supervised Input-Output Kernel Regression (IOKR) for evolutionary distant species. As training data, we use the S. cerevisiae secretory pathway protein sequences as input and their functional interactions as labels. Then we try to predict these interactions in secretory pathway protein sequences that were perturbed using different BLOSUM matrices that correspond to different genetic distances. [fig_ref] Fig 3: ROC curves for predicting PPIs in different artificial data sets with Output... [/fig_ref] the Receiver operating characteristic curves (ROC) with associated areaunder-curve (AUC) statistics for each inference method for the different evolutionary distances. As expected, all methods predict the better the smaller the distance with BLOSUM80 curves having the highest AUC and being closest to the top-left corner of the plots. The curves are averages of 20-fold cross-validation experiment.
In terms of AUC, OK3 obtains the best results, tensor kernel (PP) the second best and the IOKR methods being somewhat less accurate. However, closer examination of the method's prediction performance for top-ranked interactions (FPR < 0.1) reveals that the IOKR methods in fact have the best early precision, thus would get the top-ranked interactions more accurately predicted than the competing methods.
The AUC statistics and the ROC curves of OK3 follow a smoothly worsening pattern with respect to the increasing evolutionary distance, while the other methods manifest a step change so that BLOSUM30 is markedly worse in AUC and lies clearly below the other curves. [fig_ref] Fig 4: Precision-Recall [/fig_ref] the Precision-Recall (PR) curves of the same experiment. Here, the IOKR methods clearly perform best, having close to perfect precision regardless of the evolutionary distance until recall level of 0.5 and then a sharp drop at recall levels of 0.7-0.9 depending on the evolutionary distance. In contrast, OK3 manifests a close to one precision only for BLO-SUM80 and for recall levels up to 0.3. Pairwise kernels do not obtain a high precision and produces a pattern that is inverted with respect to the evolutionary distance, indicating a high number of false positives in the SVM classifier and possible overfitting when the evolutionary distance is small.
The ROC and PR curves together indicate IOKR as the best compromise, given that both a high overall accuracy and high initial precision are desirable for network reconstruction.
Identifying secretory pathway PPIs from full genome Next, we check how well transfer learning of the secretory pathway works in the basic case of the source and target species being the same. In this experiment, the inference models were trained on the S. cerevisiae secretion proteins and their functional interactions, and the goal is to test the ability of the models to correctly identify the secretion pathway proteins among all S. cerevisiae proteins. In this setup, the ground truth is composed of PPIs between two secretory pathway proteins as the positive class and all other interactions as the negative class (true interactions between one or two non-secretory proteins as well as missing interactions between pairs of secretory pathway proteins). [fig_ref] Fig 5: ROC curves and Precision-Recall [/fig_ref] the results of a 5-fold cross-validation experiment for the different network inference methods. In the ROC space (left pane), Pairwise kernels and the two IOKR methods are close in performance, with the semi-supervised IOKR being marginally better than the two others. OK3, however, performs significantly worse than the other three methods. In the precision-recall space (right pane), the two IOKR methods are the most robust in the lowrecall regime, with the semi-supervised variant maintaining 0.7 precision rate up to 0.6 recall rate. Pairwise kernels and OK3 demonstrate a different pattern: they suffer from a high false positive rate in the low-recall regime, but have a good precision in mid-recall regime, before tailing off.
Analyzing the ROC and PR results together, semi-supervised IOKR emerges as the best compromise, due to its good ROC behaviour and good precision in the low-recall regime. It appears that the semi-supervised aspect gives some protection for the method against false positives in the low to mid-recall levels.
## Comparison of multiple kernel learning methods
Next, we compare the different MKL methods ALIGN, ALINGF and p-norm path following on the reconstruction of the set of secretion proteins from the full genome of S. cerevisiae when using semi-supervised IOKR as predictor.
The results are shown in [fig_ref] Fig 6: ROC curves and Precision-Recall [/fig_ref] It can be seen that the MKL methods perform better than the simple sum of input kernels (UNIMKL) in terms of ROC curve as well as PR curves.
Nonetheless, the gains of MKL are smaller than we expected them to be. Looking at the ROC curves, the p-norm path following MKL outperforms the other methods, whereas for the PR measure the simpler ALIGNF outperforms all other methods with p-norm path following being the second best. Secretion network prediction for Trichoderma reesei Finally, we evaluate the PPI prediction quality in an inter-species setup, where the training data comes from S. cerevisiae and the target species is T. reesei. However, no experimental protein interaction data exists for T. reesei that could be used as the ground truth. Thus we focus on qualitative analysis of the predicted T. reesei secretion network by expert knowledge. For predicting the PPI in T. reesei we used semi-supervised IOKR and p-norm path following for learning the input kernel, since this method combination achieved the best performances in the previous experiments.
In order to validate the predicted T. reesei secretion network, its genes (T. reesei genome version 2.0 [bib_ref] Genome sequencing and analysis of the biomass-degrading fungus trichoderma reesei (syn. hypocrea..., Martinez [/bib_ref] were annotated with a combination of sequence similarity based methods: best BLASTp [bib_ref] Basic local alignment search tool, Altschul [/bib_ref] match to S. cerevisiae proteins, best BLASTp match to UniProtKB/Swiss-Prot [bib_ref] Uniprot: a hub for protein information, Consortium [/bib_ref] , Interproscan domain predictions [bib_ref] Interproscan 5: genome-scale protein function classification, Jones [/bib_ref] , PANNZER description line and GO-category predictions [bib_ref] Pannzer-high-throughput functional annotation of uncharacterized proteins in an error-prone environment, Koskinen [/bib_ref] and a manually curated set of Aspergillus niger protein secretion related genes [bib_ref] The 2008 update of the aspergillus nidulans genome annotation: a community effort, Wortman [/bib_ref].
The T. reesei secretion network contains in total 320 genes. According to the annotation described above 27 genes belong to the heterokaryon incompatibly family and are sequence wise very similar. This family contains a GTPase domain that could contain similar features as GTPases involved in secretion. 51 genes belong to other than secretion related categories of cellular function. 18 genes were annotated to be related to cell growth, cell wall synthesis and cell motility and six were found to be related to chromatin modification. In general these 24 proteins contain domains related to small molecule modifications of macromolecules such as glycosylation, phosphorylation, ubiquitinylation and methylation. Similar molecular functions are abundant in the known secretion pathway enzymes. 14 of the 51 were annotated as molecular and cellular function unknown (Column 'Class' in . Hence, manual annotation based . Unknown genes and genes without any interactions in STRING in predicted T. reesei secretion network. Column 'Gene' contains the T. reesei gene ID. 'In STRING' tells if the gene has interactions in STRING. Columns 'Btw' and 'Deg' denote the betweenness and degree network statistics of the corresponding gene. Columns 'Class' and 'Putative secretion pathway component' are author assigned classifications. 'Taxon specificity' gives the largest taxonomic group the gene was found in. on sequence similarity suggests a minimum of 75% true positive rate and a maximum of 20% false positive rate. The predicted secretion network, excluding heterokaryon incompatibility family and other than secretion related genes in order to ease visual inspection of know genes, is shown in [fig_ref] Fig 7: Predicted T [/fig_ref] An alternative layout (S1 a table format (S2 of the network are also provided as supplementary material. 16 of the 320 genes were found to have no interactions in the protein-protein interaction database STRING(Column 'In STRING' in . For 2 of these we found strong similarity based evidence that they are part of the Golgi mannosyltransferase complex and could be considered as false negative predictions by STRING.
For each gene annotated as unknown, a putative role in the secretion pathway machinery was assigned based on their position in the predicted network (Column 'Putative secretion pathway component' in . To estimate the novelty of such secretion pathway components the taxonomic distribution of the unknown genes was estimated with multi-genome protein clustering [bib_ref] Discovery of a new tyrosinaselike enzyme family lacking a c-terminally processed domain:..., Gasparetti [/bib_ref] (Column 'Taxon Specificity' in . All unknown genes were found to be restricted to the subphylum Pezizomycotina or a smaller taxon with-in Pezizomycotina.
In order to further validate the T. reesei secretion network we used a combined transcriptomics data set of public and in-house data (see Methods). Pearson correlation of the expression values of all gene pairs that have a predicted PPI (an edge in the PPI network) was computed. The average of absolute values of these correlations was found to be 0.2 with an empirical p-value of p < 0.05. This p-value was calculated by rewiring the network 1000 times with the igraph function 'rewire' [bib_ref] The igraph software package for complex network research, Csardi [/bib_ref] and counting the average of absolute correlation each time. Absolute correlations above 0.3 are highlighted in
# Discussion
Experimental measurement of protein-protein interactions is technically demanding and often different methods can give conflicting results [bib_ref] From experimental approaches to computational techniques: a review on the prediction of..., Browne [/bib_ref] [bib_ref] Protein-protein interactions: Interactome under construction, Bonetta [/bib_ref]. Also, even a reliably measured interaction might not have a detectable biological function. To circumvent such challenges we use an expert curated interaction network of functional associations derived from numerous experiments [bib_ref] Genome-scale modeling of the protein secretory machinery in yeast, Feizi [/bib_ref].
We tested several recent machine learning methods for the task of PPI prediction. Classification models tested included pairwise kernels, output kernel trees as well as supervised and semi-supervised input-output kernel regression. The methods differed in performance depending on whether ROC or PR was used as the evaluation metric. Semi-supervised IOKR proved to be the best compromise when both evaluation metrics were taken into account: it had the best PR performance and a reasonable ROC-this choice puts an emphasis on good performance in the positive class, required for reliable network reconstruction.
Multiple kernel learning methods tested included uniform kernel combination, methods based on centered kernel alignment as well as the newly proposed p-norm path following algorithm. In our tests, we found that generally p-norm path following performed best in the ROC metric while other methods were close to each other in performance. In the PR metric, ALIGNF outperformed the other methods and p-norm path following being second best. Altogether, p-norm path following seems to give the best performance, although the improvements of MKL over no MKL were smaller than expected.
To demonstrate our prediction approach we predict protein secretion network for T. reesei, an industrially important protein production organism, which has no experimentally verified PPIs to date. Novel understanding of their protein secretion network machinery could have significant impact in the generation of improved protein production strains through targeted engineering.
For T. reesei we find that the predicted network is well supported by sequence similarity based manual annotation and by transcriptomics data. Most importantly the predicted network includes 14 previously unknown genes that are taxonomically restricted to Pezizomycotina and hence could explain their exceptional protein secretion capabilities.
Finally we note that our set up does not need complex external database systems or specialized experimental data to be generated, but relies on data available through standard sequence searches, evaluated through fast machine learning models. Hence, our methods are amenable to local implementation as part of a genome annotation pipeline.
Supporting Information S1
[fig] Fig 1: Frequency distribution of percentage of amino acid sequence identity between natural S. cerevisiae sequences and (1) sets of artificial sequences created from from S. cerevisiae with different Blosum matrices, (2) natural T. reesei sequences. doi:10.1371/journal.pone.0159302.g001 [/fig]
[fig] Fig 2: Schematic representation of the duality between (A) the PPI network and (B) the adjacency matrix for the proteins in the training set (blue) and testing set (yellow) and their interactions: training interactions (black), training-testing interactions (gray) and testing interactions (white). doi:10.1371/journal.pone.0159302.g002 [/fig]
[fig] •: True negative rate (TNR): also known as specificity, is the number of true negatives divided by the number of actual negatives TN/N False positive rate (FPR): is the number of false positives divided by the number of actual negatives FP/N False negative rate (FNR): is the number of false negatives divided by the number of actual positives FN/P Precision: is the number of true positives divided by the number of predicted positives TP/ (TP + FP) [/fig]
[fig] Fig 3: ROC curves for predicting PPIs in different artificial data sets with Output Kernel trees (OK3), Tensor kernels on protein pairs (Tensor Kernel on PP), and supervised and semi-supervised Input-Output Kernel Regression (IOKR). AUROC statistic of the associated curve is depicted in the figure legend (standard deviation in parenthesis).doi:10.1371/journal.pone.0159302.g003 [/fig]
[fig] Fig 4: Precision-Recall (PR) curves for predicting PPIs in different artificial data sets with Output Kernel trees (OK3), Tensor kernels on protein pairs (Tensor Kernel on PP), and supervised and semisupervised Input-Output Kernel Regression (IOKR). AUPR statistic is shown in the legend for each curve (standard devation in parenthesis. doi:10.1371/journal.pone.0159302.g004 [/fig]
[fig] Fig 5: ROC curves and Precision-Recall (PR) curves for predicting secretory PPIs from the full S. cerevisiae genome with Output Kernel trees (OK3), Tensor kernels on protein pairs (Tensor Kernel on PP), and supervised and semi-supervised Input-Output Kernel Regression (IOKR). AUCROC and AUPR statistics are shown in the legend for each curve. doi:10.1371/journal.pone.0159302.g005 [/fig]
[fig] Fig 6: ROC curves and Precision-Recall (PR) curves for predicting secretory PPIs from the full S. cerevisiae genome with semi-supervised Input-Output Kernel Regression (IOKR) and different Multiple Kernel Learning (MKL) methods compared to no MKL (UNIMKL). AUCROC and AUPR statistics are shown in the legend for each curve. doi:10.1371/journal.pone.0159302.g006 [/fig]
[fig] Fig 7: Predicted T. reesei secretion network. A) The proteins annotated as secretory (242) and unknown (14) are included. Proteins are nodes and they are labelled with best matching S. cerevisiae protein name or if no match was found with T. reesei gene ID number. Thick edges signify either negative (red) or positive (green) absolute Pearson correlation of > 0.3 in transcriptomic data. Pink nodes do not have any interactions in STRING. B) Pie chart of functional classes of the 320 proteins included in the T. reesei secretion network. doi:10.1371/journal.pone.0159302.g007 [/fig]
[fig] S1: Fig. Alternative layout of predicted T. reesei secretion network. In this layout interactions of individual genes are easier to inspect with the cost of less clear overall structure. (TIFF) [/fig]
[table] Table: Protein feature data sources. Protein feature data sources used in training the PPI prediction models. (XLSX) S2 Table. Trichoderma reesei secretion network. Predicted protein-protein interactions of Trichoderma reesei secretion pathway. (XLSX) [/table]
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NF-κB-Dependent Role for Cold-Inducible RNA Binding Protein in Regulating Interleukin 1β
vector IL1B 3'UTR vector IL1B 3'UTR +/-LPS +/-LPS Figure S1. LPS treatment did not increased the stability of a heterologous RNA containing the 3'UTR of IL1B. The 3'UTR of IL1B was cloned downstream of the d2EGFP open reading frame that was under control of a tetracycline-regulated promoter (pTRE-d2EGFP) using a RT-PCR-based strategy and the following primers: CGGAATTCGAGAGCTGTACCCAGAGAGTC and CGGGATCCCTTCAGTGAAGTTTATTTCAG. We have used this strategy previously to study the decay of heterologous mRNAs {Melanson et al, 2011, RNA, 17, 2222}. HeLa Tet-O ® cells were transfected with either vector or the d2EGFP-IL1B and d2EGFP positive cells were identi ed through multiple rounds of uorescence -activated cell sorting (FACS) using a DakoCytomation MoFlo ow cytometer (Dako, Denmark).The d2EGFP positive pools were exposed to LPS 4 hours prior to doxycycline treatment and d2EGFP RNA levels were monitored by qRT-PCR, as previously described {Melanson, 2011 #370}. The presence of the 3'UTR of IL1B led to decreased d2EGFP expression but LPS didn't increase the basal level of d2EGFP (t=0 h in left panel). Furthermore, LPS did not stabilize the heterologous reporter mRNA (right panels). Each value represents the mean +/-SEM of three determinations.
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Media Exposure and Substance Use Increase during COVID-19
# Introduction
Social distancing and lockdown measures as a result of COVID-19 have had a negative impact on the physical and mental health for many groups in society, particularly those who are vulnerable and at high risk regardless of the pandemic [bib_ref] Trends in US Emergency Department Visits for Mental Health, Overdose, and Violence..., Holland [/bib_ref] [bib_ref] Mental Health and the Covid-19 Pandemic, Pfefferbaum [/bib_ref]. Individuals who had mental health concerns and needs prior to COVID-19, including anxiety and depression, were likely to experience an increase of their symptoms [bib_ref] The mental health impact of the COVID-19 pandemic on people with and..., Pan [/bib_ref].
Prior research has shown that media exposure is associated with increased distress, anxiety and substance use [bib_ref] Associations Between Media Exposure and Mental Distress Among U.S. Adults at the..., Riehm [/bib_ref] [bib_ref] Exposure to media and fear and worry about COVID -19, Sasaki [/bib_ref] [bib_ref] Mental health problems and social media exposure during COVID-19 outbreak, Gao [/bib_ref] [bib_ref] Media's role in broadcasting acute stress following the Boston Marathon bombings, Holman [/bib_ref] [bib_ref] Media Exposure and Marijuana and Alcohol Use Among Adolescents, Primack [/bib_ref] [bib_ref] Effectiveness of multimedia interactive patient education on knowledge, uncertainty and decisionmaking in..., Chiou [/bib_ref]. Increased audiovisual media, television, and social media have a greater impact on users' mental health [bib_ref] Mental health problems and social media exposure during COVID-19 outbreak, Gao [/bib_ref] [bib_ref] TV news images that induce anger, fear, and disgust: Effects on approach-avoidance..., Newhagen [/bib_ref]. Many studies have shown the association between television news use and negative emotional reactions [bib_ref] Disaster Media Coverage and Psychological Outcomes: Descriptive Findings in the Extant Research, Pfefferbaum [/bib_ref]. A recent study has provided evidence that higher use of social media news was associated with higher anxiety in participants in China during the COVID-19 pandemic [bib_ref] Mental health problems and social media exposure during COVID-19 outbreak, Gao [/bib_ref]. Similar findings have been demonstrated in the U.S. [bib_ref] Associations Between Media Exposure and Mental Distress Among U.S. Adults at the..., Riehm [/bib_ref]. However, other recent research has shown that media exposure in general increased fear and worry about COVID-19 in participants, with television and online media causing the most distress [bib_ref] Exposure to media and fear and worry about COVID -19, Sasaki [/bib_ref]. To date, no known studies have investigated the impact of media exposure during COVID-19 on substance use.
# Materials and methods
A nationally representative online survey of 1264 adults over 18 years of age collected responses from June 22 to July 18, during the peak of the pandemic in the United States. At that time, most states were in some form of lockdown status and were the theater of several lockdown-and social-justice-related demonstrations. This national Qualtrics panel included an over-sample of Washington state residents for a different study (N = 416) and employed demographic and regional quotas based on the 2019 U.S. census. Quality check measures eliminated duplicate responses and responders who answered in patterns, provided illogical patterns of answers, or sped through the survey in less than a third of the overall median survey duration. In addition, post-stratified sample weights were applied to adjust for the Washington State oversample and to ensure that the quota samples from the four U.S. regions reflected 2019 census estimates. This study was approved by the Institutional Review Board of Washington State University.
## Measures
Outcome variable: The outcome variable for this study was whether a participant experienced an increase in substance use since the beginning of the COVID-19 pandemic (yes vs. no). This referred to four categories of drugs including prescription drugs, nonprescription drugs, sedatives/hypnotics, and methamphetamines (i.e., four different questions, one for each drug). Those drugs were chosen because of their incidence of long-term dependencies.
Exposure variable: The primary aim was to assess whether exposure to media (cable news or social media) and COVID-19 knowledge could explain the increase in substance use noted during the pandemic. Therefore, we first created two different media exposure measures, one for cable news and one for social media. Participants were asked about the frequency of use of different media outlets, and the variable was categorized into three levels using the following criteria: no use or monthly use of media was categorized as low, weekly use of media as medium, and at least once a day as high. The following cable news media outlets were combined into one measure of cable news exposure: CNN, Fox news, and MSNBC. The same measure was created to assess exposure to social media, combining four questions about social media resources that included getting information from social media friends, influencers, community members, and what was currently trending (e.g., hashtags). The primary explanatory variable was an interaction variable with COVID-19 knowledge and each of the media exposure levels. Regarding knowledge of COVID-19, participants rated a series of statements as true or false, with correct answers totaled, based on information from the World Health Organization (2020), the Centers for Disease Control (2020), and a contemporary Research Triangle Institute (RTI), (2020); survey COVID-19 knowledge was dichotomized to high (at least four correct answers) and low knowledge (less than four correct answers) [fig_ref] Table 1: List of questions to determine COVID-19 knowledge [/fig_ref]. We used the knowledge variable together with the media exposure variable as it acted as a moderator of substance use. More specifically, we could expect individuals with high knowledge of COVID-19 to be less impacted by exposure to either cable news or social media. This is because, in general, individuals with higher knowledge will have had lower uncertainty about the pandemic, and as a result, they will have been less impacted by the media exposure [bib_ref] Effectiveness of multimedia interactive patient education on knowledge, uncertainty and decisionmaking in..., Chiou [/bib_ref]. Each of the primary explanatory variable had six levels of interaction between knowledge and media exposure: Low knowledge-Low media exposure, Low knowledge-Mid media exposure, Low knowledge-High media exposure, Mid knowledge-Low media exposure, Mid knowledge-Mid media exposure, and Mid knowledge-High media exposure. Covariates: The following variables were included in the model as covariates: Education level (high school graduate vs. non-high school graduate), ethnicity (white vs. nonwhite), age (18-39, 40-59, and 60 or over (categorical)), gender (male vs. female), income ($0-$49,999, $50,000-$74,999, and above $75,000) and political orientation (liberal vs. moderate vs. conservative). We also included a measure of rurality based on the participant's zip code. We used the Rural Urban Commuting Area (RUCA) to classify the participant's zip code place of residence based on their degree of rurality, using the following: urban, large rural, small rural, and isolated zip code.
# Analysis
Univariate analyses included the reporting measure of central tendency and variability for continuous variables and frequency distributions and percentages for categorical variables. Bivariate statistics included chi-square and the Mann-Whitney U to test for differences in demographic and exposure variables in the outcome variable groups. Multivariate analyses included generalized linear models (GLMs) with a binary logistic function to explore the association between media exposure and substance use increase during COVID-19, controlling for covariates. Associations were presented as odds ratios (ORs) with 95% confidence intervals (CIs). R was used to perform statistical modeling procedures, using a significance level of <0.05.
# Results
The characteristics of individuals who experienced an increase in substance use are shown in [fig_ref] Table 2: Characteristics of survey participants stratified by whether they experienced increased substance use... [/fig_ref]. A total of 1264 participants completed the panel survey; 51 were dropped because they did not provide information on the outcome variable, for a total of 1213 participants to be included in the analysis. Of those, 197 (16.7%) participants experienced a substance use increase; 772 (63.3%) were white, and 592 (48.8%) were male; 234 (19.3%) were over the age of 60; 475 (39.2%) described themselves as liberal (as opposed to 35.9% conservative and 18.5% moderate); 300 (24.7%) earned less than $50,000 a year, and 265 (21.8%) had only a high school degree. Geographically, 76.4% of the participants in the survey resided in urban zip codes, closely matching the actual rural/urban distribution in the US Census (United States Census Bureau, 2016) [bib_ref] Depression in Older Adults, Fiske [/bib_ref]. Finally, 197 (16.2%) reported an increase in substance use during COVID-19, and of those, only 6.4% were over the age of 60 (compared to 21.4% for ages 40-59 and 17% for ages 18-39), 18.4% were male and 13.9% were female, 22.9% earned $75,000 or more (compared to 10.7% for those who earned $50,000-$75,000 and 15.3% of those who earned less than $50,000), and 21.6% self-described as conservatives (compared to 15.9% liberal and 13% moderate). A total of 35.5% of participants with both low knowledge and high exposure experienced an increase in substance use, while only 4.6% of those with high knowledge and low exposure experienced an increase. The same trend was also observed for exposure to social media and COVID-19 knowledge; participants with both low knowledge and high exposure experienced the highest increase in substance use (30.9%), while those with high knowledge and low exposure only had a 3.9% increase [fig_ref] Figure 1: Increases in substance use during COVID-19 and the two primary explanatory interaction... [/fig_ref]. Finally, 197 (16.2%) reported an increase in substance use during COVID-19, and of those, only 6.4% were over the age of 60 (compared to 21.4% for ages 40-59 and 17% for ages 18-39), 18.4% were male and 13.9% were female, 22.9% earned $75,000 or more (compared to 10.7% for those who earned $50,000-$75,000 and 15.3% of those who earned less than $50,000), and 21.6% self-described as conservatives (compared to 15.9% liberal and 13% moderate). A total of 35.5% of participants with both low knowledge and high exposure experienced an increase in substance use, while only 4.6% of those with high knowledge and low exposure experienced an increase. The same trend was also observed for exposure to social media and COVID-19 knowledge; participants with both low knowledge and high exposure experienced the highest increase in substance use (30.9%), while those with high knowledge and low exposure only had a 3.9% increase [fig_ref] Figure 1: Increases in substance use during COVID-19 and the two primary explanatory interaction... [/fig_ref]. In the multivariable-adjusted models, an increase in substance use during COVID-19 was associated with low knowledge of COVID-19 and increased exposure to both social media and cable news [fig_ref] Table 3: Unadjusted and adjusted GLMs analyses of COVID-19 knowledge together with media exposure... [/fig_ref]. More specifically, in the social media adjusted model, participants with the highest exposure to social media (at least daily) and low knowledge of COVID-19 were 9.9 times more likely to experience an increase in substance use since the pandemic began (OR = 9.90, 95% CI = 4.71-23.61). This exact same trend was also observed in the cable news model, and participants with the highest exposure to cable news and low knowledge of COVID-19 were over 11 times more likely to experience an increase in substance use (OR = 11.64, 95% CI = 4.01-24.45). In addition, participants age 40-59 experienced much higher substance use in both the cable news and social media models (OR = 1.49, 95% CI = 1.02-2.1 and OR = 1.68, 95% CI = 1.15-2.47, respectively), and In the multivariable-adjusted models, an increase in substance use during COVID-19 was associated with low knowledge of COVID-19 and increased exposure to both social media and cable news [fig_ref] Table 3: Unadjusted and adjusted GLMs analyses of COVID-19 knowledge together with media exposure... [/fig_ref]. More specifically, in the social media adjusted model, participants with the highest exposure to social media (at least daily) and low knowledge of COVID-19 were 9.9 times more likely to experience an increase in substance use since the pandemic began (OR = 9.90, 95% CI = 4.71-23.61). This exact same trend was also observed in the cable news model, and participants with the highest exposure to cable news and low knowledge of COVID-19 were over 11 times more likely to experience an increase in substance use (OR = 11.64, 95% CI = 4.01-24.45). In addition, participants age 40-59 experienced much higher substance use in both the cable news and social media models (OR = 1.49, 95% CI = 1.02-2.1 and OR = 1.68, 95% CI = 1.15-2.47, respectively), and participants 60 years and older experienced lower substance use only in the cable news model (OR = 0.51, 95% CI = 0.27-0.93). Finally, male participants experienced a notable increase in substance use only in the social media model (OR = 1.49, 95% CI = 1.02-2.19).
# Discussion
The findings of this study showed that increased exposure to both cable news and social media were associated with substantial increases in substance use during the COVID-19 pandemic. Interestingly, older populations (60 years old and over) experienced decreased odds of substance use when exposed to cable news, while younger populations (ages 40-59) experienced a higher likelihood of substance use with exposure to both cable news and social media. To date, no known studies have investigated the impact of media exposure during COVID-19 on substance use.
Several studies have shown a clear relationship between media exposure (either television or social media) during COVID-19 and increased distress and anxiety [bib_ref] Associations Between Media Exposure and Mental Distress Among U.S. Adults at the..., Riehm [/bib_ref] [bib_ref] Mental health problems and social media exposure during COVID-19 outbreak, Gao [/bib_ref]. The results presented here confirm these studies by showing that increased media exposure not only increases anxiety, but is also likely to increase substance use. Since COVID-19 emerged in early 2020, there has been a tremendous demand for information about the pandemic. Social media platforms have been used to disseminate information and stories on COVID-19 by organizations and individuals. Some of this information has been misleading and unfounded, leading to fear and confusion, which in turn has increased anxiety and distress. Constant exposure to cable news, which tends to have better control over the content presented than social media, was also shown to induce anxiety and distress during COVID-19 and other public health crises. As a highly visual platform, increased exposure to television can also cause viewer fear and mental distress [bib_ref] TV news images that induce anger, fear, and disgust: Effects on approach-avoidance..., Newhagen [/bib_ref]. The findings in the study presented here also indicate that increased knowledge of COVID-19 could moderate the impact of media exposure and could decrease the likelihood of substance use. This is critically important, as this allows individuals to better navigate the influx of information on COVID-19, and it could act as a tool to decrease uncertainty about COVID-19 information and, in turn, reduce the likelihood of substance use.
Interestingly, while our results showed that those between 40 and 59 years of age were more likely to experience increased substance use when exposed to both cable news and social media, it also showed that those over 60 were less likely to experience substance use when exposed to cable news. This may be because mental-health problems, like depression, occur less frequently within older populations, which may, in turn, indicate a decreased likelihood of substance use [bib_ref] Depression in Older Adults, Fiske [/bib_ref]. It may also be because older populations are more likely to consume news from cable TV than social media and that cable news has a less detrimental impact on mental health. Additional research is needed to better understand the differences in the results between these age groups.
Increased dependency on illicit substance use remains a rising concern for both individuals with preexisting anxiety, stress, and substance use disorders (SUDs) and among individuals previously free of these conditions [bib_ref] Prevalence of comorbid substance use, anxiety and mood disorders in epidemi-ological surveys,..., Lai [/bib_ref]. The psychosocial impact of COVID-19 has continued to act as a severe stressor and impact the mental and general health outcomes within the public. It is especially concerning that after prolonged exposure to stress, substance use and their consequential neurobiological changes continue to persist even after the stressor is removed, further perpetuating the cycle of substance use [bib_ref] New Challenges in Addiction Medicine: COVID-19 Infection in Patients with Alcohol and..., Spagnolo [/bib_ref] [bib_ref] Addiction and stress: An allostatic view, Koob [/bib_ref]. Substance use carries a high risk of pulmonary and cardiovascular comorbidities, with complications due to the immunosuppressive qualities linked to the use of alcohol, opioids, and stimulants [bib_ref] Clinical implications of addiction related immunosuppression, Reece [/bib_ref]. The ongoing pandemic is exasperating the existing complications of treating substance and opioid use for clinicians and patients, due to the decreased access to treatment for those with substance use disorders. Thus, it remains imperative that clinicians have accessible information and guidelines to care for these vulnerable individuals during this unprecedented time and that they remain vigilant in prescribing opioid medication.
# Limitation
An important limitation that needs to be acknowledged is that our survey was limited to respondents willing and able to participate in a Qualtrics panel survey. This is especially impactful in geographic areas with limited access to broadband, which has a greater effect on rural areas and isolated communities. However, to somewhat overcome this limitation, we made sure that the rural urban distribution of our survey participants closely matched the actual rural/urban distribution in the US Census. In addition, we were not able to capture individual distress or reasons for using substances. Our survey did not include measures to examine the motivations for media use. For example, individuals could use social media to find information about the pandemic or for recreation purposes. These different motivations may have had a specific impact on people's substance use. Future research should examine these nuances to better understand the relationship between media use and substance use.
# Conclusions
Our findings point to the critical role played by media organizations in reducing uncertainty in evolving situations, such as pandemics, and the need for positive and hopeful messaging to counter the negative information often associated with such events. Our findings also show that increased knowledge has meaningful moderating effects on substance use and leads to decreased uncertainty. Given this, we recommend that during public health crises, like the COVID-19 pandemic, information from organizations such as the World Health Organization and Centers for Disease Control be prioritized and that medical professionals play a role in directing patients away from high stimulation media, such as 24-hour news and social media.
[fig] Figure 1: Increases in substance use during COVID-19 and the two primary explanatory interaction variables representing COVID-19 knowledge and media exposure (cable news and social media). [/fig]
[fig] Author: Contributions: O.A. designed the study, analyzed the data, and wrote the first draft manuscript. P.B. conceptualized and wrote the draft manuscript. D.K. wrote and edited the draft of the manuscript. S.M.M. designed and edited the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: This study was approved by the Institutional Review Board of Washington State University (IRB #18213-001). [/fig]
[table] Table 1: List of questions to determine COVID-19 knowledge. Please indicate if it is TRUE or FALSE, for each of the following statements about the COVID-19 virus: 1. Antibiotics can be used to treat the COVID-19 virus 2. People of all ages can become infected with COVID-19 3. People of all racial and ethnic groups can become infected with the COVID-19 4. Eating garlic can lower your chances of getting infected with the COVID-19 virus. 5. Most people who are infected with the COVID-19 virus die from it. 6. Most people who are infected with the COVID-19 virus recover from it. 7. Older adults or those with compromised immune systems are at a higher risk. [/table]
[table] Table 2: Characteristics of survey participants stratified by whether they experienced increased substance use during COVID-19 (n = 1213). [/table]
[table] Table 3: Unadjusted and adjusted GLMs analyses of COVID-19 knowledge together with media exposure and increase in substance use during the pandemic (n = 1213). [/table]
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10.1186/s12884-017-1239-2
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CCBY
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5307813
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28193186
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s2orc_pubmed_articles
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Afghan migrants face more suboptimal care than natives: a maternal near-miss audit study at university hospitals in Tehran, Iran
Background: Women from low-income settings have higher risk of maternal near miss (MNM) and suboptimal care than natives in high-income countries. Iran is the second largest host country for Afghan refugees in the world. Our aim was to investigate whether care quality for MNM differed between Iranians and Afghans and identify potential preventable attributes of MNM. Methods: An MNM audit study was conducted from 2012 to 2014 at three university hospitals in Tehran. Auditors evaluated the quality of care by reviewing the hospital records of 76 MNM cases (54 Iranians, 22 Afghans) and considering additional input from interviews with patients and professionals. Main outcomes were frequency of suboptimal care and the preventable attributes of MNM. Crude and adjusted odds ratios with confidence intervals for the independent predictors were examined. Results: Afghan MNM faced suboptimal care more frequently than Iranians after adjusting for educational level, family income, and insurance status. Above two-thirds (71%, 54/76) of MNM cases were potentially avoidable. Preventable factors were mostly provider-related (85%, 46/54), but patient-(31%, 17/54) and health systemrelated factors (26%, 14/54) were also important. Delayed recognition, misdiagnosis, inappropriate care plan, delays in care-seeking, and costly care services were the main potentially preventable attributes of MNM. Conclusions: Afghan mothers faced inequality in obstetric care. Suboptimal care was provided in a majority of preventable near-miss events. Improving obstetric practice and targeting migrants' specific needs during pregnancy may avert near-miss outcomes.
# Background
Disparities in maternal outcomes between migrants from low-income settings and natives in high-income countries are well documented in the literature [bib_ref] Increased risk of severe maternal morbidity (near-miss) among immigrant women in Sweden:..., Wahlberg [/bib_ref] [bib_ref] Ethnic disparity in severe acute maternal morbidity: a nationwide cohort study in..., Zwart [/bib_ref]. Migrants' vulnerability to poor pregnancy outcomes is partly explained by individual high-risk profiles such as comorbidities and socioeconomic disadvantages [bib_ref] Maternal healthcare in migrants: a systematic review, Almeida [/bib_ref] [bib_ref] The impact of socioeconomic position on severe maternal morbidity outcomes among women..., Lindquist [/bib_ref].
Furthermore, suboptimal care due to incongruent language and communication barriers, and unequal access to obstetric services are more frequent in migrant populations than European natives [bib_ref] Maternal healthcare in migrants: a systematic review, Almeida [/bib_ref] [bib_ref] Suboptimal care and maternal mortality among foreign-born women in Sweden: maternal death..., Esscher [/bib_ref].
Iran, with 79 million inhabitants, hosts refugees from neighbouring countries and accommodates an estimated one million registered and over two million unregistered migrants from Afghanistan. Essential interventions have been scaled up in this country and institutional delivery of 95% and a 75% reduction in maternal mortality ratio (25 per 100,000 live births) between 1990 and 2015 indicate healthcare achievements [bib_ref] Islamic Republic of, Unicef [/bib_ref]. Iran has faced international sanctions and financial constraints in recent decades and health resources have been inadequate to provide universal coverage for public health. Therefore, in-patient care was covered by health insurance for 90% of Iranians, while migrants were uninsured before the initiation of the new health system reform and public access to the Salamat Insurance Scheme in 2015. Our recent study in Tehran showed an increased risk of maternal near miss (MNM) among Afghan migrants through a lack of health insurance [bib_ref] Maternal near-miss at university hospital with cesarean overuse: an case-control study, Mohammadi [/bib_ref]. Iranians and Afghans have similar religious backgrounds and the majority of Afghans speak a language (Dari) similar to that of their hosts (Farsi). Therefore, the communication barriers to optimal care for migrants described in high-income settings appear to be less applicable in this context [bib_ref] Maternal healthcare in migrants: a systematic review, Almeida [/bib_ref] [bib_ref] Severe acute maternal morbidity in high-income countries, Van Roosmalen [/bib_ref]. MNM is defined as a woman who survives lifethreatening conditions during pregnancy, childbirth, or within six weeks postpartum. As high-quality care is crucial in saving lives, World Health Organization (WHO) developed a MNM audit approach in 2011 to routinely evaluate obstetric care quality in health facilities.
This study aimed at evaluating whether care quality differed between Iranian and Afghan mothers with nearmiss morbidity using the WHO approach. Additionally, we sought to identify potentially preventable factors predisposing to MNM.
# Methods
This audit study was part of an MNM project that was conducted between March 2012 and May 2014 at three hospitals affiliated with the Shahid Beheshti University of Medical Sciences in Tehran. Approximately 140 public and private hospitals provide care services in Tehran and the caesarean section (CS) rate stands at 74% [bib_ref] The trend of caesarean delivery in the Islamic Republic of Iran, Bahadori [/bib_ref]. Antenatal visits are included in primary health care and are provided free of charge for both natives and migrants.
Study sites were a secondary hospital with over 4,500 annual births and two tertiary referral hospitals with over 600 and 1,000 deliveries per year, respectively. The obstetric wards were staffed with 21 consultants and 68 residents in obstetrics and gynaecology, and 46 midwives. Consultants, residents, and midwives provide 24-hour medical staffing using national and local guidelines for obstetric care provision. Residents, under supervision of consultants, are responsible for all deliveries regardless of risk and nationality. The study hospitals provided intensive care services for adults and neonates.
At each hospital, one consultant and one resident were selected as research group members. The residents identified MNM cases under supervision of the consultants during daily morning reports. The WHO MNM criteria were used to identify cases prospectively during the first phase of the MNM project. However, these criteria were modified slightly in the case of two indicators in order to minimise inappropriate exclusion of women whose lives were seriously endangered due to obstetric complications but who did not fulfil the WHO criteria due to a limited institutional resources. As the secondary hospital had limited access to blood products (Rh-negative blood types in particular), modified criteria were the administration of four or more units of blood products and a rapid reduction of platelet count to below 75,000 platelets/ml. An acute decrease of ≥4 g/dl in the haemoglobin concentration was also applied as a near-miss criterion. [fig_ref] Figure 1: The flow diagram of the study population for maternal near-miss audit at... [/fig_ref] shows the study population.
Maternal age (<20, 20-34, ≥35 years), parity (0, 1-2, ≥3 para), and body mass index (BMI) (<18.5: underweight, 18.5-24.9: normal weight, ≥25: overweight and obese) were considered as maternal factors. Level of education (illiteracy and primary, secondary and higher), family income (low if it barely covered household expenditures, medium if it paid such bills, and high if it was more than self-reported expenses), health insurance (representing occupational status of the woman or her husband), and nationality (Iranian and Afghan according to country of birth) were identified as socioeconomic factors. Antenatal care coverage (at least four visits), timing of the near-miss event (upon or after arrival), admission status (primary or referral), previous CS, CS delivery in present pregnancy, night-shift delivery (2 o'clock pm till 8 o'clock am), severe anaemia (haemoglobin ≤10 g/dl), and comorbidity (diabetes, chronic hypertension, haematological disorders, previous pelvic operation) were assessed as medical factors.
## Audit procedure
The main researcher primarily reviewed patients' records, including admission, operation, and nurses' notes, ordering sheets, laboratory and pathology reports, and summary notes. For each case, a research form was completed with all background data including obstetric history, and clinical data related to near-miss events, as well as a copy of cardiotocoghraphy (CTG) traces and other available documents.
Three auditors, a Maternal Foetal Medicine physician and two board-certified obstetricians, comprised the audit team. During the study period, the main researcher organised audit panels and presented case histories anonymously (in terms of identity, nationality, and the hospital) to these panels. Auditors used a conceptual framework [fig_ref] Figure 2: Audit framework for evaluating the quality of obstetric care and preventability for... [/fig_ref] and performed individual case note review to evaluate obstetric care quality and preventability of near-miss events. Additionally, when documentation was insufficient for making clinical judgments, the main researcher interviewed those professionals identified as being the care providers responsible for the particular case to obtain clinical information. Thirdly, 24 near-miss mothers (16 in-person and 8 phone calls) were interviewed to provide additional ideas and input. For each case, the quality of eight care items was audited and the final decision was achieved by consensus among the three auditors (case exemplars are shown in Tables 1, 2 and 3). Antenatal care and referral systems (two items) were assessed to evaluate the quality of pre-hospital care. The quality of hospital care was analysed using a systematic approach based on six items: initial assessment, recognition, appropriate care plan, monitoring of critical conditions, provision of adequate information to women at discharge and planned follow-up, as well as proper documentation [bib_ref] Severe acute maternal morbidity: a pilot study of a definition for a..., Mantel [/bib_ref]. Pre-hospital obstetric care was labelled suboptimal if either antenatal care or referral system was inadequate (≥50% of items). Hospital care was considered suboptimal when three or more of those six items (≥50%) were inadequate. It was possible for one woman with near-miss morbidity to experience several inadequate items and preventable factors during the period of her hospitalisation.
In addition, auditors discussed preventability of near-miss events and agreed on the potential factors that could have prevented MNM or minimised the severity of the events at three levels: provider, patient, and health system [bib_ref] Audit-identified avoidable factors in maternal and perinatal deaths in low resource settings:..., Merali [/bib_ref].
# Statistical analysis
Chi-square test was used to examine and compare background factors, severe complications, and near-miss events between Iranians and Afghans. Statistical Package for the Social Sciences software, Version 21, was used for statistical analysis. Differences were considered significant where a probability of less than 0.05 was found. The association between suboptimal care and those factors that were significantly different between Iranians and Afghans was assessed by crude and adjusted odds ratios (OR, AOR) in three models. Health insurance was found protective against MNM and all Afghans and a number of Iranians were uninsured [bib_ref] Maternal near-miss at university hospital with cesarean overuse: an case-control study, Mohammadi [/bib_ref]. Therefore, a new independent variable was created and named "insurance-nationality" with three categories: insured Iranian, uninsured Iranian, and uninsured Afghan to examine the effect of lacking insurance on receiving suboptimal care for both nationalities. Model 1 represented crude associations between suboptimal care and maternal education, family income, nationality, insurance, and insurance-nationality. In Model 2, maternal education, family income, and nationality were taken into account to identify independent predictors of suboptimal care. In Model 3, "insurance-nationality" was added as a substitute for "nationality" in Model 2. The independent variables were controlled for collinearity. [fig_ref] Table 3: Statistical models of the associations between suboptimal care and socioeconomic factors of... [/fig_ref] shows that being illiterate or having primary education, low-income status, and being Afghan increased odds of receiving suboptimal care. On the contrary, health insurance protected mothers against suboptimal care. The association between suboptimal hospital care and Afghan nationality remained significant after adjusting for maternal education and income (Model 2 in [fig_ref] Table 3: Statistical models of the associations between suboptimal care and socioeconomic factors of... [/fig_ref] and even after additional adjustment for the effect of health insurance (Model 3 in [fig_ref] Table 3: Statistical models of the associations between suboptimal care and socioeconomic factors of... [/fig_ref].
Auditors determined that 71% (54/76) of MNM cases had at least one near-miss event that could have potentially been prevented. Preventability of near-miss events was not different between Iranians and Afghans (67%, 36/54 versus 81%, 18/22; p-value: 0.2). As [fig_ref] Table 4: Potentially preventable factors at three levels that attributes to 54 maternal near... [/fig_ref] shows, although provider-related factors were involved in majority of cases with preventable events, patient-and health system-related factors could also prevent the development of MNM. The following sections describe related examples of missed opportunities to optimal care.
In 82% (23/28) of MNM cases of severe postpartum haemorrhage, the amount of blood loss was neither estimated nor adequately assessed. Moreover, delayed recognition of the severity of haemorrhage and inadequate stepwise management were identified in 57% (16/28) of cases. Initial assessment in 61% (16/26) of MNM cases of hypertensive disorders was inadequate. Emergency CS due to severe pre-eclampsia was performed in 21 cases, Unavailable intensive care unit beds 1 (3) 2 (11) 3 [bib_ref] Suboptimal care and maternal mortality among foreign-born women in Sweden: maternal death..., Esscher [/bib_ref] of which 71% (15/21) occurred before stabilisation and treatment of severe hypertension. While placenta previa was the third most common obstetric complication (10%, 8/76) that led to near-miss morbidity, pre-surgical evaluation and the decision process for adopting a surgical approach was missed in 50% of cases. Three women with preterm pregnancy and co-existing epilepsy arrived at hospital with uncontrollable fits due to either discontinuation of treatment or irregular use of anticonvulsive therapy. They were delivered by emergency CS on suspicion of eclampsia while adequate history taking and initial assessment did not take place. Audits of CTG traces could only confirm abnormal CTG in 20% of those near-miss cases that delivered by CS due to foetal distress. There was no CTG documentation confirming foetal distress in 60% of cases and the opportunity for adequate assessment of CTG trace was missed in the remaining 20%. Tables 5, 6 and 7 present examples of MNM cases and the related clinical judgements.
# Discussion
Our findings demonstrate that obstetric care at hospital was more suboptimal for Afghan MNMs than Iranians. Moreover, care providers by optimal performance could potentially prevent a majority of near-miss events.
In contrary to literature, differences in background profiles, socioeconomic factors, language, and religion between Iranians and Afghans could hardly explain our findings [bib_ref] The impact of socioeconomic position on severe maternal morbidity outcomes among women..., Lindquist [/bib_ref] [bib_ref] Audit-identified avoidable factors in maternal and perinatal deaths in low resource settings:..., Merali [/bib_ref] [bib_ref] Shared language is essential: communication in a multiethnic obstetric care setting, Binder [/bib_ref]. Having a low level of education is a well-known association with increased risk of adverse maternal outcomes by delayed care-seeking and poor compliance with treatments [bib_ref] The relationship between maternal education and mortality among women giving birth in..., Karlsen [/bib_ref]. All Afghans in the present study were uninsured and had a lower level of education compared to Iranians; however, the proportion of near misses upon arrival did not differ between Iranians and Afghans. While suboptimal care was more probable for uninsured Afghans, this probability was not found for uninsured Iranians. Moreover, having insurance coverage in European countries is inadequate to protect immigrants from disproportionate suboptimal care [bib_ref] Suboptimal care and maternal mortality among foreign-born women in Sweden: maternal death..., Esscher [/bib_ref] [bib_ref] Severe acute maternal morbidity in high-income countries, Van Roosmalen [/bib_ref]. Social differentiation, isolation, and women's self-perceptions can dissociate mothers from adequate care [bib_ref] Social differentiation and embodied dispositions: a qualitative study of maternal care-seeking behaviour..., Rööst [/bib_ref]. These factors among Afghans could affect obstetric care quality but this study was unable to elaborate on such factors.
In accordance with prior studies, we found that care providers had fundamental roles in the prevention of near-miss morbidity by making timely diagnosis and successful management of obstetric complications [bib_ref] Audit-identified avoidable factors in maternal and perinatal deaths in low resource settings:..., Merali [/bib_ref] [bib_ref] Introducing maternal morbidity audit in the Netherlands, Van Dillen [/bib_ref]. Auditors noticed that although guidelines were accessible to care professionals, medical management poorly adhered to them. Literature suggests that in addition to establishing guidelines, optimal care provision requires providers' beliefs and institutional support [bib_ref] Improving maternal health and safety through adherence to postpartum hemorrhage protocol in..., Olmedo [/bib_ref]. Conducting audits in obstetrics can provide practical measures for tackling care deficiencies and may stimulate tailored interventions to improve maternal care quality [bib_ref] Critical incident audit and feedback to improve perinatal and maternal mortality and..., Pattinson [/bib_ref] [bib_ref] Clinical audits: a practical strategy for reducing cesarean section rates in a..., Mohammadi [/bib_ref].
In agreement with other publications, we found that patient-and health system-related factors were involved in preventable near-miss events [bib_ref] Maternal healthcare in migrants: a systematic review, Almeida [/bib_ref] [bib_ref] Audit-identified avoidable factors in maternal and perinatal deaths in low resource settings:..., Merali [/bib_ref]. For instance, home delivery or delayed care-seeking that may result from financial constraints at patient level could have been avoided by insurance coverage at the level of health system. Although healthy motherhood is part of women's rights to health and life, poverty seriously affects these rights when the needed care is available but unaffordable.
To our knowledge, the present study is the first attempt to apply the WHO MNM audit in Iran to evaluate care quality within a migration perspective. Using the WHO criteria was feasible to identify MNM cases prospectively in our setting, while the MNM tool failed to identify all eligible cases within a national data in the Netherlands, retrospectively [bib_ref] Validating the WHO maternal near miss tool in a high-income country, Wittevenn [/bib_ref]. Collecting first-hand data from near-miss survivors and care providers was a real strength in our study and disclosed valuable inputs Example of missed opportunities linked to care items for obstetric haemorrhage Case 1 A 21-year-old Afghan mother, 0P, in 38 weeks of gestation, was admitted to hospital with labour pains in latent phase. She was delivered by emergency CS due to foetal distress on the day shift. Ten hours after operation she was pale, had pre-shock status, and the reported haemoglobin level was 7.4 g/dl. Re-operation was performed, a very large hematoma in left broad ligament was detected, and 12 units of different blood products were transfused. She went back to the hospital two weeks after discharge due to fever and haematuria. Further examination revealed left ureter injury.
## Care items audit findings
Initial assessment Foetal heart rates were monitored and assessed inadequately.
## Recognition
No evidence was found to agree foetal distress. Intra-abdominal hematoma was recognised with delay.
## Care plan
The indicated evidence for emergency CS was missing.
## Monitoring
Postpartum controls early after CS were not documented and were inadequate for early detection of intra-abdominal bleeding.
## Preventability
Near-miss events (decreased haemoglobin, re-operation, blood transfusion) and the injured ureter could have potentially been prevented by better obstetric practice (provider-related).
that might not have been identified by reviewing the case notes alone. The audit framework enabled us to identify major obstacles to optimal care provision and facilitated determining barriers against accessing to such care. Moreover, the classification of potential preventable attributes of MNM into provider-, patient-, and health system-related factors might have a qualitative value in revealing where and how the tailored interventions could avert near-miss outcomes. As missed opportunities to better manage severe complications were found repeatedly at all of the study hospitals, the identified quality might mirror those of other university hospitals in Tehran.
The main limitation of this audit study is that our findings are based on a small sample of MNM cases. As the WHO near-miss approach underlines, the optimum number of cases for evaluating care quality has not been established. Our small sample could be an explanation for the non-significance of preventable factors between Iranians and Afghans. Another limitation is improper documentation. For instance, no records from the first institute or from professionals who referred patients to the tertiary hospitals were found, and antenatal cards were mostly unavailable. The quality of pre-hospital care was assessed based on the information extracted from the hospital admission notes and the additional input that was given by a few survivors and could thus be underestimated. We were unable to make contact with a majority of near-miss mothers for interviews. Furthermore, an interviewer who was not an obstetric professional could have received other responses or provided other input to the audits. Two auditors worked as consultant obstetricians at the study sites, one in tertiary and one in the secondary hospital, and this may have impacted on their judgements of care quality at their hospitals. However, Example of missed opportunities linked to care items for placenta previa A 36-year-old native mother, 2P, with two previous CS was admitted to hospital due to low back pain in 39 + 3 weeks of gestation. According to ultrasound examinations during antenatal visits, she had low-lying placenta previa. Emergency CS was performed two hours after admission on the night shift and the operation ended up with CS hysterectomy due to abnormal invasive placenta. More than 20 units of blood products were transfused, the mother was admitted at intensive care unit and had long-lasting intubation. Pathologic examination of uterus specimen revealed placenta increta.
## Care items audit findings
Antenatal care Despite two previous CS and low-lying placenta previa, examination of placental orientation for better obstetric plan during pregnancy was not conducted. Despite repeat CS and previa, no elective surgery was planned.
## Referral system
No timely referral from antenatal clinic to the hospital was made.
Initial assessment Despite risk for abnormal invasive placenta, no assessment at hospital was performed.
## Recognition
Recognition of abnormally invasive placenta in a high-risk mother was missed before operation room.
Care plan No evidence was found indicating acute CS on the night shift for a high-risk surgery.
## Documentation
Estimation of blood loss during operation was not documented.
Near-miss events such as the amount of administered blood, admission to intensive care unit, and long-lasting intubation were not documented in summary notes.
## Preventability
The near-miss events could have potentially become less critical and traumatic for woman and her family by better obstetric practice (provider-related). Example of missed opportunities linked with sepsis and postpartum haemorrhage Case 3 A 24-year-old Afghan mother, 3P, was admitted to hospital one week after home delivery with long-lasting bleeding, weakness, and high fever. She was in pre-shock status and the reported haemoglobin level was 5.6 g/dl. She was resuscitated with blood transfusion and was treated with intravenous antibiotic due to postpartum endometritis. Ultrasound examination was done after three days and retained placenta was detected. Fever went down after evacuation and curettage and she chose to leave the hospital before doctor's recommendation. She was interviewed afterward and said that the family could not afford the cost of hospital obstetric services.
## Care items audit findings
Initial assessment
Despite home delivery and the risk of retained placenta, history taking and initial examination of uterus cavity were incomplete.
## Recognition
Delayed recognition of retained placenta in a mother with anaemia and postpartum endometritis was identified.
## Care plan
The management of postpartum endometritis and retained placenta were inappropriate.
Documentation Previous obstetric history and risk of postpartum haemorrhage, antenatal visits and delivery process at home for index pregnancy were not documented.
## Preventability
The near-miss events could have potentially prevented by affordable safe childbirth (health system) and timely care-seeking (patient). Hospital care was also suboptimal.
MNM cases were presented anonymously at panels and auditors were unaware of the cases' nationality or the place in which they were treated.
# Conclusion
Inequalities in maternal care between Iranians and Afghan migrants were identified. The majority of nearmiss events were considered preventable and audits suggested areas for improvements. The results may draw the attention of policy makers for adopting a maternal morbidity surveillance and response system to tackle care disparities and address obstacles in obstetrics. Afghan women affected by many socioeconomic and humanitarian challenges that should gain additional attention within antenatal visits.
[fig] Figure 1: The flow diagram of the study population for maternal near-miss audit at three university hospitals in Tehran, Iran, 2012 to 2014 [/fig]
[fig] Figure 2: Audit framework for evaluating the quality of obstetric care and preventability for 76 maternal near misses at three university hospitals in Tehran, Iran, 2012 to 2014 [/fig]
[table] Table 1: Maternal, socioeconomic, and medical background factors among 54 Iranian and 22 Afghan near misses at three university hospitals in Tehran, Iran, 2012 to 2014 [/table]
[table] Table 2: Severe complications and near-miss events in 54 Iranian and 22 Afghan near-miss cases at three university hospitals in Tehran, Iran, 2012 to 2014 [/table]
[table] Table 3: Statistical models of the associations between suboptimal care and socioeconomic factors of 76 maternal near misses at three university hospitals in Tehran, Iran, 2012 to 2014Adjusted OR for socioeconomic factors while "insurance-nationality" substitutes for insurance and nationality [/table]
[table] Table 4: Potentially preventable factors at three levels that attributes to 54 maternal near misses (36 Iranians and 18 Afghans) at university hospitals in Tehran, Iran, 2012 to 2014 [/table]
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10.1080/0886022X.2022.2148537
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CCBY
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9704086
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36426736
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s2orc_pubmed_articles
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Influence of dietary protein on serum phosphorous levels in peritoneal dialysis patients with different initial transport function
Introduction: This cross-sectional study investigated the influence of dietary protein intake (DPI) on serum phosphate levels in peritoneal dialysis (PD) patients and determined the DPI cutoff required to prevent hyperphosphatemia. Methods: A total of 504 PD patients were categorized into fast (4 h dialysate/plasma [D/P] creatinine clearance !0.65) or slow (<0.65) peritoneal transporters. Serum phosphorus and peritoneal solute clearance were compared between the groups with different DPI. Results: The fast peritoneal transporters (n ¼ 233) were older, had lower serum albumin and phosphorus levels, and had higher peritoneal phosphorus clearance (all p < 0.001). Among the slow transporters (n ¼ 271), serum phosphorus levels were significantly higher among patients with DPI > 1.0 g/kg/d (p < 0.001). High DPI only increased the hyperphosphatemia risk in slow transporters (not in high transporters). DPI !1.026 g increased the hyperphosphatemia risk in those patients (area under the curve: 0.66, p ¼ 0.001). Conclusion: High DPI increases the hyperphosphatemia risk in PD patients with slower peritoneal transport function.ARTICLE HISTORY
# Introduction
Hyperphosphatemia is a common complication in individuals undergoing dialysis, which is associated with a high incidence of cardiovascular events and poor outcomes [bib_ref] Prevention and treatment of hyperphosphatemia in chronic kidney disease, Vervloet [/bib_ref]. Phosphate control interventions include dietary phosphorus restriction, the use of phosphorus binders, and adequate dialysis. The Kidney Disease Improving Global Outcomes and Kidney Disease Outcomes Quality Initiative guidelines recommend a daily protein intake of 1.2 g/kg of body weight [bib_ref] National kidney foundation K/DOQI work group. The national kidney foundation K/DOQI clinical..., Kopple [/bib_ref]. High dietary protein intake (DPI) increases the risk for hyperphosphatemia and accelerates the loss of residual kidney function [bib_ref] Better preservation of residual renal function in peritoneal dialysis patients treated with..., Jiang [/bib_ref]. Dietary protein restriction is considered to be effective for controlling serum phosphorus levels [bib_ref] Dietary phosphate restriction in dialysis patients: a new approach for the treatment..., Guida [/bib_ref]. However, low dietary protein worsens nutritional status and increases mortality in peritoneal dialysis (PD) patients [bib_ref] Is controlling phosphorus by decreasing dietary protein intake beneficial or harmful in..., Shinaberger [/bib_ref]. The role of peritoneal phosphate clearance rate, an important indicator of phosphate balance, has been under appreciated in PD patients [bib_ref] Peritoneal phosphate removal varies by peritoneal dialysis regimen: an underestimated parameter of..., Botelho [/bib_ref]. The peritoneal phosphate clearance rate is lower and serum phosphorus levels are higher in patients with low and average low peritoneal membrane function characteristics (slow peritoneal transporters) than in those with high and high average peritoneal membrane function characteristics (fast peritoneal transporters) [bib_ref] Phosphate clearance in peritoneal dialysis: automated PD compared with continuous ambulatory PD, Sawin [/bib_ref]. Very few studies have investigated the effects of DPI on serum phosphate levels in PD patients with different peritoneal transport types.
This study investigated the effect of high protein intake on the incidence of hyperphosphatemia in PD patients with different peritoneal transport types.
# Materials and methods
## Study population
Newly catheterized PD patients undergoing regular follow-up at our PD center between January 2012 and December 2019 were enrolled in this study. Patients underwent a peritoneal equilibration test (PET) within 3 months of starting PD. None of the patients had peritonitis, tumors, other hypermetabolic states, or were undergoing corticosteroid treatment. All patients received a calcium content of 1.25% dialysate and were treated using a continuous ambulatory PD model. Patients treated using automated PD and day ambulatory PD were excluded.
# Study methods
We reviewed the primary etiologies, sex, age, predialysis laboratory parameters, baseline estimated glomerular filtration rate (eGFR, calculated using the Chronic Kidney Disease Epidemiology Collaboration equation), PD adequacy at 1 month after starting dialysis, and clinical outcomes of the enrolled patients. The patients were classified as fast peritoneal transporters (4-h dialysate/plasma [D/P] creatinine clearance: !0.65) and slow transporters (4-h D/P creatinine clearance: <0.65). Standard parameters of dialysis adequacy were determined by measuring total Kt/V and liters of creatinine cleared by standard methods (L/week/1.73 m 2 ).
Residual GFR was calculated as the average of 24-h urinary urea and creatinine clearance.
The peritoneal solute clearance and urine solute clearances were calculated as follows: Pcl was expressed as mL of plasma cleared per day [bib_ref] Peritoneal protein clearance and not peritoneal membrane transport status predicts survival in..., Perl [/bib_ref].
[formula] Peritoneal [/formula]
Compliance with the prescribed protein intake was assessed by monitoring 24-h urinary urea nitrogen levels according to the Mitch-Maroni equation [bib_ref] The precision of estimating protein intake of patients with chronic renal failure, Masud [/bib_ref]. The protein equivalent of nitrogen appearance (PNA) normalized to body weight was calculated using the methods described by Bergstrom et al. [bib_ref] Calculation of the protein equivalent of total nitrogen appearance from urea appearance...., Bergstr€ Om [/bib_ref] The total 24-h peritoneal ultrafiltration (L/24-h) was recorded as the difference between the volume instilled and drained. In the steady-state, the normalized protein catabolic rate (nPCR) is equivalent to daily protein intake, which is normalized to weight (g/kg/day). This was estimated by the nPNA or nPCR, normalized to kilograms of body weight [bib_ref] Dietary phosphorus restriction in predialysis chronic kidney disease: time for a cease-fire?, Evenepoel [/bib_ref]. According to estimated DPI (eDPI) based on the nPCR at 1 month after starting PD, patients were divided into three groups: eDPI <0.80 g/kg/d (low-protein diet group, LPD Group), eDPI 0.8-1.0 g/kg/d (common protein diet group, CPD Group), and eDPI >1.0 g/kg/d (high proteins diet group, HPD Group). Patients with serum phosphate >1.78 mmol/L at 1 month after starting PD were defined as having hyperphosphatemia. Clearance was normalized to 1.73 m 2 BSA. The impact of various DPI levels on serum phosphorus levels in patients with different peritoneal transport functions was compared. The amount of phosphorus binder at the first dialysis month and the baseline blood phosphorus values were collected.
# Statistical analysis
Measurement data use the median and interquartile, count data use rate, as appropriate, and between-group differences were evaluated using the independent ttest and one-way analysis of variance. Non-normally distributed data were evaluated using the Kruskal-Wallis test. Multivariate analyses were undertaken with linear and binary logistic regression to establish a risk factor model of hyperphosphatemia and peritoneal phosphate clearance. Sex, age, peritoneal phosphorus clearance, urinary phosphorus clearance, GFR, total Kt/V, nPCR, ultrafiltration, urine volume, and dialysate dose were used as covariates in the binary logistic regression model. A receiver operating characteristic (ROC) curve was used to calculate the maximum cutoff value. Statistical significance was set at p < 0.05. All analyses were performed using IBM SPSS Statistics for Windows v. [bib_ref] High rates of protein intake are associated with an accelerated rate of..., Otero Alonso [/bib_ref].0 (IBM Corp., Armonk, NY, USA).
# Results
## Patient characteristics
A total of 504 PD patients were enrolled in this retrospective study. Baseline demographic and clinical characteristics are presented in [fig_ref] Table 1: Baseline characteristics [/fig_ref]. No significant differences in sex, baseline eGFR, predialysis serum phosphorus levels, urea levels, and creatinine levels were identified between fast and slow transporters. However, the fast peritoneal transporter group had a higher proportion of individuals with diabetes mellitus, older patients, and patients with lower serum albumin levels (p < 0.001).
Data obtained during the initial month after starting PD are shown in [fig_ref] Table 2: Comparison of baseline values in the initial dialysis month between fast and... [/fig_ref]. The mean 4-h D/P creatinine clearance was higher in fast peritoneal transporters than in slow transporters (p < 0.001). Fast peritoneal transporters had higher systolic blood pressure (p < 0.001) and lower ultrafiltration volumes (p < 0.001). There were no differences in nPCR, urine volume, dialysis adequacy indices, and residual renal function between fast and slow transporters.
Phosphorus binders were used in 160 slow transporters and 50 fast transporters. The daily dosages of calcium-based binders in 46 slow transporters were higher than those in 17 fast peritoneal transporters (p ¼ 0.009). The daily dosages of noncalcium based binders in 114 slow transporters were higher than those in 33 fast peritoneal transporters (p < 0.001). The dosage of the different phosphorus binders were converted and presented as the relative phosphate-binding coefficient (RPBC) in [fig_ref] Table 6: Results of binary logistic regression for the identification of risk factors for... [/fig_ref] [26].
## Biochemical indexes and phosphorus clearance
Compared with slow transporters, fast peritoneal transporters had lower serum urea (p ¼ 0.003), creatinine (p ¼ 0.016), albumin, uric acid (p < 0.001), and hemoglobin (p ¼ 0.003) and greater peritoneal protein clearance (p < 0.001) [fig_ref] Table 3: Comparison of biochemical indicators and phosphorus removal status of different peritoneal transporter... [/fig_ref]. Although there were no differences in baseline serum phosphorus levels, phosphorus binder dosage, and dialysis dose, the serum phosphorus levels in the initial dialysis month were significantly lower in fast peritoneal transporters than in slow transporters (p < 0.001). Peritoneal phosphorus clearance and serum phosphorus levels were negatively correlated (r ¼ 0.332, p < 0.001; [fig_ref] Figure 1: Influence of peritoneal phosphate clearance on serum phosphate levels in peritoneal dialysis... [/fig_ref]. Fast peritoneal transporters had greater peritoneal phosphorus clearance (p < 0.001) but lower urine phosphorus clearance (p ¼ 0.037) than slow transporters. Daily PD phosphorus removal was greater in fast peritoneal transporters (p < 0.001). Nevertheless, total daily urine phosphorus removal was higher in slow transporters (p ¼ 0.001). Total daily phosphorus removal rates of the two transporter groups were similar (p ¼ 0.818; [fig_ref] Table 3: Comparison of biochemical indicators and phosphorus removal status of different peritoneal transporter... [/fig_ref].
## Effect of protein intake on serum phosphorus levels in pd patients
In fast transporters, high protein intake had no significant effect on serum phosphorus levels; however, the hyperphosphatemia risk was significantly elevated in slow transporters with an eDPI of more than 1.0 g/kg [fig_ref] Table 4: Influence of eDPI on serum phosphorus levels in different peritoneal transporter types [/fig_ref]. Nonetheless, among all patients and among slow transporters, serum phosphate levels were higher in HPD Group than in CPD Group (p ¼ 0.033) and LPD Group (p ¼ 0.006). In fast transporters, no significant differences in serum phosphorus levels were identified among different eDPI groups [fig_ref] Table 4: Influence of eDPI on serum phosphorus levels in different peritoneal transporter types [/fig_ref].
## Factors influencing peritoneal phosphate clearance in pd patients
Linear logistic regression showed that peritoneal phosphate clearance was negatively associated with serum phosphorus levels (p < 0.001), GFR (p < 0.001), and urinary phosphorus clearance (p ¼ 0.001) but was positively associated with 4-h creatinine D/P assessed by PET (p < 0.001), peritoneal protein clearance (p ¼ 0.009), total peritoneal creatinine clearance (p < 0.001), total peritoneal urea clearance (p < 0.001), and dialysis dose (p < 0.001) [fig_ref] Table 5: Results of logistic linear regression for the identification of factors influencing peritoneal... [/fig_ref].
## Factors influencing serum phosphorus levels in different peritoneal transporter types
The incidence of hyperphosphatemia was 13.4% (n ¼ 68) in all the patients, including 69.1% in slow transporters, which was significantly higher than 30.9% in fast peritoneal transporters (p ¼ 0.001). The incidence of hypophosphatemia, which was defined as a serum phosphorus concentration of less than 0.81 mmol/L, was 4.1%
# Discussion
Hyperphosphatemia is an independent risk factor for vascular calcification, cardiovascular events, and all- cause mortality in PD patients [bib_ref] Peritoneal protein clearance and not peritoneal membrane transport status predicts survival in..., Perl [/bib_ref]. Dietary phosphorus restriction is considered an effective and essential measure for lowering elevated serum phosphorus levels [bib_ref] Dietary phosphorus restriction in predialysis chronic kidney disease: time for a cease-fire?, Evenepoel [/bib_ref]. However, despite dietary phosphate restriction and phosphate binder use, serum phosphate levels remain significantly high in approximately 65% of the dialysis population [bib_ref] Hyperphosphatemia in dialysis patients: is there a role for focused counseling?, Poduval [/bib_ref] [bib_ref] Achieving K/ DOQI laboratory target values for bone and mineral metabolism: an..., Aly [/bib_ref]. On the other hand, severe dietary phosphorus restriction, without dietitian guidance, may result in insufficient intake of other nutrients, including protein and calories, [bib_ref] Feasible lowphosphorus dietary patterns in maintenance hemodialysis patients: need for original research, Bover [/bib_ref] which is associated with malnutrition in dialysis patients [bib_ref] Dietary phosphate restriction in dialysis patients: a new approach for the treatment..., Guida [/bib_ref] [bib_ref] Hyperphosphatemia in dialysis patients: is there a role for focused counseling?, Poduval [/bib_ref]. This study found that fast transporters may counteract the effects of a high-protein diet on serum phosphorus level due to the higher phosphorus clearance. However, a significant increase in serum phosphorus levels was observed in HDP group those who with slower transport status. Therefore, recommending a different DPI for PD patients with different transport function statuses is necessary and may be more favorable for serum phosphorus control. Peritoneal phosphorus clearance helps maintain phosphorus balance [bib_ref] Is controlling phosphorus by decreasing dietary protein intake beneficial or harmful in..., Shinaberger [/bib_ref]. The molecular weight of phosphate (96 Da) is intermediate to that of urea (60 Da) and creatinine (130 Da), whereas the molecular radius of phosphate (2.8 Å) is closer to that of creatinine (3.0 Å) than that of urea (1.8 Å) [bib_ref] Peritoneal phosphate clearance: the effect of peritoneal dialysis modality and peritoneal transport..., Davenport [/bib_ref]. However, phosphate has hydrophilic properties and may behave like a larger molecule in terms of solute clearance which diffuses more slowly. Because peritoneal phosphate clearance is time-dependent, it may be altered by differences in peritoneal membrane transport characteristics [bib_ref] Phosphate elimination in modalities of hemodialysis and peritoneal dialysis, Kuhlmann [/bib_ref]. Our data indicate that higher 4-h D/P creatinine clearance in PET is associated with a higher peritoneal phosphorus clearance rate and lower serum phosphorus levels [fig_ref] Table 5: Results of logistic linear regression for the identification of factors influencing peritoneal... [/fig_ref] , as shown previously [bib_ref] Is controlling phosphorus by decreasing dietary protein intake beneficial or harmful in..., Shinaberger [/bib_ref]. Hyperphosphatemia seems to be more prevalent among slow transporters because of insufficient peritoneal phosphorus removal [bib_ref] Peritoneal phosphate clearance is influenced by peritoneal dialysis modality, independent of peritoneal..., Badve [/bib_ref]. In this study, 69.1% of slow transporters suffered from hyperphosphatemia, which was much higher than the rate in the fast transporters.
Our data indicated no difference in baseline serum phosphorus levels, phosphorus binder dosage, and dialysis dose; however, serum phosphorus levels in the initial dialysis month were significantly lower in fast peritoneal transporters than in slow transporters. That may be due to the higher peritoneal phosphorus clearance rate [fig_ref] Figure 1: Influence of peritoneal phosphate clearance on serum phosphate levels in peritoneal dialysis... [/fig_ref]. Despite peritoneal phosphorus clearance rate in slow peritoneal transports is weak, urinary phosphorus clearance was significantly higher than that in fast peritoneal transporters though the GFRs of the two groups in the initial dialysis month were similar . These results indicated that, early on, urinary and peritoneal phosphorus clearances were complementary during PD. For patients with better residual renal function, urinary phosphorus clearance plays an important role in regulating the phosphorus balance, particularly in patients with slower peritoneal transport function [fig_ref] Table 6: Results of binary logistic regression for the identification of risk factors for... [/fig_ref] ; Thus, retaining residual renal function is essential for serum phosphorus control [bib_ref] Phosphate clearance in peritoneal dialysis, Debowska [/bib_ref] [bib_ref] Hyperphosphatemia in chinese peritoneal dialysis patients with and without residual kidney function:..., Wang [/bib_ref]. According to our data, the albumin levels were significantly lower in fast peritoneal transporters than in slow transporters due to greater peritoneal protein clearance (p < 0.001, [fig_ref] Table 3: Comparison of biochemical indicators and phosphorus removal status of different peritoneal transporter... [/fig_ref] , and increasing the needs for protein supplementation to maintain serum albumin levels.Though a higher risk of hypoproteinemia and poor nutritional status, [bib_ref] Hyperphosphatemia in chinese peritoneal dialysis patients with and without residual kidney function:..., Wang [/bib_ref] there was no difference in serum phosphorus levels among the three eDPI groups in fast peritoneal transporters [fig_ref] Table 4: Influence of eDPI on serum phosphorus levels in different peritoneal transporter types [/fig_ref]. This means that peritoneal phosphorus clearance was sufficient to address the phosphorus load due to high dietary protein in the fast transporters [bib_ref] High rates of protein intake are associated with an accelerated rate of..., Otero Alonso [/bib_ref]. Therefor, a high DPI to improve hypoalbuminemia did not increase the hyperphosphatemia risk in fast peritoneal transporters [fig_ref] Table 6: Results of binary logistic regression for the identification of risk factors for... [/fig_ref].
According to the 2020 Clinical Practice Guidelines for Nutrition in Chronic Kidney Disease, protein intake of 1.0-1.2 g/kg/day in PD patients to supplement the protein loss in the dialysate is required [bib_ref] KDOQI clinical practice guideline for nutrition in CKD: 2020 update, Ikizler [/bib_ref]. According to our data, an eDPI >1.0 g/kg is a risk factor for hyperphosphatemia in slow transporters [fig_ref] Table 6: Results of binary logistic regression for the identification of risk factors for... [/fig_ref] and may be associated with insufficient peritoneal phosphorus clearance. Serum phosphate levels in the HPD Group were higher than those in the other groups (p < 0.001; [fig_ref] Table 3: Comparison of biochemical indicators and phosphorus removal status of different peritoneal transporter... [/fig_ref]. Moreover, slow transporters had better albumin levels than fast transporters, and it follows that there is no necessary for slow transporters to overmuch daily protein intake to maintain serum albumin levels. In contrast, blood phosphate retention is largely due to high DPI in these patients. Our study showed that both LPD Group and CPD Group significantly reduce risks of hyperphosphatemia compared with HPD Group in slow transporters. On the other hand, a higher urinary phosphorus clearance rate was linked to lower serum phosphorus levels in slow transporters [fig_ref] Table 6: Results of binary logistic regression for the identification of risk factors for... [/fig_ref]. For these patients, high protein intake leads to accelerated loss of residual renal function [bib_ref] Better preservation of residual renal function in peritoneal dialysis patients treated with..., Jiang [/bib_ref] combined with decreased urinary phosphorus clearance, which weakens the compensatory effect of the kidney on phosphorus removal. According to our study, for patients with slower peritoneal transport function, the optimal daily protein intake recommendation of 1.026 g/kg/d could minimize hyperphosphatemia risk. Unfortunately, peritoneal transport function is typically not considered during dietary education in the real world.
For slow transporters, personalized diets with reduced protein intake should be recommended unless hypoproteinemia has already occurred. However, in our date, there were trends to nutritional status worsen in the LPD than HPD groups who with slower transport function. Therefore, dietary protein intake should not lower than 0.8 g/kg/d in these patients. The key to serum phosphorus control is intervention by a nutritionist who could recommend proteins with a low phosphorus:protein ratio,such as egg white, and foods with the appropriate carbohydrates and fats, and this may have a bearing on survival. According to the patient's dietary habits, a phosphorus binder should be used during one or two meals per day to prevent an increase in serum phosphorus levels [bib_ref] Patient education for phosphorus management in chronic kidney disease, Kalantar-Zadeh [/bib_ref]. Personalized low-phosphorus diet recommendations may aid to avoiding the rapid loss of residual renal function due to high protein intake and in retaining the compensatory effects of urinary phosphate clearance [bib_ref] Hyperphosphatemia in dialysis patients: is there a role for focused counseling?, Poduval [/bib_ref]. High-phosphorus foods are related to cooking methods and smoky, bake food (maken by egg yolk or cream), convenience food, snack and processed food, which are widely existed in Chinese and Western diet. Therefore, dietitians are very necessary to direct the food cooking for patients.
In addition, ultrafiltration was significantly less in fast peritoneal transporters compared with slow transporters in our study [fig_ref] Table 2: Comparison of baseline values in the initial dialysis month between fast and... [/fig_ref]. The amount of peritoneal ultrafiltration does not affect the amount of peritoneal phosphate removal. Phosphorus transport depends on dispersion instead of convection [bib_ref] Phosphate elimination in modalities of hemodialysis and peritoneal dialysis, Kuhlmann [/bib_ref]. Longer dwell times may help control hyperphosphatemia in slow transporters [bib_ref] Phosphate clearance in peritoneal dialysis, Debowska [/bib_ref]. In addition, total phosphate removal could be increased by increasing the total dialysis dose in fast peritoneal transporters with hyperphosphatemia [fig_ref] Table 5: Results of logistic linear regression for the identification of factors influencing peritoneal... [/fig_ref]. Phosphate control can be facilitated by regulating the peritoneal prescription according to differences in peritoneal membrane transport status, residual renal function, and DPI in PD patients [bib_ref] Peritoneal phosphate clearance is influenced by peritoneal dialysis modality, independent of peritoneal..., Badve [/bib_ref].
This was a single-center cross-sectional study and only studied the influence of dietary protein on serum phosphorous levels in peritoneal dialysis patients with different initial transport function at 1 month after PD is started. Thus, the conclusion of this study may not be generalizable to the long-term effects. It is necessarily to reassess the transport status after long dialysis duration to see whether the phosphate level in slow and fast transporter status would be influenced in different protein dietary groups. Since the DPI was evaluated by nPCR, actual values of dietary protein and phosphorus intake are needed to confirm the conclusions of this study. Finally, some other variables such as vitamin D analogue use, PD modality, dialysis and phosphorus binder adherence which may play into serum phosphorus are needed to considered in research design and statistical analysis. In the future, prospective multicenter clinical studies should be designed to verify the effect of a high protein diet on blood phosphorus in patients with long-term dialysis, peritoneal sclerosis, or recurrent peritonitis and to confirm the conclusions of this study.
In summary, high dietary protein (nPCR >1.026 g/kg/ day) increased the hyperphosphatemia risk in PD patients with slow peritoneal transport function. However, it had little effect on patients with a faster status. Individualized protein dietary recommendations and treatment strategies for hyperphosphatemia in PD patients should be considered according to different peritoneal transport types, and this may lead to better control of serum phosphorus levels.
[fig] Figure 1: Influence of peritoneal phosphate clearance on serum phosphate levels in peritoneal dialysis patients. [/fig]
[table] Table 1: Baseline characteristics. eGFR, estimated glomerular filtration rate; PD, peritoneal dialysis; SD, standard deviation. [/table]
[table] Table 2: Comparison of baseline values in the initial dialysis month between fast and slow transporters.Data are presented as mean ± standard deviation, medians and quartiles. D/Pcr, dialysate/plasma creatinine; SBP, systolic blood pressure; DBP, diastolic blood pressure; nPCR, normalized protein catabolic rate; Kt/V, weekly urea clearance; GFR, glomerular filtration rate. [/table]
[table] Table 3: Comparison of biochemical indicators and phosphorus removal status of different peritoneal transporter types in the initial dialysis month. [/table]
[table] Table 4: Influence of eDPI on serum phosphorus levels in different peritoneal transporter types. [/table]
[table] Table 5: Results of logistic linear regression for the identification of factors influencing peritoneal phosphorus clearance. [/table]
[table] Table 6: Results of binary logistic regression for the identification of risk factors for hyperphosphatemia in different peritoneal transporter types. CI: confidence interval; GFR, glomerular filtration rate; nPCR, normalized protein catabolic rate; TKt/V, total weekly urea clearance. [/table]
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10.1038/s41598-018-20467-1
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CCBY
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5799249
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29403062
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s2orc_pubmed_articles
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The influence of spatial frequency content on facial expression processing: An ERP study using rapid serial visual presentation
Spatial frequency (SF) contents have been shown to play an important role in emotion perception.This study employed event-related potentials (ERPs) to explore the time course of neural dynamics involved in the processing of facial expression conveying specific SF information. Participants completed a dualtarget rapid serial visual presentation (RSVP) task, in which SF-filtered happy, fearful, and neutral faces were presented. The face-sensitive N170 component distinguished emotional (happy and fearful) faces from neutral faces in a low spatial frequency (LSF) condition, while only happy faces were distinguished from neutral faces in a high spatial frequency (HSF) condition. The later P3 component differentiated between the three types of emotional faces in both LSF and HSF conditions. Furthermore, LSF information elicited larger P1 amplitudes than did HSF information, while HSF information elicited larger N170 and P3 amplitudes than did LSF information. Taken together, these results suggest that emotion perception is selectively tuned to distinctive SF contents at different temporal processing stages.Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 Scientific RepoRts | (2018) 8:2383 |
Throughout evolution, humans have developed the ability to detect and respond to certain challenges and opportunities 1,2 , especially under the condition of limited attentional resources (i.e., in an emergency situation). The rapid decoding of visual information helps us to identify the affective states of other people and apply appropriate behavioral strategies. Moreover, different channels of spatial frequencies (SFs), which represent various periodic luminance variations across space, have different influences on the processing of visual stimuli [bib_ref] From coarse to fine? Spatial and temporal dynamics of cortical face processing, Goffaux [/bib_ref]. Influential models of visual recognition have postulated that SF contents may follow a specific temporal hierarchy in visual object recognition [bib_ref] Time course of visual perception: coarse-to-fine processing and beyond, Hegde [/bib_ref] [bib_ref] Visual objects in context, Bar [/bib_ref] [bib_ref] Integrated model of visual processing, Bullier [/bib_ref]. These models suggest that the visual system processes visual input by following a predominantly coarse-to-fine strategy (from LSFs to HSFs), which can facilitate the rapid extraction of visual information.
Additionally, studies on SF have provided some evidence for the mechanism underlying emotional face processing [bib_ref] Faces are "spatial"-holistic face perception is supported by low spatial frequencies, Goffaux [/bib_ref] [bib_ref] Emotion perception is mediated by spatial frequency content, Kumar [/bib_ref] [bib_ref] Rapid fear detection relies on high spatial frequencies, Stein [/bib_ref] [bib_ref] The importance of low spatial frequency information for recognising fearful facial expressions, Mermillod [/bib_ref]. The dual-route model of emotion processing suggested that there are two parallel routes for the processing of emotional information: a subcortical "low road" that provides fast, but crude, biologically significant signals to the amygdala, and a longer, slower "high road" that processes detailed information through cortical visual areas [bib_ref] Neural bases of the non-conscious perception of emotional signals, Tamietto [/bib_ref] [bib_ref] The role of the amygdala in human fear: automatic detection of threat, Ohman [/bib_ref]. In support of this model, Vuilleumier, et al. [bib_ref] Distinct spatial frequency sensitivities for processing faces and emotional expressions, Vuilleumier [/bib_ref] found larger amygdala and subcortical (pulvinar and superior colliculus) activation for LSF, but not for HSF information in fearful expression perception, suggesting a functional role for the subcortical pathway in providing coarse and threat-related signals. This finding is consistent with neural computational studies which have shown that LSF content, as compared with HSF content, provides more efficient information for the categorization of threat-relevant faces [bib_ref] Coarse scales are sufficient for efficient categorization of emotional facial expressions: Evidence..., Mermillod [/bib_ref] [bib_ref] Neural computation as a tool to differentiate perceptual from emotional processes: The..., Mermillod [/bib_ref].
In contrast, there is an emerging view that the processing of affective visual stimuli relies on both LSF and HSF information, and that the dual-route model needs to be revised to a more flexible model -the multiple-waves model [bib_ref] Emotion processing and the amygdala: from a 'low road' to 'many roads'..., Pessoa [/bib_ref]. This model postulates that multiple cortical regions, as well as subcortical structures, play a prominent role in the processing of ecologically relevant signals [bib_ref] Emotion processing and the amygdala: from a 'low road' to 'many roads'..., Pessoa [/bib_ref] [bib_ref] Smile through your fear and sadness transmitting and identifying facial expression signals..., Smith [/bib_ref] [bib_ref] Emotion and the brain: multiple roads are better than one, Pessoa [/bib_ref] [bib_ref] Amygdala response to emotional stimuli without awareness: facts and interpretations, Diano [/bib_ref]. A study in a patient with a damaged amygdala showed that the patient's impaired recognition of fearful faces was due to the impaired processing of the eye region of faces conveying HSF information [bib_ref] A mechanism for impaired fear recognition after amygdala damage, Adolphs [/bib_ref]. This finding demonstrates the importance of HSF information in decoding fearful expressions and implies that the amygdala is involved in this type of visual processing. Furthermore, a number of psychophysical studies have suggested that participants primarily use HSF rather than LSF information to discriminate fearful faces from other expressions [bib_ref] Rapid fear detection relies on high spatial frequencies, Stein [/bib_ref] [bib_ref] Smile through your fear and sadness transmitting and identifying facial expression signals..., Smith [/bib_ref] [bib_ref] Transmitting and decoding facial expressions, Smith [/bib_ref].
In contrast to fMRI and behavioral studies, event-related potential (ERP) techniques offer high time resolution, and the temporal characteristics of emotional face processing have been explored in numerous ERP studies. Three of the most prominently studied ERP components are P1, N170, and P3. P1 is an early visual component detected at lateral occipital electrodes, which reflects a fast, exogenous response to visual stimulation [bib_ref] Rapid extraction of emotional expression: evidence from evoked potential fields during brief..., Eger [/bib_ref] [bib_ref] Sources of attention-sensitive visual event-related potentials, Gomez Gonzalez [/bib_ref]. The face-sensitive N170 component reflects the encoding of the structure and configuration of faces [bib_ref] The speed of individual face categorization, Jacques [/bib_ref] [bib_ref] Early processing of the six basic facial emotional expressions, Batty [/bib_ref]. The late P3 component is sensitive to stimulus valence, reflecting a more elaborate processing of emotional information [bib_ref] The selective processing of briefly presented affective pictures: An ERP analysis, Schupp [/bib_ref].
However, the results of ERP studies on SF processing remain inconclusive. A number of ERP studies have suggested that LSF information of threat-related stimuli (faces and scenes) can be extracted rapidly at early stages of emotional processing, as reflected by the ERP components P1 and/or N170 [bib_ref] The role of spatial frequency information for ERP components sensitive to faces..., Holmes [/bib_ref] [bib_ref] Enhanced extrastriate visual response to bandpass spatial frequency filtered fearful faces: Time..., Pourtois [/bib_ref] [bib_ref] Dynamics of visual information integration in the brain for categorizing facial expressions, Schyns [/bib_ref] [bib_ref] Is the early modulation of brain activity by fearful facial expressions primarily..., Vlamings [/bib_ref]. In addition, an intracranial event-related potential (iERP) study found a fast amygdala response (starting 74 ms post stimulus onset) especially to LSF fearful faces, providing direct evidence for the existence of the fast, subcortical pathway to the amygdala 32 . However, You and Li 33 's study on the processing of SF-filtered threat scenes found a significant SF-emotion interaction on P1, suggesting that fear and disgust evoke opposite response patterns in LSF (localized in the dorsal visual stream) and HSF (localized in the ventral visual stream) conditions. This result indicates that both HSF and LSF information can affect the perception of threat.
The rapid serial visual presentation (RSVP) paradigm represents an appropriate task to investigate the time course of emotion processing in the context of limited attentional resources [bib_ref] Three stages of facial expression processing: ERP study with rapid serial visual..., Luo [/bib_ref] [bib_ref] Three stages of emotional word processing: an ERP study with rapid serial..., Zhang [/bib_ref] [bib_ref] Emotional noun processing: an ERP study with rapid serial visual presentation, Yi [/bib_ref] [bib_ref] The time course of emotional picture processing: an event-related potential study using..., Zhu [/bib_ref]. In this paradigm, a series of stimuli are presented sequentially and rapidly (6-20 items per second), and when the interval is around 200-500 ms, the detection of the second target (T2) is impaired by the correct detection of the first target (T1). This phenomenon is called attentional blink (AB) [bib_ref] Temporary suppression of visual processing in an RSVP task: an attentional blink?, Raymond [/bib_ref] [bib_ref] A two-stage model for multiple target detection in rapid serial visual presentation, Chun [/bib_ref] , and can be efficiently used to detect whether limited attentional resources affect the response accuracy of different emotional stimuli. Luo, et al. [bib_ref] Three stages of facial expression processing: ERP study with rapid serial visual..., Luo [/bib_ref] combined the RSVP task with emotional facial expressions (from the Chinese Facial Affective Picture System, CFAPS) and proposed a three-stage model of emotional facial expression processing. In this model, the brain distinguishes threat-relevant facial expressions (fear) from others at the first stage, which explains the augmented early visual ERP component P1/N1, elicited by fearful expressions compared with happy or neutral expressions. At the second stage, the brain distinguishes emotional (happy and fearful) and neutral facial expressions, reflected by N170/VPP. The third stage is differentiating between happy, neutral, and fearful facial expressions, as reflected by P3/N3. Interestingly, subsequent research has shown that the processing of emotional words and scenes also show similar patterns [bib_ref] Three stages of emotional word processing: an ERP study with rapid serial..., Zhang [/bib_ref] [bib_ref] Emotional noun processing: an ERP study with rapid serial visual presentation, Yi [/bib_ref] [bib_ref] The time course of emotional picture processing: an event-related potential study using..., Zhu [/bib_ref] [bib_ref] Single-trial ERP analysis reveals facial expression category in a three-stage scheme, Zhang [/bib_ref] , but the latter two processing stages are more general and stable [bib_ref] Emotional noun processing: an ERP study with rapid serial visual presentation, Yi [/bib_ref] [bib_ref] The time course of emotional picture processing: an event-related potential study using..., Zhu [/bib_ref].
Prior studies related to SF processing have tended to focus on negative emotion processing. There are very few studies have investigated the time course of SF information in the decoding of both positive and negative facial expressions. Therefore, we used RSVP paradigm to further explore the processing of SF-filtered images of happy, fearful, and neutral facial expressions with EEG measurement. Previous studies have suggested that the P1, N170, and P3 components are significantly affected by emotional valence [bib_ref] Three stages of facial expression processing: ERP study with rapid serial visual..., Luo [/bib_ref] [bib_ref] Three stages of emotional word processing: an ERP study with rapid serial..., Zhang [/bib_ref] , and emotional face processing is mediated by specific SF contents [bib_ref] Emotion perception is mediated by spatial frequency content, Kumar [/bib_ref] [bib_ref] Is the early modulation of brain activity by fearful facial expressions primarily..., Vlamings [/bib_ref]. Taking these presumptions into consideration, we hypothesized that the early perception of emotional facial expressions relies on specific SF information, and we expected to observe different neural responses to happy, neutral, and fearful faces in early ERP components (P1 and/or N170). Furthermore, we also hypothesized that different SF contents would elicit different neural processing patterns, and we expected to observe different responses to LSF and HSF information in each of the P1, N170, and P3 components.
# Results
Behavioral performance. ANOVAs for the response accuracy revealed significant main effects of facial expression and SF (F 2,58 = 12.902, p < 0.001, η 2 p = 0.308; F 1,29 = 26.446, p < 0.001, η 2 p = 0.477). Participants performed better in the HSF condition (95.8 ± 0.6%) than in the LSF condition (92.0 ± 0.8%, p < 0.001). Pairwise comparison of the main effect of facial expression showed that the accuracies of fearful (96.0 ± 0.6%, p < 0.001) and happy faces (95.1 ± 0.8%, p = 0.008) were higher than that of neutral faces (90.5 ± 1.2%), while the former two emotion conditions show no significant difference (p = 0.750). Furthermore, there was no significant interaction effect between facial expression and SF (F 2,58 = 0.900, p = 0.386, η 2 p = 0.030). ERP data analysis. P1. The P1 amplitude showed significant main effect at SF and electrode (F 1,29 = 8.914, p = 0.006, η 2 p = 0.235; F 5,145 = 12.645, p < 0.001, η 2 p = 0.304; [fig_ref] Figure 1: Grand average ERPs for LSF happy, LSF neutral, LSF fearful, HSF happy,... [/fig_ref] ; the main effect of facial expression was not significant (F 2,58 = 1.027, p = 0.357, η 2 p = 0.034). LSF faces (3.072 ± 0.253 μV) elicited larger P1 amplitudes than did HSF faces (2.015 ± 0.392 μV, p = 0.006). Largest amplitudes were elicited at O1 (3.223 ± 0.312 μV). Bilateral occipital electrode sites O1 and O2 (2.750 ± 0.274 μV) elicited larger P1 amplitudes than did central occipital electrode sites POz (1.636 ± 0.333 μV, p < 0.01) and Oz (2.049 ± 0.312 μV, p < 0.01).
N170. Facial expression, SF, and electrode each had a significant effect on the N170 amplitude (F 2,58 = 11.214, p < 0.001, η 2 p = 0.279; F 1,29 = 32.654, p < 0.001, η 2 p = 0.530; F 3,87 = 7.826, p = 0.003, η 2 p = 0.213). Happy (−6.071 ± 0.417 μV, p < 0.001) and fearful faces (−5.827 ± 0.432 μV, p = 0.011) elicited larger amplitudes than did neutral faces (−5.240 ± 0.415 μV), and HSF content (−6.601 ± 0.477 μV) elicited larger amplitudes than did LSF content (−4.824 ± 0.393 μV, p < 0.001). Larger amplitudes were elicited at the PO7 (−5.549 ± 0.541 μV, p = 0.001) electrode sites and the PO8 (−6.954 ± 0.570 μV, p = 0.002) electrode than at the P7 (−4.318 ± 0.415 μV) sites. The SF × Emotion interaction was also significant (F 2,58 = 4.527, p = 0.017, η 2 p = 0.135). The further simple effects analyses revealed that happy (−5.144 ± 0.391 μV, p = 0.001) and fearful faces (−5.177 ± 0.455 μV, p = 0.001) elicited larger amplitudes than did neutral faces (−4.152 ± 0.407 μV) in the LSF condition; happy faces P3. The P3 amplitude showed significant main effect at facial expression, SF and electrode (F 2,58 = 25.275,
[formula] p < 0.001, η 2 p = 0.466; F 1,29 = 23.049, p < 0.001, η 2 p = 0.443; F 5,145 = 7.623, p < 0.001, η 2 p = 0.208). [/formula]
There was no significant interaction effect between facial expression and SF (F 2,58 = 0.582, p = 0.552, η 2 p = 0.020). CPz (2.763 ± 0.351 μV) elicited largest amplitudes of P3, and centro-parietal electrode sites CPz and Pz (2.520 ± 0.359 μV) elicited larger P3 amplitudes than did bilateral electrode sites CP3 (1.501 ± 0.269 μV) and CP4 (1.776 ± 0.232 μV) (p < 0.05). HSF faces (2.553 ± 0.281 μV) elicited larger P3 amplitudes than did LSF faces (1.587 ± 0.246 μV, p < 0.001). The pairwise comparison showed that happy (2.122 ± 0.288 μV, p < 0.001) and fearful (2.460 ± 0.313 μV, p < 0.001) faces elicited larger P3 amplitudes than did neutral faces (1.486 ± 0.223 μV), and fearful faces elicited lager amplitudes than did happy faces (p = 0.012). The scalp topographies of the grand average ERPs across two SFs and three emotional conditions are shown in [fig_ref] Figure 3: Grand average ERP topographies of the P1, N170, and P3 components across... [/fig_ref].
# Discussion
In this study, we used ERPs and an RSVP task with SF-filtered happy, fearful, and neutral faces, to explore the time course of neural dynamics involved in the processing of facial expression conveying specific SF information.
The behavioral results show an advantage for the processing of emotional expressions (happy and fearful faces), which is in line with the common emotion effect evident in the prioritized processing of negative and positive stimuli during a deficit of attentional resources, presumably due to their biological significance [bib_ref] Affective picture processing: an integrative review of ERP findings, Olofsson [/bib_ref] [bib_ref] Distributed and interactive brain mechanisms during emotion face perception: evidence from functional..., Vuilleumier [/bib_ref] [bib_ref] A Network Model of the Emotional Brain, Pessoa [/bib_ref]. Furthermore, response accuracy on HSF stimuli was better than on LSF stimuli. Compared to previous research on sad and happy face categorization 9,44 , our results show no SF-emotion interaction in response accuracy. This difference may be due to the different presentation times of visual stimuli in different tasks, which can affect the accuracy of coarse/fine object recognition [bib_ref] Object Categorization in Finer Levels Relies More on Higher Spatial Frequencies and..., Ashtiani [/bib_ref] [bib_ref] Individual differences in spatial frequency processing in scene perception: the influence of..., Vanmarcke [/bib_ref]. More precisely, LSF information is more effective for the object recognition in a short presentation time and HSF information requires a longer exposure duration to influence participant performance. Considering that the presentation time of the target stimuli was invariant in the present RSVP task, our discussion therefore focuses on the neural time course of face processing rather than the behavioral results.
The ERP results show the time course of LSF and HSF information in the decoding of facial expression. The early visual-sensitive P1 component showed that LSF faces yielded larger neural response than did HSF faces. Considering that we carefully equalized the low-level characteristics of the images across emotion conditions, this result reveals that P1 is sensitive to SF information processing [bib_ref] Is the early modulation of brain activity by fearful facial expressions primarily..., Vlamings [/bib_ref] [bib_ref] Early ERP components differentially extract facial features: evidence for spatial frequency-and-contrast detectors, Nakashima [/bib_ref]. Unlike earlier research 34,40 , we did not find a negative bias in P1 for fearful faces, which indicated that fearful facial expressions cannot be distinguished from other expressions at the early stage in both LSF and HSF conditions. One possible explanation is that the SF filter impaired the configural and feature information of the face stimuli [bib_ref] Emotion perception is mediated by spatial frequency content, Kumar [/bib_ref] [bib_ref] Low-frequency filtering and the processing of local-global stimuli, Badcock [/bib_ref] [bib_ref] Spatial frequency and interference between global and local levels of structure, Lamb [/bib_ref] , and that the SF-filtered images are less salient and emotionally aroused than broadband images.
The face-sensitive N170 component showed that both fearful and happy faces elicited larger amplitudes than did neutral faces in the LSF condition, while happy but not fearful faces elicited larger amplitudes than did neutral faces in the HSF condition. In other words, the brain can distinguish happy and fearful from neutral facial information in the LSF condition, but can only distinguish happy from neutral information in the HSF condition. The LSF N170 result pattern is consistent with the second stage of Luo's model, while the HSF results are not. These results indicate that LSFs are important for the rapid detection of facial expressions, while HSFs do not represent efficient information for the fast detection of fear [bib_ref] Is the early modulation of brain activity by fearful facial expressions primarily..., Vlamings [/bib_ref]. Moreover, both fearful and happy face processing are mediated by LSF content at the early stage of emotion processing, which is in accordance with the idea that coarse SF contents of visual stimuli convey biologically significant information 32 . On the other hand, the rapid decoding of happy faces was also shown in HSF information processing, which argue against the common assumption that fast emotional response can be elicited only when LSF information of an emotional stimulus is presented [bib_ref] Distinct spatial frequency sensitivities for processing faces and emotional expressions, Vuilleumier [/bib_ref]. What is more, we find that happy face categorization was less impacted in the absence of certain SF channels, which could explain why happy faces are recognizable over a variety of different viewing distances, when there is either LSF or HSF information available [bib_ref] Smile through your fear and sadness transmitting and identifying facial expression signals..., Smith [/bib_ref] [bib_ref] Channel surfing in the visual brain, Sowden [/bib_ref].
The P3 component differentiated between the three types of emotional faces in both LSF and HSF conditions, which is in line with the third stage of Luo' model. This finding indicates that the brain distinguishes emotion information as positive or negative at later stages, irrespective of SF information. In other words, both happy and fearful emotion information are extracted from LSF and HSF contents at this stage.
Taken together, the divergent N170 effect and the similar P3 effect clearly indicate that the discrimination of different emotional facial expressions relies on both LSF and HSF information, but that these parallel or converging pathways process information at different speeds. These results provide evidence for the existence of parallel visual pathways in emotion processing, which is consistent with a recent proposal of multi-path processing of emotion [bib_ref] Emotion processing and the amygdala: from a 'low road' to 'many roads'..., Pessoa [/bib_ref].
Moreover, ERP components are also mediated by SF contents. At the early stage of emotional processing, as reflected by P1, LSF content elicited larger amplitudes than did HSF content. However, the results were reversed at later emotion processing stages: HSFs elicited larger N170 and P3 amplitudes than did LSFs. This SF effect in facial expression is consistent with the sequential processing (from coarse to fine) of SF information in visual object recognition [bib_ref] Time course of visual perception: coarse-to-fine processing and beyond, Hegde [/bib_ref] [bib_ref] Visual objects in context, Bar [/bib_ref] [bib_ref] Integrated model of visual processing, Bullier [/bib_ref].
# Conclusion
Through the use of an RSVP paradigm and the ERP technique, we find that the processing and detection of emotional information in a face is mediated by SF contents. LSF information elicits larger amplitudes in the early P1 component than does HSF information, while HSF information elicits larger amplitudes in the later N170 and P3 components than does LSF information. Furthermore, the face-sensitive N170 component is modulated by both facial expressions and SF channels, while P3 shows similar emotion processing patterns irrespective of SF information. More specially, the N170 effect indicates that the rapid encoding of fearful facial expression is primarily mediated by LSF information, while the fast detection of happy facial expression involves both LSF and HSF information.
# Methods
Participants. Thirty healthy undergraduate students (15 men and 15 women; mean age 19.6 years, SD = 1.59 years) from Chongqing University of Arts and Science were volunteered to participate in the experiment. All participants were healthy, right-handed, without psychiatric disorders history, and had normal or corrected normal vision. This study was approved by the Ethics Committee of Chongqing University of Arts and Sciences, and written informed consent was obtained from all participants before the experiment. All methods mentioned in this research were performed in accordance with the relevant guidelines and regulations.
Stimuli. Materials consisted of 48 face pictures (36 SF-filtered upright faces and 12 inverted faces) and 3 upright house stimuli. The original broadband face pictures were selected from the native Chinese Facial Affective Picture System (CFAPS), including 6 happy faces, 6 fearful faces, 6 neutral faces, and 12 inverted neutral faces. Please see the supplementary materials for the reason why we excluded the broadband images in this study. Furthermore, the reason why we chose only 36 target face stimuli for this study and the analysis of the training effect are also provided in the supplementary materials.
Valence and arousal ratings (ranging from 1: extremely unpleasant/extremely arousing to 9: extremely pleasant/not at all arousing) were provided by an independent sample (N = 24). The one-way repeated measures ANOVA of valence and arousal showed that the face pictures differed significantly (F 2,46 = 204.595, p < 0.001, η 2 p = 0.899; F 2,46 = 132.540, p < 0.001, η 2 p = 0.886). Valence ratings confirmed that fearful faces (3.208 ± 0.142) indeed elicited more unpleasant than did neutral (4.972 ± 0.049, p < 001) and happy faces (7.097 ± 0.171, p < 0.001), and happy faces elicited more pleasant than did neutral faces (p < 001). Arousal ratings indicated that the highest arousal was induced by fearful facial expression (6.403 ± 0.235), followed by happy faces (6.306 ± 0.265), and the least by neutral faces (2.285 ± 0.233) (the former two showed no significant difference, p = 1.000).
To generate the LSF and HSF face images, the original photograph were transformed to grayscale and equal size (260 × 300 pixels), normalized to equal luminance, and low-pass filtered at 2 cycles per degree or high-pass filtered at 6 cycles per degree, respectively. These cutoff parameters were based on previous literature 31, [bib_ref] ERP evidence for task modulations on face perceptual processing at different spatial..., Goffaux [/bib_ref] [bib_ref] Spatial content and spatial quantisation effects in face recognition, Costen [/bib_ref] [bib_ref] Angry and Mr. Smile: When categorization flexibly modifies the perception of faces..., Schyns [/bib_ref] [bib_ref] Spatial scale contribution to early visual differences between face and object processing, Goffaux [/bib_ref] [bib_ref] Attention to low-and high-spatial frequencies in categorizing facial identities, emotions and gender..., Deruelle [/bib_ref]. Procedure. To investigate the time course of LSF or HSF information in decoding facial expression, we chose a dual-target RSVP paradigm [bib_ref] Three stages of facial expression processing: ERP study with rapid serial visual..., Luo [/bib_ref] [bib_ref] Three stages of emotional word processing: an ERP study with rapid serial..., Zhang [/bib_ref] [bib_ref] Emotional noun processing: an ERP study with rapid serial visual presentation, Yi [/bib_ref] [bib_ref] The time course of emotional picture processing: an event-related potential study using..., Zhu [/bib_ref] for this study. Participants were seated in a dimly lit, quiet room and were presented with four experimental blocks of 120 trials each. As shown in [fig_ref] Figure 4: Examples of the stimuli and the RSVP paradigm used in this experiment [/fig_ref] , each trial began with a white crosshair and a blue crosshair at the center of the screen, each presented for 500 ms, following by 14 pictures (including two target stimuli) with a stimulus-onset asynchrony (SOA) of 116 ms and no blank inter-stimulus interval (ISI). The first target stimulus (T1) emerged randomly and equiprobably at the fifth, sixth, or seventh position, nextly, two distracting stimuli appeared, then the second target stimulus (T2, SOA = 232 ms), and other distracting stimuli (inverted face pictures) items were presented. All stimuli were presented in the center of the screen. To obtain the ERP components purely elicited by T2, we designed a baseline condition (a blank black screen) to remove the superposed electrical activity elicited by the distractors. The T1 (task: recognize the house that was memorized before the experiment, press key "1", "2", "3", correspondingly) was one of the three house pictures and the T2 (task: discriminate the valence in the picture, press key "1" if T2 was positive, key "2" when neutral, key "3" when negative, key "4" if T2 was absent) was one of the 36 filtered face pictures. The question would not disappear until participants pressed the index key or until 3000 ms elapsed. All subjects were required to respond to the two questions with their right hand. Stimulus presentation was controlled by E-Prime 2.0 software (Psychology Software Tools Inc., Pittsburgh, PA, USA).
Electrophysiological recording and analysis. During task performance, the Electroencephalogram (EEG) (sampling rate of 500 Hz) was recorded from a 64-channel amplifier using a standard 10-20 system (Brain Products, Gilching, Germany), with the reference on right and left mastoids. The vertical electrooculogram (EOG) was recorded from electrodes placed blow the left eye. All electrode impedances (EEG and EOG) were maintained below 5 kΩ.
The EEG data were analyzed offline for ERPs using Brain Vision Analyzer software (Brain Products, Munich, Germany). We employed an off-line re-reference to the averaged reference and filtered the data with a 30 Hz, 24 dB/octave low pass filter. ERP epochs were extracted beginning 200 ms before and ending 1000 ms after T2 stimulus, and trials were accepted only if the answer of both T1 and T2 were correct. The 200 ms pre-stimulus was used as baseline to each epoch. Thereafter, trials with EEG voltages exceeding ±75 μV (relative to baseline) were excluded from analysis. Averaged ERPs were computed for six stimulus conditions [Emotion (happy, neutral, fear) × SF (LSF, HSF)]. According to the topographical distribution and previous research [bib_ref] Three stages of facial expression processing: ERP study with rapid serial visual..., Luo [/bib_ref] [bib_ref] Three stages of emotional word processing: an ERP study with rapid serial..., Zhang [/bib_ref] , the P1, N170, and P3 components were chosen for statistical analysis in the present study. Based on the approach of collapsed localizer [bib_ref] How to get statistically significant effects in any ERP experiment (and why..., Luck [/bib_ref] , the data were collapsed across all the conditions to extract the time windows and electrode sites for each component. Time windows for amplitude calculation were centered at the peak latencies of the collapsed waveforms, with a shorter window length for earlier ERP components and a longer length for later components. More specifically, the mean amplitude of P1 component (time window: 118-148 ms) was measured and analyzed at the following six electrode sites (O1, O2, PO3, PO4, POz, and Oz); P7, P8, PO7, and PO8 were selected for statistical analysis of the N170 component (time window: 240-280 ms); P3 (time window: 426-476 ms) were analyzed at CPz, Pz, P3, P4, CP3, and CP4 electrode sites. Mean amplitudes of each component were subject to a three-way repeated measures analyses of variance (ANOVAs; with Greenhouse-Geisser corrections) with SF (two levels: HSF, LSF), emotion (three levels: happy, neutral, fear), and electrode position. Significant ANOVA effects were followed by pairwise comparisons to contrast the effects of individual conditions (using the Bonferroni method).
[fig] Figure 1: Grand average ERPs for LSF happy, LSF neutral, LSF fearful, HSF happy, HSF neutral, and HSF fearful faces recorded at the indicated electrode sites. The gray area represents the time window of each component. Scientific RepoRts | (2018) 8:2383 | DOI:10.1038/s41598-018-20467-1 [/fig]
[fig] Figure 2: The interaction effect of facial expression by SF on the N170 amplitude. Bars represent the standard error of the mean. *p < 0.05; **p < 0.01. [/fig]
[fig] Figure 3: Grand average ERP topographies of the P1, N170, and P3 components across two SFs and three emotion conditions. Scientific RepoRts | (2018) 8:2383 | DOI:10.1038/s41598-018-20467-1 [/fig]
[fig] Scientific: RepoRts | (2018) 8:2383 | DOI:10.1038/s41598-018-20467-1 Filtering was performed in Matlab (Mathworks, Natick, MA), using a set of two order Butterworth filter. After the filtering, we used SHINE toolbox 57 to ensure all the filtered image sets were equaled on luminance [92 (0.07) on a 0-255 scale] and contrast [64(0.13)]. All stimuli were presented on a 17-inch LCD monitor (resolution: 1280 × 800; refresh rate: 60 Hz), 80 cm from the participants' eyes, and with the viewing angle as 7.6 × 9°. [/fig]
[fig] Figure 4: Examples of the stimuli and the RSVP paradigm used in this experiment. (A) Each trial contained 12 inverted faces (IF) and two target stimuli (T1 and T2), followed by two questions (Q1 and Q2) at the end of each trial. The T1 emerged randomly and equiprobably at the fifth, sixth, seventh position, T1 and T2 were presented within an interval of 232 ms. (B) Example for the two questions in each trial. [/fig]
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10.3892/or.2014.3544
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s2orc_pubmed_articles
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Suppression of mucin 2 promotes interleukin-6 secretion and tumor growth in an orthotopic immune-competent colon cancer animal model
Mucin 2 (MUC2) is the major secreted mucin of the large intestine and is expressed by adenomas and mucinous carcinomas. Since colon cancer is associated with a proinflammatory microenvironment and dysregulated MUC2 expression, the aim of this study was to characterize the effects of MUC2 gene expression in colon tumor progression using colonic cancer cells. CT26 colon cancer cells were stably transfected with MUC2 siRNA (MUC2 RNAi) or a control construct containing a nonspecific sequence (scrambled RNAi). Expression of MUC2 was significantly decreased in the MUC2 RNAi cell clones. Although MUC2 suppression did not affect the cell growth of colon cancer cells in vitro, MUC2 knockdown promoted tumor growth in an orthotopic colon cancer model in vivo. MUC2 silencing also increased interleukin (IL)-6 secretion by colon cancer cells. IL-6 neutralization attenuated tumor formation by MUC2 RNAi cells; it also increased CD8 T cell infiltration into the peritoneum. Taken together, to the best of our knowledge, this is the first study indicating that the immune response to cancer cells plays an important role in tumor growth regulated by MUC2. Furthermore, given the effects of MUC2 on IL-6 secretion, its targeting may represent a potentially useful strategy to treat colonic carcinomas.
# Introduction
Mucins are the major glycoproteins of the gastrointestinal tract; secretory mucin 2 (MUC2) is the main component of the protective mucus layer. Although MUC2 expression is decreased in colorectal adenocarcinoma, colonic mucinous carcinomas are characterized by overexpression or ectopic expression of MUC2 [bib_ref] Overexpression or ectopic expression of MUC2 is the common property of mucinous..., Hanski [/bib_ref]. In contrast, the suppressive effect of MUC2 in colorectal cancer (CRC) was demonstrated in MUC2-null mice [bib_ref] Colorectal cancer in mice genetically deficient in the mucin Muc2, Velcich [/bib_ref] , suggesting that it has a protective effect in the colon. Although MUC2 overexpression was specifically associated with mucinous carcinoma, its effects on tumor progression remain unclear.
Intestinal inflammation is a crucial component of tumor development and metastasis [bib_ref] Inflammation and cancer, Coussens [/bib_ref] , and polymorphonuclear neutrophils and macrophages may affect tumor development and progression in CRC [bib_ref] Inflammation and cancer, Coussens [/bib_ref]. The effects of interleukin (IL)-6 on tumor growth are multifaceted. In previous studies, tumorinfiltrating macrophages (TIMs) were found to express a high level of IL-6 (4,5), which is implicated in CRC carcinogenesis. In prostatic carcinoma, IL-6 acts as a growth factor [bib_ref] Elevated levels of circulating interleukin-6 and transforming growth factor-beta 1 in patients..., Adler [/bib_ref]. In CRC patients, IL-6 levels were significantly higher than these levels in healthy controls [bib_ref] Prognostic significance of circulating IL-10 and IL-6 serum levels in colon cancer..., Galizia [/bib_ref] and were correlated with tumor stage, tumor size, liver metastasis and poor survival [bib_ref] Serum levels of cytokines in patients with colorectal cancer: possible involvement of..., Ueda [/bib_ref] [bib_ref] Serum interleukin-6 level reflects the tumor proliferative activity in patients with colorectal..., Kinoshita [/bib_ref] [bib_ref] Serum interleukin-6 levels reflect the disease status of colorectal cancer, Chung [/bib_ref]. Moreover, macrophage secretion of IL-6 decreased MUC2 expression in a human colon cancer cell line through phosphorylation of STAT3 [bib_ref] Macrophagederived interleukin-6 up-regulates MUC1, but down-regulates MUC2 expression in the human colon..., Li [/bib_ref]. IL-6 can also regulate the immune microenvironment surrounding the tumor [bib_ref] Inflammation amplifier, a new paradigm in cancer biology, Atsumi [/bib_ref]. However, the effects of MUC2 expression on IL-6 secretion by colon cancer cells have not been determined. Therefore, we employed RNA interference (RNAi) to suppress MUC2 expression in mouse colon carcinoma cells, and evaluated the effects of MUC2 suppression on IL-6 production and tumor growth.
# Materials and methods
Cell culture. CT26 colon carcinoma cells (BALB/c mouse origin) were kindly provided by Dr M.D. Lai of the National Cheng Kung University (Tainan, Taiwan). CT26 cells were Suppression of mucin 2 promotes interleukin-6 secretion and tumor growth in an orthotopic immune-competent colon cancer animal model maintained in high glucose Dulbecco's modified Eagle's medium (DMEM) (gibco-Invitrogen) containing 10% FBS (HyClone Laboratories, Logan, UT, USA) and 1% penicillin/streptomycin. All cell lines were stored in a humidified 37˚C incubator with 5% CO 2 .
Mice. Eight-week-old BALB/c (H 2 d ) mice and nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mice, NOD.CB17-PRKDC, were purchased from the Laboratory Animal Center of National Cheng Kung University and were maintained under pathogen-free conditions. All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of the National Cheng Kung University.
RNA interference. siRNA targeting mouse MUC2 was constructed within the pHsU6 vector as described previously (15) using the following target sequences: 5'-GCTATGT GCCTGGCTCTAA-3' (RNAi-1), 5'-GCAACAAGTGCACC TT CTT-3' (RNAi-2) and 5'-GCTGCCCTACAAACTGTTT-3' (RNAi-3). Cells were cotransfected with the pHsU6 vectors containing the shRNA target sequences, a nonspecific sequence (scrambled shRNA), and pCMV-neo using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). The stable transfectants were selected with G418 (Calbiochem, San Diego, CA, USA). We established three clones of MUC2-suppressed cells, including CT26 MUC2 RNAi-1, CT26 MUC2 RNAi-2 and CT26 MUC2 RNAi-3. The clonal cell lines were maintained in complete medium with 250 µg/ml G418. To monitor the efficacy of MUC2 silencing, the expression of MUC2 in CT26 stable transfectants was analyzed by western blotting.
Western blot analysis. A MUC2-specific antibody was obtained by inoculating rabbits with the mouse MUC2 peptide, CVRTRRSSPRFLgRK (c-terminal position 911-924). Peptides used in this study were synthesized and purified by Genemed Synthesis (San Antonio, TX, USA). Total cell lysates were prepared and analyzed by SDS-PAgE as previously described [bib_ref] Establishment of an orthotopic transplantable gastric cancer animal model for studying the..., Shan [/bib_ref]. Immunodetection was performed using a horseradish peroxidase (HRP)-based SuperSignal Chemiluminescent Substrate (Pierce, Rockford, IL, USA). For quantification, the bands were measured by the AlphaImager 2200 system (Alpha Innotech, San Leandro, CA, USA) and normalized by the density obtained for β-actin.
Cell proliferation assay. Cells (1x10 3 /well) were seeded in quadruplicate onto 96-well plates and incubated at 37˚C under 5% CO 2 . At 24, 48 and 72 h, viable cell numbers were measured using the CellTiter 96 Aqueous One Solution Cell Proliferation assay (Promega, Madison, WI, USA) according to the manufacturer's instructions. The proliferation curves were constructed by calculating the mean value of absorbance at 490 nm with an Ultra Multifunctional Microplate Reader (Tecan, Durham, NC, USA).
Orthotopic model of colon adenocarcinoma. Eight-week-old female BALB/c mice were anesthetized by intraperitoneal injection of zoletil (50 mg/kg; Parnell Laboratories, Alexandria, NSW, Australia) and xylazine (10 mg/kg; Troy Laboratories, Glendenning, NSW, Australia). After making a small median abdominal incision in the mice under anesthesia, cecums were exteriorized and 1x10 6 cells (scramble RNA, MUC2 RNAi-1, RNAi-2 or RNAi-3 tumor cells) in 0.05 ml of PBS were injected into the middle wall of the greater curvature of the cecal wall using a 1-cc U-100 insulin disposable syringe (Becton-Dickinson, Franklin Lakes, NJ, USA). Bipolar (ICC, ER BE) coagulation was used when the needle was removed from the wall of the cecum. Each cecum was then returned to the peritoneal cavity, and the abdominal wall and skin were closed with a Dexon 4-0 surgical suture (Ethicon, Bridgewater, NJ, USA). The mice were sacrificed 11-17 days after the tumor cell implantation or when moribund. The whole cecums from mice were excised, washed, removed of remaining diet, and weighed. For IL-6 antibody neutralization, BALB/c mice were pretreated with an injection of 100 µg/mouse of IL6-neutralizing antibody or isotype control antibody (both from BD Pharmingen, San Diego, CA, USA) 2 days prior to tumor cell injection. After tumor cell injection, mice were treated with IL-6 neutralizing antibody or control antibody every 3 days by intraperitoneal injection as previously described (17).
Cytokine array and enzyme-linked immunosorbent assay (ELISA). To collect culture supernatants, scrambled control and CT26 MUC2 RNAi-1 cells were cultured for 48 h in serum-free medium after which the supernatants were collected and measured using Mouse Cytokine Antibody Array II kit (RayBiotech, Norcross, GA, USA) and an IL-6 ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturers' instructions. The signal intensity of the array was scanned and quantified by densitometry using an AlphaImager 2200 system (Alpha Innotech) and normalized to the positive control.
Flow cytometric analysis. To characterize the immune cells of the peritoneal fluid in vivo, peritoneal fluid was isolated and subjected to flow cytometry as previously described [bib_ref] Establishment of an orthotopic transplantable gastric cancer animal model for studying the..., Shan [/bib_ref] [bib_ref] Polarization of tumorassociated neutrophil phenotype by TgF-beta: 'N1' versus 'N2' TAN, Fridlender Zg [/bib_ref]. The following fluorescent-labeled antibodies were purchased from BD Bioscience: rat anti-Ly6G-FITC, anti-CD4-PE, anti-CD8-PE, anti-CD11b-PE and isotype controls (FITC and PE). Data analysis was evaluated using a flow cytometer (FACScan; Becton-Dickinson).
Statistical analysis. Data are expressed as means ± SD. Statistical analyses were performed using the Student's t-test. A P-value <0.05 was considered significant for all comparisons.
# Results
## Suppression of muc2 expression in mouse ct26 colon carcinoma cells by rnai.
To study the role of MUC2 in colon cancer, we used shRNA to suppress the expression of MUC2 in the CT26 murine colon cancer cell line, which expresses high levels of MUC2. Three shRNAs targeting different sites on MUC2 were transfected into CT26 cells, and the stable transfectants were established by G418 selection. The vector control was established by transfecting CT26 cells with an empty vector, and the scrambled RNA (SR) transfectant contains a different shRNA sequence with the same base composition of RNAi-1. Stable cells expressing three RNAi target sequences were established: MUC2 RNAi-1, MUC2 RNAi-2 and MUC2 RNAi-3 [fig_ref] Figure 1: Mucin 2 [/fig_ref]. Western blot analysis confirmed that the SR did not alter MUC2 protein levels [fig_ref] Figure 1: Mucin 2 [/fig_ref]. However, MUC2 expression was significantly decreased in the MUC2 RNAi-1, MUC2 RNAi-2 and MUC2 RNAi-3 CT26 cells [fig_ref] Figure 1: Mucin 2 [/fig_ref].
## Muc2 suppression does not affect cell proliferation in vitro.
To determine the effects of MUC2 suppression on cell growth, cells were seeded at a low density, and growth rates were determined for the parental cell line and its derived cell clones at 24, 48 and 72 h. MUC2 silencing did not inhibit the in vitro proliferation of CT26 cells [fig_ref] Figure 1: Mucin 2 [/fig_ref].
## Effect of muc2 suppression on ct26 tumor growth in vivo.
To investigate the role of MUC2 in a native tumor environment, we examined the effects of MUC2 knockdown in an orthotopic immune-competent animal model. One million SR control or MUC2 shRNA-expressing cells were implanted orthotopically in BALB/c mice, and macroscopic tumor nodules indicative of tumor formation were detected [fig_ref] Figure 2: Mucin 2 [/fig_ref]. The tumor weight of mice injected with SR cells was significantly lower than the tumor weights of the mice injected with RNAi-1 [fig_ref] Figure 2: Mucin 2 [/fig_ref] , RNAi-2 [fig_ref] Figure 2: Mucin 2 [/fig_ref] and RNAi-3 cells [fig_ref] Figure 2: Mucin 2 [/fig_ref] at day 17. These results demonstrated that knockdown of MUC2 promoted the tumor growth of colon cancer cells in vivo, suggesting that MUC2 plays an important role in the tumorigenicity of colon cancer.
Since the growth of the MUC2 shRNA transfectants was not altered in vitro, we hypothesized that the changes in tumor growth in vivo may result from altered tumor-microenvironment interaction. To assess the potential immunological effects of MUC2 on tumor progression, tumor growth was measured in the immune-deficient NOD/SCID mice. In the absence of NK cells, macrophages, B and T cells, the mean tumor mass of mice implanted with the SR cells was similar to the mean tumor mass of mice implanted with MUC2 RNAi-1 cells [fig_ref] Figure 3: In vivo orthotopic growth of CT26-scramble RNA [/fig_ref] , suggesting that the effects of MUC2 were dependent upon the presence of a competent immune system.
## Suppression of muc2 increases il-6 secretion by ct26 colon cancer cells.
given the importance of a functional immune system for the effects of MUC2 on tumor growth, we further investigated whether cancer cell-secreted cytokines were involved in the tumor microenvironment. Specifically, the cytokine profile consisting of 32 different factors in the conditioned medium of SR and MUC2 RNAi-1 cells was compared 48 h after serum-deprivation. Conditioned medium from MUC2 RNAi-1 cells had significantly increased IL-6, regulated on activation, normal T cell expressed and secreted (RANTES) and granulocyte colony-stimulating factor (gCSF) expression and decreased vascular endothelial growth factor (VEGF) expression compared to the SR conditioned medium [fig_ref] Figure 4: Cytokine secretion into condition medium after mucin 2 [/fig_ref]. IL-6 secretion by SR and MUC2 RNAi-1 cells 48 h after serum-deprivation was further quantified by ELISA. MUC2 RNAi-1 and MUC2 RNAi-2 cells secreted significantly higher levels of IL-6 than SR cells [fig_ref] Figure 4: Cytokine secretion into condition medium after mucin 2 [/fig_ref]. Therefore, MUC2 expression by colon cancer cells alters IL-6 secretion.
## Il-6 neutralization attenuates tumor formation by ct26 muc2 knockdown cells.
To confirm the biological effect of IL-6 in vivo, we tested whether an IL-6 neutralization antibody could inhibit tumorigenic growth. Mice pretreated with either an IL-6-neutralizing antibody or control IgG antibody were injected with MUC2 RNAi-1 cells, and then treated every 3 days with either IL-6 or control antibody. The growth of MUC2 RNAi-1 tumors was reduced with the IL-6 neutralizing antibodies on day 11 [fig_ref] Figure 5: Mucin 2 [/fig_ref] and B) and day 14 [fig_ref] Figure 5: Mucin 2 [/fig_ref]. These experiments suggest that MUC2 knockdown enhanced IL-6 secretion and promoted tumor growth.
## Il-6 neutralization enhances the cd8-mediated immune response in ct26 cells after muc2 silencing.
To elucidate the possibility that MUC2 regulates IL-6 secretion and induces a local immune response in colon cancer cells, we analyzed Ly6G + CD11b + neutrophil, Ly6G -CD11b + macrophage, CD4 T cell and CD8 T cell levels in the peritoneal fluid of mice with MUC2 RNAi-1 cell tumors treated with control or IL-6 neutralization antibodies. Although there was no significant difference in the proportion of Ly6g + CD11b + neutrophils, Ly6G -CD11b + macrophages and CD4 T cells in the peritoneal fluid between the two groups on day 14 [fig_ref] Figure 7: Interleukin-6 [/fig_ref] , the peritoneal fluid of the MUC2 RNAi-1 tumor-bearing mice treated with IL6-neutralizing antibody had a significantly greater proportion of CD8 T cells than MUC2 RNAi-1 tumor-bearing mice treated with the control antibody on day 14 [fig_ref] Figure 7: Interleukin-6 [/fig_ref]. Thus, an IL-6 neutralizing antibody could inhibit the in vivo growth of MUC2 knockdown tumors, increasing CD8 T cell influx in the peritoneal cavity.
# Discussion
In the present study, we examined the effects of MUC2 on tumor cell growth, IL-6 secretion and the immune response in colon cancer. To the best of our knowledge, this is the first study to demonstrate that downregulation of MUC2 expression enhances IL-6 secretion as well as tumor growth. Thus, MUC2 had a protective effect during tumorigenesis. This observation is consistent with previous studies in which loss of MUC2 expression was associated with progression and metastasis in CRC [bib_ref] Significance of MUC1 and MUC2 mucin expression in colorectal cancer, Ajioka [/bib_ref] [bib_ref] MUC2 gene expression is found in noninvasive tumors but not in invasive..., Yonezawa [/bib_ref] [bib_ref] Expression of MUC1 and MUC2 mucins and relationship with cell proliferative activity..., Li [/bib_ref] [bib_ref] Mucin production by human colonic carcinoma cells correlates with their metastatic potential..., Bresalier [/bib_ref] [bib_ref] Expression of MUC2-mucin in colorectal adenomas and carcinomas of different histological types, Blank [/bib_ref]. Moreover, CRC patients with a high MUC2/carcinoembryonic antigen (CEA) mRNA ratio in their lymph nodes had a significantly better prognosis than those with a low ratio [bib_ref] Lymph node CEA and MUC2 mRNA as useful predictors of outcome in..., Ohlsson [/bib_ref]. In addition, MUC2-positive CRC was found to be correlated with reduced disease recurrence and prolonged survival as well as a low incidence of liver and nodal metastasis [bib_ref] Loss of E-cadherin and MUC2 expressions correlated with poor survival in patients..., Kang [/bib_ref] [bib_ref] Loss of MUC2 expression predicts disease recurrence and poor outcome in colorectal..., Elzagheid [/bib_ref].
In previous studies, MUC2 suppressed inflammation in the intestinal tract and inhibited intestinal tumorigenesis [bib_ref] Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic..., Van Der Sluis [/bib_ref]. In the present study, the secretion of IL-6, RANTES and GCSF was increased in the MUC2-knockdown tumor cells in comparison to the SR tumor cells. Both IL-6 and RANTES were associated with tumor progression, metastasis and macrophage activation in previous studies [bib_ref] Breast cancer cellderived cytokines, macrophages and cell adhesion: implications for metastasis, Eichbaum [/bib_ref] [bib_ref] Elevated expression of the CC chemokine regulated on activation, normal T cell..., Luboshits [/bib_ref] [bib_ref] Correlation of tissue and plasma RANTES levels with disease course in patients..., Niwa [/bib_ref] [bib_ref] Serum interleukin-6 levels correlate to tumor progression and prognosis in metastatic breast..., Gj [/bib_ref] [bib_ref] Circulating interleukin-6 predicts survival in patients with metastatic breast cancer, Salgado [/bib_ref]. In particular, IL-6 is an inflammatory cytokine released by T cells, macrophages and several cancer cell types [bib_ref] Elevated levels of circulating interleukin-6 and transforming growth factor-beta 1 in patients..., Adler [/bib_ref] [bib_ref] The biology of interleukin-6, Kishimoto [/bib_ref]. IL-6 production by tumor cells may act as an autocrine and/ or paracrine growth factor during carcinogenesis, and various studies have proposed promising targets for CRC therapy using anti-IL-6 and anti-IL-6R antibodies as well as soluble gp130Fc (sgp130Fc) and selective small-molecule JAK inhibitors that suppress the IL-6/STAT3 pathway [bib_ref] Therapeutic strategies for the clinical blockade of IL-6/gp130 signaling, Jones [/bib_ref]. The increased secretion of IL-6 by MUC2-knockdown tumor cells and enhanced tumor growth observed in the present study suggest that the protective mechanisms of MUC2 in colon cancer may be associated with its effects on suppressing the IL-6/STAT3 signaling pathway.
Inflammatory cells of the innate and adaptive immune system may affect different stages of tumor progression or metastasis in colon cancer. Since MUC2 silencing increased IL-6 secretion, we analyzed neutrophil, macrophage, CD4 T cell and CD8 T cell levels in the peritoneal fluid of mice. In this study, mice bearing MUC2-knockdown tumors and treated with IL-6 neutralizing antibodies displayed increased CD8 T cell infiltration into the peritoneal fluid. To the best of our knowledge, this is the first study to demonstrate that the immunoregulatory effects of MUC2 may represent a novel therapeutic method for treating cancer. Alternatively, tumorassociated antigens (TAAs) have served as a target for CTL immunotherapy as DNA vaccines have a therapeutic effect on established tumors through activation of a CD8 T cell-dependent pathway [bib_ref] A novel cancer therapy by skin delivery of indoleamine 2,3-dioxygenase siRNA, Yen [/bib_ref] [bib_ref] Therapeutic HER2/Neu DNA vaccine inhibits mouse tumor naturally overexpressing endogenous neu, Lin [/bib_ref]. Thus, it is intriguing that MUC2 may induce a systemic antitumor immune response and may have therapeutic value in the treatment of IL-6-secreting cancers.
In conclusion, the therapeutic effects of MUC2 may be mediated at least in part by decreasing IL-6 secretion, inhibiting tumorigenicity, and inducing CD8 T cells in vivo.
[fig] Figure 1: Mucin 2 (MUC2)-specific shRNA decreases MUC2 protein expression in CT26 cell clones. (A) RNAi targets on the coding sequence of MUC2. (B) MUC2-specific shRNA decreased MUC2 protein expression in the MUC2 RNAi-derived cell clones as detected by western blot analysis. β-actin was used as an internal control. * P<0.05, when compared to a nonspecific sequence scrambled RNA (SR). P, parental cells; RNAi-1, RNAi-2 and RNAi-3, MUC2-specific shRNA-expressing cells. Results were obtained from three independent experiments. (C) MUC2 silencing in CT26 cell clones did not inhibit cell proliferation in vitro. The mean value ± SD of absorbance at 490 nm is shown for three independent experiments. [/fig]
[fig] Figure 2: Mucin 2 (MUC2) silencing in CT26 cell clones enhances tumorigenicity in vivo. (A) The macroscopic appearance of the tumor masses after the orthotopic injection of scramble RNA (SR) and MUC2 RNAi-1 cell clones. The tumor weights of (A) MUC2 RNAi-1, (B) MUC2 RNAi-2 and (C) MUC2 RNAi-3 tumor-bearing mice were significantly increased on day 17 as compared to the tumor weights of the SR tumor-bearing mice. * P<0.05 compared to SR mice. Results are expressed as the mean tumor weight, and the data are shown for two independent experiments. [/fig]
[fig] Figure 4: Cytokine secretion into condition medium after mucin 2 (MUC2) silencing in CT26 cell clones. (A) Cytokine array of conditioned media from scrambled RNA (SR) control (left panel) and MUC2 RNAi-1 cells (right panel) after 48 h in culture. (B) Cytokine assay of the conditioned media shows that the secretion of interleukin-6 (IL-6), regulated on activation, normal T cell expressed and secreted (RANTES) and granulocyte colony-stimulating factor (gCSF) was increased while secretion of vascular endothelial growth factor (VEgF) was decreased in the RNAi-1 group as compared with the SR group. Data are shown for three independent experiments. (C) Conditioned medium from CT26 parental cells, SR, MUC2 RNAi-1 and MUC2 RNAi-2 cells was analyzed for IL-6 secretion using ELISA. Results are expressed as the mean pg/ml ± SD, and the data are shown for three independent experiments. [/fig]
[fig] Figure 3: In vivo orthotopic growth of CT26-scramble RNA (SR) and CT26 mucin 2 (MUC2) RNAi-1 tumors in non-obese diabetes/severe combined immunodeficiency (NOD/SCID) mice. The macroscopic appearance of the tumor masses after the orthotopic injection of the SR and MUC2 RNAi-1 cell clones into NOD/SCID mice. [/fig]
[fig] Figure 5: Mucin 2 (MUC2)-knockdown tumor growth was retarded with interleukin-6 (IL-6) neutralizing antibody treatment. (A) The macroscopic appearance of the tumor masses after the orthotopic injection of MUC2 RNAi-1 cell clones in mice injected every 3 days with 100 µg of either the Igg1 control antibody (RNAi-1-C) or the IL-6 neutralizing antibody (RNAi-1/IL-6-N). The mice were sacrificed (B) 11 days or (C) 14 days after the tumor cell implantation and tumor weight was determined. [/fig]
[fig] Figure 7: Interleukin-6 (IL-6) neutralization increases the proportion of CD8 T cells in the peritoneal fluid of mice bearing mucin 2 (MUC2) RNAi-1 tumors. MUC2 RNAi-1 tumor-bearing mice were injected every 3 days with 100 µg of either the Igg1 control antibody (RNAi-1-C) or the IL-6 neutralizing antibody (RNAi-1/IL-6-N). CD4 and CD8 T cells were evaluated in the peritoneal fluid of RNAi-1-C and RNAi-1/IL-6-N mice. Flow cytometry was performed on the peritoneal fluid from (A and C) RNAi-1-C and (B and D) RNAi-1/IL-6-N mice. The numbers shown are the percentage of total cells. Igg1 control antibody, RNAi-1-C; IL6-neutralizing antibody, RNAi-1/IL-6-N. The percentages of (E) CD4 and (F) CD8 T cells isolated from the peritoneal fluid of RNAi-1-C and RNAi-1/IL-6-N mice were analyzed by flow cytometry 14 days after tumor cell implantation. [/fig]
[fig] Figure 6: Ly6G + CD11b + and Ly6G -CD11b + cells were evaluated in the peritoneal fluid of RNAi-1-C and RNAi-1/interleukin-6 (IL-6)-N mice. Flow cytometry was performed on the peritoneal fluid from (A) RNAi-1-C and (B) RNAi-1/IL-6-N mice. The numbers shown are the percentage of total cells. R2, Ly6g -CD11b + macrophages; R4, Ly6G + CD11b + neutrophils; IgG1 control antibody, RNAi-1-C; IL6-neutralizing antibody, RNAi-1/IL-6-N. The percentages of (C) neutrophils (Ly6G + CD11b + ) and (D) macrophages (Ly6G -CD11b + ) isolated from the peritoneal fluid of RNAi-1-C and RNAi-1/IL-6-N mice were analyzed by flow cytometry 14 days after the tumor cell implantation. [/fig]
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10.1093/nar/gky622
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CCBY
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6144786
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30007325
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s2orc_pubmed_articles
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DNA binding with a minimal scaffold: structure–function analysis of Lig E DNA ligases
DNA ligases join breaks in the phosphodiester backbone of DNA by catalysing the formation of bonds between opposing 5 P and 3 OH ends in an adenylationdependent manner. Catalysis is accompanied by reorientation of two core domains to provide access to the active site for cofactor utilization and enable substrate binding and product release. The general paradigm is that DNA ligases engage their DNA substrate through complete encirclement of the duplex, completed by inter-domain kissing contacts via loops or additional domains. The recent structure of a minimal Lig E-type DNA ligase, however, implies it must use a different mechanism, as it lacks any domains or loops appending the catalytic core which could complete encirclement. In the present study, we have used a structure-guided mutagenesis approach to investigate the role of conserved regions in the Lig E proteins with respect to DNA binding. We report the structure of a Lig-E type DNA ligase bound to the nicked DNA-adenylate reaction intermediate, confirming that complete encirclement is unnecessary for substrate engagement. Biochemical and biophysical measurements of point mutants to residues implicated in binding highlight the importance of basic residues in the OB domain, and inter-domain contacts to the linker.
# Introduction
DNA ligases catalyze the formation of phosphodiester bonds between adjacent 5 P and 3 OH ends in the backbone of double-stranded DNA and have a conserved core structure comprising the catalytic adenylation (AD) domain and an oligonucleotide-binding (OB) domain joined by a flexible linker region [bib_ref] DNA ligases: structure, reaction mechanism, and function, Tomkinson [/bib_ref]. Catalysis proceeds in a three-step reaction, with step 1 involving nucleophilic attack, by a conserved lysine residue on the ␣-phosphate of an adenylated cofactor, which can be ATP or NAD+, generating a covalent enzyme-adenylate that is poised to bind the nicked DNA substrate. In step 2, the adenosine monophosphate (AMP) moiety is transferred to the 5 phosphate of the nicked DNA activating it for attack by the 3 OH in step 3 forming a new phosphodiester bond that seals the break across the DNA backbone. This catalytic process is accompanied by large-scale reorientations of the AD and OB domains to expose the catalytic site for cofactor and substrate binding and clamp around the DNA during steps 2 and 3 of catalysis [bib_ref] DNA ligases: structure, reaction mechanism, and function, Tomkinson [/bib_ref]. In most cases, complete encirclement of the duplex is completed through participation of appending DNA binding domains, which are either N-terminal in the case of ATP-dependent DNA ligases or C-terminal in the NAD-dependent form [bib_ref] DNA ligases: progress and prospects, Shuman [/bib_ref]. Alternately, smaller viral ligases for example the Chlorella virus (ChlV-Lig) and presumably T7 ATP-dependent DNA ligases use flexible loops which become ordered upon DNA interaction [bib_ref] Structural basis for nick recognition by a minimal pluripotent DNA ligase, Nair [/bib_ref]. In some cases additional subunits are required for optimal activity, such as the Ku end-binding protein which activate the non-homologous end-joining bacterial ligase Lig D [bib_ref] Bacterial DNA repair by non-homologous end joining, Shuman [/bib_ref]. For the larger ligases, this DNA binding domain is indispensable for productive substrate binding and ligase activity [bib_ref] Human DNA ligase I completely encircles and partially unwinds nicked DNA, Pascal [/bib_ref]. Likewise, mutation or deletion of the Chlorella virus ligase latch decreases DNA affinity and activity [bib_ref] Analysis of the DNA joining repertoire of Chlorella virus DNA ligase and..., Odell [/bib_ref].
The minimal Lig E-type DNA ligases comprise a clade of phylogenetically distinct ATP-dependent DNA ligases found almost exclusively in proteobacteria [bib_ref] Analysis of the distribution and evolution of the ATP-dependent DNA ligases of..., Williamson [/bib_ref]. The majority of these enzymes lack any appending domains and possess a predicted N-terminal signal sequence for localization in the periplasm, the removal of which increases both protein stability and activity. The recently-determined crystal structure of the enzyme-adenylate of Lig E from Psychromonas sp. strain SP041 (Psy-Lig) revealed a compact structure with no flexible loops or other elements that would be sufficient to surround the circumference of the DNA helix [bib_ref] Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding..., Williamson [/bib_ref]. Despite lacking the known binding determinants of larger ATP-dependent DNA ligases, Psy-Lig and other Lig Es from Neisseria meningitidis, and Haemophilus influenza are able to seal phosphorylated double-strand DNA substrates at comparable rates to their larger counterparts [bib_ref] Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding..., Williamson [/bib_ref] [bib_ref] Characterization of an ATP-dependent DNA ligase encoded by Haemophilus influenzae, Cheng [/bib_ref] [bib_ref] Mechanistic and kinetic study of the ATP-dependent DNA ligase of Neisseria meningitidis, Magnet [/bib_ref]. Analysis of the Psy-Lig structure suggests it may engage its DNA substrate using well-ordered residues on the DNAbinding faces of the AD and OB domain. Modelling studies and sequence alignment indicate several highly-conserved basic residues on the surface of the OB domains that are positioned to interact with the DNA [bib_ref] Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding..., Williamson [/bib_ref]. In addition, potentially important inter-domain contacts have been identified in the structure which could facilitate binding by prearranging the domains in a favourable orientation [bib_ref] Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding..., Williamson [/bib_ref].
To further explore the mechanism of Lig E DNA ligases we have carried out site-directed mutagenesis of Psy-Lig and determined the structure of a close homolog from the marine gamma proteobacterium Alteromonas mediterranea Lig E (Ame-Lig) bound to nicked adenylated DNA. The Lig E class of ATP-dependent DNA ligases represent the most minimal DNA ligases characterized to date, and as such, provide a simplified model to study the structureactivity relationship of these enzymes; information that may be especially useful information for guiding protein engineering endeavours.
# Materials and methods
## Cloning and expression
Wild-type (WT) ame-lig, the triple ame-lig 3xmut (K/Q/Rloop mutant) and quadruple psy-lig OB-Ala mutant were ordered as codon-optimized synthetic constructs from the Thermofisher GeneArt service. In the case of ame-lig the gene sequence encoded the predicted mature form of Lig E of A. mediterranea (WP 020742910) where a signal peptide with a predicted cleavage site between positions 21 and 22 was omitted from the sequence. The psy-lig OB-Ala construct was identical to the Psy-Lig WT [bib_ref] Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding..., Williamson [/bib_ref] with the exception of point mutations described below. Synthetic genes were supplied sub-cloned into pDONR221 and were transferred into the pDEST17 plasmid (Invitrogen) as described previously [bib_ref] Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding..., Williamson [/bib_ref]. Site-directed mutagenesis of Psy-Lig to create single and double mutants was carried out on the pDONR221 construct using the Stratagene kit Quickchange II kit (Agilent) and the oligonucleotides listed in [fig_ref] Table 1: Data collection and refinement statistics for Ame-Lig bound to nicked DNA-adenylate [/fig_ref]. The Ame-Lig loop deletion mutant ( -Loop) was generated by amplification of pDONR221::ame-lig using primers listed [fig_ref] Table 1: Data collection and refinement statistics for Ame-Lig bound to nicked DNA-adenylate [/fig_ref] to generate ame-lig 5 and 3 fragments excluding the loop-encoding region, and generating a new BamHI site at the junction. Digestion and ligation using BamHI and T4 DNA ligase (New England Biolabs) yielded the -Loop ame-lig fragment. WT ame-lig was excised from pDONR221::ame-lig through the pDONR221 ApaI site and ame-lig HindIII sites, and the -Loop ame-lig fragment ligated into the gel-purified backbone. Mutations made in pDONR221 constructs, were verified by Sanger sequencing with M13 primers before exchange into expression vectors. Protein expression and purification was carried out as previously described [bib_ref] Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding..., Williamson [/bib_ref].
## Crystallization, structure determination and analysis
Single oligonucleotides listed in [fig_ref] Table 1: Data collection and refinement statistics for Ame-Lig bound to nicked DNA-adenylate [/fig_ref] were resuspended at 9 mM in annealing buffer (50 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA), mixed 1:1:1 to give a final duplex concentration of 3 mM and incubated at 85 - C before cooling overnight. Ame-Lig (275 M) was incubated with 1.2 molar equivalents of nicked duplex and 5 mM additional EDTA for 30 minutes on ice to form theand AIMLESS [bib_ref] How good are my data and what is the resolution?, Evans [/bib_ref]. The complex structure was solved by molecular replacement using Phaser-MR (17) with chlorella virus DNA-protein complex and Psy-Lig enzyme-adenylate as search models, and further refined in COOT [bib_ref] Coot: model-building tools for molecular graphics, Emsley [/bib_ref]. Data collection and statistics are listed in [fig_ref] Table 1: Data collection and refinement statistics for Ame-Lig bound to nicked DNA-adenylate [/fig_ref]. Structural comparisons were done using the PDBeFold server with default settings [bib_ref] Secondary-structure matching (SSM), a new tool for fast protein structure alignment in..., Krissinel [/bib_ref]. Protein-DNA and interdomain interactions were detected using the NuProPlot program [bib_ref] NuProPlot: nucleic acid and protein interaction analysis and plotting program, Pradhan [/bib_ref] and the RING server (21) respectively.
## Ligation assays
Ligase activity of Ame-Lig and Psy-Lig mutants were measured by molecular beacon (MB) assay as previously described [bib_ref] Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding..., Williamson [/bib_ref] [bib_ref] Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons, Tang [/bib_ref]. Unless otherwise stated, the reaction conditions were 2.0 nM enzyme, 300 nM substrate, 0.1 mM ATP, 10 mM MgCl 2 , 1.0 mM 1,4-dithiothreitol (DTT), 100 mM NaCl, 50 mM Tris pH 8.0 at 30 - C.
## Microscale thermophoresis binding assays (mst)
The fluorescently-labelled 40-mer nicked substrate was made by annealing 80 nM of FAM-labelled 3 OH oligo with a 5-fold excess of complement and 5 phosphate oligos in 50 mM Tris, 100 mM NaCl, 1.0 mM DTT. Oligos are listed in [fig_ref] Table 1: Data collection and refinement statistics for Ame-Lig bound to nicked DNA-adenylate [/fig_ref]. The solution was incubated at 85 - C for 5 min and cooled to 25 - C before use. Psy-Lig mutants were titrated into the DNA substrate over a concentration range of 0.1-10 M and incubated at 4 - C for 30 min prior to measurement. The final reaction mixture contained 40 nM nicked DNA substrate, 5.0 mM EDTA, 1.0 mM ATP, 0.5% Tween, 50 mM Tris, 100 mM NaCl, 1.0 mM DTT. MST analysis was carried out on the Monolith NT.115Pico using standard capillaries with instrument settings MST power 40%, excitation power 1%, temperature 25 - C.
## Electrophoretic mobility shift assays (emsa)
DNA binding by Psy-Lig mutants was measured by EMSA using the 18-mer singly-nicked substrate described previously [bib_ref] Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding..., Williamson [/bib_ref] or a 40-mer linear substrate prepared as described above and listed in Supplementary Table S1. 80 nM substrate was incubated with a 40×, 20× or 10× molar excess of enzyme, 0.1 mM ATP, 50 mM Tris pH 8.0, 50 mM NaCl, 5.0 mM ethylenediaminetetraacetic acid (EDTA) at 15 - C for 30 min. Samples were electrophoresed through an 8% polyacrylamide gel and visualized on a Pharox FX Plus imager (Biorad).
## Differential scanning fluorimetry (dsf)
DSF measurements for thermal stability of Psy-Lig mutants in the final purification buffer (50 mM Tris pH 8.0, 200 mM NaCl, 1.0 mM DTT, 5% glycerol) were carried out as described previously [bib_ref] Thermofluor-based high-throughput stability optimization of proteins for structural studies, Ericsson [/bib_ref] with final protein concentrations of 10 M (standard) or 2 M (with DNA). Unfolding between 5 and 90 - C was measured in the presence of: no additives; 100-500 mM NaCl; 0.1 mM ATP; 10 mM MgCl 2 or DNA. The latter condition used doublestranded nicked MB substrate without the TAMRA (5carboxytetramethylrhodamine) fluorophore or DABCYL (N-[4-(4-dimethylamino)phenylazo]benzoic acid) quencher and was added at an equimolar concentration to the protein together with 5 mM EDTA and 0.1 mM ATP.
## Circular dichroism (cd) spectrometry
The recombinant proteins were dialyzed overnight at 4 - C against CD-buffer (10 mM Tris pH 8.0 and 100 mM NaF). The samples were filtered through a 0.45 m pore size filter (Spin X Costar) to remove precipitate and diluted to a final concentration of 0.15 mg/ml. Data was collected on a J-810 CD spectrophotometer (Jasco) at 25 - C using a 1 mm path length cuvette using the following settings: sensitivity 100 mdeg, datapitch 0.5 nm, scan speed 50 nm/min, response 2.0 s, bandwidth 1 nm, accumulation three scans, units CD mdeg. Three sample scans were recorded and averaged. Three scans of buffer were also recorded, averaged and subtracted. . Structural alignment between Ame-Lig and other ligases that contain only the catalytic core domains; Psy-Lig, PDB 4D05; ChlV-Lig, 2Q2T, T7 DNA ligase, 1A0I. The domain boundary is between first lysine of motif V and the next residue. Q-score, quality score of the Ca-alignment between 1 and 0 with 1 being the highest score; Z-score, significance of the alignment based on Gaussian statistics; root-mean-square deviation (R.M.S.D.) between C-␣ atoms in the three-dimensional alignment
# Results
## Lig e partially encircles nicked dna
The overall structure of the Ame-Lig-DNA complex resolved to 2.3Å clearly reveals that this Lig E-type ligase binds its substrate without complete encirclement of the DNA duplex [fig_ref] Figure 1: Two views of the overall structure of Ame-Lig bound to 21-bp nicked... [/fig_ref]. The complex contains one monomer in the asymmetric unit with the ends of the DNA engaged in crystal contacts forming a DNA filament through the crystal. As with other DNA-ligase intermediates, the core catalytic domains of Ame-Lig are oriented to form a C-shaped clamp about the DNA with the nick positioned above the AMP-binding pocket. Inspection of the electron density reveals a phosphodiester bond between the 5 P of the nicked strand and the alpha phosphate of the AMP, indicating that the step two intermediate has been crystalized after transfer of the cofactor from the catalytic lysine K34 to the DNA (Supplementary [fig_ref] Figure 1: Two views of the overall structure of Ame-Lig bound to 21-bp nicked... [/fig_ref]. DNA encirclement by Ame-Lig encompasses ∼245 - of the helix circumference, and the interaction buries 3837Å 2 of contact surface. Continuous electron density is observed for most the protein chain from I7 to R290 with the exception of residues 20-21, and a 26 residue section in the AD domain (S94-S119). This section, located between helices ␣3 and ␣4, is presumed to form an unstructured loop. The importance of this region, which is absent in most other Lig Es, was tested by mutating three positively-charged residues (K91, Q98, R106) to alanine (hereafter Ame-Lig 3x mut) or replacing the non-conserved section of loop (L98-T115) with the truncated sequence G-S-G (Supplementary [fig_ref] Figure 2: Ame-Lig binding to DNA [/fig_ref] and B). Both variants had equivalent activity to the WT, indicating that this region is dispensable for regular ligation activity of Ame-Lig (Supplementary [fig_ref] Figure 2: Ame-Lig binding to DNA [/fig_ref]. Comparisons of structural homology made by superposition of individual domains indicates the Ame-Lig has similarity with Psy-Lig enzyme-adenylate , indicating that comparison of these structures gives a good indication of variations between the DNA-bound and apo forms of Lig E.
## Interactions of lig e with dna
The DNA-binding footprint of Ame-Lig is 8 nucleotides (nt29-nt36) on the nicked strand and 12 nucleotides on the complement (nt5-nt16), thus smaller than any previouslydescribed DNA-bound ligase structure [fig_ref] Figure 2: Ame-Lig binding to DNA [/fig_ref]. No- tably, several positions within these margins are not contacted by Ame-Lig. This includes a three nucleotide stretch between nt6 and nt8 which is on the exposed face of the helix not encircled by the core domains as well as nt10 and nt15 of the complement and nt35 of the 5 nick strand. Both Ame-Lig and Psy-Lig are fully active and able to ligate a range of DNA breaks including single nicks, 4 base-pair cohesive ends and mis-matches at the nick site, and to a lesser extend gaps, although little or no activity is observed with blunt substrates for Psy-Lig and Ame-Lig respectively ((12) and Supplementary [fig_ref] Figure 3: Active site of Ame-Lig [/fig_ref]. This indicates that, despite lacking the extensive DNA circumferential engagement or extensive contact surface of larger DNA ligases, the minimal Lig E class of ATP-dependent DNA ligases are able to form robust interactions with the DNA substrate. . Corresponding regions of Chlorella virus ligase (ChlV-Lig) and Human Lig 3 (Hu-Lig3) are shown for reference. Naming of sequences is as follows: Hin-lig, Haemophilus influenzae; Nme-lig, Neisseria meningitidis; Vch-lig, Vibrio cholera; Vib-lig, Aliivibrio salomicida; ChlV, Chlorella virus; Hu-lig3, Human ligase3.
In total Ame-Lig makes direct contacts to 14 nucleotides in the nicked duplex [fig_ref] Figure 2: Ame-Lig binding to DNA [/fig_ref] and C). As with other DNA ligases, the AD domain binds across the broken DNA strands with the nick positioned over the AMP-binding pocket in the center [fig_ref] Figure 2: Ame-Lig binding to DNA [/fig_ref]. R50 bridges both strands, contacting the 5 PO 4 and the backbone phosphate of the 3 OH of the nick. The 3 OH strand runs from the outer edge of the domain along a positively-charged groove created by the 3-␣2 loop and the ␣3 helix. A second ionic interaction to the phosphate of nt31 is made by the R39 side-chain, while one position upstream, the phosphate of nt30 makes two contacts to N51, both from the side-chain and main-chain nitrogens, as well as to O␥ 1. The hydrogen bond between N53 N␦ and the phosphate oxygen of nt29, three nucleotides upstream of the nick, marks the outermost contact with the 3 OH strand of the nick. The nt30 sugar makes a Van der Waals contacts from L98. The nt30 sugar also makes a Van der Waals contact with Leu98. ADdomain contacts to the 5 PO 4 strand of the nick are limited to a hydrogen bond from Q16 in the 1 strand with the Nε2 forming a hydrogen bond with the vicinal phosphate of nt34, and an ionic interaction between R50 to both 5 phosphates on nt31 and nt32 [fig_ref] Figure 1: Two views of the overall structure of Ame-Lig bound to 21-bp nicked... [/fig_ref].The more extensive contacts between the AD-domain and the DNA-bound AMP that are essential for tethering the DNA in the active site are described below. The outer edges of the AD-domain form contacts to each end of the complementary strand including S86 and S90 from the ␣3helix which inserts into the major groove between the complement and 3 OH DNA strands, as well as K15 and R203 from the the 1 strand the 7--the 8 loop respectively.
The complementary strand, runs the length of the OB domain in a 5 to 3 direction from the top, along a channel formed by side chains of K227, S236 and K248 on the major groove side, and K229, T251, S285, F283 and T276 in the minor groove [fig_ref] Figure 2: Ame-Lig binding to DNA [/fig_ref]. The central span of the complement backbone is contacted by K227, K229, K248, G250, T276 and N278, and extensive interactions are made between the OB domain and five of the six complementary nucleotides opposite the nick, including hydrogen bonds from side-chains of T276 and N278 to nt14 and between the main-chain carbonyl of G250 the sugar moiety of nt11. A second basic pocket accommodates a two-nucleotide section of backbone from the 5 PO 4 strand including an ionic interaction between the side chain of K271 and nt33, while the backbone of nt34 forms a hydrogen bond with the backbone nitrogen of E255 on the periphery of the domain. Specific side-chain contacts to the 5 strand of the break include F283 positioned in the minor groove between the complement and 5 PO 4 strand which stacks against the sugar of nt33, and S285 which forms a hydrogen bond to the ribose sugar of nt34 two-and three-nucleotides downstream of the nick respectively [fig_ref] Figure 2: Ame-Lig binding to DNA [/fig_ref]. The margins of contact between Ame-Lig and the complementary strand are seven nucleotides up-and five nucleotides downstream of the nick position and are formed between side-chains of K15 and R203 to the phosphate of nt5, and side-chains of S86 and S90 which hydrogen bond with the phosphate and the sugar respectively of nt16.
## Nick binding interactions and conformations are conserved
Extensive contacts between the DNA-bound AMP moiety are essential for positioning the 5 PO 4 strand for nucleophilic attack, and for tethering the complex in the active site. These include hydrogen bonds from the side chain of motif V residue K209 of to the alpha phosphate, motif III residue E73 to the 2 OH group of the ribose sugar, and the conserved R50 to the 3 OH [fig_ref] Figure 3: Active site of Ame-Lig [/fig_ref]. The rings of the adenine base are sandwiched between M191 of motif IV and F133 of motif IIIa. Motif I provides three further polar contacts from the sidechains of S32 to the exocyclic N6, K34 of the adenine ring and AMP phosphate and R39 to the ribose 2 OH.
The two terminal base pairs of the nick are distorted into an RNA-like A structure with the sugar moiety adopting a C3 endo conformation and shortened distances between backbone phosphates, while the remainder of the duplex adopts the preferred DNA-B form. As is the case with other previously-described ligase-DNA intermediates, this distortion is a consequence of two conserved phenylalanines F81 and F283 stacking against the sugars of the 5 PO 4 and 3 OH nucleotides. The characteristic DNA bending observed in other ligase structures is also preserved here, with the DNA portions of the Ame-Lig and ChlV-Lig structures (both nicked and sealed) being remarkably superimposable [fig_ref] Figure 5: Binding affinity of Psy-Lig mutants for nicked DNA [/fig_ref]. This bending is less pronounced when compared with larger Human ligase 1 (Hu-Lig1) and Human Ligase 3 (Hu-Lig3) structures, presumably due to participation of their DNA-binding domains.
Examination of the diphosphate bond linking the AMP to the 5 PO 4 indicates we have captured the step 2 intermediate immediately after transfer of the AMP from K34 to the 5 PO 4 of nt32 and before reorientation of the AMPnucleotide phosphodiester bond. In the Ame-Lig structure the 5 PO 4 is orientated away from the adjacent terminus at a distance of more than 5Å from the 3 OH of nt31. For nucleophilic attack to occur, the activated phosphate must reorient almost 90 - about the diphosphate-bond axis as seen in the app-DNA intermediate captured in the Human lig-ase1 (Hu-Lig1) structure, bringing it within 3Å of the 3 OH [fig_ref] Figure 3: Active site of Ame-Lig [/fig_ref]. In this way the AMP-Lig structure describes an additional conformation on the reaction trajectory towards formation of the new phosphodiester backbone bond [fig_ref] Figure 3: Active site of Ame-Lig [/fig_ref]. Participation of the conserved arginine R39 (R42 in ChlV-Lig and R589 in Hu-Lig1) may be important in this transition as its coordination changes from the phosphate backbone of the 3 terminal nucleotide in the pre-
Step-2 ChlV-Lig structure (2q2t) to the 3 OH of the AMP in the post-Step-2 Ame-Lig form, and then to the bridging oxygen of the DNA-AMP phosphodiester in the Hu-Lig1 structure, presumably stabilizing this rotated conformation. The linear product-bound configuration of ChlV-Lig (2q2u) shows the sidechain has returned to the orientation observed in the DNA-bound enzyme adenylate form.
No metal ions were resolved in the present structure, which is consistent with addition of EDTA during crystallization conditions to prevent step 3 catalysis. Attempts to determine metal binding sites or later reaction intermediates by soaking with MgCl 2 prior to cryoprotection were unsuccessful, giving rise to poorly diffracting crystals. Two views of the phosphodiester bond between the 5 phosphate of the nick and the AMP highlighting the difference in orientation between the Ame-Lig structure (3 terminus colored light orange) and Hu-Lig1 (3 terminus coloured olive). Note that the latter structure was crystalized with a 3 deoxy nucleotide at the nick terminus to prevent nick joining. (C) Steps of nick-sealing captured in different ligase-DNA complexes by X-ray crystallography. The DNA-bound enzyme adenylate of ChlV-Lig (4), the step 2 intermediates from Ame-Lig and Hu-Lig1 (8) before and efter phosphodiester bond rotation, and ChlV-Lig after step-3 nick sealing before product release.
## Unique dna binding interactions form conserved motifs in lig e
The majority of protein-DNA contacts are conserved between the minimal Ame-Lig, the small latch-bearing ChlV-Lig and the large Hu-Lig1 and Hu-Lig3 enzymes with their helical DNA-binding domains. However, additional binding interactions identified in the Ame-Lig structure appear to be highly conserved among the Lig E-type ATPdependent DNA ligases and may account for their ability to bind DNA without a latch or DNA-binding domain. These include the lysine-bearing motif at the apex of the OB domain between the 9 and 10 strands, which is replaced by the 29-residue latch in ChlV-Lig. As seen in the previous Psy-Lig enzyme-adenylate structure, this short interstrand segment of Ame-Lig forms a 3 10 helix positioning the lysine pair in a fork straddling the complement strand with the side-chain of K227 positioned alongside the complement backbone and that of K229 pointing into the minor groove between the complement and the 5 PO 4 strand. The latter residue forms two hydrogen bonds to nt9 from the side-chain amine to the ribose O4 , and from the mainchain amide to the backbone phosphate as well as extensive Van der Waals interactions [fig_ref] Figure 2: Ame-Lig binding to DNA [/fig_ref]. A long-range polar contact to the base of nt35 with a length of 4.7Å is also possible, making this the only residue to interact with both strands of the duplex. The short-range contacts of K227 are less extensive, limited to a main-chain interaction with the phosphate of nt11, which is complementary to the 5´position of the nick, and Van der Waals interactions; however long-range polar contacts (5.0Å) are possible from the side- A second key region of interactions on the OB domain are the polar side chains of K248 T251 and E255 which point into the minor groove, while this 11-␣7 loop is stabilized by a series of hydrophobic interactions between F247, L249 and F253 on the reverse side. K248 has main-chain interactions with the phosphate of nt13, the sugar of nt12, and its side-chain has a long-range ionic interaction with the phosphate of nt12 (5.0Å) and nt11 (5.0Å). Nucleotide11 and nt12 are complementary to the 5 and 3 ends of the nick respectively. The side-chain hydroxyl of T251, is in hydrogen bonding distance of both O4 of nt34, and the base of nt33, one position upstream of the nick. L89 which makes Van der Waals contacts with the ribose sugar of nt30 adjacent to the 3 end of the nick occupies an equivalent position to R80 of Psy-Lig in the AD domain.
A multiple alignment of more than 500 Lig-E sequences reveals that these two motifs G-K-G-K-Aromatic and F-Basic-I-G-S-G-F-x-D are highly conserved [fig_ref] Figure 2: Ame-Lig binding to DNA [/fig_ref]. Structural alignment indicates a lower degree of conservation in other ligases, for example the first motif containing a lysine fork is not found in ChlV-Lig, the Human ligases 3 and 4 (Hi-Lig3 and Hu-Lig-4) or ligases from Sulfolobus solfataricus and T7, while the second larger pattern is absent in Hu-Lig3, Hu-Lig 4 and T7 structures.
## Structure-guided mutagenesis reveals essential binding interactions
The importance of specific side-chain to DNA interactions of Lig E activity was further investigated for a sub-set of positions by mutating single residues to alanine in the close homolog Psy-Lig. The choice of positions to be mutated was based on a combination of modelled DNA-binding predictions from the enzyme-adenylate structure of Psy-Lig in our previous work [bib_ref] Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding..., Williamson [/bib_ref] , and on analyses of the Ame-Lig-DNA complex. It includes the two lysines in the small loop motif that replaces the ChlV latch, K191 and K193 (Ame-Lig K227 and K229), as well as K212 and S215 (Ame-Lig K248 and T215) in in 11 of the OB domain. A single position R80 (Ame-Lig L89) in the AD-domain was also investigated for its DNA-binding contribution, as well as K25A, the site of covalent adenylation.
These mutations decreased the specific ligase activity of Psy-Lig by up to 50%, and in the case of K191A by more than 80%. K25A was, as expected, catalytically inactive. Increased NaCl concentration has a marked effect on ligase activity of mutations to DNA-binding residues R80, K191, K193, K212 and S215 relative to the WT. This indicates that these residues contribute significantly to the collective electrostatic interaction between protein and DNA as removal of single positions renders the remaining interactions more sensitive to charge screening by salt ions [fig_ref] Figure 4: NaCl dependence of Psy-Lig point mutants [/fig_ref]. The Psy-Lig K191A/K212A double-mutant was equally defective in ligation to the single K191A mutant, however a variant where all four positions on the OB-domain were mutated (K191/K193/K212/S215, hereafter OB-Ala) was completely inactive. The effect of these mutations on DNA binding further indicates a key role for these residues, with EMSA confirming that binding is significantly reduced in both the single (K191A) and double (K191A/K212A) mutants and entirely abolished for the OB-Ala variant [fig_ref] Figure 5: Binding affinity of Psy-Lig mutants for nicked DNA [/fig_ref]. K25A was able to bind DNA [fig_ref] Figure 5: Binding affinity of Psy-Lig mutants for nicked DNA [/fig_ref] , although with decreased affinity [fig_ref] Figure 5: Binding affinity of Psy-Lig mutants for nicked DNA [/fig_ref]. EMSA experiments with un-nicked DNA indicate that wild-type Psy-Lig binds the linear product, but very poorly.
DNA-binding affinities quantified by MST show that all mutations had a negative impact on binding affinity, with increases of between two-and 7-fold for EC50 values [fig_ref] Figure 5: Binding affinity of Psy-Lig mutants for nicked DNA [/fig_ref] , [fig_ref] Table 3: Binding affinity of Psy-Lig mutants to nicked DNA measured by MST [/fig_ref]. The biggest effect on measurable affinity was seen for the single K212A mutant, while no binding was measurable by MST for any single (K191A), double (K191A/K212A) or quadruple (OB-Ala) variants of K191. Binding of wild-type Psy-Lig to linear DNA was also not detectable by MST.
## Linker region plays a role in dna binding
In our previous report on the enzyme-adenylate structure of Psy-Lig, two conformers denoted Psy-LigA (partially open) and Psy-LigB (fully-open) were observed in the asymmetric unit, both in the 'open' conformation with the OB-domain deflected away from the adenylation site on the AD-domain [bib_ref] Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding..., Williamson [/bib_ref]. With the closed DNA-bound structure of Ame-Lig we can describe a third conformation in which the AD-and OB-domains are swivelled to wrap about the DNA [fig_ref] Figure 6: Comparison of Lig E domains in DNA-bound and apo-conformations [/fig_ref]. This open-to-closed transition has been described pre-viously for the enzyme-adenylate and DNA-bound forms of ChlV-Lig and is considered to be essential for ensuring that the active site in its adenylated form is exposed and available to receive nicked DNA [bib_ref] Crystal structure of eukaryotic DNA ligase-adenylate illuminates the mechanism of nick sensing..., Odell [/bib_ref]. In Psy-Lig, residues in the linker form different hydrogen bonding patterns between the two open A-and B-conformations. These residues are conserved or equivalently substituted in Ame-Lig, and are involved in a different set of interactions in the closed structure.
The most open structure is Psy-LigB where the OB domain is tilted furthest from the AD active site. Here, K175 and K176 of the linker form interactions with the ADdomain through a hydrogen bond with N10 and a salt bridge with E152 respectively. In the partially open conformation Psy-LigA, these interactions are rearranged by a one-residue turn about the linker giving rise to a new interaction between K176 and N144 of the AD-domain, while K175 interacts instead with E152. Its former bonding partner N10 forms a hydrogen bond with D178 of the linker, directly proximal to the OB domain and an inter-domain interaction is formed between R246 of the OB domain and Q13 of the AD-domain. In the DNA-bound Ame-Lig structure the two interactions between the linker and the ADdomain equivalent to those observed in Psy-LigA are retained; K212-E218 (Psy-Lig K175-N152) and E180-N212 (Psy-Lig N144-K176). The linker-AD domain interaction between Q16 and E214 (Psy-Lig N10-E178) and the interdomain bond between Q22 and R282 (Psy-Lig Q13-R246) are lost upon domain reorientation to the closed form. R282 (Psy-Lig E152) instead forms a new interdomain bond with E188 [fig_ref] Figure 6: Comparison of Lig E domains in DNA-bound and apo-conformations [/fig_ref] while Q16 hydrogen bonds to the phosphodiester backbone of the DNA substrate as described above. Q16 and Q22 are now solvent exposed and our inability to resolve two intervening residues S21 and R22 in the Ame-Lig structure likely reflects the flexibility of this loop.
In addition to these polar interactions, the domain orientations of Psy-LigB, the most-open configuration, are stabilized by inter-domain pi-pi stacking from a trio of phenylalanines F140 to F177 and F247. The latter has an addi-tional inter-domain Van der Waals contact, with Y11 of the AD-domain. In Ame-Lig an acidic residue E176 is found in place of the central AD-domain phenylalanine (F140 in Psy-Lig), therefore no stacking interaction is possible between Ame-Lig Y17 and F123 (Psy-Lig Y11 and F177). However, Ame-Lig F283, which has Van der Waals contacts with DNA in the bound form, and Y17 would form equivalent interaction to the Psy-Lig Y11-F247 pair when Ame-Lig assumes a fully-open conformation [fig_ref] Figure 6: Comparison of Lig E domains in DNA-bound and apo-conformations [/fig_ref]. These aromatic interactions are not present in either the less-open Psy-LigA conformation or the DNA-bound closed Ame-Lig conformation.
In summary, all three conformations represented by Psy-LigB, Psy-LigA and Ame-Lig appear to be stabilized by non-covalent interdomain interactions; two hydrogen bonds and at least one aromatic interaction in the most open (Psy-LigB) state, four hydrogen bonds in the less-open (Psy-LigA) state, and two hydrogen bonds, in addition to DNA-mediated interactions, in the closed (Ame-Lig) form.
To investigate the role of inter-domain interactions in facilitating DNA binding, individual mutations were made to Psy-Lig, N144 and K176 (Ame-Lig E180 and Q212); the two residues observed to form hydrogen bonds between the ␣6 helix of the AD-domain and the inter-domain linker in the enzyme-adenylate structure. The conserved Psy-Lig R69 (Ame-Lig, R78) of the 4-␣3 loop of the AD-domain was also mutated as it forms hydrogen bonds with both the ␣6 helix through the main-chain oxygen of G149 (Ame-Lig S185), and to E24 (Ame-Lig E33) of motif I, directly adjacent to the catalytic lysine residue, thus it has potential to coordinate interactions between these key regions. Mutation at either position of the Psy-Lig linker-AD hydrogen bond resulted in a >40% decrease in ligation, while the R69A mutant retained 80% of WT activity [fig_ref] Figure 4: NaCl dependence of Psy-Lig point mutants [/fig_ref]. Moderate decreases in DNA-binding affinity were detected by MST for all three mutants [fig_ref] Figure 5: Binding affinity of Psy-Lig mutants for nicked DNA [/fig_ref] , however no NaCl sensitivity relative to the WT Psy-Lig was observed, consistent with these mutants disrupting the protein structure, rather than binding electrostatics with DNA [fig_ref] Figure 4: NaCl dependence of Psy-Lig point mutants [/fig_ref]. The more severe effect of mutating the K176 member of the pair than N144 may be rationalized as K176 participates in Hbonds in both the Psy-LigA (partially open) and Psy-LigB (fully-open) forms, suggesting that the both conformations may play a role in DNA engagement. Furthermore, the impact of removing the side-chain of Psy-Lig R69 which forms an intra-domain interaction between ␣-6 and the adenylation motif suggests this is important for maintaining the correct protein conformation for DNA binding.
## Mutant stability depends on cofactor and metal ion concentration
Correct folding of all Psy-Lig mutants was confirmed by CD spectroscopy (Supplementary [fig_ref] Figure 3: Active site of Ame-Lig [/fig_ref]. Under standard conditions measured by DSF, both mutants and WT exhibited a major unfolding event between 40 and 45 - C, with the exception of the K25A catalytic mutant which unfolded below 30 - C [fig_ref] Figure 7: Stability of Psy-Lig mutants measured by DSF [/fig_ref]. NaCl concentration had no impact on thermal stability indicating that the decrease in activity of DNA binding-site mutants was not due to destabilization of the protein under high salinity conditions (data not shown). DSF did however indicate mutant-specific differences in stability depending on the presence of cofactors or substrates. Addition of ATP and MgCl 2 increased stability of the WT and most mutants by approximately 5 - C [fig_ref] Figure 7: Stability of Psy-Lig mutants measured by DSF [/fig_ref]. Addition of MgCl 2 alone was stabilizing for K191A, K212A and OB-Ala, but ATP alone was destabilizing in all cases. Interestingly, for several mutants (N144A, K176A, K193A and K212A) omission of any additives, or addition of ATP or MgCl 2 separately lead to the appearance of an additional lower temperature transition (Supplementary [fig_ref] Figure 3: Active site of Ame-Lig [/fig_ref]. All mutants except the double mutant K191A/K212A exhibited a significant increase in stability in the presence of DNA/ATP, which in the case of K25A was >20 - C. Unlike the other mutants, K25A consistently lacked an unfolding transition >30 - C, except in the presence of DNA, suggesting that the thermal stability of Psy-Lig is influenced by adenylation state.
To test whether impaired adenylation could account for the loss of activity, Psy-Lig mutants were assayed at increased ATP concentrations. However, addition of a 5-fold excess of ATP under otherwise standard reaction conditions did not lead to noticeable increases in activity or rescue poorly-active variants, indicating that neither mutations targeting DNA-binding residues or linker interactions decreased affinity for ATP (Supplementary [fig_ref] Figure 3: Active site of Ame-Lig [/fig_ref].
# Discussion
Here, we unequivocally demonstrate that Lig E binds DNA stably without participation of appending DNAbinding domains or ordering of unstructured loops. The DNA-binding footprint of Ame-Lig, (8 nucleotides and 12 nucleotides on the nicked and complement strands respectively) is two nucleotides fewer on the 3 OH strand than the previously-characterized minimal ATP-dependent DNA ligase from Chlorella virus (ChlV-Lig) and lacks additional contacts within these boundaries; in particular the three nucleotide stretch on the open duplex face of the complement. In the ChlV-Lig structure, contacts to these positions are supplied by residues within the latch module, and by the DNA-binding-domains of larger ligases such as Hu-lig1, Hu-lig3 or the NAD+-dependent housekeeping bacterial ligases [bib_ref] Human DNA ligase I completely encircles and partially unwinds nicked DNA, Pascal [/bib_ref] [bib_ref] Last stop on the road to repair: structure of E. coli DNA..., Nandakumar [/bib_ref] [bib_ref] Human DNA ligase III recognizes DNA ends by dynamic switching between two..., Cotner-Gohara [/bib_ref].
The present the crystal structure of Ame-Lig bound to adenylated DNA combined with structure-guided mutagenesis study of Psy-Lig confirms that the Lig E class of ATP-dependent DNA ligases binds DNA without complete encirclement of the duplex using a combination of specific charge-pair interactions on the well-structured globular OB domain of the protein. Several of these interactions are unique with respect to previously-described ligases and highly conserved among other Lig Es. In particular, the G-K-G-K-F motif that replaces the latch region of ChlV-Lig [fig_ref] Figure 2: Ame-Lig binding to DNA [/fig_ref] and contains interacting lysines Ame-Lig K227 and K229 (Psy-Lig K191 and K193), and the F-Basic-I-G-S-G-F-x-D position on the OB domain near to the linker which contains Ame-Lig K248 and T251(Psy-Lig K212 and S215; [fig_ref] Figure 2: Ame-Lig binding to DNA [/fig_ref]. Mutagenesis studies demonstrate that these charged residues on the OB-domain are important for Lig E-DNA interaction, and mutation of all four in parallel completely abolished DNA binding. A clear point is that mutation of residues interacting with complementstrand nucleotides that are base-paired to the ends of the nick are significantly more deleterious than those at more distal sites. Specifically, mutations to Psy-Lig K191 (Ame-Lig K229) and Psy-Lig K212 (Ame-Lig K248) are the most severe individual mutations tested here, although they are not additive. The importance of the long-range polar side chain interactions of Ame-Lig K229 (Psy-Lig K191) with DNA are underscored as mutation at this position caused an 80% decrease in activity, complete loss of salt tolerance and extremely defective binding (below the detection limit of MST). These interactions involve bridging phosphates to Nucleic Acids nucleotides complementary to both the 5 and 3 positions of the nick. The side chain amide of Ame-Lig K248 (Psy-Lig K212) is the only short-range polar contact to the complementary nucleotide base-paired to the 3 position of the nick, with binding further strengthened by side-chain Van der Waals interactions. Mutation of Psy-Lig K212A gave the most significant effect on binding affinity that could be quantified MST and caused loss of salt tolerance. Contribution of other interactions can be rationalized from the Ame-Lig structure; Ame-Lig K229 (Psy-Lig K193) has polar interactions with both the complementary and 5 strands via a hydrogen bond to sugar and long-range polar interaction respectively. Ame-Lig T251 (Psy-Lig S215) makes the only contact with the base moiety of the DNA through its sidechain, an interaction that is conserved in its serine counterpart in the DNA-bound ChlV-Lig structure and has additional interactions with an adjacent ribose sugar.
In addition to illuminating the mode of DNA interaction by minimal Lig E ligases, the Ame-Lig structure has captured a second conformation of the Step-2 intermediate im-mediately after DNA adenylation, prior to re-orientation of the DNA-AMP phosphodiester bond for Step-3 catalysis. This demonstrates inversion of the phosphate centre of the AMP, but suggests that shortening of the interphosphate distances prior to Step-2 occurs through movement of the catalytic lysine-AMP phosphoamide bond toward the 5 PO 4 rather than movement of the 5 PO 4 as was previously proposed [bib_ref] Structural basis for nick recognition by a minimal pluripotent DNA ligase, Nair [/bib_ref]. Unanswered questions remain about the final Step-3 catalysis, which would be partially resolved by the capture of states immediately prior to the post-Step-3 ChlV-Lig structure (2q2u) where the enzyme remains engaged with the linear product but the 'spent' AMP cofactor has diffused from the active site. In particular a structure with the cofactor retained in the binding pocket, or where added metal ions can be resolved would be highly informative, especially if combined with more extensive mutagenesis studies to, for example elucidate the role of the conserved R39 in 5 PO 4 orientation and AMP leaving.
The most salient difference between the Lig E DNAbound structure and the minimal viral ChlV-Lig is there is no ordering of unstructured regions for Lig E upon interaction with DNA and no circumferential encirclement. Although Ame-Lig has a probable loop region, this was not observed to participate in DNA binding from the crystal structure, and deletion or mutation of this segment had no impact on steady-state ligase activity. The lack of disordered-to-order transitions or other local structural changes is underscored by the high structural identity between Ame-Lig and Psy-Lig domains when these are aligned individually, suggesting that the DNA-interaction surface of the enzyme-adenylate is pre-organized for substrate binding. As a consequence, the most significant region of plasticity in Lig Es during the binding process is the linker region.
Changes in the hydrogen-bonding pattern of the linker region are implicated in coordinating rearrangement of the domains relative to each other to form the C-shaped clamp observed in the Ame-Lig structure. Residues of the linker region which comprises nucleotidyl transferase motif V are already known to be important for activity: the first conserved lysine of motif V (K173 in Ame-Lig, K209 in Psy-Lig) forms hydrogen bonds with the a-phosphate of the AMP in the enzyme-adenylate, and both conserved lysines (Psy-Lig K173/K175 and Ame-Lig 209/K211) stabilize the step 2 intermediate by coordinating non-bridging oxygens on the AMP phosphate in the DNA-bound structure (4). The present study highlights an additional role of stabilizing contacts between the linker and the domains including hydrogen bonds and salt bridges, as well as polar interactions. It is notable that although the individual residues involved in linker-domain bonds are not strictly conserved among homologs, the potential for charge-pair or hydrophobic interactions is generally preserved, providing further support for a functional role of these interactions (Supplementary [fig_ref] Table 3: Binding affinity of Psy-Lig mutants to nicked DNA measured by MST [/fig_ref]. The significant effect of mutating Psy-Lig K176A which features in interactions of both the B (most open) and A (partially-open) forms of enzyme-adenylate suggests that both conformers play a role in activity. Superposition of the AD-domain of the Psy-Lig B conformation with DNA-bound structure of Ame-Lig form places a 12 residue loop of Psy-Lig in the major groove of the DNA substrate where polar residues are positioned to interact with backbone phosphates (Supplementary [fig_ref] Figure 6: Comparison of Lig E domains in DNA-bound and apo-conformations [/fig_ref]. It is possible that these interactions help position the DNA at the enzyme-adenylate binding interface, and the rearrangement of hydrogen bonds described here acts as a conformational switch between the open and closed states, reorienting the domains. Such additional interactions may be necessary to ensure tight binding in the absence of an intrinsic nicksensing mechanism such as the latch of ChlV-Ligase or human ligases. Surprisingly the K25A mutant was able to bind to DNA, albeit with decreased affinity. This is in contrast with ChlV-Lig, which requires adenylation at the active site for nick sensing, but has been reported for other viral ligases such as T4 where an analogous lysine to leucine substitution at the catalytic site could bind nicked DNA, and T7 DNA-ligase where a truncated protein that eliminated the catalytic lysine was still able to bind DNA [bib_ref] Crystal structure of eukaryotic DNA ligase-adenylate illuminates the mechanism of nick sensing..., Odell [/bib_ref] [bib_ref] Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7, Subramanya [/bib_ref] [bib_ref] Chlorella virus DNA ligase: nick recognition and mutational analysis, Sriskanda [/bib_ref]. The T7 DNA-ligase is especially interesting in this respect, as like the bacterial Lig E-type ligases, it lacks an extensive DNA binding do-main or long latch-like insert. The crystal structure of the apo-enzyme show it possesses two unstructured loops in the AD-and OB-domains, each 9 residues long, which may be involved in DNA binding but are situated between different secondary structure elements compared with the ChlV-Lig latch or the unstructured Ame-Lig loop [bib_ref] Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7, Subramanya [/bib_ref]. Incredibly, truncation studies show that the individual domains of T7 DNA ligase have intrinsic double-strand binding capacity suggesting and their specificity for high-affinity nick binding appears to be cooperative [bib_ref] Functional domains of an ATP-dependent DNA ligase, Doherty [/bib_ref]. This suggests that in DNA ligases where complete duplex encirclement is not a prerequisite for ligase-DNA engagement, intrinsic binding determinants residing in individual domains may impart these with independent DNA-binding abilities, and it would be interesting to investigate whether the same applies to the Lig E class of DNA ligases.
It is interesting to consider the implications of these findings for understanding the evolution of DNA ligases. In foundational papers describing minimal ATP-dependent DNA ligase interaction with DNA, the Shuman group propose that the larger cellular ligases evolved by domain fusion to minimal pluripotent enzymes such as ChlV-Lig, presumably with acquisition of large helical domains accompanying loss of nick-sensing latches and loops [bib_ref] Analysis of the DNA joining repertoire of Chlorella virus DNA ligase and..., Odell [/bib_ref]. Our previous phylogenetic analyses of Lig E proteins indicate these may have evolved from a T4 phage-like ancestor that possessed an N-terminal DNA-binding domain and that proteobacterial Lig Es represent a minimised form as a result of domain truncation [bib_ref] Analysis of the distribution and evolution of the ATP-dependent DNA ligases of..., Williamson [/bib_ref]. It would be extremely interesting to examine the structural basis of such bacteriophage ATP-dependent DNA ligases binding to their substrates; to date no protein structures exist for T4 phage-like ATPdependent DNA ligases either with or without DNA.
In summary, the Lig E-type ATP-dependent DNA ligases, epitomised by the Ame-Lig and Psy-Lig proteins studied here, preserve all essential attributes of functional DNA ligation in a minimal scaffold. This includes robust substrate engagement, discrimination between nicked and linear DNA, bending of the nicked duplex, and distortion of the terminal nucleotides to the A-form.
## Data availability
Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data bank under accession number 6GDR.
[fig] Figure 1: Two views of the overall structure of Ame-Lig bound to 21-bp nicked DNA-adenylate with the protein represented as a surface (A and C) or cartoon showing secondary structural elements (B and D). Detail of the active site is shown in panel E. [/fig]
[fig] Figure 2: Ame-Lig binding to DNA. Throughout the figure the text and structure are coloured as follows: the AD-domain red and the OB-domain cyan, the 3 OH oligo green, the 5 P oligo orange and the complement strand purple. Polar contacts are indicted by dashed grey lines. (A) Schematic of interactions for Ame-Lig predicted by NuProPlot (20). Default settings were applied (maximum distance 3.25Å, Van der Waals distance 3.80Å, minimum angle 85 • ) and confirmed by manual inspection. (B) Interactions of the complement and 5 PO 4 strand with the OB domain. (C) Interactions of the nicked and complement strands with residues in the AD domain. (D) DNA interactions of conserved binding motif GKGKF. (E) DNA interactions with conserved motif KLGTG. (F) Conservation of motifs among a selection of aligned Lig Es (right) and sequence logo built from 542 Lig Es (Supplementary [/fig]
[fig] Figure 3: Active site of Ame-Lig. (A) side-chain interactions with the adenylated nick. AMP is shown in blue, AD-domain side-chain in red, OB-side chains in teal. Hydrogen bonds are indicated by dashed yellow lines. (B) [/fig]
[fig] Figure 4: NaCl dependence of Psy-Lig point mutants. Ligase activity is measured by MB assay, each data point is normalized to WT activity at the same NaCl concentration. Measurements are the mean of three replicate experiments; error bars represent the standard deviation from the mean. Raw data, including the NaCl dependence of the WT enzyme are given in SupplementaryFigure S3E. [/fig]
[fig] Figure 5: Binding affinity of Psy-Lig mutants for nicked DNA. (A) EMSA with FAM-labeled DNA incubated with a 40×, 20× or 10× molar excess of DNA ligase. Bound DNA is shown in the upper panel, free DNA from the same lane in the lower. WT Psy-Lig binding to linear DNA is shown in an adjacent panel. (B) MST binding curves of Psy-Lig AD-domain mutants (left) and OB-domain mutants (right). [/fig]
[fig] Figure 6: Comparison of Lig E domains in DNA-bound and apo-conformations. AD-domain is shown in red, OB domain is shown in cyan/blue. Polar contacts are indicted by dashed gray lines. (A) Overall domain conformations. (B) Rearrangement of hydrogen bonding patterns between three conformations. (C) Comparison of hydrophobic interactions between three conformations. [/fig]
[fig] Figure 7: Stability of Psy-Lig mutants measured by DSF. In the case that multiple transitions were observed, T m values are given for the higher temperature, with the exception of K25A which had a single low T m . (A) Difference in T m with additives; reference condition is 0.1 mM ATP, 10 mM MgCl 2 . Asterisks mark conditions where no high-temperature transition was observed. (B) T m of mutants in buffer C (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM DTT, 5% glycerol) without any additives. [/fig]
[table] Table 1: Data collection and refinement statistics for Ame-Lig bound to nicked DNA-adenylate (PDB entry 6GDR) [/table]
[table] Table 3: Binding affinity of Psy-Lig mutants to nicked DNA measured by MST. Values are the average of four replicates, error is given as standard deviation of the mean [/table]
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10.3390/ma15093297
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CCBY
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9102866
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35591632
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s2orc_pubmed_articles
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Assessment of the Influence of Additives on the Mechanical Properties and Machinability of Al-11%Si Cast Alloys: Application of DOE and ANOVA Methods
Citation: Zedan, Y.; Songmene, V.; Samuel, A.M.; Samuel, F.H.; Doty, H.W. Assessment of the Influence of Additives on the Mechanical Properties and Machinability of Al-11%Si Cast Alloys: Application of DOE and ANOVA Methods. Materials 2022, 15, 3297. https://
# Introduction
Aluminum-silicon alloys are normally employed for the fabrication of automotive components due to their excellent mechanical behavior. Their relatively low density, compared to those made of steel or cast iron, has made them more convenient for use in transmission cases and intake manifolds, as well as engine block parts [bib_ref] Effect of Solution Heat Treatment and Additives on the Microstructure of Al-Si..., Moustafa [/bib_ref]. In turn, the enhanced quality of aluminum alloy workpieces requires intensive investigations in terms of their microstructure, as well as from the point of view of ambient-and high-temperature performance.
The main micro-constituents that have been observed in this microstructure are eutectic Si particles. The size and distribution of these particles depend on two main parameters: chemical treatment by Sr and the application of high solidification rates [bib_ref] Effect of Transition Metals Addition on Tensile Properties of Al-Si-Cu-Based Alloys at..., Alyaldin [/bib_ref]. In addition, other phases that would be observed are Fe-based intermetallics (with β-platelet or α-Chinese script morphologies), together with Mg 2 Si and Al 2 Cu, and other complex phases [bib_ref] Influence of the Microstructure on Surface Integrity in Turning-Part II: The Influence..., Grum [/bib_ref] [bib_ref] Intermetallic phases in Al-Si based cast alloys: New perspective, Samuel [/bib_ref]. Zedan et al. [bib_ref] Effects of Fe Intermetallics on the Machinability of Heat-Treated Al-(7-11) % Si..., Zedan [/bib_ref] and Pathak et al.examined the influence of Fe-based intermetallics on the machinability of Al-Si alloys with different levels of Fe and Mn. The results revealed that Fe phases, in particular sludge, would cause significant deterioration of the cutting tools used, coupled with a marked increase in the machining power required and hence in machining costs.
When Cu and Mg constitute a significant part of the employed aluminum alloys, their interaction with other elements in the alloy could result in improved properties. In addition, wearing of the cutting tool is not an issue except when their volume fraction is as high as 50%, particularly in regard to the formation of insoluble Al-Cu-Mg phases [bib_ref] Artificial Aging Behavior of 319-Type Cast Aluminum Alloys with Mg and Sr..., Tavitas-Medrano [/bib_ref] [bib_ref] Effect of Metallurgical Parameters on the Hardness and Microstructural Characterization of As-Cast..., Tash [/bib_ref] [bib_ref] Investigating the Machinability of Al-Si-Cu cast alloy containing bismuth and antimony using..., Barzani [/bib_ref] [bib_ref] Enhancing strength, ductility and machinability of an Al-Si cast alloy by friction..., Guru [/bib_ref]. The application of the statistical design of experiments (DOE) method enabled the designers to understand the role of the factors that would determine the design of the final product. The use of the DOE method has been the subject of many studies, leading to marked achievements in the area of the development of computer science [bib_ref] Computerized Simulation of Interference in Thread Milling of Non-Symmetric Thread Profiles, Fromentin [/bib_ref].
Several researchers [bib_ref] Designed Experimentation: Microstructural Optimization of Al AA 512 for the PM Process..., Major [/bib_ref] [bib_ref] Investigation of the effects of machining parameters on the thrust force and..., Kaplan [/bib_ref] [bib_ref] Study on Machining Parameters for Thrust Force and Torque in Milling AA7039..., Karabulut [/bib_ref] [bib_ref] Design-of-experiments application in machining titanium alloys for aerospace structural components, Khanna [/bib_ref] [bib_ref] Predicting the effects of microstructural features on strain localization of a two-phase..., Ji [/bib_ref] have investigated the behavior of Al-based alloys using the DOE method. These researchers reported that tool life is primarily influenced by the materials and strength of the workpiece. A model was designed by Othman et al. [bib_ref] Optimization of Tool Life and Surface Roughness for Hypereutectic Al-Si Alloys in..., Othman1 [/bib_ref] and Khorasani et al. [bib_ref] Tool Life Prediction in Face Milling Machining of 7075 Al by Using..., Khorasani [/bib_ref] , which indicated that the thrust force and torque required for drilling the last hole is approximately 50% higher than those required for drilling the first hole.
In the present study, an attempt was made to investigate the effect of compositional variations, including modification of the Cu content, Fe content and Sr content, on the mechanical properties and machinability of heat-treated Al-11%Si near-eutectic alloy. The responses measured in the experiments were hardness, yield stress, ultimate tensile strength, percentage elongation, total drilling forces, drilling power, and drill life as a function of the number of the drilled holes up to the point of drill fracture. A three-factor, two-level full factorial design was adopted for analyzing the results. A procedure was developed to establish the relationship among the investigated parameters by incorporating (i) a standardized Pareto chart; (ii) main and interaction graphs; and (iii) the analysis of various variables (ANOVA) method.
## Scheme of investigation
Several factors could influence the mechanical properties of Al-Si alloys and their machinability performance, such as (i) the percentage composition of the alloying element, (ii) the heat treatment, (iii) the melt treatment, and (iv) the casting mode. Prior to the present investigation, the role of alloying elements in determining the final microstructure and hence the mechanical properties of alloys was examined. The main elements studied were Fe, Mn, Cu, and Mg. Consequently, alloys with potential applications were considered for the following purposes:
## 1.
Identifying the important factors which influence the characteristics of Al-Si casting alloys; 2.
Finding the upper and lower limits of the factors identified; 3.
Developing the experimental design matrix using the design of experiments method; 4.
Conducting the experiments as per the design matrix; 5.
Developing regression equations between the response variable and the independent factor; 6.
Assessing the factors and their effects using a standardized Pareto chart; and 7.
Analyzing the results using analysis of variance (ANOVA).
## Developing the experimental design matrix
For this study, three parameters were varied for two levels: Cu (2.25% and 3.5%), Fe (0.5% and 1%), and Sr-level (0 and 200 ppm). To carry out the experiments, the statistical design of experiments method was used. This significantly reduces the number of experiments and the time required, compared to experiments assessing one factor at a time. These designs are labeled 2 n, , where n is the number of factors that may be evaluated in the full factorial design, i.e., 2 3 = 8 trials in the experiment. [fig_ref] Table 1: Experimental settings for independent variables [/fig_ref] represents the notations, units, and levels of factors which were varied during the present study.
## Evaluation of response variables
The measured responses in these experiments were the mechanical properties and machining behavior of investigated alloys. The mechanical properties evaluated were hardness, yield stress (YS), ultimate tensile strength (UTS), and percentage elongation (%El), whereas the machining response variables were the total drilling force, drilling power, and drill life (which was defined by the number of holes drilled up to the point of drill fracture). [fig_ref] Table 2: Response variables and codes [/fig_ref] presents the parameters analyzed, along with their codes. The mechanical properties were evaluated using hardness and tensile tests, as provided in detail in Section 3, in which we present the experimental procedures. On the other hand, the machinability response variable used in this work was evaluated as follows:
## 1.
Total Cutting Force and Power A Kistler model 9255B, 6-component piezoelectric quartz crystal dynamometer was used during drilling tests for the online measurement of the cutting forces and moments. The total cutting force and moment were calculated using the Matlab signal processing program. The signals obtained were processed in such a way that the mean components of the cutting force (Fx m , Fy m , and Fz m ) and moment (Mx m , My m , and Mz m ), as well as their corresponding standard deviations (σ Fxm , σ Fym , σ Fzm , σ Mxm, σ Mym , and σ Mzm ), were calculated for each hole using the following set of equations:
The standard deviations σ Ftm , σ Ftm of the total mean cutting force and moment, respectively, were calculated as follows [bib_ref] Effect of Solution Heat Treatment and Additives on the Microstructure of Al-Si..., Moustafa [/bib_ref] :
[formula] σ [/formula]
Eventually, the drilling force and moment and their standard deviations were found for each test block as the mean values calculated over the respective values of 180 holes drilled in the same block. Consequently, the drilling power was calculated in this study by employing the following expression: Pc = (π*Mz m *n)/30 [bib_ref] Effect of Transition Metals Addition on Tensile Properties of Al-Si-Cu-Based Alloys at..., Alyaldin [/bib_ref] where n represents drill speed.
## Tool life criteria
In this study, each alloy condition was tested with a new drill until it broke. It should be mentioned here that each drilling test was carried out at least two or three times to validate the results regarding drill life. A drill life of 2500 holes, i.e., 14 test blocks, was targeted for each alloy condition.
Based on the previous machinability studies carried out by our group, the drilling tests were carried out as follows: if the drill broke down during the drilling process, one of two options was followed: (i) drilling was halted and then the test was changed to another alloy condition, or (ii) in the case that the drill broke as a result of the presence of a defect or large inclusion, the test was resumed for the remaining blocks of the same alloy condition using a new drill. All alloy conditions were tested under the same drilling conditions.
## Experimental procedures
All experiments were conducted on experimental Al-11%Si alloy, which was received in the form of 12.5 kg ingots. The chemical composition of the base alloy was 10.8% Si, 2.24% Cu, 0.31% Mg, 0.46% Fe, 0.49% Mn, 0.014% Sr, and 0.057% Ti, with Al as a balance. Melting was carried out in an SiC crucible with a 120 kg capacity, using an electrical resistance furnace in which the melting temperature was maintained at 750 - C ± 5 - C. At this temperature, measured amounts of Cu, Fe, and Sr were added. All melts were degassed using pure dry argon injected into the melt for~15 min by means of a rotating graphite degassing impeller (125 rpm), to ensure homogenous mixing of the additions.
For each set of melt conditions, identical castings were prepared for tensile and machining testing. The melt was poured at~735 - C into the following molds, which had been preheated to 450 - C:
(i) An ASTM B-108 permanent mold (five bars per each condition); (ii) A waffle-plate graphite-coated metallic mold to obtain castings for machinability test blocks (eighteen machinability test blocks per each condition).
The tensile and machinability specimens were solution heat-treated at 495 - C for 8 h, then quenched in warm water at 65 - C, followed by artificial treatment at 180 - C for 5 h (i.e., the bars were T6 tempered). Both the solution and aging heat treatments were carried out in a forced-air Blue M electric furnace, equipped with a programmable temperature controller accurate to within ±2 - C.
Hardness measurements were carried out on the heat-treated samples using a Brinell hardness tester, with a steel ball of 10 mm in diameter and a load of 500 kg applied for 30 s. Four blocks were randomly selected from among the eighteen test blocks prepared for each alloy condition. The average hardness value for the four blocks selected per alloy was then obtained and designated as representing the hardness value for that alloy condition.
Tensile testing was conducted at room temperature using an MTS servo-hydraulic universal testing machine. The average yield strength (YS), ultimate tensile strength (UTS), and elongation to fracture (%El) values obtained from the five samples tested were considered to be the values representing a specified alloy/condition. It should be kept in mind that all of these alloys were mechanically tested in order to acquire an understanding of the effects of the additives on the mechanical properties at the same specific T6 heattreated conditions which were applied to the machinability test blocks.
Drilling tests were performed using a Makino A88E high-speed horizontal machining center with maximum power of 40 HP (30 kW) and a maximum rotation speed of 18,000 rpm under fixed machining conditions in terms of speed, feed, length of cut, tool type, and coolant, as applied to the examination of the alloys under discussion. The drilling tests were carried out at rotational speeds of 11,000 rpm using a feed rate of 1.117 m/min, with each hole being 28.38 mm deep, as provided in [fig_ref] Table 3: Cutting parameters applied for machinability testing [/fig_ref]. A synthetic metalworking fluid concentrate composed of 5% cutting fluid + 95% liquid, known as CIMTECH ® 310, was pumped at high pressure through the drill to ensure adequate cooling and chip evacuation.
## Assessing the factors and their effects
Assessing the factors and their effects on the mechanical properties and machining performance of experimental Al-11%Si alloys was carried out through the use of (i) a mathematical model; (ii) a standardized Pareto chart; and (iii) the analysis of variance (ANOVA) technique.
## Mathematical modeling
The purpose of developing the mathematical model relating the response variables (mechanical properties and machining behavior) and the metallurgical factors (percentage composition of the alloying element and modification level) was to conduct a quantitative analysis to acquire an understanding of the effects of the variables and their interactions on the properties of Al-Si casting alloys. According to the two-level experimental design, a non-linear object may be approximated by means of a nonlinear regression function, as shown in Equation [bib_ref] Influence of the Microstructure on Surface Integrity in Turning-Part II: The Influence..., Grum [/bib_ref]. The aim of the analysis was to find out the effect of independent variables on the response
[formula] Y = b 0 + b 1 X 1 + b 2 X 2 + b 3 X 3 + b 1 b 2 X 1 X 2 + b 1 b 3 X 1 X 3 + b 2 b 3 X 2 X 3 (4) [/formula]
where Y is the response variable (hardness, yield stress, ultimate tensile strength, percentage elongation, total drilling force, drilling power, tool life); b 0 , b 1 , b 2 , and b 3 are constants representing the effects of the main variable; and b 1 b 2 , b 1 b 3 , and b 2 b 3 represent the respective interaction factor. X 1 , X 2 , and X 3 are coded values of the factors of copper (Cu), iron (Fe), and strontium (Sr) content, respectively. For the convenience of recording and processing the experimental data, the upper and lower levels of factors or parameters are coded as +1 and −1. The coded value of any intermediate level can be calculated by using the following expression:
[formula] X i = X − X max+X min 2 X max−X min 2(5) [/formula]
where X max is the upper level of the parameter, X min is the lower level of the parameter, and X i is the required coded values of the parameter of any value of X from X min to X max . In the present study, STATGRAPHICS Centurion XVI software was employed to calculate the principal parameters and the interactions between the independent variables and the response variables using an experimental Al-11%Si alloy. In this case, the level of variables changed from level −1 to level +1. In addition, coded values were inserted into Equation (4) in order to compute the levels of response variables. The equations are non-linear, along with multiple binary and ternary coefficients. Both experimental factors and the response variables are listed in [fig_ref] Table 4: Experimental parameters and average response variables for trial experiments [/fig_ref]. The standard deviation associated with the average value of the response variables is also reported. By processing the data provided in [fig_ref] Table 4: Experimental parameters and average response variables for trial experiments [/fig_ref] , regression Equations (6)- [bib_ref] Computerized Simulation of Interference in Thread Milling of Non-Symmetric Thread Profiles, Fromentin [/bib_ref] were developed for the hardness, YS, UTS, %El, Ft m , Pc, tool life, and the variation of a number of different factors as follows:
[formula] Y 1 (BHN) = 114.75 + 1.75X 1 + 0.25X 2 + 0.5X 3 − 0.25X 1 X 2 + 4X 1 X 3 + X 2 X 3 (R 2 = 90.99%) (6) Y2 (Y.S) = 350 + 10X 1 + 5.25X 2 + 7.75X 3 + 17.25X 1 X 2 + 5.75X 1 X 3 − 4X 2 X 3 (R 2 = 80.68%) (7) Y 3 (UTS) = 383.5 + 11X 1 + 2X 2 − 4X 3 + 17.5X 1 X 2 −5.5X 1 X 3 − 4.5X 2 X 3 (R 2 = 94.199%) (8) Y 4 (El) = 0.6975 − 0.0675X 1 − 0.0525X 2 + 0.105X 3 + 0.0375X 1 X 2 − 0.06X 1 X 3 − 0.03X 2 X 3 (R 2 = 94.79%)(9) [/formula]
Y5 (Ft m ) = 480 + 18.5X 1 + 22X 2 + 48.5X 3 + 16.5X 1 X 2 + 3X 1 X 3 + 11.5X 2 X 3 (R 2 = 93.522%) (10)
[formula] Y6 (Pc) = 2.187 − 0.325X 1 − 0.285X 2 + 0.51X 3 − 0.2775X 1 X 2 + 0.103X 1 X 3 + 0187X 2 X 3 (R 2 = 91.528%)(11) [/formula]
Y7 (Tool life) = 1152 + 93X 1 + 86.25X 2 + 92.25X 3 + 341.25X 1 X 2 − 414.25 X 1 X 3 − 339.5X 2 X 3 (R 2 = 89.81%) [bib_ref] Computerized Simulation of Interference in Thread Milling of Non-Symmetric Thread Profiles, Fromentin [/bib_ref] where the values of X 1 , X 2 , and X 3 can be decoded using the following relations: X 1 = (%Cu − 2.75)/0.75, which ranged between 2.25% and 3.5%; X 2 = (%Fe − 0.75)/0.25, which ranged between 0.5% and 1%; X 3 = (Sr − 100)/100, which ranged between 0 and 200 ppm. In general, the influence of the addition of copper (Cu) on the response variables Y H ,Y YS , Y UTs , Y %El , Y Ftm , Y Pc , and Y tool life is represented by the coefficients b 1 , b 1 b 2 , and b 1 b 3 . A comparison of the values of these coefficients indicates that (i) the coefficient b 1 is of crucial importance; (ii) when considering the effect of the addition of (Fe) on the response variables, the coefficients b 2 , b 1 b 2 , and b 2 b 3 should be taken into account; in this particular case, the coefficient, b 2 , is of major significance; and (iii) the addition of strontium (Sr) has an effect on the response variables as a result of the coefficients b 3 , b 1 b 3 , b 3 b 2 ; b 3 appears to have a greater influence on the values of the response variables.
The regression equations which were created for this study show varying degrees of accuracy. Correlation coefficients (R 2 , R 2 Adj ) are given in [fig_ref] Table 5: Multiple regression coefficients [/fig_ref]. For example, the value of R 2 = 0.9099 for hardness indicates that 90.9% of the total variations are explained by model and 9.1% is accounted for either by variables which are assumed to be constant or by the inability of the data to be modeled by a quadratic equation. The adjusted R 2 value is a statistic that is adjusted for the "size" of the model, that is, the number of factors (terms). The value of R 2 Adj = 0.85944 indicates that 85.94% of the total variability is explained by the model after considering the significant factors. As shown in [fig_ref] Table 5: Multiple regression coefficients [/fig_ref] , the models for hardness, UTS, %El, Ft m , and Pc had high multiple correlation coefficients, whereas those for YS and tool life were slightly low, suggesting that these two variables were sensitive to some factors which were not included within the scope of this study. The validity of the equations was checked by performing random experiments in the range of the variation of Cu, Fe, and Sr contents. Tables 6 and 7 provide a comparison between the calculated values of the mechanical and machining properties obtained from Equations (6)- [bib_ref] Computerized Simulation of Interference in Thread Milling of Non-Symmetric Thread Profiles, Fromentin [/bib_ref] and the values obtained experimentally from the random runs. An examination of the results indicates that there was a close match between the properties obtained by performing random experiments and those calculated using the respective regression equations. The preceding operation was carried out by inserting the reduced values of the parameters corresponding to the random experiments into the respective equations. The closeness of the match indicates that the equations were sufficiently accurate within an acceptable range of variations in the variables. From the proposed two-level factorial design experiments on the properties of neareutectic Al-11%Si alloys, it is obvious that by using this type of polynomial regression equation, the effect of each of the individual variables and those of their interactions on the mechanical and machining properties may be obtained. Modifications may suitably be applied to the equation model in order to clarify the responses of the properties of the samples beyond the specified range. Finally, for a better understanding of the effects of individual variables and their interaction on the mechanical and machining properties, a higher level of factorial experimental design was suggested, wherein the influence of other parameters, such as heat treatment, casting mode, cooling rate, and so forth, could be examined.
## Standardized pareto chart
A standardized Pareto chart is a horizontal bar chart plotting values in descending order. The length of each bar is proportional to the values of the estimated effect. [fig_ref] Figure 1: Pareto charts of the standardized effects for hardness data [/fig_ref] shows the Pareto chart of the standardized effects for the hardness data with a confidence level of 95%, in which the most significant effects corresponded to the interaction effect between the Cu content and Sr level (X 1 X 3 ). Next in significance were the Cu-content (X 1 ) and the interaction effect between the Fe content and Sr level (X 2 X 3 ). However, the effects of the coefficients X 3 , X 1 X 2 , and X 2 were found to be insignificant. It can be noted that, of the three alloying elements, Cu had the greatest effect by increasing the YS and UTS values. Cu is thus a superior strengthener and its addition as an alloying element is desirable, whereas the Sr level and Fe content also increased the strength but this effect was mild.
It should be noted that the presence of a number of binary interactions indicates the formation of various intermetallic compounds. Therefore, several interaction effects were present, and they may have had a significant effect on the strength value. These interaction effects included the interaction between Cu and Fe, namely, X 1 X 2 , and the interaction between Cu and the Sr level, namely, X 1 X 3 . [fig_ref] Figure 3: Regression model for yield stress [/fig_ref] shows a three-dimensional (3D) representation of the response surface of YS as function of the coded values of Cu content (X 1 ) and Fe content (X 2 ) for alloys containing medium level of Sr (100 ppm). These results point to the maximum values for YS occurring at high levels of Cu (+1) and Fe (+1), whereas the minimum values of YS occurred at low levels of Cu and Fe for alloys containing 100 ppm of Sr. The average values of yield stress were calculated for all combinations. Using these values, interaction graphs were drawn for each combination.shows the interaction coefficient (X 1 X 2 ) between Cu content and Fe content, in which the yield stress was found to increase significantly with an increase in the Cu content from a low level (−1) to a high level (+1) at a high level of Fe (+1), i.e., alloys containing 1% Fe. On the other hand, the yield stress decreased slightly with an increase in the Cu content at a low level of Fe, i.e., alloys containing 0.5% Fe.represents the interaction coefficient (X 1 X 3 ) between the Cu content and the Sr level; the yield stress was found to increase significantly with an increased level of Cu content from 2.25% to 3.5% in the modified alloy which contained a high level of Sr. On the other hand, the yield stress increased slightly with an increase in the Cu content in the non-modified alloy. It was also observed that the modified alloys exhibited yield stress values higher than those of non-modified alloys within all levels of Cu contents, as shown in. [fig_ref] Figure 6: Pareto charts of the standardized effects for [/fig_ref] ,b show the relative significance of the independent parameters on the cutting forces and cutting power (Pc). The Sr level (X 1 ) had the most significant effect, followed by the Fe content (X 2 ) and Cu content (X 3 ). Next in significance were the interactions between these parameters. [fig_ref] Figure 7: Regression model for total cutting force [/fig_ref] shows the regression model for cutting force as a function of the Cu content (X 1 ) and Sr level (X 3 ) for alloys containing 0.5% Fe. This figure once again points to the fact that the maximum values for the cutting force prevailed with Sr and Cu contents greater than 0 ppm and 2.5%, respectively. On the other hand, the minimum cutting force occurred at low levels of Sr and Cu. As also observed from the main effects plot of total cutting force as function of Cu, Fe, and Sr contents, the cutting force was found to increase significantly with an increase in the level of Sr for a constant level of Cu and Fe. The cutting force was also found to increase with an increase in the level of Cu and Fe and a constant level of Sr, as clearly shown in [fig_ref] Figure 8: Main effects plots for total cutting force [/fig_ref].
The same procedures were applied on tool life, resulting in a Pareto charts with a confidence level of 95%, as shown in [fig_ref] Figure 9: Pareto chart of the standardized effects for tool life data [/fig_ref]. It was also observed that the binary interactions between X 1 , X 2 , and X 3 had significant effects on the tool life. As shown in this figure, the interaction coefficients of X 1 X 3 , X 1 X 2 and X 2 X 3 were the most significant in comparison to independent variables X 1 , X 2 , and X 3 . This fact may be attributed to the formation of complex insoluble phases between Cu, Fe, Si, and Al.
## Analysis of variance (anova) technique
An ANOVA summary table is commonly used to summarize the testing of a regression model, testing of significant factors and their interaction, and lack-of-fit testing. If the pvalues in the ANOVA table are less than 0.05, then the factors (and the interaction of factors) are said to be significant. Finally, the % F-value column is used in the ANOVA summary table and this often serves as a rough but effective indicator of the relative importance of each model term.
The results of the ANOVA for yield stress are shown in [fig_ref] Table 8: Analysis of variance [/fig_ref] ; this analysis was carried out for a level of significance of 5%, i.e., for a confidence level of 95%. As shown in Tables 8-10, the interaction coefficients (X 1 X 2 ), the Cu content (X 1 ), and the Sr-level (X 3 ) contributed 34.5%, 20%, and 15.5% to the total variability of the model, respectively. These results show that the alloying elements interacted with each other to a significant degree. Similarly, the results of the analysis of variance (ANOVA) for total cutting force and tool life are shown in Tables 7 and 8, respectively.
# Discussion
The properties of alloys may be improved by adding Cu, Fe, and Sr to the alloys. Mohamed et al. [bib_ref] Influence of Additives on the Microstructure and Tensile Properties of Near-Eutectic Al-10.8%Si..., Mohamed [/bib_ref] reported on the changes in the microstructure (i.e., with respect to intermetallics and silicon particle characteristics) with the addition of Fe, Mn, Cu, Sr, and Mg to Al-10.8%Si near-eutectic alloys. The results showed that increasing the level of Mg and Cu in the Sr-containing alloys produced larger Si particle sizes, thus, in effect, diminishing the modifying influence of Sr. Among intermetallics, Al 2 Cu phase particles were more or less completely dissolved in the Al matrix after solution heat-treatment, whereas the β-Fe phase underwent partial dissolution and Al 2 Cu 2 Mg 8 Si 6 , α-Fe intermetallic phases and sludge phases persisted after 8 h of solution time in all the samples.
Based on the statistical analysis, the corresponding hardness data indicated that the decrease in the hardness values of Sr-modified alloys compared to the non-modified alloys was mainly the result of changes in the morphology of the eutectic Si particles, from brittle coarse acicular plates in the non-modified alloy to a rounded fibrous form, as shown in [fig_ref] Figure 1: Pareto charts of the standardized effects for hardness data [/fig_ref]. Furthermore, Sr led to a depression in the eutectic temperature, causing a shift of the eutectic point to a higher Si content, resulting in an increase in the amount of soft α-Al formed. It was also found that an increase in the Fe content resulted in a slight increase in the hardness values, which can be attributed to the formation of hard and brittle (metastable) intermetallic phases of Al 2 Cu and Al-Cu-Mg and also to an increased bonding of silicon particles with the matrix, in which the thermal energy is enough to precipitate such intermediate phases which are coherent with the matrix, as shown in the SEM micrograph and EDX analysis presented in [fig_ref] Figure 1: Pareto charts of the standardized effects for hardness data [/fig_ref]. The results shown here prove that of the three alloying elements, Cu exerted the greatest effect by increasing the yield stress and the ultimate tensile strength values. We observed from the experiments that the elongation (%El) was highly sensitive to the alloy composition. With regard to the increased level of Cu in modified alloys, we found that the ductility was considerably lower. Such a result may be attributed to the influence of Sr on severity, as displayed by the Al 2 Cu phase segregation, resulting in the formation of large amounts of the coarse block-like form of the phase. It can also be noted that as the percentage of Fe increased beyond 0.75%, the elongation decreased to a significant degree, a fact which may be attributed to the presence of the β-Fe phase in the structure of the alloy, containing a high level of Fe, as shown clearly in [fig_ref] Figure 1: Pareto charts of the standardized effects for hardness data [/fig_ref]. The high stress concentrations at the sharp edges of the β-Fe phase, as well as the weak bonding between the β-phase and the Al matrix, enhance crack initiation and thus decrease the ductility of this alloy. This observation is in agreement with the work of Ojolo and Ogunkomaiy [bib_ref] A study of effects of machining parameters on tool life, Ojolo [/bib_ref] who reported that increasing Cu and Mg contents generally increased strength and decreased ductility, whereas increasing the Fe content (at an Fe/Mn ratio = 0.5) dramatically lowered the ductility and strength of low-Si alloys. The morphology of eutectic silicon in the Al-Si alloys has a major influence on the machining behavior. Through our analysis, we found that an increase in the Sr level has the greatest effect in terms of increasing the total cutting force and power values. In other words, the non-modified alloy (with a low level of Sr) generated lower drilling forces compared to Sr-modified alloys, which may be explained by the fact that the non-modified acicular silicon structure provided an easy path for fracture, resulting in decreases in the cutting forces during the machining. The higher drilling force and power observed with an increasing level of Cu and Fe may be attributed to an increase in the volume fraction of Cu and Fe intermetallics with the increase in the Cu and Fe content.
The interactions between the alloying elements play a prominent role in affecting the machining behavior of Al-Si casting alloys [bib_ref] Effect of intermetallics and drill materials on the machinability of Al-Si cast..., Zedan [/bib_ref]. In the present study, the three interaction factors between the parameters had significant effects on tool life, including the interaction of the Cu content and Fe content (X 1 X 2 ), the Cu content and Sr level (X 1 X 3 ), and the Fe-content and Sr level (X 2 X 3 ). This fact may be attributed to the formation of complex insoluble phases between Cu, Fe, Si, and Al, resulting in the formation of large amounts of coarse undissolved phases with an increase in Cu and Fe contents. From the machinability point of view, such undissolved phase particles represent the abrasive area of the matrix, with the potential to cause tool breakage. It has been reported that tool wear can be increased by as much as 50% through the presence of substantial quantities of undissolved Al-Cu and Al-Cu-Mg-Si phases. These results are in agreement with the work of Khorasani et al. [bib_ref] Tool Life Prediction in Face Milling Machining of 7075 Al by Using..., Khorasani [/bib_ref] , who reported that the dominant variables influencing tool life in the Al-Si alloys are the morphology of eutectic silicon; the inhomogeneities of the alloy structure; and an interrupted regime of cutting, resulting from the coarse undissolved particles.
The machining characteristics of the Al-11%Si alloy depend mainly on the shape, size, and distribution of α-Al dendrites, the eutectic Si morphology, and Al 2 Cu particles in the interdendritic region. [fig_ref] Figure 1: Pareto charts of the standardized effects for hardness data [/fig_ref] -c show that the addition of 1% Cu to the base alloy (coded the M1 alloy), thereby producing the M5 (M1 + 1.0% Cu) alloy, had only a slightly diminishing effect on the drilling force and moment, compared to the case of the M1 alloy. On the other hand, the increase in the level of Cu and Mg from 2.2% and 0.3% in the M1 alloy to 3.4% and 0.6%, creating the M6 alloy, had a noticeable effect in terms of increasing the mean total drilling force and mean total drilling moment, by 25% and 20%, respectively, compared to the M1 alloy. It can also be clearly observed that the Mg-free M1 alloy (coded the M9 alloy) displayed a significant decrease in the total drilling force and in the total drilling moment compared to the M1 reference alloy; specifically, the M9 alloy required an average of 50% lower mean total drilling force, ranging from 35% to 65%, and exhibited an average of 52% lower total drilling moment, ranging from 35% to 69%, as shown in [fig_ref] Figure 1: Pareto charts of the standardized effects for hardness data [/fig_ref]. [fig_ref] Figure 1: Pareto charts of the standardized effects for hardness data [/fig_ref]. Effects of the addition of Cu, Mg, and Sr on the machinability of M1, M5, M6, M0, and M9 alloys in terms of (a) mean total drilling force, (b) mean total drilling moment, and (c) mean power cutting required for the drilling of 90 holes.
# Conclusions
The selection of an alloy with certain specific properties is extremely exhausting and time consuming, particularly because the classic methods have not always led to the development of a quantitative relationship between the mechanical and machinability of the alloy on the one hand, and their chemical composition and melt treatments on the other. Therefore, if two or more variables are mofidied, it can become difficult to quantify the effect that any interaction between different variables would have on the alloy's mechanical and machining properties. By using an experimental design (DOE) with only eight runs and the resulting regression equations, valuable information on the relationships of three independent variables-namely, Cu, Fe, and Sr contents-with the mechanical and machining properties of the near-eutectic T6-treated Al-11%Si alloy was obtained. Through an analysis of the results obtained, the following conclusions may be drawn:
## 1.
Based on the statistical analysis, the corresponding hardness data indicated that the decrease in the hardness value of Sr-modified alloys compared to the non-modified alloys was mainly the result of changes in the morphology of the eutectic Si particles, from brittle coarse acicular plates in the non-modified alloy to a rounded fibrous form; 2.
The results proved that of the three alloying elements, Cu had the greatest effect in terms of increasing the yield stress and ultimate tensile strength values. This fact may be attributed to the formation of the hard and brittle (metastable) intermetallic phases Al 2 Cu and Al-Cu-Mg. It was also found that an increase in the Fe content resulted in a slight increase in hardness values; 3.
The elongation percentage of alloys was effected by three elements, with Fe and Cu having the greatest effect and Sr having the least; 4.
The morphology of eutectic silicon in the Al-Si alloys has a major influence on the machining behavior. Through our analysis, we found that an increase in the Sr level had the greatest effect in terms of increasing the total cutting force and power values; 5.
The higher drilling force and power with an increased level of Cu and Fe may be attributed to an increase in the volume fraction of Cu-and Fe-intermetallics with an increase in the Cu and Fe content; 6.
The presence of a number of binary interactions indicated the formation of various intermetallic compounds. Therefore, several interaction effects were present, and they may have had the most significant effect on the tool life. These interaction effects included those of the Cu content and Fe content (X 1 X 2 ), the Cu content and Sr level (X 1 X 3 ), and the Fe content and Sr level (X 2 X 3 ); 7.
The validity of the equation was checked and the results indicated that there was a close match between the properties obtained by performing random experiments and those calculated by means of the respective regression equations. The closeness of the match indicates that the equations were sufficiently accurate over the range of variables. Data Availability Statement: Data will be made available upon request.
## Conflicts of interest:
The authors declare no conflict of interest.
[fig] Figure 1: Pareto charts of the standardized effects for hardness data. [/fig]
[fig] Figure 2a ,: b show the effect of alloying elements on the yield stress (YS) and ultimate tensile strength (UTS) values. [/fig]
[fig] Figure 2: Pareto charts of the standardized effects for (a) yield stress data and (b) ultimate tensile strength data. [/fig]
[fig] Figure 3: Regression model for yield stress (YS) as a function of Cu and Fe content in heat-treated Al-11%Si alloys containing 100 ppm Sr. [/fig]
[fig] Figure 4: (a) Interactive effect of %Cu (X 1 ) and %Fe (X 2 ); (b) interactive effect of %Cu (X 1 ) and Sr-level (X 3 ) on the yield stress values (YS in MPa). [/fig]
[fig] Figure 5: shows that the elongation percentage (%El) was highly sensitive to alloy composition. In thisFigure,it can be observed that most of the variables contributed negatively to the elongation percentage. Both Cu and Fe appeared to affect the elongation adversely, whereas the Sr level (X3) showed a positive effect on the elongation percentage. [/fig]
[fig] Figure 6: Pareto charts of the standardized effects for (a) total cutting force data and (b) cutting power data. [/fig]
[fig] Figure 7: Regression model for total cutting force (F tm ) as a function of Cu and Sr content of heattreated Al-11%Si alloy containing 0.5% Fe. [/fig]
[fig] Figure 8: Main effects plots for total cutting force (F tm in N) as a function of Cu, Fe, and Sr content for heat-treated Al-11%Si alloys. [/fig]
[fig] Figure 9: Pareto chart of the standardized effects for tool life data. [/fig]
[fig] Author: Contributions: Conceptualization, H.W.D.; methodology: Y.Z.; writing-original draft preparation, Y.Z. and F.H.S.; supervision: V.S. and F.H.S.; project administration: V.S.; writing-review and editing, A.M.S. All authors have read and agreed to the published version of the manuscript.Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. [/fig]
[table] Table 1: Experimental settings for independent variables. [/table]
[table] Table 2: Response variables and codes. [/table]
[table] Table 3: Cutting parameters applied for machinability testing. [/table]
[table] Table 4: Experimental parameters and average response variables for trial experiments (runs) used for the factorial design. Sr); b M: modified alloy with 200 ppm Sr; c (F tm ) and Pc: total cutting force and power average for the first 180 holes in order to neglect the effect of tool wear on cutting force values. [/table]
[table] Table 5: Multiple regression coefficients. [/table]
[table] Table 6 Table 7: Mechanical and machining properties values calculated using Equations (6)-(12). Values of mechanical and machining properties obtained from random experiments. [/table]
[table] Table 8: Analysis of variance (ANOVA) for yield stress data (YS in MPa).Table 10. Analysis of variance (ANOVA) for tool life data (number of holes). [/table]
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10.1155/2011/541851
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CCBY
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3135208
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21776386
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s2orc_pubmed_articles
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Individual Rac GTPases Mediate Aspects of Prostate Cancer Cell and Bone Marrow Endothelial Cell Interactions
The Rho GTPases organize the actin cytoskeleton and are involved in cancer metastasis. Previously, we demonstrated that RhoC GTPase was required for PC-3 prostate cancer cell invasion. Targeted down-regulation of RhoC led to sustained activation of Rac1 GTPase and morphological, molecular and phenotypic changes reminiscent of epithelial to mesenchymal transition. We also reported that Rac1 is required for PC-3 cell diapedesis across a bone marrow endothelial cell layer. In the current study, we queried whether Rac3 and RhoG GTPases also have a role in prostate tumor cell diapedesis. Using specific siRNAs we demonstrate roles for each protein in PC-3 and C4-2 cell adhesion and diapedesis. We have shown that the chemokine CCL2 induces tumor cell diapedesis via Rac1 activation. Here we find that RhoG partially contributes to CCL2-induced tumor cell diapedesis. We also find that Rac1 GTPase mediates tight binding of prostate cancer cells to bone marrow endothelial cells and promotes retraction of endothelial cells required for tumor cell diapedesis. Finally, Rac1 leads to β1 integrin activation, suggesting a mechanism that Rac1 can mediate tight binding with endothelial cells. Together, our data suggest that Rac1 GTPase is key mediator of prostate cancer cell-bone marrow endothelial cell interactions.
# Introduction
Skeletal metastases represent a major clinical problem for men suffering from prostate cancer (PCa). Nearly 80% of men who die from this disease have significant spread of the cancer to bone [bib_ref] Treatment of tumor osteopathy in cancer of the prostate, Alcover [/bib_ref] [bib_ref] Molecular genetics of prostate cancer, Abate-Shen [/bib_ref]. Like all cancers, PCa cells must successfully complete a series of ordered steps, known as the metastatic cascade, to form a distant tumor [bib_ref] Different deficiencies in the prevention of tumorigenic-low-metastatic murine K-1735b melanoma cells from..., Aukerman [/bib_ref] [bib_ref] Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. clowes..., Fidler [/bib_ref]. One key step in the PCa metastatic cascade is the process of extravasation from the circulation into the bone microenvironment [bib_ref] Cell adhesion and chemotaxis in prostate cancer metastasis to bone: a minireview, Cooper [/bib_ref] [bib_ref] Preferential adhesion of prostate cancer cells to a human bone marrow endothelial..., Lehr [/bib_ref]. The process of PCa cell extravasation can be subdivided into a number of substeps, which include arrest, binding, adhesion, and spreading on bone marrow endothelial cells, migration along the endothelial barrier, tumor cell diapedesis, and invasion into the bone stromal compartment [bib_ref] Preferential adhesion of prostate cancer cells to a human bone marrow endothelial..., Lehr [/bib_ref] [bib_ref] Prostate cancer cell adhesion to quiescent endothelial cells is not mediated by..., Cooper [/bib_ref] [bib_ref] Preferential adhesion of prostate cancer cells to bone is mediated by binding..., Cooper [/bib_ref]. Although many of these substeps have been well studied for leukocyte extravasation, relatively little is known about the process of PCa tumor cell extravasation across a bone marrow endothelium (reviewed in [bib_ref] Stepping out of the flow: capillary extravasation in cancer metastasis, Miles [/bib_ref].
The Rho GTPases are a group of proteins that comprise a subfamily of the Ras-superfamily of monomeric GTPbinding proteins that act as molecular switches regulating the cytoskeleton promoting cell migration [bib_ref] Rho GTPases and their effector proteins, Bishop [/bib_ref] [bib_ref] Rho GTpases and the actin cytoskeleton, Hall [/bib_ref] [bib_ref] Rho GTPases and cell migration, Ridley [/bib_ref] [bib_ref] Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes..., Nobes [/bib_ref] [bib_ref] Distinct patterns of actin organization regulated by the small GTP-binding proteins Rac..., Ridley [/bib_ref]. Furthermore, the Rho proteins are implicated in cancer progression and metastasis (reviewed in [bib_ref] RHO-GTPases and cancer, Sahai [/bib_ref]. Previously, we have suggested potential roles for individual Rho GTPases in PCa extravasation [bib_ref] RhoC GTPase is required for PC-3 prostate cancer cell invasion but not..., Yao [/bib_ref] [bib_ref] CCL2 induces prostate cancer transendothelial cell migration via activation of the small..., Van Golen [/bib_ref] [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref]. Specifically, we demonstrated that RhoC GTPase is required for invasion in response to insulin-like growth factor I and type I collagen [bib_ref] RhoC GTPase is required for PC-3 prostate cancer cell invasion but not..., Yao [/bib_ref] [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref]. Upregulation of the integrin heterodimer α2β1 in LNCaP cells selected for their ability to bind to type I collagen led to increased RhoC activation and cellular invasion upon 2 Journal of Signal Transduction integrin ligation [bib_ref] Type I colagen receptor (alpha2 beta1) signaling promotes the growth of human..., Hall [/bib_ref] [bib_ref] Type I collagen receptor (αβ) signaling promotes prostate cancer invasion through RhoC..., Hall [/bib_ref]. Downregulation of RhoC in PC-3 human PCa cells through introduction of either a dominant negative (dn)RhoC or a RhoC-specific shRNA led to a significant decrease in the cells ability to invade either collagen or Matrigel-coated filters [bib_ref] RhoC GTPase is required for PC-3 prostate cancer cell invasion but not..., Yao [/bib_ref] [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref]. However, these cells underwent changes reminiscent of epithelial to mesenchymal transition (EMT). Concordant with EMT, the cells displayed increased random linear motility, which was due to increased and sustained levels of Rac1 GTPase expression and activation. Further, we demonstrated that active Rac1 GTPase is required for PC-3 cell diapedesis across a BMEC layer [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref].
The Rac GTPase branch of the Rho subfamily is comprised of four members, Rac1, Rac2, Rac3, and RhoG. Rac1 and RhoG are ubiquitously expressed, while Rac2 is primarily expressed in hematopoietic cells, and Rac3 is expressed mainly in nervous tissue but can be found expressed at lower levels in most other tissues (reviewed in [bib_ref] Rho-family GTPases: it's not only Rac and Rho (and i like it), Wennerberg [/bib_ref]. Seminal experiments demonstrated a role for Rac1 in the formation of lamellipodia [bib_ref] Rho GTpases and the actin cytoskeleton, Hall [/bib_ref] [bib_ref] Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes..., Nobes [/bib_ref] [bib_ref] Distinct patterns of actin organization regulated by the small GTP-binding proteins Rac..., Ridley [/bib_ref] [bib_ref] The small GTP-binding protein rac regulates growth factor-induced membrane ruffling, Ridley [/bib_ref] [bib_ref] The cellular functions of small GTP-binding proteins, Hall [/bib_ref]. Recent evidence suggests a role for Rac3 GTPase in cellular adhesion and neurite outgrowth [bib_ref] Rac1 and rac3 have opposing functions in cell adhesion and differentiation of..., Hajdo-Milasinovic [/bib_ref] [bib_ref] Normal levels of Rac1 are important for dendritic but not axonal development..., Gualdoni [/bib_ref] [bib_ref] Roles of the Rac1 and Rac3 GTPases in human tumor cell invasion, Chan [/bib_ref]. RhoG GTPase has been shown to signal in a parallel pathway to Rac1, being regulated by some of the same upstream regulatory proteins such as Vav2 and activating some of the same downstream effectors as Rac1 [bib_ref] RhoG signals in parallel with Rac1 and Cdc42, Wennerberg [/bib_ref]. RhoG has also been shown to act as a hierarchical GTPase; activation of RhoG can lead to the activation of Rac1 through direct interaction with the Dock180-ELMO [bib_ref] RhoG activates Rac1 by direct interaction with the Dock180-binding protein Elmo, Katoh [/bib_ref]. Therefore, Rac1-mediated cell migration can be regulated through direct activation of RacGEFs or via RhoG GTPase [bib_ref] Activation of Rac1 by RhoG regulates cell migration, Katoh [/bib_ref].
Rac1 GTPase plays an intimate role in monocyte and macrophage diapedesis [bib_ref] Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis, Terashima [/bib_ref]. Monocytes are recruited to the sites of inflammation via stimulation by the chemokine CCL2 (a.k.a. MCP-1) [bib_ref] Monocyte chemoattractant protein-1 expression correlates with macrophage infiltration and tumor vascularity in..., Ohta [/bib_ref]. Binding of CCL2 to its putative receptor CCR2 leads to clustering of the novel actin regulatory protein PCNT1 to the cells leading edge and regulation of migration through activation of Rac1 [bib_ref] Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis, Terashima [/bib_ref]. Activation of Rac1 is required to form lamellipodia, which in turn is required for monocytes to sense junctions between endothelial cells [bib_ref] Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis, Terashima [/bib_ref]. BMECs from PCa patients secrete high levels of CCL2 [bib_ref] CCL2 as an important mediator of prostate cancer growth in vivo through..., Loberg [/bib_ref]. Stimulation of PC-3 cells with CCL2 results in activation of Rac1 GTPase and EMT consistent with what is observed when RhoC activity is downregulated through introduction of a dnRhoC or shRNA to RhoC [bib_ref] RhoC GTPase is required for PC-3 prostate cancer cell invasion but not..., Yao [/bib_ref] [bib_ref] CCL2 induces prostate cancer transendothelial cell migration via activation of the small..., Van Golen [/bib_ref] [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref]. Furthermore, CCL2 stimulation drives PC-3 tumor cell diapedesis across a BMEC layer via PCNT1 [bib_ref] CCL2 induces prostate cancer transendothelial cell migration via activation of the small..., Van Golen [/bib_ref].
Levels of Rac1 and Rac3, but not Rac2, are shown to be increased in prostate cancer patient samples compared to normal prostate [bib_ref] Prognostic relevance of increased Rac GTPase expression in prostate carcinomas, Engers [/bib_ref]. However, the role that these GTPases play in PCa tumor cell metastasis has not been thoroughly studied. Similarly, there is no information on the contribution of RhoG to PCa progression. In the current study we investigate the roles of Rac1, Rac3, and RhoG GTPases in the process of prostate tumor cell diapedesis across a bone marrow endothelial cell layer. All three Rac proteins have an influence on tumor cell diapedesis across a bone marrow endothelial cell monolayer. Further, we demonstrate that Rac1 GTPase has a significant effect on PCa cell diapedesis, while Rac3 has a negative effect on tumor cell diapedesis. In addition, RhoG has a partial effect on CCL2-stimulated diapedesis. Finally, Rac1 regulates binding of prostate cancer cells to the bone marrow endothelial cells. Our data suggest that Rac1 is required for the activation of β1 integrins leading to binding of the prostate cancer cell to the BMEC. This is the first study to demonstrate roles for different isoforms of Rac GTPase in the PCa metastatic phenotype.
# Materials and methods
## Cell lines and cell
Culture. PC-3 PCa cell lines were obtained from American Type Culture Collection (Manassas, Va) and maintained in Ham's F-12 medium with 1.5 g/L sodium pyruvate, 2 mM L-glutamine, and 10% FBS (Invitrogen/Gibco, Carlsbad, Calif). C4-2 cells were a gift from Dr. Robert Sikes (University of Delaware) and maintained in T-medium containing 10% FBS (Invitrogen/Gibco). Human bone marrow endothelial cells (BMECs) were a gift from Dr. Graca Almeida-Porada (University of Nevada School of Medicine, Reno, Nevada). Cultures were maintained in Medium 199 with Earles's salts, L-glutamine, 2,200 mg/L sodium bicarbonate, 25 mM HEPES (Invitrogen/Gibco) buffer, 10% FBS, 1% pen/strep, endothelial cell growth supplement (BD Biosciences, Bedford, Mass), and 7500 u/500 mL media of heparin (Sigma-Aldrich, St. Louis, Mo). All cell lines were maintained at 37 - C in a 90% : 10% air : CO 2 incubator. C3 exotransferase was introduced into cells as previously described using a lipid transfer-mediated method [bib_ref] RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells, Van Golen [/bib_ref] and treated for 2 h before analysis. Rac1 inhibitor NSC23766 (Calbiochem, San Diego, Calif) treatment was performed by adding directly to tissue culture medium to a final concentration of 100 μM 1 h prior to analysis. Prostate cancer cells were stimulated with 100 ng/mL recombinant human (rh)CCL2 (MCP-1) in tissue culture medium (Millipore-Chemicon Inc., Billeceria, Mass) for 30 min during the Rac activation assays and kept in the presence of the chemokines during the diapedesis assays.
## Sirnas. specific sirnas for human rac1 and rac3
GTPases were a gift from Dr. Marc Symons and described previously [bib_ref] Roles of the Rac1 and Rac3 GTPases in human tumor cell invasion, Chan [/bib_ref]. RhoG siRNA and scrambled control siRNAs were synthesized by integrated DNA technologies. RhoG siRNA target sequences were (1) 5 -TGCCCTGATGTG-CCCATCCTGCTGGTGGG-3 and (2) 5 -ACGTGCCTG-CTCATCTGCTACACAACTAA-3 . The Rac1, Rac3, and RhoG siRNA duplexes were formed by adding 30 μL of each RNA oligo solution together with 15 μL of 5x annealing buffer (100 mM NaCl and 50 mM Tris-HCl pH 7.5) to give a final volume of 75 μL and a final concentration of 20 μM; incubated for 2 min in water bath at 95 - C; allowed to cool to room temperature. Additional experiments were performed using ON-TARGET plus SMARTpool siRNAs Rac1, Rac3, and RhoG siRNAs that were obtained from Dharmacon (Dharmacon/Thermo Scientific, Layfette, Colo). siRNAs were transfected into prostate cancer cells using FuGene6 (Roche, Indianapolis, Ind) or GeneSilencer Reagent (Genlantis, San Diego, Calif) per the manufacturers instructions and cells used 72 h after transfection. For rescue experiments, mutations were generated using the QuickChange II Site-Directed Mutagenesis kit (Stratagene) according to the manufactures recommendations. To fully abolish the effect of siRNAs, two nucleotides in the siRNA-targeted area were changed in both Rac3 and RhoG GTPases. To create a RhoG fast cycling mutant, glutamine 63 was converted to lysine.
## Reverse transcriptase and real-time quantitative pcr.
Total RNA was harvested from cells and converted to cDNA as previously described [bib_ref] RhoC GTPase is required for PC-3 prostate cancer cell invasion but not..., Yao [/bib_ref]. PCR primers were designed using the primer design feature on the Evocycler PCR program (Evogen Ltd., UK). Primer design parameters were set to optimally produce PCR products between 100 and 150 bp in size. Primer sequences are found in Supplemental (see in Supplementary Material available online at doi:10.1155/2011/541851). RT-PCR was performed on an Evocycler EPx (Evogen Ltd.) using Fast SYBR Green chemistry (Applied Biosystems Inc., Foster City, Calif) per the manufacturers recommendations for 30 cycles (98 - C for 15 s, 67 - C for 15s, and 72 - C for 30s), and PCR products visualized on a virtual gel and band intensities were normalized to GAPDH using the Evocycler PCR program.
For quantitative (q)PCR, RNA was isolated from the cell lines using TRIzol Reagent (Invitrogen, Carlsbad, Calif). cDNA was synthesized from this RNA using the Promega Reverse Transcription kit (Promega Corp., Madison, Wis). Appropriate primers (Integrated DNA Technologies, Inc., Coralville, Iowa) were diluted to a final concentration of 10 μM. The cDNA synthesized from the isolated RNA was diluted to a final concentration of 4 ng/μL. Reactions were prepared as a bulk "master mix" using the ABI SYBR Green PCR Master Mix (Applied Biosystems Inc., Foster City, Calif) for each target gene/primer pair used. Three no-template controls were included for each primer pair being used. A 5 μL aliquot of cDNA was pipetted into each well of the ABI 96-well plate, and 20 μL of the reaction master mix was added to it. Plates were covered with ABI adhesive cover, centrifuged at 1000 rpm to mix the contents, and run on an ABI 7000 real-time qPCR machine housed in the Center for Translational Cancer Research (University of Delaware).
## Tumor cell diapedesis assays.
Tumor cell diapedesis assays were performed as previously described [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref]. Briefly, 100,000 HBME cells were added to the top chamber of either uncoated or Matrigel-coated Transwells 24 h prior to the assay and allowed to form a confluent monolayer. PC-3 and C4-2 cells were harvested, labeled with Calcein AM (Invitrogen/Molecular Probes) per manufacturers recommendations, and resuspended in serum-free medium containing 0.1% BSA at a concentration of 3.75 × 10 5 cells/mL, and 0.5 mL was added to the top chambers. The chambers were incubated for 24 h at 37 - C in a 10% CO 2 incubator. Medium was aspirated from the top chamber, and excess Matrigel and cells were removed from the filter using a cotton swab. Filters were cut away from the inserts, mounted on microscope slides, and visualized on a fluorescent microscope and number of invaded cells counted.
## Rac gtpase activation assay. activation of total rac
GTPase proteins was performed using a GLISA pan-Rac activation assay kit (Cytoskeleton Inc., Denver, Colo) as previously described [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref]. Briefly, prostate cancer cells were grown to 75% confluence in a 100 mm dishes and serum starved for 24 h. On the day of the assay, cells were harvested using nonenzymatic cell dissociation buffer (Sigma-Aldrich), washed twice with ice-cold PBS, and resuspended in 65 μL GLISA lysis buffer. Protein lysates were transferred to icecold 1.5 mL centrifuge tubes and clarified by centrifugation at 10,000 rpm for 2 min. Protein concentrations were determined using the supplied Precision Red advance protein assay and 1.0 mg/mL protein used for the GTPase activation assay per manufacturers recommendations. After antibody and horseradish peroxidase detection reagent incubation, signals were detected on a Benchmark Plus microplate spectrophotometer at 490 nm (Bio-Rad Laboratories, Hercules, Calif).
## Atomic force microscopy.
All AFM experiments were conducted with a Bioscope II (Vecco, Santa Barbara, Calif) using silicon-nitride tips (Vecco; spring constant 0.06 N/m). Unbinding force measurements were conducted with tips functionalized with collagen or fibronectin (Becton-Dickinson, Franklin Lakes, NJ) at concentrations of 50 μg/mL and 15 μg/mL, respectively. Likewise, 35 mm tissue culture dishes (Corning Inc., Corning, NY) were coated with collagen or fibronectin and sterilized under ultraviolet light overnight. PC-3 cells were transfected with siRNA specific for Rac1, Rac3, or RhoG using FuGene6 (Roche) or GeneSilencer Reagent (Genlantis) and plated on the prepared dishes 8 h prior to experimentation. BMECs were cultured in RPMI 1640 media (Hyclone/Thermo Scientific) supplemented with 10% FBS. The functionalized AFM tip was dropped onto a single live BMEC cell and after attachment was verified, the loaded tip was gently lowered onto the center of a PC-3 cell. The unbinding force interaction between the two live cells was measured. The unbinding force is the force required to separate two adhesion molecules and is measured in picoNewtons (pN). The number of events for a particular unbinding force is the number of molecules separated at each force. Specifically, 250 unbinding events were captured per cell site with 4 areas probed per cell, and 3 separate cells were probed per treatment. Force curves were generated at a frequency of 1 Hz in a relative trigger mode.
AFM stiffness measurements were based on recording the elastic response of cells, BMECs and PC-3s using an AFM tip. The AFM was operated in the force-volume mode for recording a set of loading/unloading load displacement curves at a frequency of 1.03 Hz and a forward/reverse velocity of 4.11 μm/sec. The resultant measurement is the dynamic elastic modulus (a.k.a. the Young's modulus), which measures the stiffness of the cell. The Young's modulus is the ratio of stress to strain and is thus represented by units of pressure, Pascals (Pa). Cell stiffness changes are due to morphologic changes resulting from alterations in cytoskeletal structure (reviewed in [bib_ref] Connections between single-cell biomechanics and human disease states: gastrointestinal cancer and malaria, Suresh [/bib_ref]. The elastic modulus was measured with individual BMECs, individual PC-3 cells, and the duo: PC-3 cells attached to plated BMECs and BMECs attached to plated PC-3 cells. The elastic modulus for the BMEC/PC-3 combinations was generated for the plated cell, and the attached cells separately. Each force-volume map consists of 256 data points per sample site with 3 separate sites measured per experimental condition, 3 separate times.
## Transendothelial electrical resistance (teer).
Transendothelial electrical resistance (TEER) measurements were done using Epithelial Voltohmmeter (EVOM; World Precision Instruments Inc., Sarasota, Fla) following manufacturers directions. Briefly, BMECs were plated at a concentration of 1.3 × 10 6 cells/mL on 12-well 0.4 μ polycarbonate membrane inserts (CLS3401; Corning Transwell) and were maintained until day 4 (we determined empirically that the TEER for the BMEC monolayer was optimum on day 4 after plating due to maturation of cell junctions). On day 4, tissue culture medium was removed from the top chamber, an equal concentration of PC-3 cells was added to the BMEC monolayer and TEER measured at specified intervals.
## Fluorescence-activated cell sorting (facs) analysis.
Prostate cancer cells were cultured in T25 flasks (Corning Inc., Edison, NJ), detached, washed, and resuspended in 5% bovine serum albumin (BSA; Sigma-Aldrich) in phosphate buffered saline (PBS; Sigma-Aldrich). All washes and resuspensions were also performed in 5% BSA containing PBS. One set of control and siRac1-transfected prostate cancer cells were each further treated with CCL2 (100 ng/mL) for 30 min, washed, and resuspended. The several states of β1 activation were queried with two conformationsensitive antibodies N29 (BD Biosciences, Franklin Lakes, NJ) and HUTS-21 (BD Biosciences) in addition toa total β1 conformation-insensitive antibody, MAR4 (Chemicon, Billerica, Mass). All antibodies were used at a final concentration of 10 μg/mL, and all incubations were conducted in the dark and at 37 - C. Cells were analyzed using an FACS Calibur cytometer (BD Biosciences), equipped with 488 nm and 633 nm lasers. Analyses were performed on 10,000-gated events, and the numeric data were processed with Cellquest software (Becton Dickinson).
# Statistical analysis.
All experiments were performed a minimum of three separate times with individual transfections consisting of no less than three replicates per experiment. Statistical analysis of the combine experiments was performed using GraphPad Prism and by the University of Delaware College of Agriculture and Natural Resources Statistics Laboratory. A one-way ANOVA analysis was used with Bonferroni's post hoc analysis for comparison between multiple groups. A Students t-test was used for comparison between two groups. Significance was defined as a P value < .001. Data is represented as mean ± standard deviation.
# Results
## Active rac gtpases affect prostate cancer cell transendothelial migration. previously, we demonstrated that rac1
GTPase was required for tumor cell transendothelial cell migration. However, we did not thoroughly explore if other Rac family members contributed to diapedesis [bib_ref] RhoC GTPase is required for PC-3 prostate cancer cell invasion but not..., Yao [/bib_ref] [bib_ref] CCL2 induces prostate cancer transendothelial cell migration via activation of the small..., Van Golen [/bib_ref] [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref]. The expression of Rac1 and Rac3 GTPases is increased in PCa patient tumors; however, it is unknown if RhoG is expressed [bib_ref] Prognostic relevance of increased Rac GTPase expression in prostate carcinomas, Engers [/bib_ref]. Using quantitative (q)PCR, we demonstrate detectable message for Rac1, Rac3 and RhoG in the PC-3 cells. As shown in, normalized Rac1 mRNA expression levels were on average 8-fold higher than both Rac3, and RhoG, suggesting that Rac1 is the predominant Rac GTPase expressed in the PC-3 cells. Expression levels were confirmed when the products of a semiquantitative PCR were visualized by virtual gel (Supplemental. Band intensity for each product on the virtual gel is automatically normalized to the corresponding GAPDH. Similar expression levels were observed for C4-2 prostate cancer cells (Supplemental . Due to the lack of specific antibodies, particularly for Rac3, protein expression levels were not assessed by Western blot analysis.
To elucidate the role of each Rac protein in transendothelial cell migration, we selectively downregulated the expression of Rac GTPase isoforms using siRNA.is the results of isoform-specific Rac message depletion using siRNA duplexes. Expression of each Rac mRNA was significantly reduced by a minimum of 80% compared to PC-3 cells treated with an appropriate scrambled control. Each siRNA specifically reduced its target without affecting other Rac GTPases or affecting cell growth (growth data not shown). Similar results were seen when alternate siRNAs were used for each Rac isoform.
PC-3 cells were treated with the pharmacologic RacGEF inhibitor NSC23766 (iRac) or Rac-specific siRNA and the effect on total Rac activity determined. As expected, both the NSC23766 inhibitor and Rac1-specific siRNA reduced total active Rac levels by ∼60% compared to untransfected control. Interestingly, knockdown of RhoG led to a significant 45% reduction in total Rac activity suggesting that RhoG may activate Rac1 during physiologic process such as diapedesis. In contrast to Rac1 and RhoG, knockdown of Rac3 resulted in a significant 52% increase in total Rac activity compared to control.
Since Rac GTPases are required for transendothelial cell migration across a BMEC layer [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref] , we next tested the individual role of Rac1, Rac3, and RhoG in PCa diapedesis across a BMEC layer. As expected, downregulation of Rac1 led to a significant decrease in diapedesis. However, inhibition of RhoG and Rac3 had no effect on inhibiting tumor cell diapedesis. In contrast to Rac1, depletion of Rac3 led to a 70% increase in transendothelial migration, suggesting that Rac3 limits PCa diapedesis similar to what has been shown for RhoA in PCa invasion [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref]. The increase in diapedesis observed when RhoG was depleted approached but did not achieve significance compared to untransfected or scrambled controls.(1) and (2), specific for Rac1, Rac3, or RhoG, were compared. Messenger RNA was harvested and SYBR green-based qPCR performed using primers specified in Supplemental .
Relative expression levels were normalized to GAPDH expression from the corresponding sample and expressed as arbitrary units (a.u.). (b) is the effect of the RacGEF inhibitor NSC23766 (iRac), siRNA specific for Rac1, Rac3, and RhoG, or scrambled control (siScr) on total Rac activation. Cells were treated with 100 μM NSC23766 for 1 h or 20 μM siRac1, siRac3, or siRhoG. Activation of total Rac was performed using GLISA. Cells treated with iRac or transfected with siRNA to Rac isoforms were compared with untransfected (UT) and representative siRNA-scrambled control (siScr). Each analysis was performed in triplicate with individual transfections. (c) BMECs were layered onto a Matrigel-coated filter and allowed to form a monolayer; 0.5 mL of a suspension of 3.75 × 10 5 PC-3 cells/mL were added to the BMECs and allowed to undergo diapedesis for 24 h. Treated and transfected cells were compared with untransfected or scrambled controls. (d) Introduction of an RNAi-insensitive Rac3 into siRac3-treated PC-3 cell. Shown in all four panels is the mean ± S.D. of at least triplicate analysis with significance being * P < .001. Noncapped lines above the bars represent that the siRNA group is significantly different from controls.
RhoG on tumor cell diapedesis. Supplemental demonstrates a similar trend for the C4-2 prostate cancer cells. Depletion of Rac1 led to a significant decrease in transendothelial cell migration. However, depletion of Rac3 or RhoG increased tumor cell diapedesis. Rescue experiments reversed the trends of the siRNAs in the C4-2 cells.
## The chemokine ccl2 stimulates diapedesis via rhog
GTPase. The chemokine CCL2 is produced by BMECs and stimulates Rac1-mediated tumor cell diapedesis [bib_ref] CCL2 induces prostate cancer transendothelial cell migration via activation of the small..., Van Golen [/bib_ref] [bib_ref] CCL2 as an important mediator of prostate cancer growth in vivo through..., Loberg [/bib_ref]. Since RhoG appears to have an effect on Rac activation, we next set out to determine if CCL2-stimulated diapedesis could be affected by depletion of RhoG. As shown in , CCL2 treatment increased diapedesis 3-fold UN/UT UT siScr siRac1(1) siRac1 (2) siRhoG (1) siRhoG (2) siRhoG (1) Introduction of a siRNA-resistant RhoG fully rescued CCL2induced diapedesis in RhoG-depleted cells. However, introduction of a RhoGQ63L fast cycling mutant did not rescue the cells ability to cross an endothelial cell layer when Rac1 was depleted. Finally, Supplemental demonstrates that CCL-2-induced diapedesis is inhibited in C4-2 cells when RhoG is depleted. Again, introduction of a siRNAresistant RhoG fully restores the cells ability to cross the BMEC layer. demonstrates that CCL2-induced total Rac activation is decreased by ∼40% when RhoG is depleted from the PC-3 cells, suggesting that CCL2 may activate Rac1 directly and also indirectly through RhoG GTPase. Concordant to what is observed in the diapedesis assay, introduction of a siRNA-resistant RhoG restores actives levels of total Rac similar to controls. Restoration of Rac activity and PCa diapedesis in the rescue experiments were not due to overexpression of nonphysiologic levels of ectopic RhoG. As shown in , during rescue, mRNA levels of RhoG were increased 4-fold over the RhoG-depleted cells. These expression levels were still well under what is observed for the siScr control cells. Similar results were observed for the C4-2 cells and in the RNAi-insensitive Rac1 rescued cells. On average, an ∼70% transfection efficiency was observed for each construct in both the PC-3 and C4-2 cells.
## Rac1 gtpase mediates the interaction between pc-3 cells and bmecs.
We previously demonstrated that downregulation of Rac1 does not significantly affect PC-3 cell binding to BMECs [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref]. However, anecdotal evidence suggested that Rac1 depletion leads to decreased binding strength of the PC-3 cells to BMECs. To quantitate binding strength, we used atomic force microscopy (AFM) to measure the unbinding force of PC-3 cells bound to BMECs after Rac1, Rac3, or RhoG depletion. For the siScr control, siRac3-and siRhoGtreated PC-3 cells, a number of individual unbinding events occurred over time suggesting tight binding of multiple adhesion molecules is involved in cell-cell contact. In contrast, down-regulation of Rac1 led to a significant decrease in the number and frequency of unbinding events that occurred, suggesting fewer and weaker cell-cell contacts. shows that depletion of Rac1 led to a significant average 85% decrease in the unbinding force of the PCa cells to the bone marrow endothelial cells. Interestingly, downregulation of RhoG did not affect the ability of the PC-3 cells to bind to the BMECs, suggesting that RhoG activation of Rac1 is not involved in cell-cell binding.
In a system resembling initial contact during diapedesis, PC-3 cells were allowed to bind to a BMEC monolayer, and the dynamic elastic modulus (a.k.a. Young's modulus) was measured using AFM. Because of the pronounced effect of Rac1 depletion on PCa cell adhesion to BMECs seen in , we compared siScr control and siRac1-transfected PC-3 cells.shows that the elasticity was essentially unchanged for the siRNA-scrambled control and siRac1 PC-3 cells alone. Compared to the unbound cells, the siRNA control PC-3 cells became more elastic (a decrease in the Young's modulus) when bound to BMECs suggesting that they begin to spread onto the endothelial cell monolayer. In contrast, the PC-3 cells transfected with siRNA to Rac1 were significantly less elastic (increase in the Young's modulus) than the unbound PC-3 cells when bound to BMECs suggesting that they remain in a rounded configuration as we previously reported [bib_ref] RhoC GTPase is required for PC-3 prostate cancer cell invasion but not..., Yao [/bib_ref]. are measurements of the dynamic elastic modulus or elasticity of PC-3 and BMECs in contact with one another. Elasticity is given as the Young's modulus and is a ratio of cell stress and strain and is measured in Pascals. BMECs were grown as a monolayer and control (siScr) or siRac1 expressing PC-3 cells were allowed to bind to the BMEC monolayer, and the elasticity of the PC-3 cells (a) and the BMECs (b) was measured by AFM. Data are the result of over 10,000 data points and represented as mean ± S.D. with significance being * P < .001. (c) are measurements of transendothelial electrical resistance (TEER). BMECs were grown on a monolayer, PC-3 cells were added to the monolayer, and the electrical resistance was measured every 10 min up to 1 h (i) and the final measurement at 24 h (ii).
by the PC-3 cells. BMECs had a significant 30% increase in elasticity when in contact with control PC-3 cells suggesting reorganization of the actin cytoskeleton. In contrast, the BMECs had no change in theirdynamic elastic modulus when bound to Rac1-depleted PC-3 cells.
In a variation of this experiment, we allowed individual BMECs to come into contact with a PC-3 cell monolayer (Supplemental . Again, the elasticity of the PC-3 cells was essentially unchanged due to downregulation of Rac1. There was a significant and consistent 30% increase in elasticity of the BMECs when they came in contact with the control PC-3 cells. However, there was no change in elasticity of the BMECs when they came into contact with PC-3 cells that had depleted Rac1.(i) demonstrates a marked change in transendothelial electrical resistance (TEER) across the BMEC layer. TEER is a measure of the integrity of tight junctions between cells. Decreased TEER is indicative of cellular retraction. Addition of PC-3 cells to a confluent BMEC layer led to a significant, time-dependent decrease in TEER. TEER levels were fully restored, in a time-dependent manner by 24 h(ii)). Taken together these data suggest that the BMECs undergo cytoskeletal changes that influence their elasticity when they interact with PC-3 cells.
## Active rac1 gtpase leads to stimulation of β1
Integrins. Active Rho GTPases are known to lead to expression and activation of integrins [bib_ref] Integrins and GTPases in tumour cell growth, motility and invasion, Keely [/bib_ref]. Two integrin heterodimers are associated with binding to VCAM-1 and ICAM-1 on endothelial cells, α4β1 and αLβ2, respectively. With this in mind, we set out to determine if Rac1 GTPase influenced the activation state of integrins leading to BMEC binding. Since β2 integrins are not associated with prostate cancer and the role of the β1 integrins is established in PCa/BMEC interactions [bib_ref] Preferential adhesion of prostate cancer cells to a human bone marrow endothelial..., Lehr [/bib_ref] [bib_ref] Preferential adhesion of prostate cancer cells to bone is mediated by binding..., Cooper [/bib_ref] [bib_ref] Stepping out of the flow: capillary extravasation in cancer metastasis, Miles [/bib_ref] , we focused on the expression and activation of the β1 subunits. To determine this fluorescenceactivated cell sorting (FACS), analysis was performed using a set of antibodies that recognize total and active levels of the β1 subunit. The MAR4 antibody recognizes total β1 integrin subunit regardless of activation state. The α4β1 heterodimer can exist in 3 conformations, closed headpiece/bent (inactive), closed headpiece/extended (partially activated, recognized by the N29 antibody), and open headpiece/extended (fully active, recognized by the HUTS21 antibody). [fig_ref] Figure 5: Effect of Rac1 GTPase on β1 integrin activation [/fig_ref] demonstrates that unstimulated PC-3 cells have similar levels of total and partially activated β1 integrin as compared to Rac1-depleted PC-3 cells. When the cells were treated with CCL2, thus leading to increased Rac1 activation, there was no change in total and partially activated levels of β1 integrin. However, significantly more fully activated β1 integrin was detected in the control but not Rac1-depleted PC-3 cells. For simplicity, the results shown are from one siRNA; however, near identical results were obtained with alternate siRNAs. [fig_ref] Figure 5: Effect of Rac1 GTPase on β1 integrin activation [/fig_ref] demonstrates that the decrease in CCL2stimulated β1 integrin activity due to Rac1 depletion can be rescued by expression of an RNAi-insensitive Rac1 GTPase. Similarly, depletion of RhoG GTPase led to a significant decrease in CCL2-induced active β1 integrin expression as compared to scrambled control. Expression of a siRNAresistant RhoG led to a significant increase in β1 integrin activation. In both cases, the RNAi-insensitive GTPases restored CCL2 activation of β1 integrin to levels comparable to the control cells.
# Discussion
The Rho GTPases comprise a subfamily of the Ras superfamily of monomeric GTP-binding proteins [bib_ref] Rho-family GTPases: it's not only Rac and Rho (and i like it), Wennerberg [/bib_ref]. Like Ras, the Rho proteins transiently move from an inactive to active to an inactive state via the GTPase cycle. This cycle is controlled by a number of regulatory proteins, which in turn regulate Rho signal transduction via effector proteins [bib_ref] GEFs, GAPs, GDIs and effectors: taking a closer (3D) look at the..., Geyer [/bib_ref] [bib_ref] GEF means go: turning on Rho GTPases with guanine nucleotide-exchange factors, Rossman [/bib_ref] [bib_ref] GTPase-activating proteins and their complexes, Gamblin [/bib_ref] [bib_ref] Regulation of morphology by rho p21 and its inhibitory GDP/GTP exchange protein..., Miura [/bib_ref] [bib_ref] The small GTPase Rho: cellular functions and signal transduction, Narumiya [/bib_ref]. This coordinate regulation of the Rho proteins allows for cytoskeletal reorganization leading to changes in cell shape and motility [bib_ref] Rho GTPases and cell migration, Ridley [/bib_ref] [bib_ref] Rho family proteins: coordinating cell responses, Ridley [/bib_ref]. Overexpression and/or aberrant activation of individual Rho GTPases has been shown in a number of cancers and is thought to drive metastatic progression [bib_ref] RHO-GTPases and cancer, Sahai [/bib_ref]. Although one Rho protein may be the predominant GTPase in a cancer, other GTPases must also become active to reorganize the actin cytoskeleton and drive migration.
RhoC GTPase is expressed in several cancers and promotes metastasis [bib_ref] A novel putative low-affinity insulin-like growth factor-binding protein, LIBC (lost in inflammatory..., Van Golen [/bib_ref] [bib_ref] Genomic analysis of metastasis reveals an essential role for RhoC, Clark [/bib_ref] [bib_ref] RhoC is dispensable for embryogenesis and tumor initiation but essential for metastasis, Hakem [/bib_ref] [bib_ref] Up-regulation of small GTPases, RhoA and RhoC, is associated with tumor progression..., Horiuchi [/bib_ref] [bib_ref] A definitive role of RhoC in metastasis of orthotopic lung cancer in..., Ikoma [/bib_ref] [bib_ref] RhoC-GTPase is a novel tissue biomarker associated with biologically aggressive carcinomas of..., Kleer [/bib_ref] [bib_ref] Expression of RhoC is associated with metastasis of gastric carcinomas, Kondo [/bib_ref] [bib_ref] Higher expression of RhoC is related to invasiveness in non-small cell lung..., Shikada [/bib_ref] [bib_ref] Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of..., Suwa [/bib_ref] [bib_ref] Expression and significance of RhoC gene in hepatocellular carcinoma, Wang [/bib_ref]. Previously, we demonstrated that RhoC GTPase expression and activation is required for PCa invasion [bib_ref] RhoC GTPase is required for PC-3 prostate cancer cell invasion but not..., Yao [/bib_ref] [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref] [bib_ref] Type I colagen receptor (alpha2 beta1) signaling promotes the growth of human..., Hall [/bib_ref] [bib_ref] Type I collagen receptor (αβ) signaling promotes prostate cancer invasion through RhoC..., Hall [/bib_ref]. When RhoC expression or activation is downregulated, the PCa cells undergo Rac GTPase-mediated EMT [bib_ref] RhoC GTPase is required for PC-3 prostate cancer cell invasion but not..., Yao [/bib_ref] [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref]. Decreased Rho expression or activity leads to increased expression and sustained activity of Rac1. Furthermore, Rac expression and activation was found to be required for tumor cell diapedesis across a human BMEC layer. We believe that together RhoC and Rac are needed to drive PCa extravasation from the vasculature into the bone marrow environment.
There are four members of the Rac branch of the Rho subfamily: Rac1, Rac2, Rac3, and RhoG. Rac1 and Rac3, but not Rac2, are shown to have increased expression in PCa, while expression of RhoG has not been examined [bib_ref] Prognostic relevance of increased Rac GTPase expression in prostate carcinomas, Engers [/bib_ref]. In the current study, we set out to determine the individual roles of Rac1, Rac3, and RhoG in tumor cell diapedesis. Rac1 levels are significantly higher than either Rac3 or RhoG suggesting that it is the predominant Rac GTPase in these cells. The relative levels of Rac1 and Rac3 in PCa are similar to what has been shown in glioblastoma cells [bib_ref] Roles of the Rac1 and Rac3 GTPases in human tumor cell invasion, Chan [/bib_ref]. Rac3 GTPase has clearly been shown to be involved in adhesion of tumor and normal cells of neural origin [bib_ref] Rac1 and rac3 have opposing functions in cell adhesion and differentiation of..., Hajdo-Milasinovic [/bib_ref] [bib_ref] Roles of the Rac1 and Rac3 GTPases in human tumor cell invasion, Chan [/bib_ref]. Normal and malignant prostate has a neuroendocrine component; therefore, the question arises if Rac3 expression plays a role in neuroendocrine differentiation of PCa [bib_ref] Neuroendocrine cells in the normal, hyperplastic and neoplastic prostate, Noordzij [/bib_ref] [bib_ref] Neuroendocrine cells in tumour growth of the prostate, Abrahamsson [/bib_ref] [bib_ref] Neuroendocrine differentiation in prostatic carcinoma, Di Sant'agnese [/bib_ref]. We have clear AFM data that implicates Rac3 in binding of PCa cells to fibronectin and to a lesser extent, collagen I (unpublished data). Binding to laminin would be the next logical choice to examine. This aspect may also begin to explain the apposing effect that Rac3 has on Rac1 and transendothelial cell migration. We found that downregulation of Rac3 led to an increase in total Rac activity, independent of an increase in total Rac protein levels. A similar observation was made previously; downregulation of RhoC increased Rac1 activity [bib_ref] RhoC GTPase is required for PC-3 prostate cancer cell invasion but not..., Yao [/bib_ref] [bib_ref] Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell..., Sequeira [/bib_ref]. However, this was accompanied by an increase in total Rac1 protein.
Expression of RhoG is found ubiquitously throughout the body, but its expression in PCa has not been studied. We found that RhoG, although expressed in low levels, has an effect on total Rac activation. Inhibition of RhoG led to a significant decrease in Rac activation, but diapedesis was slightly increased. In contrast, CCL2-stimulation of PCa cells transfected with siRNA specific for RhoG significantly decreased diapedesis. Coexpression of a siRNAresistant RhoG led to restoration of the cells ability to cross the endothelial cell layer. Furthermore, depletion of RhoG led to a significant decrease in CCL2-stimulated Rac activation, suggesting that CCL2 activates Rac1. Expression of a fast cycling RhoG in Rac1-depleted cells did not rescue the cells ability to undergo diapedesis. This also suggests that Rac1-mediated diapedesis may be regulated through direct activation of Rac1 or indirectly via RhoG GTPase and the different effects that RhoG has on PCa diapedesisis intriguing. Without CCL2 stimulation, RhoG appears to act like Rac3 and limit diapedesis, even after decreasing total Rac activation. Upon CCL2 stimulation, RhoG appears to play a role in activating Rac1 thereby decreasing diapedesis. This may suggest a specific RhoG GEF(s) that are activated by CCL2. Also of interest is the fact that ectopic expression of an RNAi-insensitive RhoG led to a significant decrease in unstimulated diapedesis suggesting a balance of RhoG expression required for migration.
Our results measuring BMEC stiffness using AFM showed specific differences that were consistent over a large array of experimental attempts suggesting a specific biological interaction. The PC-3/BMEC interaction is of particular interest; PC-3 cells, whether adhered to substrate or attached to a BMEC, maintain a constant measured elastic modulus. The BMECs, however, when in contact with a PCa cell consistently, undergo a 30% decrease in stiffness. This apparent conferred decrease in stiffness points to a change in the internal cytoskeletal architecture of the BMEC. Depletion of Rac1 in the PC-3 cells led to a significant decrease in the strength of binding to the BMECs. The elasticity of the BMEC cell was not decreased when bound to a Rac1depleted PC-3 cell indicating a Rac1-mediated interaction between the two cells. This interaction may be due, at least in part, to binding mediated by β1 integrins. We demonstrate that unstimulated PC-3 cells have partially activated β1 integrins that become fully activated upon CCL2 stimulation and activation of Rac1. Clearly, β1 integrins are required for binding of PCa cells to extracellular matrix [bib_ref] Prostate cancer cell adhesion to quiescent endothelial cells is not mediated by..., Cooper [/bib_ref] [bib_ref] Type I colagen receptor (alpha2 beta1) signaling promotes the growth of human..., Hall [/bib_ref] [bib_ref] Type I collagen receptor (αβ) signaling promotes prostate cancer invasion through RhoC..., Hall [/bib_ref] [bib_ref] Characterization of integrin subunits, cellular adhesion and tumorgenicity of four human prostate..., Witkowski [/bib_ref] [bib_ref] Specific alterations in the expression of alpha 3 beta 1 and alpha..., Dedhar [/bib_ref]. Studies in the literature suggest a role for β1 integrins in binding PCa to BMECs [bib_ref] Preferential adhesion of prostate cancer cells to a human bone marrow endothelial..., Lehr [/bib_ref] [bib_ref] Prostate cancer cell adhesion to quiescent endothelial cells is not mediated by..., Cooper [/bib_ref] [bib_ref] Primary prostatic epithelial cell binding to human bone marrow stroma and the..., Lang [/bib_ref] [bib_ref] Interactions of human prostatic epithelial cells with bone marrow endothelium: binding and..., Scott [/bib_ref]. One report suggests that the use of a β1 integrin-blocking antibody did not affect PC-3 cell binding to the human bone marrow endothelial cell line HBME-1 but was responsible for mediating PCa interactions with fibronectin [bib_ref] Prostate cancer cell adhesion to quiescent endothelial cells is not mediated by..., Cooper [/bib_ref]. However, other studies show a role for β1 integrins in binding to other bone marrow endothelial cells [bib_ref] Primary prostatic epithelial cell binding to human bone marrow stroma and the..., Lang [/bib_ref] [bib_ref] Interactions of human prostatic epithelial cells with bone marrow endothelium: binding and..., Scott [/bib_ref]. Rho GTPases such as Rac1 are implicated in bidirectional signaling with integrins activating Rho proteins and the active Rho proteins promoting integrin dimer activation increasing binding strength [bib_ref] Integrins and GTPases in tumour cell growth, motility and invasion, Keely [/bib_ref] [bib_ref] Integrin-linked kinase activity regulates Rac-and Cdc42-mediated actin cytoskeleton reorganization via α-PIX, Filipenko [/bib_ref] [bib_ref] Integrin regulation of membrane domain trafficking and Rac targeting, Grande-Garcia [/bib_ref].
These mechanisms are similar to leukocyte diapedesis, where, after initial binding, the interaction between the leukocyte and endothelial cell increases leading to dynamic cytoskeletal changes and endothelial cell retraction [bib_ref] Trading spaces: Rap, Rac, and Rho as architects of transendothelial migration, Wittchen [/bib_ref] [bib_ref] Rap1 GTPase inhibits leukocyte transmigration by promoting endothelial barrier function, Wittchen [/bib_ref]. Although this has been suggested for PCa diapedesis, this is the first time this has been shown experimentally. A complete understanding of how these different Rac proteins are activated and how they contribute to tumor cell diapedesis may have profound implications for any strategies targeting the extravasation process.
[fig] Figure 1: (d) demonstrates that expression of a siRNA-resistant Rac3 in Rac3-downregulated cells results in a significant decrease in diapedesis. Similarly, re-expression of RhoG led to a significant decrease in diapedesis compared to cells depleted of RhoG. This suggests a negative effect of Rac3 and possibly Effect of Rac depletion on total Rac activation and tumor cell diapedesis across a bone marrow endothelial cell monolayer. (a) Rac isoform expression and isoform-specific depletion in PC-3 cells. Individual siRNAs [/fig]
[fig] Figure 2, Figure 3: Effect of Rac depletion on tumor cell diapedesis across a BMEC monolayer. (a) PC-3 cells were treated with 100 ng/mL CCL2 in a diapedesis assay. Control untransfected (UT) and siRNA control (siScr) cells demonstrated increased diapedesis compared with untreated/untransfected (UN/UT) PC-3 cells. The ability of cells to undergo CCL2-stimulated diapedesis after depletion of Rac1, RhoG, or treatment with iRac was compared to UT and siScr. Rescue experiments of RhoG-depleted cells were performed by the introduction of a siRNA-insensitive RhoG. Rac1-depleted cells were rescued with the introduction of fast cycling RhoG (RhoGQ63L). (b) Depletion of RhoG led to a decrease of total Rac activation in PC-3 cells treated with 100 ng/mL CCL2. Rescue experiments were performed by introducing a siRNA-insensitive RhoG GTPase. Shown are means ± S.D. of at least triplicate analysis representing individual transfections, with significance being P < .001; ( * ) signifies a significant difference between siRNA-transfected cells and stimulated controls, while ( ∧ ) signifies a significant difference between siRNA-transfected and -rescued cells.across a BMEC layer in untransfected (UT) and siScr control cells compared to untreated/untransfected cells (UN/UT).Contrary to what we observed for unstimulated diapedesis inFigure 1(c), there was an approximate 45% decrease in PC-3 diapedesis across the endothelial cell layer when RhoG was depleted using siRNAs (P < .001). Similarly, direct depletion of Rac1 or treatment with the inhibitor NSC23766 led to a significant decrease in transendothelial cell migration. Interaction of prostate cancer cells with bone marrow endothelial cells BMECs was attached to the AFM tip and the unbinding forces of PCa cells measured. PC-3 cells were transfected with siRNAs specific for individual Rac isoforms. Shown are the results from one set of siRNAs. (a) Effect on the frequency of unbinding events and forces (pN) occurring between BMECs and PC-3 cells after depletion of each Rac isoform. (b) Average unbinding force occurring between BMECs and PC-3 cells. The average unbinding force is the physical force required to pull two adhered cells apart. Data are compiled from 3000 data points and are the mean ± S.D. with significance being * P < .001. [/fig]
[fig] Figure 4: (b) compares the elasticity of the cells in the BMEC monolayer Bone marrow endothelial cells react to PCa cell binding [/fig]
[fig] Figure 5: Effect of Rac1 GTPase on β1 integrin activation. (a) Comparison of total, partially and fully activated β1 integrinsin control and Rac1 depleted PC-3 cells as assessed by FACS analysis. Cells were transfected with either siRNA-scrambled control or Rac1 siRNA(2) and left unstimulated or treated with 100 ng/mL CCL2. FACS analysis was performed after incubating fixed cells with the antibodies MAR4 (for total β1 integrin), N29 (for partially active β1 integrin), and HUTS21 (for fully active β1 integrin). (b) is a rescue experiment demonstrating that introduction of either an RNAi-insensitive Rac1 or RhoG GTPase leads to restoration of active β1 integrin levels in CCL2-treated PC-3 cells. Shown are the results of triplicate experiments showing the percentages of 10,000 gated events with significance being * P < .001. Capped lines signify a comparison and significance between siRNA-depleted cells and cells rescued with siRNA-insensitive constructs. [/fig]
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10.7717/peerj-cs.1478
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CCBY
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10403162
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37547407
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s2orc_pubmed_articles
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What does Chinese BERT learn about syntactic knowledge?
Pre-trained language models such as Bidirectional Encoder Representations from Transformers (BERT) have been applied to a wide range of natural language processing (NLP) tasks and obtained significantly positive results. A growing body of research has investigated the reason why BERT is so efficient and what language knowledge BERT is able to learn. However, most of these works focused almost exclusively on English. Few studies have explored the language information, particularly syntactic information, that BERT has learned in Chinese, which is written as sequences of characters. In this study, we adopted some probing methods for identifying syntactic knowledge stored in the attention heads and hidden states of Chinese BERT. The results suggest that some individual heads and combination of heads do well in encoding corresponding and overall syntactic relations, respectively. The hidden representation of each layer also contained syntactic information to different degrees. We also analyzed the fine-tuned models of Chinese BERT for different tasks, covering all levels. Our results suggest that these fine-turned models reflect changes in conserving language structure. These findings help explain why Chinese BERT can show such large improvements across many language-processing tasks.
# Introduction
Bidirectional Encoder Representations from Transformers (BERT) [bib_ref] BERT: pre-training of deep bidirectional transformers for language understanding, Devlin [/bib_ref] , a type of pre-trained language model, has been widely used in the natural language processing (NLP) community [bib_ref] Transfer learning in biomedical natural language processing: an evaluation of BERT and..., Peng [/bib_ref] [bib_ref] Evaluation of BERT and ALBERT sentence embedding performance on downstream NLP tasks, Choi [/bib_ref]. BERT has greatly improved the effects of many NLP tasks [bib_ref] Glue: a multi-task benchmark and analysis platform for natural language understanding, Wang [/bib_ref]. Therefore, researchers have started to explore the cause of BERT's excellent performance [bib_ref] A primer in BERTology: what we know about how BERT works, Rogers [/bib_ref] and what knowledge BERT learned from the corpus during pre-training [bib_ref] Perturbed masking: parameter-free probing for analyzing and interpreting BERT, Wu [/bib_ref]. In other words, there has been a focus on the interpretability of the model [bib_ref] Dis-cover AI minds to preserve human knowledge, Ranaldi [/bib_ref]. Most of the work in this area has centered on the knowledge, such as lexicon [bib_ref] On the systematicity of probing contextualized word representations: the case of Hypernymy..., Ravichander [/bib_ref] , syntax [bib_ref] What does BERT look at?, Clark [/bib_ref] , and reasoning competence [bib_ref] How does BERT answer questions? A layerwise analysis of transformer representations, Aken [/bib_ref] learned by English BERT.
Unlike English, Chinese sentences involve a sequence of characters without explicit word boundaries [bib_ref] Does Chinese BERT encode word structure, Wang [/bib_ref]. Relatively little research has been conducted on the interpretability of Chinese BERT [bib_ref] Does Chinese BERT encode word structure, Wang [/bib_ref] [bib_ref] Discourse probing of pretrained language models, Koto [/bib_ref] [bib_ref] CLiMP: a benchmark for chinese language model evaluation, Xiang [/bib_ref]. Chinese BERT stores the information about the relationships between characters, and previous works have studied the word structure captured by Chinese BERT [bib_ref] Does Chinese BERT encode word structure, Wang [/bib_ref]. However, no research has ever explored whether Chinese BERT has determined the relationship between words composed of characters, as well as the syntactic information by which words can be organized into sentences. The research on the syntactic knowledge encoded in Chinese BERT can not only reveal the reasons why the model has achieved superb performance in many NLP tasks, but also guide the design of a more targeted model. Therefore, this work aimed to explore the syntactic ability of this model, Chinese BERT.
We designed a series of probing experiments, shown in [fig_ref] Figure 1: Illustration of syntactic probes [/fig_ref]. Our probs can be classified into two parts: for original BERT and for fine-tuned BERT. For probing the original Chinese BERT, each attention head of Chinese BERT was firstly detected. When a sentence was input, the attention information between words was represented by each head of each layer, which is the attention matrix of the sentence. We tested whether a specific head existed so that a certain type of dependency relationship could be better determined and exceed the baseline. We then explored whether the attention head was sensitive to the relative position in syntactic relations. According to the particular linguistic phenomena in Chinese, we investigated Chinese BERT's ability in some typical sentence structures, such as ''bèi'' construction, ''bǎ'' construction, and sentences using particles ''zhe'', ''le'', and ''guò'' to express aspects. Next, we combined all heads in the model to detect the prediction performance on the entire syntactic relationship. Additionally, we studied the syntactic knowledge learned by the hidden state of each layer. Following [bib_ref] What you can cram into a single $ & !#* vector: probing..., Conneau [/bib_ref] , we designed three syntactic tasks in the Chinese version and developed the corresponding datasets, namely tree depth, bigram shift, and dependency relation. By adding a simple classifier on the hidden state, we explored whether syntactic knowledge was learned by hidden representations, according to the results of the classifier on the three syntactic tasks. For probing fine-tuned Chinese BERTs, we fine-tuned Chinese BERT to downstream tasks at different levels. By comparing our results with the original Chinese BERT, we explored whether there were changes in the syntactic knowledge stored in the fine-tuned models.
Our experiments showed that no individual attention head could effectively learn the overall syntactic relationship, but some heads did capture the corresponding relationships. By combining attention heads, BERT could parse a sentence well, meaning that BERT's attention heads encoded a large amount of syntactic knowledge. In addition, some attention heads were able to learn certain linguistic phenomena in Chinese. Through probing relative positions, we found that the performance of heads became worse as the distance between the dependent word and head word increased. As for hidden states, syntactic information was embedded in each layer to various degrees. When fine-tuning into downstream tasks, we observed the changes in conserving syntactic knowledge. Part-of-speech (POS) tagging strengthened syntactic information in Chinese BERT to some extent, while natural language inference (NLI) enabled Chinese BERT to forget plenty of knowledge in the language's structure.
To our best knowledge, we are the first to investigate syntactic knowledge in Chinese BERT from different perspectives, including attention heads, hidden states, and downstream tasks. In addition, although our research took the most representative Chinese language model, Chinese BERT, as research object, our approaches and thoughts could be generalized to study other Chinese language models.
Our contribution can be summarized as the following:
(1) By referring previous work, we made out a series of comprehensive probes on attention heads about Chinese syntactic knowledge. Then we provided detailed analysis of these probing results.
(2) We modified the previous probing measure, which could be more applicable to Chinese with a character-based sequence.
(3) We evaluated linguistic phenomena learned by attention heads, and tested the impact of relative position on capturing syntactic knowledge.
(4) We released the Chinese datasets about three syntactic tasks: Bigram Shift (BShift), Tree Depth (TreeDepth), and Dependency Relation (DepRel).
# Related work
Researchers have proposed many methods to investigate the syntactic knowledge that English BERT has learned. [bib_ref] What does BERT look at?, Clark [/bib_ref] probed each attention head for various syntactic relationships by calculating accuracy in terms of the attention weights of the mostattended-to other word of each input word, and then they combined all attention heads to measure the overall dependency parsing ability. [bib_ref] A structural probe for finding syntax in word representations, Hewitt [/bib_ref] used a structural probe to investigate whether syntax trees were embedded into a word representation space of the neural network by way of linear transformation. They concluded that the syntactic trees could be relatively recovered. In addition to exploring attention heads, some researchers have studied syntactic knowledge stored in hidden states. designed a classifier on the span representations to probe syntactic knowledge in BERT. They concluded that BERT encodes syntax more than semantics.fed complete sentences into BERT while masking out the single focus verb and then asked BERT for word predictions of the masked position. It was determined that BERT learns significant knowledge of syntax, particularly subject-verb agreement. [bib_ref] BERTology for Machine Translation: What BERT Knows about Linguistic Difficulties for Translation, Dai [/bib_ref] used some syntactic probing tasks to analyze the performance of BERT's syntactic dependencies and demonstrated that BERT ''knows'' about these knowledge. In addition, they also found that BERT's ability to recognize syntactic dependencies often decreases after fine-tuning for NMT tasks. Besides, found that syntactic knowledge could be acted as a point to test the connection between the empirism in real world and the knowledge derived from BERT. Based on the probing works in English, [bib_ref] Cross-Linguistic Syntactic Difference in Multilingual BERT: How Good is It and How..., Ningyu [/bib_ref] evaluated the cross-lingual syntactic relations in mBERT. They overlaid a linear classifier to decode the syntactic relation between head word and dependent word of each language.Then visualized the output representations of each classifier to analyze and summarize relations among languages. The above research was an insightful reference for our study.
Another line of work has studied the linguistic knowledge that Chinese BERT has encoded. [bib_ref] Does Chinese BERT encode word structure, Wang [/bib_ref] investigated word features in Chinese BERT according to attention weight and some probing tasks, including Chinese Word Segmentation (CWS) and various-level downstream tasks in NLP. They found that some attention heads can implicitly capture word structure, and different Chinese tasks rely on word information to different degrees. [bib_ref] Discourse probing of pretrained language models, Koto [/bib_ref] introduced seven discourserelated probing tasks to explore the discourse structure that Chinese BERT has learned. By adding an MLP layer on top of the model, they tested the accuracy of the classifier on predicting the competence of Chinese BERT comprehending discourse structure. [bib_ref] CLiMP: a benchmark for chinese language model evaluation, Xiang [/bib_ref] constructed the corpus of Chinese linguistic minimal pairs (CLiMP) to study the knowledge that Chinese language models have acquired, including 16 grammatical contrasts in Mandarin, covering nine major Mandarin linguistic phenomena. However, their work did not explore what syntactic relationship Chinese language models have learned. They still determined the competence of models' language understanding in terms of the accuracy of the classifier on representation. Based on those works, we explored the syntactic knowledge of Chinese BERT across various aspects, including attention heads, hidden-state representation, and downstream tasks. The experimental results also showed Chinese BERT's abilities more thoroughly.
# Background: chinese bert
We chose Chinese BERT, a very representative transformer-based model [bib_ref] Attention is all you need, Vaswani [/bib_ref] , as the target for analysis. Chinese BERT [bib_ref] BERT: pre-training of deep bidirectional transformers for language understanding, Devlin [/bib_ref] is pre-trained on Chinese simplified and traditional text from a Chinese Wikipedia dump of about 0.4 billion tokens.
In this work, we used the PyTorch implementation of Chinese BERT. All our experiments were based on the BERT-based-Chinese model. This model contained 12 layers, and each layer had 12 attention heads (110M parameters). Given a Chinese sentence s = c 1 , c 2 , . . . , c n , c i delegated a token in the sentence. An attention head took as input vectors a sequence of e = [e 1 , e 2 , . . . , e n ], which corresponded to n tokens. For each token vector ei, an attention head transformed it into query (q i ), key (k i ), and value (v i ) vectors. An output vector (h i ) could be obtained via a weighted sum of value vectors based on attention distribution (α), a kind of weight matrix between all pairs of tokens. Attention distribution can be calculated using the dot product with a softmax function between the query and key vectors.
[formula] α ij = exp(q T i k j ) n l=1 exp(q T i k l ) h i = n j=1 α ij v j . [/formula]
The output vector h i represents the hidden state of a head about token c i . The hidden states of all heads from the same layer can be concatenated to obtain a hidden representation h i about token c i .
[formula] h i = [h 1 i ,h 2 i ,...,h n i ] [/formula]
where h j i represents the hidden state of j-th head ot token i. When preprocessing the input text, the special tokens [CLS] and [SEP] were added to the beginning and end of each sentence, respectively. Chinese BERT is pretrained on two tasks: masked language modeling (MLM) and next sentence prediction (NSP). The MLM task predicts the words masked randomly in the input, while NSP determines whether a sentence is subsequent to another in the original document.
## Probing tasks
It has been reported that BERT can implicitly encode linguistic knowledge [bib_ref] What does BERT learn about the structure of language, Jawahar [/bib_ref]. To identify what knowledge Chinese BERT has learned, some experiments have been designed to probe it. In this work, we first adopted two Chinese Dependency Treebanks as golden datasets for experiments and evaluation. Then we designed two types of probing tasks: attention-based tasks and hidden-state-based tasks. Attention-based tasks include probing individual attention heads, relative positions, and linguistic phenomena in Chinese which the attention head has learned. Hidden-state-based tasks evaluate the syntactic competence stored in the hidden state according to three syntactic tasks.
## Datasets
Different treebanks exist, with divergence in their annotation guidelines and corpus sources. We chose two representative Chinese dependency treebanks for our experiments: the Chinese Universal Dependencies treebank 2.11(UD 2.11) (https://universaldependencies. org/) and Chinese Dependency Treebank 1.0 (CDT 1.0) (https://catalog.ldc.upenn.edu/ LDC2012T05).
Universal Dependency is an open community covering nearly 200 treebanks in over 100 languages. We selected all Chinese treebanks from Universal Dependencies 2.11. The Chinese Universal Dependencies treebanks contain 8,460 sentences (161,856 words). The annotation guidelines can be found in [bib_ref] Universal dependencies, Marneffe [/bib_ref]. Chinese Dependency Treebank 1.0 was released by the Harbin Institute of Technology Research Center for Social Computing and Information Retrieval (HIT-SCIR). From the People's Daily newswire stories published between 1992 and 1996, 49,996 Chinese sentences (902,191 words) were randomly selected. For more details about the annotation guidelines, please refer to. We shuffled the data for subsequent experiments.
## Probing individual attention heads
## Setup
In this subsection, we probed which individual heads could best learn dependency relations. When we input a sentence into Chinese BERT, we obtained the attention matrix about characters in this sentence for each head. Considering that no explicit word boundary exists in Chinese sentences, we used the word segment of datasets in 'Datasets' as the standard. Then, we summed the columns and averaged the rows corresponding to the constituent characters of the standard words:
[formula] α w p →w q = 1 |w p | c i ∈w p c j ∈w q α c i →c j [/formula]
where, w p and w q are the words in the input sentence. c i and c j are the constituent characters in words w p and w q , respectively. α ∈(0, 1) n×n is the attention weight of a certain head regarding the input sentence. | w p | is the number of characters in w p . [fig_ref] Figure 2: An example sentence parsed by dependency relations and maximum attention weights [/fig_ref] shows that an example sentence parsed by dependency relations and expressed by attention weights from head 6-6. If an attention head learned a certain dependency relation well, this head had a higher probability of allocating the maximum weight to the head word in each row of the attention matrix. During the evaluation, we ignored the direction between the dependent word and head word, and tested the performance of each attention head on each dependency relation and overall relations. We used the undirected unlabeled attachment score (UUAS) as our evaluation:
[formula] UUAS = correct k i |rel i | [/formula]
where, |rel i | is the number of dependency relation i in the datasets, and correct k i is the number of correct predictions of relation i for a given head k.
## Baselines
We adopted positional offset and Random BERT as baselines. For the positional offset baseline, we determined the most common position where the head word could occur for each attention word. For the Random BERT baseline, we used a BERT-base model with randomly initialized weights.
# Results
Tables 1 and 2 show the results of our probing method and baselines on UD2.11 and CDT1.0, respectively. The number in the parentheses in the line ''positional offset'' is the offset location with the best performance (e.g., (-1) means the head word was located to the left of the dependent word). The number in the parentheses in the line ''Chinese BERT'' denotes the best performance head, i-j denotes the j-th head in the i-th layer. The 17 most common relations are shown in [fig_ref] Table 1: UUAS on UD2 [/fig_ref] and all relations are shown in [fig_ref] Table 2: UUAS on CDT1 [/fig_ref]. From the two tables, we found that Chinese BERT >Positional Offset >Random BERT in terms of performance. This indicated that the attention heads in Chinese BERT learned some dependency relations implicitly, while Random BERT captured very little syntactic knowledge (<10%). Meanwhile, positional offset performed similarly on some dependency relations, such as ''compound'' in [fig_ref] Table 1: UUAS on UD2 [/fig_ref] and ''RAD'' in [fig_ref] Table 2: UUAS on CDT1 [/fig_ref]. This could be because the head word appeared fixed in the distance of the dependent word. The attention head could only learn positional or distance information between the two words to achieve general performance.
In addition, we also found that some dependency heads did significantly learn some specific syntactic relations, sometimes achieving high accuracy, such as ''obj'' and ''aux'' in [fig_ref] Table 1: UUAS on UD2 [/fig_ref] , and ''VOB'' and ''POB'' in [fig_ref] Table 2: UUAS on CDT1 [/fig_ref]. However, no single heads performed well on the total relations. The best single heads only obtained 35.1 UUAS on the two datasets. This finding is similar to the work of [bib_ref] What does BERT look at?, Clark [/bib_ref] on English treebanks. We also found that most of the heads with the best performance in the specific dependency relations were located in the middle layers (layers 5-9). This was due to the fact that Chinese BERT encodes how to organize words into a sentence mostly in the middle layer, similar to English BERT [bib_ref] What does BERT learn about the structure of language, Jawahar [/bib_ref].
## Probing relative position
## Setup
According to the previous subsection, we found that positional offset could also achieve good performance on some dependency relations. Therefore, we investigated whether the distance between dependent words and head words could affect the performance of Chinese BERT in capturing syntactic knowledge. UUAS was still used as our evaluation metric for this experiment.
## Baselines
We adopted Random BERT as a baseline. For the full details, please refer to 'Probing individual attention heads'. [fig_ref] 3: 他去北京了 [/fig_ref] shows the accuracy of relative positions on UD2.11 and CDT1.0, respectively. We also found that Chinese BERT apparently exceeds Random BERT in different positional distributions. In addition, the performance of Chinese BERT decreased as the distance increased. This indicates that positional information between words is important for Chinese BERT. The closer the distance between the head word and dependent word, the better Chinese BERT can capture the dependency relation between the two. Among all relative positions, Chinese BERT achieves very high performance (>99%) when the head word and dependent word are next to each other (±1). Furthermore, in order to analyze the influence of positional distribution on different dependency relations, we calculated the accuracy of relative positions on the common relations of the two datasets, shown in . From this figure, we can easily see that the relation between model performance and dependency distance still exists in most dependency relations.
# Results
## Probing linguistic phenomena in chinese
## Setup
Based on the findings from the previous subsections, we were very interested in exploring particular linguistic phenomena existing in Chinese, as well as determining whether Chinese BERT had captured them. Hence, we designed a test suite for evaluation. Our test suite covered two sentence constructions unique in Chinese and three auxiliary words for expressing aspects in Chinese sentences. For sentence construction, we chose bǎ (把) construction and bèi (被) construction. For auxiliary words, -zhe (着), -le (了), and -guò (过) were adopted.
The particle bǎ (把) is commonly used in Chinese. It can change the word order from ''subject -verb -object'' to ''subject -bǎ -object -verb'' [bib_ref] The semantic processing of syntactic structure in sentence comprehension: an ERP study, Ye [/bib_ref]. The construction is always used to express the result of the action on the object. Different from English, the bèi (被) construction is used to express passive voice in Chinese. Due to lack of morphological inflection, the particle bèi as a fixed word is used before an agent to express passive voice [bib_ref] Cue competition between animacy and word order: acquisition of chinese notional passives..., Wang [/bib_ref]. The basic structure consists of ''object -bèi -subject -verb -other components''. The particles -zhe (着), -le (了), and -guò (过) in Chinese can come after a verb to express aspects in Chinese sentences. The durative aspect can be reflected by the marker -zhe, which describes an enduring or continuing situation. The perfective aspect can be expressed by the markers -le and -guò. The perfective particle -le expresses a situation in its entirety, an event bounded at the beginning and the end, while the other perfective particle -guò presents an event that has been experienced at some indefinite time [bib_ref] The development of aspectual marking in child Mandarin Chinese, Chen [/bib_ref]. We give some example sentences for illustration:
(1) 他把杯子打破了 (He ba cup break le.) He broke the cup.
(2) 杯子被他打破了 (Cup bei he break le.) The cup was broken by him. The door is open. In our experiment, we probed the model's competence at capturing these phenomena through an attention matrix. Specifically, we measured whether those particles allocated the maximum attention weight to their dependency heads.
## Baselines
We adopted positional offset and Random BERT as baselines. For the full details, please refer to 'Probing individual attention heads'.
# Results
Our experimental results are displayed in . The number in the parentheses is the specific head with the best performance. The performance still illustrates: Chinese BERT >Positional Offset >Random BERT. Meanwhile, we also saw that the results of Chinese BERT and Positional Offset were the same on the particles -zhe (着) and -guò (过). By analyzing the corpus, we found that -zhe and -guò followed the main verb most of time, indicating that Chinese BERT could only learn some positional information used in predicting the dependency relations of the two particles. In particular, we discovered that the two constructions (bèi and bǎ) were learned very well by the heads in the middle layers (layers 4-6), while the three particles (-zhe, -le, and -guò) were captured best by the heads in the lower layers (layers 2-4). This indicates that the structure information about sentences exists in the middle layers. Some lexical or morphological knowledge is embedded in the lower layers [bib_ref] What does BERT learn about the structure of language, Jawahar [/bib_ref].
## Probing attention head combinations
## Setup
In 'Probing individual attention heads' we found that some single attention heads were good at learning the corresponding dependency relations, but no heads could capture the Accuracy on Chinese linguistic phenomena. The values with the bold style are the maximum values in each column among these methods or models. whole dependency structures of sentences. Hence, we considered to combine all heads to perform sentence parsing. We followed the setting from [bib_ref] What does BERT look at?, Clark [/bib_ref] by training a classifier combing with all attention heads linearly:
## Datasets
## Model bǎ(''把 把 把'') bèi(''被 被 被'') -zhe(''着 着 着'') -le(''了 了 了'') -guo(''过")
[formula] UUAS = soft max( 144 k=1 w k α k ij ) [/formula]
where softmax is a function for classification, 144 is the number of heads in Chinese BERT, w k are weights for training, and a k ij is the attention weight of word i on word j produced by head k. We refer to this method as ''Attn''.
Additionally, we also considered the impact of words in carrying out parsing tasks. We incorporated word embeddings from [bib_ref] Directional skip-gram: explicitly distinguishing left and right context for word embeddings, Song [/bib_ref] into the classifier. This method is called ''Attn + embeddings''.
## Baselines
Similar to [bib_ref] What does BERT look at?, Clark [/bib_ref] , ''Random Initial Attention + embeddings'', ''Right Branching'', and ''Distances + Embeddings'' were adopted as baselines in this experiment. ''Random Initial Attention + embeddings'' used a randomized network and incorporated the pre-trained word embeddings for head and dependent words. Meanwhile, ''Right Branching'' predicts that the head word was always on the right of the dependent. ''Distances + Embeddings'' is used to replace the attention matrix of Chinese BERT with pre-trained word and positional embeddings, and randomly initialized other weights.
# Results
Results are exhibited in . We can see that both ''Attn + embeddings'' and ''Attn'' achieved better performances than the baselines on the two datasets. The accuracy of ''Attn'' was higher than 50%, and ''Attn + embeddings'' obtained nearly 70% accuracy. These results are similar to the findings in English [bib_ref] What does BERT look at?, Clark [/bib_ref] [bib_ref] A structural probe for finding syntax in word representations, Hewitt [/bib_ref]. This indicates that the attention heads of Chinese BERT did acquire many organizational structures in language. ''Attn + embeddings'' outperformed ''Attn''(∼15%), which proves that specific vocabulary contributes to Chinese BERT capturing dependency relations. Together with the findings from individual attention heads, we believe that Chinese BERT encodes abundant information in syntax by a way of indirect supervision, even though the word boundaries do not exist in the Chinese language. Accuracy on dependency parsing. The values with the bold style are the maximum values in each column among these methods or models.
# Methods
## Ud(%) cdt(%)
Random Init Attn + embeddings 11.47 11.01
## Right
## Probing hidden state
## Setup
Besides probing attention heads, we explored the ability of hidden representation in capturing syntactic knowledge. Because no suitable datasets for testing Chinese syntax were available, we designed three Chinese syntactic tasks by imitating the work in English [bib_ref] What you can cram into a single $ & !#* vector: probing..., Conneau [/bib_ref] : Bigram Shift (BShift), Tree Depth (TreeDepth), and Dependency Relation (DepRel).
In the BShift task, we inverted two random adjacent characters and let the model predict whether the sentence was inverted. In the TreeDepth task, the depth of the dependency tree of a sentence was predicted. The task DepRel refers to the prediction of the dependency relation of a phrase consisting of two words.
As shown in [fig_ref] Figure 5: Probing syntactic knowledge in hidden states [/fig_ref] , we overlaid a one-layer MLP on the hidden state of each layer to construct a classifier. After trying different parameter combinations, an optimal set of parameters were finally determined. We then only trained each classifier one epoch, so that the classifier was forced to pay attention to information encoded in the hidden state representation as much as possible.
## Baselines
We adopted Random BERT as a baseline. For the complete details, please refer to 'Probing individual attention heads'. [fig_ref] Table 5: Accuracy on three syntactic tasks [/fig_ref] displays the prediction result of each layer. The number in parentheses denotes the baseline from Random BERT. The bold numbers are the maximum values among all 12 layers in each task. According to the results, the best performances of Chinese BERT were all achieved in the final layer (layer 12). Chinese BERT still outperformed Random BERT in most of the layers across the three tasks. Compared to probing in the attention head, the performance of the hidden state from Random BERT was not very poor. This indicates that these hidden states contain some information that will contribute to predicting syntactic knowledge. Interestingly, we found the prediction results from Random BERT were better than Chinese BERT's corresponding to lower layers (layers 1-3) in the TreeDepth task. This could be because a relatively complete structure is needed to be captured in the TreeDepth task. However, Chinese BERT may not encode structural information well in the lower layers. Therefore, its performance was outperformed by Random BERT.
# Results
## Fine-tuning on downstream tasks
When Chinese BERT is fine-tuned into downstream tasks, does its syntactic knowledge change? In order to explore this question, we selected tasks with different levels to fine-tune Chinese BERT. These tasks covered low-level tasks, such as word segment and POS tagging, and high-level tasks involving semantic comprehension, including NLI and question matching. We carried out the experiments on the following datasets: Word segment (WS). We adopted CTB8.0as the dataset. POS tagging. CTB8.0 was usedas the dataset. NLI. Original Chinese NLI (OCNLI) [bib_ref] OCNLI: original chinese natural language inference, Hu [/bib_ref] is a CLUE task used to infer whether a premise sentence entails, contradicts, or is neutral towards a hypothesis sentence. Question matching (CQ). We used the Large-scale Chinese Question Matching Corpus (LCQMC) [bib_ref] LCQMC: a large-scale Chinese question matching corpus, Liu [/bib_ref] , which is a large-scale Chinese corpus. We refer to these fine-tuned models as WS-BERT, POS-BERT, NLI-BERT, and CQ-BERT. These fine-tuned BERTs will be compared with the original Chinese BERT in the following experiments.We ran each downstream task three times and stored the model parameters. And our probing results are the averages of every three experiments.The findings will be described as follows.
## Probing individual attention heads for fine-tuned berts
We still adopted positional offset and Random BERT as baselines. [fig_ref] Figure 6: UUAS for fine-tuned models and Chinese BERT [/fig_ref] shows the UUAS of the individual heads on the overall relations for these different BERTs. One can easily see that the performance of NLI-BERT decreased dramatically (≈27%), suggesting that inference tasks do not need syntactic knowledge. Additionally, WS-BERT and CQ-BERT showed small loss consistently, which indicates that the two tasks could also forget some language structures during training. POS-BERT showed a little improvement compared to Chinese BERT. This may be because this task needed some relation information from surrounding words so that the POS of the current word could be identified more accurately. The accuracy results of the common relations of individual heads on fine-tuned BERTs are displayed in [fig_ref] Figure 7: UUAS on common relations for fine-tuned models and Chinese BERT [/fig_ref]. Our findings from the overall relation are still roughly suitable to these frequent dependency relations. However, there exists some different cases. POS-BERT outperformed Chinese BERT on VOB and SBV. SBV and VOB act as the subject and object for a verb in a sentence, respectively [bib_ref] Universal dependencies, Marneffe [/bib_ref]. These relations could be useful for POS-BERT to determine the POS of a word. Also, Chinese BERT performed better than CQ-BERT on nmod, a kind of nominal modifier. This indicates that this relation could may not be necessary for the CQ task.
## Relative position for fine-tuned berts
The accuracy of relative positions for fine-tuned models and Chinese BERT is displayed in [fig_ref] Table 6: Accuracy of relative positions for fine-tuned models and Chinese BERT [/fig_ref]. We found that NLI-BERT only reserves some dependency knowledge in the relative position following fine-tuning. Compared with Chinese BERT, other BERTs maintain the performance when the dependent word and head word are next to each other. While the distance extends to two, these fine-tuned models improved their competence on capturing dependency relation. However, when the distance becomes longer, they are mostly exceeded by Chinese BERT. The reason could be that these fine-tuned BERTs pay more attention to local information between words. Therefore, when the dependent word and head word are very close, these fine-tuned models can obtain better results. For exploring the changes of fine-tuned BERTs in frequent relations, we carried out the corresponding experiments on the two datasets (Figs. 8 and 9). In general, the margin between WS-BERT and Chinese BERT grew as the relative position became longer. This demonstrates that WS-BERT's ability to preserve common syntactic relations decreases as the distance increases. Additionally, the performance gap between Chinese BERT and CQ-BERT in most relations remained small, indicating that dependency knowledge is not forgotten by CQ-BERT. POS-BERT's performance surpassed Chinese BERT on some relations, such as nsubj, obj, VOB, SBV, and COO, which suggests that these relations are important for POS tagging task. [fig_ref] Table 7: Accuracy on dependency parsing for fine-tuned models and Chinese BERT [/fig_ref] shows the results of the dependency parsing accuracy after combining attention heads. The differences in performance among these BERTs were similar to the results in the previous subsections. Notably, POS-BERT outperformed Chinese BERT on CDT, but displayed a loss in performance on UD. We believe that this phenomenon is related to the smaller size of the UD dataset, so that the classifier failed to learn the information encoded in the fine-tuned BERTs.
## Probing attention head combinations for fine-tuned berts
Probing hidden state for fine-tuned BERTs [fig_ref] Figure 1: Illustration of syntactic probes [/fig_ref] displays the best performance among all 12 layers of each BERT on three syntactic tasks. NLI-BERT still performed very poor. Chinese BERT still outperformed WS-BERT on all tasks, which indicates that the syntactic knowledge in hidden states of WS-BERT could be forgotten to some extent. Very interestingly, both POS-BERT and CQ-BERT showed improvement on BShift and DepRel. However, only CQ-BERT surpassed Chinese BERT on Tree Depth. The reason could be that POS-BERT might capture some local information about the relations between words. Hence, POS-BERT is very suitable to BShift and DepRel tasks. CQ-BERT can learn the organization structure of the whole sentence better, Therefore, this model can acquire more obvious progress on the TreeDepth task.
# Conclusion
We explored the competence of Chinese BERT in encoding syntactic knowledge across two aspects: attention heads and hidden states. We observed that certain attention heads learned specific dependency relations and syntactic phenomena. By combining attention heads, we succeeded in parsing the sentences. Hidden states also reflected some competence in encoding syntactic knowledge. When Chinese BERT was fine-tuned into different downstream tasks, we found some changes of different models in preserving language structure. POS tagging reinforced syntactic information in Chinese BERT to some extent, while NLI enabled Chinese BERT to lose knowledge in learning sentence structure. Those findings above can guide the design of model distillation algorithms in term of those heads encoding syntactic knowledge. Furthermore, we can be aware that whether syntactic knowledge is of importance when finishing a specific NLP downstream task.
Meanwhile, some specific syntactic information can be introduced more precisely to improve the task performance according to our findings.
[fig] Figure 1: Illustration of syntactic probes. Full-size DOI: 10.7717/peerjcs.1478/fig-1 [/fig]
[fig] Figure 2: An example sentence parsed by dependency relations and maximum attention weights. Full-size DOI: 10.7717/peerjcs.1478/fig-2 [/fig]
[fig] Figure 3 3, Figure 4: Accuracy of relative positions. Full-size DOI: 10.7717/peerjcs.1478/fig-Accuracy of relative positions on common relations. Full-size DOI: 10.7717/peerjcs.1478/fig-4 [/fig]
[fig] 3: 他去北京了 (He go Beijing le.) He went to Beijing. (4) 他去过 北京 (He go guo Beijing.) He has been to Beijing. (5)门开着 (Door open zhe.) [/fig]
[fig] Figure 5: Probing syntactic knowledge in hidden states. Full-size DOI: 10.7717/peerjcs.1478/fig-5 [/fig]
[fig] Figure 6: UUAS for fine-tuned models and Chinese BERT. Full-size DOI: 10.7717/peerjcs.1478/fig-6 [/fig]
[fig] Figure 7: UUAS on common relations for fine-tuned models and Chinese BERT. Full-size DOI: 10.7717/peerjcs.1478/fig-7 [/fig]
[fig] Figure 8: Accuracy of relative positions on UD2.11 for fine-tuned models and Chinese BERT. Full-size DOI: 10.7717/peerjcs.1478/fig-8 [/fig]
[fig] Figure 9: Accuracy of relative positions on CDT1.0 for fine-tuned models and Chinese BERT. Full-size DOI: 10.7717/peerjcs.1478/fig-9 [/fig]
[table] Table 1: UUAS on UD2.11. The values with the bold style are the maximum values in each column among these methods or models. [/table]
[table] Table 2: UUAS on CDT1.0. The values with the bold style are the maximum values in each column among these methods or models. [/table]
[table] Table 5: Accuracy on three syntactic tasks. The values with the bold style are the maximum values in each column among these methods or models. [/table]
[table] Table 6: Accuracy of relative positions for fine-tuned models and Chinese BERT. The values with the bold style are the maximum values in each column among these methods or models. [/table]
[table] Table 7: Accuracy on dependency parsing for fine-tuned models and Chinese BERT. The values with the bold style are the maximum values in each column among these methods or models. [/table]
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Deep learning for fast low-field MRI acquisitions
Low-field (LF) MRI research currently gains momentum from its potential to offer reduced costs and reduced footprints translating into wider accessibility. However, the impeded signal-to-noise ratio inherent to lower magnetic fields can have a significant impact on acquisition times that challenges LF clinical relevance. Undersampling is an effective way to speed up acquisitions in MRI, and recent work has shown encouraging results when combined with deep learning (DL). Yet, training DL models generally requires large databases that are not yet available at LF regimes. Here, we demonstrate the capability of Residual U-net combined with data augmentation to reconstruct magnitude and phase information of undersampled LF MRI scans at 0.1 T with a limited training dataset (n = 10). The model performance was first evaluated in a retrospective study for different acceleration rates and sampling patterns. Ultimately, the DL approach was validated on prospectively acquired, fivefold undersampled LF data. With varying performances associated to the adopted sampling scheme, our results show that the approach investigated can preserve the global structure and the details sharpness in the reconstructed magnitude and phase images. Overall, promising results could be obtained on acquired LF MR images that may bring this research closer to clinical implementation.OPEN
Low-field (LF) magnetic resonance imaging (MRI) has recently regained attention, with multiple initiatives aiming not only to democratize MRI worldwide, but also to complement conventional high-field MRI. Mostly driven by simpler and scalable magnet construction, LF MRI offers increased accessibility from reduced purchasing and maintenance costs, cryogenic-free infrastructure, reduced susceptibility artifacts, higher T1 contrast, and potential for smaller footprint designs [bib_ref] Low-field MRI: How low can we go? A fresh view on an..., Sarracanie [/bib_ref] [bib_ref] Safety and efficiency of low-field magnetic resonance imaging in patients with cardiac..., Schukro [/bib_ref]. Yet, LF MRI suffers from lower sensitivity due to an intrinsically lower nuclear spin polarization. Consequently, the associated lower signal-to-noise ratio (SNR) per unit time and volume almost invariably requires averaging and hence prolonged acquisition times that may restrict its clinical relevance. Technical progress achieved over the past decades in diverse areas of MRI, e.g. power electronics, radio frequency detection, sequence programming, and image processing, have contributed to bring LF back to the surface 1,2 but further acceleration remains paramount to push the achievable SNR and voxel resolution per unit time, aiming at a broad deployment in radiology departments and beyond.
Regardless of magnetic field strength, MRI requires one to encode the signal originating from 1 H nuclei to make an image, that may result in long imaging times. k-space undersampling strategies have been largely used to accelerate acquisitions, but typically lead to information loss or reconstruction artifacts after Fourier Transform (FT). Restoring the unaltered image becomes then an ill-posed, inverse problem 5 where regularization methods involving prior knowledge such as the sparsity of MR data in a certain domain (e.g. Wavelet domain) can be leveraged, as done in Compressed Sensing (CS) [bib_ref] Compressed sensing MRI, Lustig [/bib_ref]. Yet, the application of CS in clinical routine is still limited as it requires complex parameter tuning, sometimes very specific to an application or image type, and suffers from long online reconstruction times. An alternative approach to accelerating MR acquisitions is parallel imaging (PI) [bib_ref] SENSE: Sensitivity encoding for fast MRI, Pruessmann [/bib_ref]. As opposed to CS, PI is broadly spread and employed in clinical routine. In PI methods, the undersampled k-space is acquired using multiple receiver coils and the spatial dependence of their B − 1 field is leveraged to turn the initial ill-posed to a well-posed inverse problem that can be solved by a direct matrix inversion. As noise is severely amplified at high undersampling rates though (characterized by the g-Factor), a limited speed-up rate of fourfold (maximum) is generally observed. Relying on body noise regime dominance to exploit the spatial dependency of coil elements and spatially altered SNR, PI shall however not be indicated to accelerate MRI below a certain magnetic field strength. Indeed, below 5 MHz, sample noise may no longer dominate in the acquisition chain [bib_ref] Perspectives with cryogenic RF probes in biomedical MRI, Darrasse [/bib_ref] and such a reconstruction paradigm no longer holds.
Deep Learning (DL) is an emerging field of research that has shown promising results in a variety of domains, including MRI. In fact, DL has provided a new paradigm to solve ill-posed inverse problems, and particularly the reconstruction of undersampled MR acquisitions where superior performance to widespread CS and PI acceleration methods was shown [bib_ref] Image reconstruction by domain-transform manifold learning, Zhu [/bib_ref] [bib_ref] DAGAN: deep de-aliasing generative adversarial networks for fast compressed sensing MRI reconstruction, Yang [/bib_ref] [bib_ref] Learning a variational network for reconstruction of accelerated MRI data: Learning a..., Hammernik [/bib_ref]. Here, we hypothesize that DL could be a serious means to accelerated LF MRI. www.nature.com/scientificreports/ In DL approaches, neural networks made of numerous layers (i.e. referred to as "deep") are used to learn the complex functions that directly map an input data to a corresponding output. DL models, and especially data-driven approaches with a large number of free parameters require large training set to avoid what is called overfitting [bib_ref] Deep MRI reconstruction: Unrolled optimization algorithms meet neural networks, Liang [/bib_ref]. Overfitting occurs when a model learns from a training set which variety is limited, hence failing to generalize to images too different from the training set. Considering such limitation, initiatives were proposed to provide large-scale, open access databases of processed MR images [bib_ref] The multimodal brain tumor image segmentation benchmark (BRATS), Menze [/bib_ref] [bib_ref] Automated mining of large-scale lesion annotations and universal lesion detection with deep..., Yan [/bib_ref] [bib_ref] The Alzheimer's disease neuroimaging initiative, Mueller [/bib_ref] [bib_ref] The WU-Minn human connectome project: An overview, Essen [/bib_ref] or raw space 20 to researchers in the MRI community. Interestingly, two aspects in undersampled DL-MR works often seem overlooked, namely prospective validation and phase image reconstruction. Prospective validation is a key step though, to properly assess the performance of proposed methods on undersampled data acquired in real conditions (i.e. not subsampled a posteriori). The integration of complex data inherent to MRI could additionally open new perspectives for the application of DL with phase-contrast MR techniques such as magnetic susceptibility mapping, or further flow and motion encoding. It may also provide more accurate outcomes as MRI data does not limit itself to most used magnitude information. Yet only little research has reported on phase data reconstruction, none of which were validated prospectively [bib_ref] A. accelerated phase contrast magnetic resonance imaging via deep learning, Nath [/bib_ref]. Meanwhile, it should be noted that several studies were inherently limited by their model architecture that cannot handle complex input data, or by the nature of the training set constituted solely of magnitude images.
At LF, few attempts have yet been reported that leverage DL for undersampled MRI data reconstruction 23 . Schlemper et al. developed a non-uniform variational model to simultaneously reconstruct and recover artifacts from undersampling (3.5-fold) and hardware for a point-of-care scanner (64 mT). In a different work, Koonjoo et al. exploited a model named AUTOMAP to improve SNR at low field, respectively 6.5 mT and 47 mT.
In general, DL approaches face a major challenge at LF: there is no large-scale LF database acquired at the same magnetic field strength. Indeed, the number of scanners/clinical exams at LF are still very limited and there is no consensus as to what field regime is LF MRI. Consequently, LF databases remain private and rather small both on global (worldwide) and laboratory scales. The above-mentioned LF studies relied on an open access conventional (i.e., high-field) MR database from the human connectome project (HCP) [bib_ref] The WU-Minn human connectome project: An overview, Essen [/bib_ref] to train and test their model. The latter was pre-processed ad hoc by adding noise 23,24 and artifacts that would result from patient motion to simulate LF brain data, and account for the limited gradients linearity specific to the system used 23 . Despite such processing, peculiarities such as noise regime and image contrast at LF can be quite different from what is expected at conventional fields (1.5-3 T). Typically, the magnetic properties of biological tissue change at low field, leading to a superior T1 dispersion that implies different soft tissue contrast, and noise in the reception chain may no longer be dominated by the sample but instead by thermal noise from the electronics. Therefore, training with preprocessed conventional field data might very well have a negative impact on reconstruction outcomes. First, this can translate in decreased model performance because of the discrepancy between training and test sets [bib_ref] Assessment of the generalization of learned image reconstruction and the potential for..., Knoll [/bib_ref]. Second, it might lead to an undesired contrast transfer from conventional field to LF DL reconstructed MR images. Finally, we will note that for both LF DL approaches referenced above, the phase information was not considered as the HCP database contains only magnitude images.
In the proposed work, we explore the capability of a data-driven DL approach to accelerate LF MR acquisitions (here, at 0.1 T) while maintaining both magnitude and phase information. A particular emphasis was brought to tackle challenges associated with small datasets typically encountered at low field. Here, a relatively small dataset (n = 10) of human wrist images was collected and data augmentation was employed to mitigate data scarcity while preventing the deviation between training and testing sets. With data augmentation, the size of a small dataset is artificially expanded based on basic image manipulations (e.g. rotation, translation, cropping, noise injection, etc.) or on deep learning (e.g. generative adversarial networks) [bib_ref] A survey on image data augmentation for deep learning, Shorten [/bib_ref]. We used 2-channel U-net 27 , one of the state-of-the-art deep learning architectures [bib_ref] Deep learning for undersampled MRI reconstruction, Hyun [/bib_ref] , as an image domain learning model. Its performance was first evaluated on magnitude and phase reconstructed images for three different acceleration rates (R = 3, 4 and 5). Then, in an attempt to preserve high frequencies at the highest acceleration rate (R = 5), the model performance was investigated for undersampling schemes describing different k-space coverage. Finally, the proposed approach was validated on both retrospective and acquired, prospective 3D LF data, keeping a keen eye towards clinical transfer.
# Materials and methods
Low-field data. A total of 10 fully sampled, 3D spoiled gradient echo (GRE) in vivo MR images of the human hand and wrist were acquired at 0.1 T, on a compact, biplanar system using a custom-built transmit/receive coil tuned at F 0 = 4.256 MHz [bib_ref] Low-field dedicated and desktop magnetic resonance imaging systems for agricultural and food..., Constantinesco [/bib_ref]. All data were collected with the following imaging parameters: matrix size = 128 × 115 × 9, voxel size = [1.2 × 1.2 × 6.3] mm 3 , TE/TR = 7.2/31 ms, bandwidth = 17 kHz, flip angle (FA) = 70°, number of averages (NA) = 28 (acquisition time = 14 min 56 s). The dataset was split by randomly choosing 80% (8 sets) for training and 20% (2 sets) for validation. Retrospective and prospective test data consisted each of 6 additional sets of 3D images acquired with the same protocol described above. All MRI experiments were conducted following the local ethics regulations and informed consent was obtained from all subjects. The study was approved by the Ethikkommission Nordwest-und Zentralschweiz (EKNZ) (project-ID 2022-00348).
Data preprocessing. Each 3D k-space was zero-filled to reach 128 × 128 × 9 matrix dimension and fit the square input/output dimensions required by U-net. Even though training with non-square input dimensions (128 × 115) is feasible, we chose to avoid the potential problem of stride and padding size adjustments that could be encountered in this case. Each k-space was then inverse Fourier transformed to generate a complex 3D image. To avoid potential overfitting caused by the rather small size of our dataset and improve the model performance, data augmentation was employed. The Keras libraryThe 'augmented' 3D complex image set was then Fourier transformed back to k-space domain and retrospectively undersampled on both the first and second phase encode directions (k y and k z ). Afterwards, data normalization was performed by dividing each 3D k-space with its corresponding standard deviation. Pairs of input/output, undersampled and fully sampled complex images were finally obtained following an inverse Fourier transform to go back to the image domain.
Model architecture and training details. In undersampled MRI, the aim is to find an optimal reconstruction function f : x → y , which maps an undersampled image x to a fully sampled image y . f can be formulated as follows:
with L being the loss function. x is generated retrospectively using the acquired fully sampled k-space: x = iFT(U(k)) , where U denotes the sampling pattern and k the fully acquired k-space. To train our model, the mean squared error (MSE) was used as a loss function. Subsequently, the later formula can be expressed as follows:
where i indicates the ith sample in the set and N is the total number of samples used to compute the loss. In this work, the deep convolutional neural network architecture U-Net was used as our reconstruction function f . U-net consists of an encoding path providing low-level features followed by a decoding path that enables precise localization of these features. Feature localization is further improved thanks to concatenation operations that binds the encoding path to the decoding path (cf. . It is a multiscale model with a large receptive field that can capture globally distributed artifacts.
In detail, U-net input/output dimensions were set to 128 × 128 × 2. The two channels correspond to the real and imaginary parts of the input/output image, hence preserving the complex nature of the data. The encoding path involves a sequence of two convolution operations (size: 3 × 3, stride 1), followed by a ReLU activation function and max-pooling operation (size: 2 × 2, stride 2). This last step halves the spatial dimensions. This sequence is repeated four times and the number of filters doubles after each sequence. Similar to the encoding path, the decoding path consists of four sequences where each sequence involves an up-sampling operation to restore image size followed by two convolution operations. The decoding path additionally involves a concatenation operation, where a higher resolution feature map from the encoding path is concatenated to its corresponding feature map in the decoding path to recover details lost in the encoding path. The last layer is a convolution layer with 2 filters (size 1 × 1) used to map a 64-channel layer into real and imaginary outputs.
Lee et al. showed that a residual block can improve the reconstruction performance of a U-net model [bib_ref] Deep residual learning for compressed sensing MRI, Lee [/bib_ref]. The residual block introduced in reference [bib_ref] Deep Residual learning for image recognition, He [/bib_ref] consisted of a shortcut connection that performs an identity map www.nature.com/scientificreports/ of U-net input and adds it to the output resulting in a 'residual U-net' . Using the residual block instead of the original alias-free full image simplifies the topology structure which translates in easier and more accurate learning [bib_ref] Deep residual learning for compressed sensing MRI, Lee [/bib_ref]. This method was used, later on, in many MR accelerated acquisition studies [bib_ref] DAGAN: deep de-aliasing generative adversarial networks for fast compressed sensing MRI reconstruction, Yang [/bib_ref] [bib_ref] KIKI-net: Cross-domain convolutional neural networks for reconstructing undersampled magnetic resonance images, Eo [/bib_ref] and is similarly employed in our work. RMSProp was adopted as an optimizer with an adaptive learning rate starting from 10 -3 . The number of epochs was set arbitrarily to 2000. However, the model converged before reaching 2000 epochs without overfitting. The validation was done every epoch to avoid the potential overlooking of overfitting phenomena. Training and validation loss curves are shown in supplementary material (cf. . Additionally, repeatability experiments were carried out (cf. supplementary material). The pipeline was implemented in Python3 using the Keras library and TensorFlow 38 as a backend. The learning computation was carried out on an Intel Xeon workstation associated with a Graphics Processing Unit (GPU) GeForce GTX 1080 Ti, and took a total time of 1.5 h.
[formula] (1) f = argmin f L f (x) − y (2) f = argmin f N 1 �f x i − y i � [/formula]
Conducted studies. The phase encoding directions (k y and k z ) were undersampled following 2D Gaussian sampling distributions, while a fixed number of k-space center lines (CL) was fully sampled to preserve low spatial frequency information and SNR. The study was divided into two parts: a retrospective analysis based on the results of two different experiments, and a prospective analysis performed on data acquired according to the retrospective outcomes.
Retrospective study. In a first experiment, the performance of the DL model was assessed for different acceleration rates. Fully sampled k-spaces were retrospectively undersampled using three Gaussian sampling patterns with the same number of CL = 7, the same variances σ y and σ z along the two-phase directions ( σ y = 0.10 and σ z = 0.20), but different acceleration rates R = 3, 4 and 5 (cf. [fig_ref] Figure 2: Model performance for different acceleration rates [/fig_ref].
In a second experiment, the performance of residual U-net was investigated for the highest acceleration rate (R = 5) while changing the variance of the Gaussian sampling patterns, hence leading to various artifacts in the reconstructed images. Indeed, when a Gaussian sampling pattern is used, the lower σ y and σ z , the closer to a low pass filter the sampling pattern, and the blurrier the resulting zero-filled images. Similar to noise artifacts, blurriness is globally distributed over the image. If σ y and σ z are high, high-frequencies are better preserved but at the cost of less-distributed or localized artifacts that we term 'local' artifacts. Here, four different sampling patterns were compared with fixed R = 5 and σ z = 0.2, and a) CL = 23 / σ y = 0, b) CL = 7 /σ y = 0.10, c) CL = 7/ σ y = 0.15, and ultimately d) CL = 7/ σ y = 0.20 (cf. [fig_ref] Figure 4: Impact of sampling patterns on the reconstruction performance of the magnitude images [/fig_ref].
Prospective study. A set of test data was acquired using a fivefold undersampled k-space acquisition (acquisition time ~ 3 min). An optimal sampling pattern was chosen according to the results of the previous experiment.
## Metrics.
Most commonly accepted metrics in the field of image reconstruction were used to evaluate the model performance, namely Peak SNR (PSNR), structure similarity index (SSIM) [bib_ref] Image quality assessment: From error visibility to structural similarity, Wang [/bib_ref] , and normalized root MSE (NRMSE).
- PSNR represents the ratio between the maximum intensity across a reference image f and the MSE of the DL reconstructed image f :
where M is the number of pixels in the image. - The SSIM index attempts to quantify the perceptual differences between two images. The comparison is based on three features: luminance, contrast and structure. These features are evaluated at different image locations by using a sliding window. The resulting SSIM between two image patches A and B is given by:
µ and σ are the mean and the variance values of an image patch. C 1 and C 2 are two constants to stabilize the division. The reported SSIM in the rest of the document is the mean SSIM calculated as follows: 1
[formula] P P i=1 SSIM , [/formula]
where P is the total number of patches.
- NRMSE is the normalized root mean squared error. When normalized by the mean value of the reference image f , NRMSE is given by:
Additionally, to assess the sharpness of the reconstructed images, 2D kernels for edge detection were employed in vertical and horizontal directions ( G x and G y ). The gradient magnitude G is given in Eq. 6 and as opposed to the above metrics, G is a reference-free metric. www.nature.com/scientificreports/ This metric was only considered on magnitude images. In fact, in phase-contrast images, the contribution of phase wraps dominates compared to that of image details in the gradient calculation, translating in very challenging data interpretation. In general, considering that the phase is encoded between -pi and pi, we believe that only NRMSE and SSIM are appropriate metrics for phase images and were chosen as such in all further analyses.
[formula] (3) PSNR = 20log 10 max(f ) 1 M M i=1 (f (i) − f (i)) 2 (4) SSIM(A, B) = (2µ A µ B + C 1 )(σ AB + C 2 ) (µ 2 A + µ 2 B + C 1 )(σ 2 A + σ 2 B + C 2 ) (5) NRMSE = 1 M M i=1 (f (i) − f (i)) [/formula]
A binary mask defined as a Region-of-Interest (ROI) for each image was generated. More specifically, a preliminary mask was first created based on pixels thresholding of the magnitude of the reference image. Then, a closing operation followed by an opening operation [bib_ref] Noise removal and enhancement of binary images using morphological operations, Jamil [/bib_ref] , which are mathematical morphological operations, were performed in order to extract the ROI and remove the isolated noisy pixels. The resulting mask was used such that the background contribution (i.e., noise in the image) was excluded when computing all reconstruction metrics. Indeed, U-net generally leads to reconstructed images with a noticeably lower background noise than the reference images. This naturally leads to an underestimation of the quality of the reconstructed images when the entire field-of-view is considered.
Images of the 3D datasets that did not contain signal (i.e., noise only) were not included in the calculation of our reconstruction metrics.
Finally, to determine differences between groups or methods, non-parametric tests were carried out for each metric (Kruskal-Wallis and Wilcoxon), where a p-value < 0.05 was in general considered statistically significant (when relevant, p-values were adjusted using the Bonferroni correction to account for multiple comparisons).
# Results
Retrospective study. The model performance was first evaluated at different acceleration rates with a Gaussian sampling pattern acting as a low pass filter (CL = 7, σ y = 0.10 and σ z = 0.20). An example of the corresponding reconstructions is shown in [fig_ref] Figure 2: Model performance for different acceleration rates [/fig_ref]. Residual U-net was able to reduce the blurriness in zero-filled Fourier transformed magnitude images for the three acceleration rates and provided smooth magnitude reconstructed images with sharper boundaries as illustrated by the gradient intensity profile shown in . In particular, the gradient metric indicated that residual U-net could better recover image sharpness and preserve the structures edges especially at high acceleration rates (see . For instance, the sharpness of the magnitude images improved in average over the test set by almost 13% at R = 5. However, higher acceleration rate progressively resulted in lower image quality where high spatial-frequencies and hence small details were more likely to be missed in the reconstructed magnitude images (cf. red circles in [fig_ref] Figure 2: Model performance for different acceleration rates [/fig_ref]. Regarding phase images, interestingly, residual U-net was able to preserve not only the image sharpness but also most edges that were smoothed out in zero-filled phase images especially for high acceleration rates (cf. [fig_ref] Figure 2: Model performance for different acceleration rates [/fig_ref]. As for magnitude images, the model performance was lower with higher acceleration rates. Our observations are supported by the quantitative results calculated over test data and summarized in . The statistical analysis on phase and magnitude metrics showed that DL reconstruction is significantly better than FT reconstruction for all acceleration rates (p-values < 0.01667) (cf. Tables S3 and S4 in supplementary material).
The impact of different sampling patterns on the reconstruction performance at the highest acceleration rate R = 5 was also explored and the results are summarized in . [fig_ref] Figure 4: Impact of sampling patterns on the reconstruction performance of the magnitude images [/fig_ref] show respectively magnitude and phase reconstruction examples corresponding to the four sampling patterns. Sampling pattern (a) led to . Example illustrating the model performance in preserving edges. Gradient profiles along the red line in reference (green), FT (red) and DL (blue) magnitude images with a fivefold acceleration rate (CL = 7, σ y = 0.10). www.nature.com/scientificreports/ highly blurred images after FT with local aliasing-type of artifacts in both the background and the ROIs. The proposed model enhanced the edges sharpness and corrected the artifacts in the background, yet could neither recover the lost higher frequencies nor correct for the local artifacts inside the ROI (cf. red arrows in [fig_ref] Figure 4: Impact of sampling patterns on the reconstruction performance of the magnitude images [/fig_ref]. Sampling pattern (b) led to blurred Fourier transformed images with subtle, local artifacts. Therefore, the model reconstructed sharp and nearly artifact-free magnitude images. The non-centered sampling patterns (c) and (d), as expected, led to sharper zero-filled images. However, local artifacts were still visible in the ROIs depending on the MR images. Regarding phase images, larger σ y led to higher details recovery. The local artifacts seen on magnitude images are less obvious in phase images. Apart from sampling pattern (a) or σ y = 0 which translated in the lowest performance, the rest of the sampling patterns show, overall, similar quantitative results. Nevertheless, the sampling pattern (b) or σ y = 0.10 demonstrates improved PSNR, SSIM and NRMSE on magnitude images (cf. Tables S5 and S6 in supplementary material) and led to nearly artifact-free images of 3D LF wrist/hand test data. Therefore, this pattern was used to evaluate the performance of the model on prospectively acquired data.
[formula] (6) G = mean G 2 x + G 2 y [/formula]
Prospective study. [fig_ref] Figure 6: Reconstruction results of 3 different prospectively undersampled MR data with the Gaussian... [/fig_ref] shows three examples of fivefold undersampled MR data. A similar behavior to retrospective undersampling was observed; namely, edges were sharper, and edges lost in the Fourier transformed phase images were recovered. [fig_ref] Table 3: Results of prospectively acquired MR images [/fig_ref] shows an increase in gradient values of 9.4% for magnitude images. . Summary of performance obtained on the magnitude and phase images for different acceleration rates. The stars indicate when differences in metrics between U-net and FT are statistically significant (* for p < 0.01667; ** for p << 0.001; the comparison not showing statistical significance had p-value = 0.073 for the phase image). . Summary of performance obtained on magnitude and phase images for different sampling patterns. Acceleration rate = 5. www.nature.com/scientificreports/
## Magnitude
## Magnitude
# Discussion
In this work, we investigated the potential of residual U-net, a data-driven model, to reconstruct 3D LF undersampled MR images in combination with small databases. Data augmentation with basic image transformations was adopted to mitigate the small size of the training dataset. The performance of this approach for three different acceleration rates was first assessed on retrospectively undersampled LF data. Quantitative results showed a statistically significant improvement when U-net reconstruction was used instead of conventional iFT. More specifically, the proposed DL approach was able to recover the image sharpness, even for high acceleration rates. We believe that sharpness was improved as a consequence of a reduced global distributed artifact -blurriness in the present case-generated by the sampling pattern rather than an explicit learning of the anatomical edges or details. The global artifact correction was most likely the result of data augmentation coupled to the U-net architecture nature. Indeed, the model learned at each iteration a newly-generated undersampled real/imaginary data having the same global artifact and learned to correct for it without overfitting details of the image. Moreover, considering its large receptive field, U-net was often used to remove incoherent globally distributed artifacts such as noise or streaking artifacts [bib_ref] Deep convolutional neural network for inverse problems in imaging, Jin [/bib_ref]. Thus, the large receptive field of U-net proves once more to be suitable for correcting a global distributed artifact. Phase images were reconstructed with equal performance as magnitude images, thus opening new perspectives for MR techniques relying on phase-contrast imaging. The edges appearing smoothed in zero-filled FT phase-contrast images were sharper using DL reconstruction. One can think that this could be caused by an explicit anatomical learning. However, since this information was already present in the DL model inputs (i.e., the real and imaginary components of Fourier transformed image as illustrated in [fig_ref] Figure 4: Impact of sampling patterns on the reconstruction performance of the magnitude images [/fig_ref] , we hypothesize that it originates from a reduced global blurriness www.nature.com/scientificreports/ of real and imaginary components inherited from the k-space sampling pattern. Accordingly, in the context of pathological cases, we would assume that, as long as the pathology information is present in a "blurred" form in the DL model input, it can be restored without artifact. However, assuming that pathology would take the shape of very small structural changes lost from the undersampling scheme used, there could indeed be a risk that the model forces the reconstructions toward a healthy wrist appearance. That said, a solution could be to settle on a minimum resolution loss that would limit such a risk, to recover structures of a minimum size. Then of course, as for any imaging modality and regardless of DL reconstruction pipelines, a general lack of resolution may prevent to recognize small pathological structures that would be lost from partial volume effects. In the latter case though, DL is not the limiting factor. www.nature.com/scientificreports/ One limitation of our DL reconstruction comes from some remaining smoothness in both magnitude and phase images though. This could be due to 1-the undersampling patterns inevitably missing high spatial frequencies in k-space, and/or 2-the MSE loss function that tends to reduce the noise over flat intensity regions, translating in noiseless textures on the reconstructed image. For the employed sampling pattern (CL = 7, σ y = 0.10), this loss of higher spatial frequencies is further exacerbated by the higher acceleration rate (fivefold), both in the U-net reconstructed magnitude and phase images, as illustrated by the lower metrics shown in .
In line with this hypothesis, an attempt to maintain the high spatial frequencies in the reconstructed images was conducted by investigating different sampling patterns ( σ y values 0, 0.10, 0.15 and 0.20) for R = 5. As expected, higher σ y led to better details preservation in both reconstructed magnitude and phase images. However, U-net was not able to correct for the local artifacts generated by the different sampling patterns, especially inside the ROIs of the magnitude images. These generated artifacts are highly dependent on the sampling pattern when dealing with small learning dataset. This in turn means that the proposed approach cannot be easily generalized. Additional concerns on model generalization arise from the homogenous sequence parameters that we deliberately used so far for the training set. Indeed, as the knowledge in this specific field of research is still in its www.nature.com/scientificreports/ infancy, we believe it is important to limit the degrees of freedom that might influence the model performance.
In future work, we will explore different solutions for enhanced flexibility by using sampling patterns less prone to local artifacts such as variable density Poisson-disc. And, for clinical relevance, we will study the generalization ability of our model with respect to different sequence parameters. Additional improvement can be obtained using other types of loss functions. Although we employed in this work one of the most commonly used MSE, future work includes exploring the use of perceptual loss to improve the reconstruction performance [bib_ref] Perceptual losses for real-time style transfer and super-resolution, Johnson [/bib_ref] [bib_ref] MR image reconstruction using deep learning: Evaluation of network structure and loss..., Ghodrati [/bib_ref].
Finally, as opposed to most undersampled MR studies using DL, we prospectively tested our trained model on undersampled wrist MR images using the highest acceleration rate (fivefold) and σ y = 0.10. The results on magnitude and phase images confirmed that our model performs similarly on both simulated and acquired data.
This key aspect has two major advantages. First, our method could be directly used in concrete applications to reconstruct full MR complex data without further optimization steps. Second, it could also be used to fine-tune with confidence the sampling pattern on simulated data without the need of collecting extra data.
As future steps, further investigations will be carried out to compare the effectiveness of simulated data against data augmentation, as well as to assess the potential added value of transfer learning approach 46 based on natural or MR datasets compared to the present work. Ultimately, different promising network architectures will be considered in future work such as model-driven 5 methods (unrolled optimization algorithms) and transformers 47 .
# Conclusion
In this work, we demonstrated the potential of residual U-net for accelerating LF 3D MRI acquisitions. Promising results were obtained not only on magnitude but also on phase reconstructed images for three to fivefold acceleration rates. Although a relatively small (n = 10) dataset was used for training, data augmentation based on geometric image manipulations successfully mitigated data scarcity and allowed complex-valued MR data recovery. The global structure and image sharpness were preserved in the reconstructed magnitude and phase images. Additionally, our results indicated that the model performances are tied to the adopted sampling pattern. Nevertheless, promising results were obtained not only on simulated data but also on acquired fivefold accelerated MR data. This highlights the substantial potential of emerging DL approaches to significantly speed up LF MR acquisitions and ultimately to bring LF MR closer to clinical applications.
## Data availability
The data that support the findings of this study are available on request from the corresponding author (R. Ayde) for noncommercial, research purposes.
[fig] 2, Figure 1: The chosen Residual U-net used to reconstruct LF undersampled data. Scientific Reports | (2022) 12:11394 | https://doi.org/10.1038/s41598-022-14039-7 [/fig]
[fig] Figure 2: Model performance for different acceleration rates: (a) threefold, (b) fourfold and (c) fivefold. The red circles point out the missed details with higher acceleration rates in the magnitude images. Scientific Reports | (2022) 12:11394 | https://doi.org/10.1038/s41598-022-14039-7 [/fig]
[fig] Figure 4: Impact of sampling patterns on the reconstruction performance of the magnitude images. (a) CL = 23 σ y = 0, (b) CL = 7 σ y = 0.10, (c) CL = 7 σ y = 0.15 and (d) CL = 7 σ y = 0.20. Acceleration rate = 5. Although the model succeeded in fully removing local artifacts in the background, red arrows show examples of local artifacts still present inside the ROIs. Scientific Reports | (2022) 12:11394 | https://doi.org/10.1038/s41598-022-14039-7 [/fig]
[fig] Figure 5: Impact of the sampling pattern on the reconstruction performance of the phase images. (a) CL = 23 σ y = 0, (b) CL = 7 σ y = 0.10, (c) CL = 7 σ y = 0.15 and (d) CL = 7 σ y = 0.20. Acceleration rate = 5. Scientific Reports | (2022) 12:11394 | https://doi.org/10.1038/s41598-022-14039-7 [/fig]
[fig] Figure 6: Reconstruction results of 3 different prospectively undersampled MR data with the Gaussian sampling pattern: CL = 7, σ y = 0.10 . Acceleration rate = 5. [/fig]
[table] Table 3: Results of prospectively acquired MR images. https://doi.org/10.1038/s41598-022-14039-7 [/table]
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A function-based typology for Earth’s ecosystems
[bib_ref] Ecological forecasts: An emerging imperative, Clark [/bib_ref] [bib_ref] Which ecosystems provide which services? A meta-analysis of nine selected ecosystem services..., Bordt [/bib_ref] [bib_ref] The IUCN Red List of ecosystems: motivations, challenges, and applications, Keith [/bib_ref] [bib_ref] The IUCN Red List of ecosystems: motivations, challenges, and applications, Keith [/bib_ref] [bib_ref] Catastrophic shifts in ecosystems, Scheffer [/bib_ref] [bib_ref] Summary for Policymakers of the, Díaz [/bib_ref] [bib_ref] Which ecosystems provide which services? A meta-analysis of nine selected ecosystem services..., Bordt [/bib_ref]
## Conceptual foundations
We developed a conceptual model to inform the construction of the Global Ecosystem Typology, consistent with the six design principles, and to serve as a template for describing the units of classification. The model [fig_ref] Figure 1 |: The generic model of ecosystem assembly underlying the Global Ecosystem Typology [/fig_ref] frames working hypotheses about the processes (or 'drivers') that shape ecosystem properties and the interactions among drivers and properties. Ecosystem properties are attributes of ecosystems and their component biota that result from assembly processes. They include aggregate ecosystem functions (productivity, stocks and fluxes), ecological processes (for example, trophic networks), structural features (for example, 3D spatial structure and diversity) and species-level traits of characteristic organisms (for example, ecophysiology, life histories and morphology).
Our model postulates five groups of ecological drivers that may shape ecosystems by acting both as assembly filters and evolutionary pressures [fig_ref] Figure 1 |: The generic model of ecosystem assembly underlying the Global Ecosystem Typology [/fig_ref] and Supplementary Information, Appendix 2, for details). Filters are biotic and abiotic processes that determine community assembly from a species pool, given initial occupancy or dispersal (based on community assembly theory [bib_ref] Rethinking community assembly through the lens of coexistence theory, Hillerislambers [/bib_ref]. Evolutionary pressures are agents of selection that influence ecosystem function and constituent species traits, typically over longer time scales, through evolution and extinction within a dynamic species pool [bib_ref] Ecological and evolutionary perspectives on community assembly, Mittelbach [/bib_ref].
'Resource drivers' (Supplementary Information, Appendix 2, page 2) supply water, oxygen, nutrients, carbon and energy, the resources essential for life. The 'ambient environment' (Supplementary Information, Appendix 2, page 2) includes environmental features (for example, temperature, pH, salinity) that continually influence the availability of resources or the ability of organisms to acquire them. The model distinguishes these continuous factors from 'disturbance regimes' (Supplementary Information, Appendix 2, page 2), which are sequences of discrete events with different intensities and patterns of occurrence (for example, fires, floods, storms and earth mass movement) that destroy living biomass, liberate and redistribute resources, and regulate life-history processes. 'Biotic interactions' (Supplementary Information, Appendix 2, page 3) include competition, predation, pathogenicity, mutualisms and facilitation, which operate at local scales but may shape ecosystem properties at landscape and seascape scales (for example, reef-building symbioses). 'Human activities' (Supplementary Information, Appendix 2, page 3) are a special class of biotic interaction that influence ecosystem disassembly and reassembly through resource appropriation, physical restructuring, movement of biota, and climate change [bib_ref] The broad footprint of climate change from genes to biomes to people, Scheffers [/bib_ref]. These anthropic processes operate largely, but not exclusively, through effects on other drivers. Although our model portrays humans as integral drivers of ecosystem assembly, we separated human activity from other biotic interactions to highlight connections between ecosystems and socio-economic systems that drive anthropogenic change [bib_ref] How a socio-ecological metabolism approach can help to advance our understanding, Erb [/bib_ref] , and the need to assess and mitigate the human impacts on biodiversity and ecosystem functioning.
Interactions may exist among drivers, modulating their effects on ecosystem properties [fig_ref] Figure 1 |: The generic model of ecosystem assembly underlying the Global Ecosystem Typology [/fig_ref] and Supplementary Information, Appendix 2, page 4). For example, resource levels may influence ecosystem assembly directly through niche partitioning or indirectly through alteration of biotic interactions [bib_ref] Refining the stress-gradient hypothesis for competition and facilitation in plant communities, Maestre [/bib_ref]. Similarly, feedbacks exist between ecosystem properties and drivers. For example, human land-use intensification initiates changes in ecosystems that, in turn, influence human social structure, markets and consumption patterns, driving changes in resource appropriation and further change in ecosystem properties [bib_ref] How a socio-ecological metabolism approach can help to advance our understanding, Erb [/bib_ref] Boxes represent abiotic (resources, the ambient environment and disturbance regimes) and biotic (biotic interactions and human activity) drivers that filter assemblages and form evolutionary pressures that in turn, shape ecosystem-level properties (inner green circle). The range of major organizational scales at which drivers operate are shown in parentheses, followed by a list of the major expressions of the drivers. The species pool is the set of 'available' traits on which the assembly filters and evolutionary pressures operate over short and longer time frames, respectively. Species pools are dynamic products of vicariance, dispersal and evolution that depend on biogeographic context and history. The outer green circle (dashed line) represents the contemporary dispersal filter that mediates the biota currently subjected to local selection by the abiotic and biotic filters and pressures. The inner green circle represents the properties (aggregate ecosystem functions and species-level traits) that characterize the ecosystem. ecosystems in our typology (Supplementary Information, Appendices 3 and 4, pages 52-186) reflect our hypotheses about how drivers influence ecosystem properties directly, or indirectly through interactions with other drivers. The model posits that ecosystems share convergent ecological processes and functional properties if they are shaped by similar drivers-and conversely, major changes to these drivers (or their interactions) cause disassembly, transformation and ultimately ecosystem collapse, with consequent losses of biodiversity, ecosystem function and services [bib_ref] Catastrophic shifts in ecosystems, Scheffer [/bib_ref].
Convergences in ecosystem properties are axiomatic to a functionally based ecosystem typology because they underpin robust generalizations and predictions about ecosystem responses to environmental change and management. Convergences in species traits may arise from common evolutionary origins and niche conservatism [bib_ref] Phylogenetic biome conservatism on a global scale, Crisp [/bib_ref] [bib_ref] Freezing and water availability structure the evolutionary diversity of trees across the, Segovia [/bib_ref] , but similarities in ecological drivers (selection pressures and assembly filters) may also produce functional convergences in independent lineages. These convergences are enablers of a functional classification framework represented in the upper three levels of our typology. Functional constraints may be imposed by the species pool, which is a dynamic outcome of vicariance, dispersal and evolution, depending on ecosystem location and biogeographic history [bib_ref] How was the Australian flora assembled over the last 65 million years?..., Crisp [/bib_ref].
Only a few ecological drivers are likely to be important in shaping the key properties of any particular ecosystem, despite the vast array of potential drivers on Earth and the complex interactions among them. This principle was critical to design of assembly models of each ecosystem functional group and for developing a parsimonious global typology (Supplementary , principle 6).
## Typology structure
Our ecosystem typology, adopted by the IUCN at the 2020 World Conservation Congress 22,23 has six hierarchical levels, enabling applications at different thematic scales (Methods and [fig_ref] Figure 3 |: Hypothesized relationships of functional groups differentiated along gradients of selected assembly filters [/fig_ref]. Three upper levels (Supplementary differentiate functional groupings and three lower levels (Methods and Supplementary Information, Appendix 3, pages 19 and 20) accommodate differences in biotic composition among functionally convergent ecosystems. The scalable hierarchical structure .1, principle 4) and the explicit description of properties and drivers enables units at any thematic level to be mapped at different spatial scales. These units may be tracked through different temporal scales according to needs of specific applications and constraints arising from the resolution of available data.
Level 3 units of the typology (ecosystem functional groups, described in Supplementary Information, Appendix 4, pages 52-186 and summarized in Extended Data Tables 1-4) are fundamental to generalizations and predictions about ecosystems with similar functional properties, and therefore have key roles in global synthesis and knowledge transfer for ecosystems. Their distribution across landscapes and seascapes is governed by the expression of ecological drivers along temporally variable multidimensional gradients [bib_ref] Revising the biome concept for understanding and predicting global change impacts, Moncrieff [/bib_ref] [bib_ref] The freshwater biome gradient framework: predicting macroscale properties based on latitude, altitude,..., Dodds [/bib_ref] [fig_ref] Figure 3 |: Hypothesized relationships of functional groups differentiated along gradients of selected assembly filters [/fig_ref]. Interactions between the drivers that operate at different spatial scales in this multidimensional space determine the dominant filters and evolutionary pressures that shape ecosystem properties in different parts of the biosphere (see Methods, 'Hierarchical levels' and Supplementary Information, Appendix 3 for key drivers that differentiate ecosystem functional groups along landscape and seascape gradients visualized in Figs. 2 and 3).
## Applications for ecosystem management
Decisions about effective action to conserve biodiversity and sustain ecosystem services require evidence of which ecosystems are most exposed to risks of collapse [bib_ref] The IUCN Red List of ecosystems: motivations, challenges, and applications, Keith [/bib_ref] and which ecosystems contribute most to particular human benefits 5 . These analyses are conspicuously lacking in global ecosystem assessments [bib_ref] Summary for Policymakers of the, Díaz [/bib_ref] , but the IUCN Global Ecosystem Typology and a rapidly growing body of spatial datahave established an ecologically robust and powerful capability, and signal a growing readiness for such syntheses.
The IUCN Global Ecosystem Typology facilitates integrated assessment of Earth's ecosystems, enabling a more powerful and complete evaluation of progress towards biodiversity targets and sustainable development goals than previously possible. This fills a significant gap, exemplified by the limited range of ecosystems assessed in the Convention on Biological Diversity (CBD) Global Biodiversity Outlook 5and the IPBES Global Assessment 12 . It will also strengthen the evidence base for setting science-and knowledge-based specific, measurable, ambitious, realistic and time-bound (SMART) biodiversity targets in the forthcoming post-2020 CBD global biodiversity framework and for reviewing progress towards them 2 . The United Nations Statistical Commission recently adopted the IUCN typology as a reference classification for extending the System of Environmental Economic Accounting (SEEA) framework to Ecosystem Accounts 28 , meeting a long-recognized need for a spatially explicit, functionally based ecosystem typology to underpin natural capital accounting [bib_ref] Recording environmental assets in the national accounts, Obst [/bib_ref].
Integrating both functions and biota into the hierarchical structure of the typology confers versatility for diverse applications in ecosystem management and conservation [fig_ref] Figure 4 |: Current and potential applications of the Global Ecosystem Typology to conserve biodiversity... [/fig_ref] and Supplementary Information, Appendix 6). Our typology and developing archive of maps (see caveats in Supplementary Information, Appendix 4) provide a globally consistent framework for advancing the IUCN Red List of Ecosystems [bib_ref] The IUCN Red List of ecosystems: motivations, challenges, and applications, Keith [/bib_ref] [bib_ref] Scientific foundations for an IUCN Red List of ecosystems, Keith [/bib_ref] and Key Biodiversity Areas 31 , as well as broadly based nature education [bib_ref] Ask not what nature can do for you: a critique of ecosystem..., Bekessy [/bib_ref].
Diagnostic models of ecosystem dynamics, as developed in Red List assessments [bib_ref] Scientific foundations for an IUCN Red List of ecosystems, Keith [/bib_ref] , with improved ecosystem and threat distribution data, will strengthen capacity to forecast state changes that result in loss of ecosystem function, services and biota. Ecosystem groupings based on convergent drivers, properties and environmental relationships will reveal similarities in threats and mechanisms of degradation, and therefore inform the development of ecosystem-specific management strategies for recovery. Embracing the dynamic nature of ecosystems and its dependency on ecological processes is a key feature that differentiates the IUCN Global Ecosystem Typology from other ecological typologies . This will enable policy and management actions to be targeted towards causes of ecosystem degradation, with knowledge transfer and adaptive learning 33 about local ecosystems from functionally similar ecosystems elsewhere (Supplementary .
## Limitations and the way forward
We expect progressive improvements in future versions of the IUCN Global Ecosystem Typology as knowledge increases. Several aspects of the typology warrant further development to address uncertainties. In particular, models of assembly for each ecosystem functional group represent working hypotheses, for which available empirical evidence varies greatly (Methods, 'Limitations'). Redressing research biases across different ecosystem types and among different assembly filters will help improve not only the assembly models, but also the distinctions between ecosystem functional groups and units within other levels of the typology.
By highlighting poorly known systems in the atmosphere, deep sea floors, subterranean freshwaters, lithosphere and beneath ice, and by prompting researchers and other users to ask where particular ecosystems belong in the scheme, we foresee the typology promoting research to fill significant knowledge gaps that will improve outcomes of its application and inform future amendments of its structure, as well as descriptions of its units.
Ecosystem mapping is another component of the information base that urgently requires further development, as the currently available indicative global maps for ecosystem functional groups vary Article substantially in accuracy and precision (Methods, 'Limitations'). Many uses of the typology [fig_ref] Figure 3 |: Hypothesized relationships of functional groups differentiated along gradients of selected assembly filters [/fig_ref] do not require a full set of comprehensive and globally consistent maps because they are non-spatial (that is, knowledge transfer and framing generalizations), national in scope, or specific to particular ecosystem groups (for example, forests, coral reefs and mangroves). Reliable global maps of suitable resolution, however, are pivotal to the global synthesis of ecosystems, as required for systematic reporting on CBD targets and some other applications 2 .
By decoupling the mapping process from prior development of the classification, our approach liberates the definition of ecosystem units from constraints imposed by the current availability of spatial data and allows for progressive improvement in maps (Supplementary Information, Appendix 4, page 13). New technologies in cloud computing and artificial intelligence, improved global environmental data and deepening time archives of satellite images are paving the way [bib_ref] Satellite remote sensing of ecosystem functions: opportunities, challenges and way forward, Pettorelli [/bib_ref] [bib_ref] The role of satellite remote sensing in structured ecosystem risk assessments, Murray [/bib_ref]. High-resolution maps, some with extended time series, that match the concepts of ecosystem functional groups have been produced for contrasting ecosystem groups such as tidal mudflats [bib_ref] The global distribution and trajectory of tidal flats, Murray [/bib_ref] ; whereas generic data cubes for forest cover [bib_ref] High-resolution global maps of 21st-century forest cover change, Hansen [/bib_ref] and surface water [bib_ref] High-resolution mapping of global surface water and its long-term changes, Pekel [/bib_ref] suggest that global high-resolution time-series mapping should be possible for most ecosystem functional groups within the next decade. Future versions of the typology will progressively improve map standards to support applications that depend on spatial analysis. Improved mapping of threats and degradation is similarly required to support ecosystem assessments [bib_ref] Filling in biodiversity threat gaps, Joppa [/bib_ref] , particularly in marine environments. We acknowledge the limitations associated with discrete representation of continuous ecological patterns in nature (Supplementary Information, Appendix 3, page 23). Even though our descriptive framework recognizes core and transitional units, its discrete structure generates boundary and other uncertainties among ecosystems that are ultimately unavoidable, even with extensive description or splitting of classes [bib_ref] A taxonomy and treatment of uncertainty for ecology and conservation biology, Regan [/bib_ref]. However, this fallibility is outweighed by a classificatory approach founded in deep-seated cognitive processes that govern how humans understand and manage environmental, social, economic and cultural dimensions of their conscious universe by dividing it into parts. This will facilitate the widespread uptake of the IUCN typology for effective storage, retrieval and transfer of ecosystem information.
The hierarchical structure of our typology should enable global imperatives to be linked directly with on-ground, nature-based solutions [bib_ref] Core principles for successfully implementing and upscaling Nature-based solutions, Cohen-Shacham [/bib_ref] , supporting international mandates for sustainable development and biodiversity conservation. Viewing Earth's ecosystems through a dynamic functional lens, rather than through largely biogeographic or biophysical ones, will enable a more powerful and direct basis to address the dual goals of conserving biodiversity and sustaining ecosystem services.
## Online content
Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-022-05318-4.
## 1.
Open
# Methods
We developed the IUCN Global Ecosystem Typology in the following sequence of steps: design criteria; hierarchical structure and definition of levels; generic ecosystem assembly model; top-down classification of the upper hierarchical levels; iterative circumscription of the units and ecosystem-specific adaptations of the assembly model; full description of the units; and map compilation. Some iteration proved necessary, as the description and review process sometimes revealed a need for circumscribing additional units.
## Design criteria and other typologies
Under the auspices of the IUCN Commission on Ecosystem Management, we developed six design principles to guide the development of a typology that would meet the needs for global ecosystem reporting, risk assessment, natural capital accounting and ecosystem management: (1) representation of ecological processes and ecosystem functions;
(2) representation of biota; (3) conceptual consistency throughout the biosphere; (4) scalable structure; (5) We assessed 23 existing ecological classifications with global coverage of terrestrial, freshwater, and/or marine environments against these principles to determine their fitness for IUCN's purpose (Supplementary Information, Appendix 1). These include general classifications of land, water or bioclimate, as well as classifications of units that conform with the definition of ecosystems adopted in the United Nations Convention on Biological Diversity 45 or an equivalent definition in the IUCN Red List of Ecosystems [bib_ref] Scientific foundations for an IUCN Red List of ecosystems, Keith [/bib_ref]. We reviewed documentation on methods of derivation, descriptions of classification units and maps to assess each classification against the six design principles (Supplementary .2 for details).
## Typology structure and ecosystem assembly
We developed the structure of the Global Ecosystem Typology and the generic ecosystem assembly model at a workshop attended by 48 terrestrial, freshwater and marine ecosystem experts at Kings College London, UK, in May 2017. Participants agreed that a hierarchical structure would provide an effective framework for integrating ecological processes and functional properties .1, design principle 1), and biotic composition (principle 2) into the typology, while also meeting the requirement for scalability (principle 4). Although neither function nor composition were intended to take primacy within the typology, we reasoned that a hierarchy representing functional features in the upper levels is likely to support generalizations and predictions by leveraging evolutionary convergence. By contrast, a typology reflecting compositional similarities in its upper levels is less likely to be stable owing to dynamism of species assemblages and evolving knowledge on species taxonomy and distributions. Furthermore, representation of compositional relationships at a global scale would require many more units in upper levels, and possibly more hierarchical levels. Therefore, we concluded that a hierarchical structure recognizing compositional variants at lower levels within broad functionally based groupings at upper levels would be more parsimonious and robust (principle 6) than one representing composition at upper levels and functions at lower levels.
Workshop participants initially agreed that three hierarchical levels for ecosystem function and three levels for biotic composition could be sufficient to represent global variation across the whole biosphere. Participants developed the concepts of these levels into formal definitions , which were reviewed and refined during the development process.
To ensure conceptual consistency of the typology and its units throughout the biosphere (principle 3), we drew from community assembly theory to develop a generic model of ecosystem assembly.
The traditional community assembly model incorporates three types of filters (dispersal, the abiotic environment and biotic interactions) that determine which biota from a larger pool of potential colonists can occupy and persist in an area. We extended this model to ecosystems by: (1) defining three groups of abiotic filters (resources, ambient environment and disturbance regimes) and two groups of biotic filters (biotic interactions and human activity); (2) incorporating evolutionary processes that shape characteristic biotic properties of ecosystems over time; (3) defining the outcomes of filtering and evolution in terms of all ecosystem properties including both ecosystem-level functions and species-level traits, rather than only in terms of species traits and composition; and (4) incorporating interactions and feedbacks among filters and selection agents and ecosystem properties to elucidate hypotheses about processes that influence temporal and spatial variability in the properties of ecosystems and their component biota. In community assembly, only a small number of filters are likely to be important in any given habitat. In keeping with this proposition, we used the generic model to identify biological and physical features that distinguish functionally different groups of ecosystems from one another by focusing on different ecological drivers that come to the fore in structuring their assembly and shaping their properties.
## Hierarchical levels
The top level of classification and Extended Data Tables 1-4) defines five core realms of the biosphere based on contrasting media that reflect ecological processes and functional properties: terrestrial; freshwaters and inland saline waters (hereafter freshwater); marine; subterranean; and atmospheric. Biome gradient concepts 25 highlight continuous variation in ecosystem properties, which is represented in the typology by transitional realms that mark the interfaces between the five core realms (for example, floodplains (terrestrial-freshwater), estuaries (freshwater-marine), and so on). In Supplementary Information, Appendix 3 (pages 3-16) and .1, we describe the five core realms and review the hypothesized assembly filters and ecosystem properties that distinguish different groups within them. The atmospheric realm is included for comprehensive coverage, but we deferred resolution of its lower levels because its biota is poorly understood, sparse, itinerant and represented mainly by dispersive life stages [bib_ref] Bioaerosols in the Earth system: climate, health and ecosystem interactions, Fröhlich-Nowoisky [/bib_ref].
Functional biomes (level 2) are components of the biosphere united by one or more major assembly processes that shape key ecosystem functions and ecological processes, irrespective of taxonomic identity [fig_ref] Figure 3 |: Hypothesized relationships of functional groups differentiated along gradients of selected assembly filters [/fig_ref] , page 17). Our interpretation aligns broadly with 'functional biomes' described elsewhere [bib_ref] Revising the biome concept for understanding and predicting global change impacts, Moncrieff [/bib_ref] [bib_ref] The freshwater biome gradient framework: predicting macroscale properties based on latitude, altitude,..., Dodds [/bib_ref] [bib_ref] Biome: evolution of a crucial ecological and biogeographical concept, Mucina [/bib_ref] , extended here to reflect dominant assembly filters and processes across all realms, rather than the more restricted basis of climate-vegetation relationships that traditionally underpin biome definition on land. Hence, the 25 functional biomes (Supplementary Information, Appendix 4, pages 52-186 and https://global-ecosystems.org/) include some 'traditional' terrestrial biomes 47 , as well as lentic and lotic freshwater systems, pelagic and benthic marine systems, and anthropogenic functional biomes assembled and usually maintained by human activity [bib_ref] Anthropogenic transformation of the biomes, Ellis [/bib_ref].
Level 3 of the typology defines 110 ecosystem functional groups described with illustrated profiles in Supplementary Information, Appendix 4 (pages 52-186) and at https://global-ecosystems.org/. These are key units for generalization and prediction, because they include ecosystem types with convergent ecosystem properties shaped by the dominance of a common set of drivers (Supplementary Information, Appendix 3, pages [bib_ref] How a socio-ecological metabolism approach can help to advance our understanding, Erb [/bib_ref] [bib_ref] Refining the stress-gradient hypothesis for competition and facilitation in plant communities, Maestre [/bib_ref] [bib_ref] Phylogenetic biome conservatism on a global scale, Crisp [/bib_ref]. Ecosystem functional groups are differentiated along environmental gradients that define spatial and temporal variation in ecological drivers [fig_ref] Figure 3 |: Hypothesized relationships of functional groups differentiated along gradients of selected assembly filters [/fig_ref] and Supplementary [fig_ref] Figure 3 |: Hypothesized relationships of functional groups differentiated along gradients of selected assembly filters [/fig_ref]. For example, depth gradients of light and nutrients differentiate functional groups in pelagic ocean waters [fig_ref] Figure 3 |: Hypothesized relationships of functional groups differentiated along gradients of selected assembly filters [/fig_ref] and Extended Data , influencing assembly directly and indirectly through predation. Resource gradients defined by flow regimes (influenced by catchment precipitation and evapotranspiration) and water chemistry, modulated by environmental gradients in temperature and geomorphology, differentiate functional groups of freshwater ecosystems [bib_ref] The freshwater biome gradient framework: predicting macroscale properties based on latitude, altitude,..., Dodds [/bib_ref] [fig_ref] Figure 3 |: Hypothesized relationships of functional groups differentiated along gradients of selected assembly filters [/fig_ref] and Extended Data . Terrestrial functional groups are distinguished primarily by gradients in water and nutrient availability and by temperature and seasonality [fig_ref] Figure 3 |: Hypothesized relationships of functional groups differentiated along gradients of selected assembly filters [/fig_ref] and Extended Data , which mediate uptake of those resources and regulate competitive dominance and productivity of autotrophs. Disturbance regimes, notably fire, are important global drivers in assembly of some terrestrial ecosystem functional groups.
Three lower levels of the typology distinguish functionally similar ecosystems based on biotic composition. Our focus in this paper is on global functional relationships of ecosystems represented in the upper three levels of the typology, but the lower levels [fig_ref] Figure 1 |: The generic model of ecosystem assembly underlying the Global Ecosystem Typology [/fig_ref] are crucial for representing the biota in the typology, and facilitate the scaling up of information from established local-scale typologies that support decisions where most conservation action takes place. These lower levels are being developed progressively through two contrasting approaches with different trade-offs, strengths and weaknesses. First, level 4 units (regional ecosystem subgroups) are ecoregional expressions of ecosystem functional groups developed from the top-down by subdivisions based on biogeographic boundaries (for example, in ref. [bib_ref] An ecoregion-based approach to protecting half the terrestrial realm, Dinerstein [/bib_ref] that serve as simple and accessible proxies for biodiversity patterns 51 . Second, level 5 units (global ecosystem types) are also regional expressions of ecosystem functional groups, but unlike level 4 units they are explicitly linked to local information sources by bottom-up aggregation [bib_ref] Using tree species inventories to map biomes and assess their climatic overlaps..., Silva De Mirandao [/bib_ref] , are often developed independently of one another, and thus may involve inconsistencies in methods and thematic resolution of units (that is, broadly defined or finely split). Aggregation of level 6 units to broader units at level 5 based on compositional resemblance is necessary to address inconsistencies among different subglobal classifications and produce compositionally distinctive units suitable for global or regional synthesis.
Integrating local classifications into the global typology, rather than replacing them, exploits considerable efforts and investments to produce existing classifications, already developed with local expertise, accuracy and precision. By placing national and regional ecosystems into a global context, this integration also promotes local ownership of information to support local action and decisions, which are critical to ecosystem conservation and management outcomes (Supplementary Information, Appendix 3, page 20). These benefits of bottom-up approaches come at the cost of inevitable inconsistencies among independently developed classifications from different regions, a limitation avoided in the top-down approach applied to level 4.
## Circumscribing upper-level units
We formed specialist working groups (terrestrial/subterranean, freshwater and marine) to develop descriptions of the units within the upper levels of the hierarchy, subdividing realms into functional biomes, and biomes into ecosystem functional groups. We used definitions of the hierarchical levels and the conceptual model of ecosystem assembly [fig_ref] Figure 1 |: The generic model of ecosystem assembly underlying the Global Ecosystem Typology [/fig_ref] to maintain consistency in defining the units at each level during iterative discussions within and between the working groups.
Working groups agreed on preliminary lists of functional biomes and ecosystem functional groups by considering variation in major drivers along ecological gradients [fig_ref] Figure 3 |: Hypothesized relationships of functional groups differentiated along gradients of selected assembly filters [/fig_ref] and [fig_ref] Figure 3 |: Hypothesized relationships of functional groups differentiated along gradients of selected assembly filters [/fig_ref] based on published literature, direct experience and expertise of working group members, and consultation with colleagues in their respective research networks. After the workshop, working groups sought recent global reviews of the candidate units and recent case studies of exemplars to shape descriptions of the major groups of ecosystem drivers and properties for each unit. Circumscriptions and descriptions of the units were reviewed and revised iteratively to ensure clear distinctions among units, with a total of 206 reviews of descriptive profiles undertaken by 60 specialists, a mean of 2.4 reviews per profile . The working groups concurrently adapted the generic model of ecosystem assembly [fig_ref] Figure 1 |: The generic model of ecosystem assembly underlying the Global Ecosystem Typology [/fig_ref] to represent working hypotheses on salient drivers and ecosystem properties for each ecosystem functional group.
## Incorporating human influence
Very few of the ecological typologies reviewed in Supplementary Information, Appendix 1 integrate anthropogenic ecosystems in their classificatory frameworks. Anthropogenic influences create challenges for ecosystem classification, as they may modify defining features of ecosystems to a degree that varies from negligible to major transformation across different locations and times. We addressed this problem by distinguishing transformative outcomes of human activity at levels 2 and 3 of the typology from lesser human influences that may be represented either at levels 5 and 6, or through measurements of ecosystem integrity or condition that reflect divergence from reference states arising from human activity.
Anthropogenic ecosystems grouped within levels 2 and 3 were thus defined as those created and sustained by intensive human activities, or arising from extensive modification of natural ecosystems such that they function very differently. These activities are ultimately driven by socio-economic and cultural-spiritual processes that operate across local to global scales of human organization. In many agricultural and aquacultural systems and some others, cessation of those activities may lead to transformation into ecosystem types with qualitatively different properties and organizational processes (see refs.for cropland and urban examples, respectively). Indices such as human appropriation of net primary productivity [bib_ref] Quantifying and mapping the human appropriation of net primary production in earth's..., Haberl [/bib_ref] , combined with land-use maps [bib_ref] A comprehensive global 5 min resolution land-use data set for the year..., Erb [/bib_ref] , offer useful insights into the distribution of some anthropogenic ecosystems, but further development of indices is needed to adequately represent others, particularly in marine, and freshwater environments. Beyond land-use classification and mapping approaches [fig_ref] Figure 1 |: The generic model of ecosystem assembly underlying the Global Ecosystem Typology [/fig_ref] , a more comprehensive elaboration of the intensity of human influence underpinning the diverse range of anthropogenic ecosystems requires a multidimensional framework incorporating land-use inputs, outputs, their interactions, legacies of earlier activity and changes in system properties [bib_ref] How a socio-ecological metabolism approach can help to advance our understanding, Erb [/bib_ref].
Where less intense human activities occur within non-anthropogenic ecosystem types, we focused descriptions on low-impact reference states. Therefore, human activities are not shown as drivers in the assembly models for non-anthropogenic ecosystem groups, even though they may have important influences on the contemporary ecosystem distribution. This approach enables the degree and nature of human influence to be described and measured against these reference states using assessment methods such as the Red List of Ecosystems protocol [bib_ref] Scientific foundations for an IUCN Red List of ecosystems, Keith [/bib_ref] , with appropriate data on ecosystem change.
## Indicative distribution maps
Finally, to produce spatially explicit representations of the units at level 3 of the typology (principle 5), we sought published global maps (sources in Supplementary that were congruent with the concepts of respective ecosystem functional groups. Where several candidate maps were available, we selected maps with the closest conceptual alignment, finest spatial resolution, global coverage, most recent data and longest time series. The purpose of maps for our study was to visualize global distributions. Prior to applications of map data to spatial analysis, we recommend critical review of methods and validation outcomes reported in each data source to ensure fitness for purpose [fig_ref] Figure 4 |: Current and potential applications of the Global Ecosystem Typology to conserve biodiversity... [/fig_ref].
Extensive searches of published literature and data archives identified high-quality datasets for some ecosystem functional groups (for example, T1.3 Tropical-subtropical montane rainforests; MT1.4 Muddy shorelines; M1.5 Sea ice) and datasets that met some of these requirements for a number of other ecosystem functional groups (see .1 for details). Where evaluations by authors or reviewers identified limitations in available maps, we used global environmental data layers and biogeographic regionalizations as masks to adjust source maps and improve their congruence to the concept of the relevant functional group (for example, F1.2 Permanent lowland rivers). For ecosystem functional groups with no specific global mapping, we used ecoregions [bib_ref] An ecoregion-based approach to protecting half the terrestrial realm, Dinerstein [/bib_ref] [bib_ref] Freshwater ecoregions of the world: a new map of biogeographic units for..., Abell [/bib_ref] [bib_ref] Marine ecoregions of the world: a bioregionalization of coastal and shelf areas, Spalding [/bib_ref] as biogeographic templates to identify broad areas of occurrence. We consulted ecoregion descriptions, global and regional reviews, national and regional ecosystem maps, and applied in situ knowledge of participating experts to identify ecoregions that contain occurrences of the relevant ecosystem functional group (for example, T4.4 Temperate woodlands) (see .1 for details). We mapped ecosystem functional groups as major occurrences where they dominated a landscape or seascape matrix and minor occurrences where they were present, but not dominant in landscapeseascape mosaics, or where dominance was uncertain. Although these two categories in combination communicate more information about ecosystem distribution than binary maps, simple spatial overlays using minor occurrences are likely to inflate spatial statistics. The maps are progressively upgraded in new versions of the typology as explicit spatial models are developed and new data sources become available (see ref.for a current archive of spatial data).
The classification and descriptive profiles, including maps, for each functional biome and ecosystem functional group underwent extensive consultation, and targeted peer review and revision through a series of four phases described in Supplementary Information, Appendix 5 (pages 2-4). The reviewer comments and revisions from targeted peer review are documented in .1. In all, more than 100 ecosystem specialists have contributed to the development of v2.1 of the typology.
# Limitations
Uneven knowledge of Earth's biosphere has constrained the delimitation and description of units within the typology. There is a considerable research bias across the full range of Earth's ecosystems, with few formal research studies evaluating the relative influence of different ecosystem drivers in many of the functional groups, and abiotic assembly filters generally receiving more attention than biotic and dispersal filters. This poses challenges for developing standardized models of assembly for each ecosystem functional group. The models therefore represent working hypotheses, for which available evidence varies from large bodies of published empirical evidence to informal knowledge of ecosystem experts and their extensive research networks. Large numbers of empirical studies exist for some forest functional groups, savannas, temperate heathlands in Mediterranean-type climates, coral reefs, rocky shores, kelp forests, trophic webs in pelagic waters, small permanent freshwater lakes, and others (see references in the respective profiles [fig_ref] Figure 4 |: Current and potential applications of the Global Ecosystem Typology to conserve biodiversity... [/fig_ref]. For example, Bondreviewed empirical and modelling evidence on the assembly and function of tropical savannas that make up three ecosystem functional groups, showing that they have a large global biophysical envelope that overlaps with tropical dry forests, and that their distribution and dynamics within that envelope is strongly influenced by top-down regulation via biotic filters (large herbivores and their predators) and recurrent disturbance regimes (fires). Despite the development of this critical knowledge base, savannas suffer from an awareness disparity that hinders effective conservation and management [bib_ref] Biome Awareness Disparity is BAD for tropical ecosystem conservation and restoration, Silveira [/bib_ref]. In other ecosystems, our assembly models rely more heavily on inferences and generalizations of experts drawn from related ecosystems, are more sensitive to interpretations of participating experts, and await empirical testing and adjustment as understanding improves. Empirical tests could examine hypothesized variation in ecosystem properties along gradients within and between ecosystem functional groups and should return incremental improvements on group delineation and description of assembly processes.
High-quality maps at suitable resolution are not yet available for the full set of ecosystem functional groups, which limits current readiness for global analysis. The maps most fit for global synthesis are based on remote sensing and environmental predictors that align closely to the concept of their ecosystem functional group, incorporate spatially explicit ground observations and have low rates of omission and commission errors, 'high' spatial resolution (that is, rasters of 1 km 2 (30 arcsec) or better), and time series of changes. Sixty of the maps currently in our archive 27 aligned directly or mostly with the concept of their corresponding ecosystem functional group, while the remainder were based on indirect spatial proxies, and most were derived from polygon data or rasters of 30 arcsec or finer (Supplementary . Maps for 81 functional groups were based either on known records, or on spatial data validated by quantitative assessments of accuracy or efficacy. Therefore, we suggest that maps currently available for 60-80 of the 110 functional groups are potentially suitable for global spatial analysis of ecosystem distributions. Although, a significant advance on broad proxies such as ecoregions, the maps currently available for ecosystem functional groups would benefit from expanded application of recent advances in remote sensing, environmental datasets, spatial modelling and cloud computing to redress inequalities in reliability and resolution. The most urgent priorities for this work are those identified in .1 as relying on indirect proxies for alignment to concept, qualitative evaluation by experts and coarse resolution (>1 km 2 ) spatial data.
## Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
## Data availability
Descriptions, images and interactive maps for the typology are updated periodically at https://global-ecosystems.org/. The spatial data for this study are available at Zenodo (https://doi.org/10.5281/ zenodo.3546513).
[fig] Figure 1 |: The generic model of ecosystem assembly underlying the Global Ecosystem Typology. [/fig]
[fig] Figure 3 |: Hypothesized relationships of functional groups differentiated along gradients of selected assembly filters. a, The Tropical forests biome (T1), with temperature, elevation and water availability gradients. b, The Rivers and streams biome (F1), with stream gradient and temporal flow pattern. c, The Marine pelagic biome (M2), with depth and current gradients. In a, a third filter related to an edaphic environmental gradient differentiates group T1.4 from T1.1, but is not shown here (seeSupplementary Information, Appendix 4, for details on the respective functional groups). [/fig]
[fig] Figure 4 |: Current and potential applications of the Global Ecosystem Typology to conserve biodiversity and sustain ecosystem services. The typology provides a common ecosystem vocabulary and supports consistent treatment of ecosystems across applications where policy links exist between multiple initiatives. Details are presented in Supplementary Information, Appendix 4. Photo credit: Keith Ellenbogen (Ecosystem monitoring and management); Getty Images (Environmental education); KBA World Database of Key Biodiversity Areas at www.keybiodiversityareas.org; United Nations Sustainable Development Goals at: www.un.org/sustainabledevelopment. [/fig]
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No evidence of handling‐induced mortality in Serengeti's African wild dog population
The disappearance of an endangered African wild dog population from Serengeti National Park (SNP) led to international debate centered around one question: were researchers to blame? The "Burrows' hypothesis" postulated that stress induced by research-related immobilization and handling reactivated a latent rabies virus, eliminating the population. Insufficient data inhibited hypothesis testing, but since wild dogs persisted alongside SNP and have been studied since 2005, the hypothesis can be tested 25 years after its proposition. To be supported, wild dog immobilization interventions should have resulted in high mortality rates. However, 87.6% of 121 handled wild dogs (2006-2016) survived >12 months post-handling. Some argued that viral reactivation would necessitate long-term stress. Following immobilization, 67 animals were captured, transported, and held in a translocation enclosure. Despite the longer-term stress, 95.5% survived >12 months. Furthermore, the stable number of wild dog packs in the ecosystem over the past decade, and lack of recolonization of SNP, strongly oppose Burrows' hypothesis. Instead, factors such as heightened levels of interspecific competition are likely to have contributed to the wild dog disappearance and subsequent avoidance of the Serengeti plains. Handling and radio telemetry are invaluable when studying elusive endangered species, yielding information pertinent to their conservation and management, and had no effect on Serengeti wild dog survival. K E Y W O R D S animal welfare, canid conservation, ethics, immobilization, radio telemetry | 1111 JACKSON et Al. National Park (SNP), Tanzania. Studied since 1964, this population of
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Large carnivores have suffered significant global population declines, and protected areas are becoming increasingly important for their continued survival in the face of anthropogenic threats [bib_ref] The size of savannah Africa: A lion's (Panthera leo) view, Riggio [/bib_ref] [bib_ref] Status and ecological effects of the world's largest carnivores, Ripple [/bib_ref]. However, even within large protected areas carnivore populations can decline precipitously to the point of local extinction [bib_ref] Surveys of lions Panthera leo in protected areas in Zimbabwe yield disturbing..., Groom [/bib_ref].
Factors driving such events may not be immediately apparent, even when populations have been the subject of long-term research and
monitoring. An understanding of potential extinction-causing factors, including the role of well-intended human interventions, are vital to facilitate informed conservation and management actions within protected areas [bib_ref] Detecting declines of apex carnivores and evaluating their causes: An example with..., Rosenblatt [/bib_ref] [bib_ref] Edge effects and the extinction of populations inside protected areas, Woodroffe [/bib_ref].
A case in point is that of the African wild dog population that formerly inhabited the grassland plains in Serengeti endangered wild dogs declined and eventually disappeared entirely from the study area in 1991 [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] [bib_ref] Local extinction in a small and declining population -Wild dogs in the..., Ginsberg [/bib_ref]. The cause of the wild dogs' decline and eventual demise was extensively debated among scientists at the time [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] [bib_ref] Population-dynamics, intervention and survival in African wild dogs (Lycaon pictus), Burrows [/bib_ref] [bib_ref] Radiocollaring and stress hormones in African wild dogs, Creel [/bib_ref] [bib_ref] Handling-induced stress and mortalities in African wild dogs (Lycaon pictus), Devilliers [/bib_ref] [bib_ref] Wild dogs in the Serengeti ecosystem: What really happened, East [/bib_ref] [bib_ref] Stress hormones and radiocollaring of African wild dogs, East [/bib_ref] [bib_ref] Aspects of rabies infection and control in the conservation of the African..., Gascoyne [/bib_ref] [bib_ref] Handling and survivorship of african wild dog (Lycaon-pictus) in 5 ecosystems, Ginsberg [/bib_ref] [bib_ref] Local extinction in a small and declining population -Wild dogs in the..., Ginsberg [/bib_ref] [bib_ref] Assessing the risks of intervention: Immobilization, radio-collaring and vaccination of African wild..., Woodroffe [/bib_ref]. Yet more than 25 years later, consensus has not been reached. A controversial hypothesis implicated researchers and their handling of wild dogs as the driver of the extinction. Prior to their demise, serum samples showed that some of the population had been exposed to rabies and certain animals had significant rabies-neutralizing antibody titers. This led [bib_ref] Rabies in wild dogs, Burrows [/bib_ref] to postulate that the stress associated with the immobilization and handling of wild dogs (in the absence of vaccination) for research purposes caused immunosuppression, resulting in the reactivation of a latent form of the rabies virus, thereby causing the death of the entire study population. Despite staunch opposition (see Methods), the hypothesis was vehemently defended by its proponents and the debate remains unresolved [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] [bib_ref] Population-dynamics, intervention and survival in African wild dogs (Lycaon pictus), Burrows [/bib_ref] [bib_ref] Wild dogs in the Serengeti ecosystem: What really happened, East [/bib_ref] [bib_ref] Stress hormones and radiocollaring of African wild dogs, East [/bib_ref].
The implications of this hypothesis extend beyond wild dogs in the Serengeti; not only was immobilization of wildlife periodically suspended in certain countries immediately thereafter, but also the notion of researcher-induced extinction continues in the scientific literature [bib_ref] The ethical dimensions of wildlife disease management in an evolutionary context, Crozier [/bib_ref] [bib_ref] Evolutionary perspectives on wildlife disease: Concepts and applications, Vander Wal [/bib_ref]. More recently,attributed wild dog population declines in two southern African populations to researcher intervention, thereby perpetuating the validity of the original hypothesis. Furthermore, three handled packs in three different southern African protected areas succumbed to disease during 2016-2017; could this also have been due to researcher intervention? The hypothesis' validity not only has ethical implications for research activities but is particularly important for the conservation and management of this and other endangered species. Therefore, resolving whether research intervention was to blame more than 25 years after the event is challenging, yet critically important.
Although much of the scientific literature referred to the disappearance of the wild dogs from SNP as a population "extinction", the population never went extinct within the broader region [bib_ref] Population-dynamics, intervention and survival in African wild dogs (Lycaon pictus), Burrows [/bib_ref] [bib_ref] A historical perspective of the Maasai-African wild dog conflict in the Serengeti..., Lyamuya [/bib_ref] [bib_ref] Inferring the ancestry of African wild dogs that returned to the Serengeti-Mara, Marsden [/bib_ref].
Packs persisted in the adjoining Loliondo Game Controlled Area (LGCA) and Ngorongoro Conservation Area (NCA) to the east of SNP and numerous packs have occupied these areas for the past decade. Sporadic sightings of transient groups as well as global positioning system (GPS) collar data (unpublished data) indicates that these packs occasionally enter SNP, thereby confirming accessibility and connectivity across the entire region, and that the resident wild dogs represent a single population [bib_ref] Inferring the ancestry of African wild dogs that returned to the Serengeti-Mara, Marsden [/bib_ref]. The wild dog population thus survived in the eastern parts of the ecosystem and has been the subject of research and monitoring since 2005.
## | how to revisit and test the hypothesis 25 years later?
Low-intensity monitoring of study animals and the collection of few biological samples prevented a conclusive, data-derived consensus on the cause of the Serengeti wild dog disappearance. Researchers found no evidence of handling-induced mortality in other ecosystems [bib_ref] Handling and survivorship of african wild dog (Lycaon-pictus) in 5 ecosystems, Ginsberg [/bib_ref] , yet the proponents of Burrows' hypothesis discredited such studies, stating that "If intervention caused immunosuppression and increased disease-mediated mortality among adult pack members, then logically any test of this idea should be conducted on data from ecosystems where (a) wild dogs contact pathogens that are lethal to adults; (b) rates of exposure to lethal pathogens are similar to that for rabies in the Serengeti; (c) types and levels of interventions sustained by packs are similar to those applied in the Serengeti" [bib_ref] Stress hormones and radiocollaring of African wild dogs, East [/bib_ref]. Using data from wild dogs in the same ecosystem would therefore present an ideal study opportunity to test the hypothesis.
Since wild dogs did survive in the Serengeti ecosystem and have been the subject of an active research program that included immobilization and radio collaring, we aimed to test Burrows' hypothesis while satisfying the above-mentioned criteria. Firstly, rabies and canine distemper virus are prevalent in the ecosystem, including
LGCA and NCA [bib_ref] Maintenance of a microparasite infecting several host species: Rabies in the Serengeti, Cleaveland [/bib_ref] [bib_ref] The conservation relevance of epidemiological research into carnivore viral diseases in the..., Cleaveland [/bib_ref] F I G U R E 1 The Serengeti-Mara Ecosystem in northern Tanzania and southern Kenya. Black circles denote the location of resident packs observed during chance encounters or tracking of very high frequency (VHF) collared individuals between 2005 and 2009. The approximate location of the former study area is indicated by the large dashed circle [bib_ref] Aspects of rabies infection and control in the conservation of the African..., Gascoyne [/bib_ref] [bib_ref] Fatal canine distemper infection in a pack of African wild dogs in..., Goller [/bib_ref] [bib_ref] Exploring reservoir dynamics: A case study of rabies in the Serengeti ecosystem, Lembo [/bib_ref] [bib_ref] A canine distemper virus epidemic in Serengeti lions (Panthera leo), Roelke-Parker [/bib_ref] , and are lethal to adult wild dogs [bib_ref] Fatal canine distemper infection in a pack of African wild dogs in..., Goller [/bib_ref] [bib_ref] Rabies in African wild dogs (Lycaon pictus) in the Madikwe Game Reserve, Hofmeyr [/bib_ref].
During a five-year period alone , 128 cases of rabid domestic dogs were reported in LGCA and NCA [bib_ref] Exploring reservoir dynamics: A case study of rabies in the Serengeti ecosystem, Lembo [/bib_ref].
Secondly, contact with domestic dogs increases exposure to rabies [bib_ref] Contact with domestic dogs increases pathogen exposure in endangered African Wild dogs..., Woodroffe [/bib_ref] and therefore, rates of exposure are expected to be at least the same or higher than those experienced 20 to 30 years ago in SNP. This is because (a) the domestic dog population, a reservoir host for rabies [bib_ref] Exploring reservoir dynamics: A case study of rabies in the Serengeti ecosystem, Lembo [/bib_ref] , has increased in size [bib_ref] Interactions between domestic and wild carnivores around the greater Serengeti ecosystem, Craft [/bib_ref] , with an annual growth rate of up to 8% in certain parts of the ecosystem [bib_ref] Ecology and demography of free-roaming domestic dogs in rural villages near Serengeti..., Czupryna [/bib_ref] and (b) the surviving wild dog population occurs sympatrically with domestic dogs, and it is thus reasonable to assume that the current wild dog population has even greater rates of exposure to the rabies virus than the portion of the wild dog population formally resident within SNP, which is almost entirely free of domestic dogs. Several cases of rabies have been reported from the areas adjoining SNP post-1991 [bib_ref] Exploring reservoir dynamics: A case study of rabies in the Serengeti ecosystem, Lembo [/bib_ref] , and over 40% of village members in NCA reported the presence of wild dogs at their households [bib_ref] Ecology and demography of free-roaming domestic dogs in rural villages near Serengeti..., Czupryna [/bib_ref] , confirming the sympatric occurrence of domestic dogs and wild dogs. Thirdly, wild dogs are exposed to similar types of interventions, that is immobilization and collaring , and the same types of handling that allegedly resulted in individual and pack mortality in SNP [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref].
To ensure that the underlying mechanistic basis of Burrows' hypothesis was met, we used data from the same Serengeti wild dog population and assessed whether handling led to any detectable increases in mortality. Between 2006 and 2016, 121 wild dogs were immobilized and handled within the Serengeti Ecosystem, 45 of which were radio-collared. [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] reported that "handled individuals were significantly less likely to survive for 12 months after the date of first handling". We therefore assessed the survival of the handled wild dogs for 12 months post-handling in an attempt to determine whether increased mortality was evident following short-term and longer-term handling interventions. Additionally, we assessed whether there had been any recolonization of wild dogs in SNP because if researcher intervention alone was responsible for the disappearance of wild dogs on the Serengeti plains, then recolonization over the past 25 years would have been probable given the persistence of numerous wild dog packs immediately alongside the former study area and the cessation of wild dog research within SNP.
## | material s and me thods
## | study area and wild dog populations
The Serengeti-Mara Ecosystem LGCA and SNP and in addition to wildlife is home to sizeable Maasai and livestock populations. When the Serengeti wild dog study commenced in 1964, a 3,000 km 2 study area dominated by grassland plains was selected [bib_ref] Social organization of African wild dogs (Lycaon pictus) on the Serengeti Plains, Frame [/bib_ref]. In 1973, the study area was expanded northwards to include a total of 5,200 km 2 and comprised 4,200 km 2 of short and medium grassland and 1,000 km 2 of surrounding open Acacia woodlands [bib_ref] Social organization of African wild dogs (Lycaon pictus) on the Serengeti Plains, Frame [/bib_ref].
Following their disappearance from the Serengeti plains, the wild dog population survived in LGCA and NCA. Locals in LGCA and NCA saw wild dogs regularly for several decades, both before and after their disappearance from the Serengeti plains .
Genetic evidence further indicates that these wild dogs are genetically similar to the wild dogs formally resident on the Serengeti plains, ruling out recolonization from elsewhere [bib_ref] Inferring the ancestry of African wild dogs that returned to the Serengeti-Mara, Marsden [/bib_ref]. In combination, this evidence confirms that the Serengeti wild dog population did not go extinct, but survived in the eastern part of the ecosystem, with the population currently comprised of 120 animals in ten packs. The Tanzania Wildlife
Research Institute (TAWIRI) has monitored this wild dog population since 2005, including the use of radio collars, allowing us to test specific predictions related to Burrows' hypothesis.
## | a timeline of wild dog research and recorded mortality
A timeline of published records on wild dog handling, mortality, and other events relevant to the populations' persistence in the Serengeti Ecosystem are presented in . In 1970, the Serengeti study population numbered an estimated 95 wild dogs in 12 packs, the vast majority of which occurred on the open grassland plains in SNP [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] [bib_ref] Local extinction in a small and declining population -Wild dogs in the..., Ginsberg [/bib_ref]. Little research was conducted during the early 1980s, but work recommenced in 1985 and saw researchers immobilize and collar wild dog packs.
Between 1985 and 1990, a total of five Serengeti packs died or disappeared, and rabies was confirmed in one instance [bib_ref] Response to burrows, Gascoyne [/bib_ref]. These deaths occurred two to five months after handling by researchers for radio collar deployment.
In an attempt to protect the remaining packs, a vaccination program was initiated in 1990. Despite this, the remaining seven packs died five to 12 months after vaccination and the wild dog population was declared locally extinct [bib_ref] Rabies in wild dogs, Burrows [/bib_ref] [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref].
Although [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] maintained that the final demise was due to rabies, no samples were collected in the Serengeti, and there is no evidence that rabies caused the post-vaccination wild dog deaths [bib_ref] Response to burrows, Gascoyne [/bib_ref]. Instead, others suggested that it might have been canine distemper virus, which had emerged in the ecosystem at a similar time [bib_ref] African wild dogs (Lycaon pictus) endangered by a canine distemper epizootic among..., Alexander [/bib_ref]. If true, this would also account for why rabies vaccinations failed to protect the remaining wild dogs against the disease outbreak.
Stress, and the potential inhibitory effect that elevated glucocorticoid levels may have on the immune system, are central to hypothesis. No data on glucocorticoids levels, or fluctuations therein following handling, are available for the Serengeti wild dogs. Stress per se is not necessarily harmful; short-term stress responses are adaptive and may be beneficial to an organism's survival, while a prolonged stress response may be harmful [bib_ref] Radiocollaring and stress hormones in African wild dogs, Creel [/bib_ref]. While handling does increase wild dog stress levels in the short-term, there is no evidence that this is persistent enough to result in disease reactivation [bib_ref] Handling-induced stress and mortalities in African wild dogs (Lycaon pictus), Devilliers [/bib_ref].
## | testing burrows' hypothesis
## | prediction 1: effects of handling-induced stress on survival
The handling referred to by Burrows was all of a relatively short duration, yet deemed sufficient to evoke the reactivation of latent rabies [bib_ref] Population-dynamics, intervention and survival in African wild dogs (Lycaon pictus), Burrows [/bib_ref]. Consequently, for Burrows' hypothesis to be supported, the presence and potential exposure to rabies (as well as canine distemper virus) should see the handling of wild dogs in the Serengeti Ecosystem result in high rates of mortality following stress-induced viral reactivation. To assess this prediction, we collated all available data on wild dog immobilizations conducted within the Serengeti Ecosystem post-1991. All immobilizations during this period were carried out by TAWIRI veterinarians, and drugs were administered via darting rifle; the same technique utilized prior to 1991. A total of 121 wild dogs were immobilized between June 2006 and July 2016, 45 of which were radio-collared. We assessed individual survival to 3, 6, and 12 months post-immobilization.
## | prediction 2: effects of short-term and longterm stress on survival
Opponents to Burrows' hypothesis argued that short-term stress from relatively brief immobilizations would be insufficient to reactivate latent viruses, and that longer-term (chronic) stress would be required for this to potentially occur [bib_ref] Handling-induced stress and mortalities in African wild dogs (Lycaon pictus), Devilliers [/bib_ref]. Between 2012 and 2016, six wild dog packs were immobilized and captured in
LGCA, and thereafter relocated to SNP. The comparatively long-term handling that included immobilization, physical handling while loading into crates, relocation by road, and prolonged confinement in enclosures prior to release into an unfamiliar area, would be likely to result in far greater and prolonged stress levels than comparatively brief immobilization and radio collaring.
F I G U R E 2 A timeline highlighting dates of events or observations relevant to understanding and assessing the potential causes of the localized disappearance of the Serengeti wild dog population. Sources: 1. [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] , 2. [bib_ref] Social organization of African wild dogs (Lycaon pictus) on the Serengeti Plains, Frame [/bib_ref] , 4., 5., 6. [bib_ref] African wild dogs (Lycaon pictus) endangered by a canine distemper epizootic among..., Alexander [/bib_ref] , 8. , 10.Consequently, in accordance with Burrows' hypothesis, high rates of mortality would be predicted for these animals. To contrast the potential effects of short-versus long-term stress (handling), we assessed survival to 12 months post-handling of wild dogs that were handled and relocated (n = 67) with those that were not relocated but released after a short handling period (n = 54).
## | prediction 3: recolonization
For the past decade, several wild dog packs have occupied the area immediately alongside the Serengeti plains where wild dogs were studied between 1964 and 1991. Following the wild dogs' disappearance from SNP, four to six packs persisted in
LGCA and NCA [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref]. The number of packs more than doubled in subsequent years, with 13 packs recorded in LGCA alone during 2012. Wild dog populations have the ability to rapidly increase in size [bib_ref] Demography of a recovering African wild dog (Lycaon pictus) population, Woodroffe [/bib_ref] , and dispersal and pack formation have been recorded in the population . New packs need to establish territories [bib_ref] Population dynamics of African wild dogs, Fuller [/bib_ref] and the lack of wild dogs on the Serengeti plains post-1991 would have provided the necessary territorial voids immediately alongside the other resident packs [bib_ref] The effect of relatedness and pack size on territory overlap in African..., Jackson [/bib_ref] [bib_ref] Sex-biased dispersal in African wild dogs, Lycaon pictus, Mcnutt [/bib_ref]. Accordingly, we predict that if handling alone was responsible for the extinction [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] , the former study area would have been recolonized by the packs and dispersing groups occurring and originating immediately alongside because the reason for the demise of the SNP packs (researcher intervention) had been eliminated. Furthermore, six packs were reintroduced into SNP between 2012 and 2016, further increasing the probability of recolonization. Successful recolonization would support [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] s argument that ecological factors, such as interspecific competition, did not play a significant role in the study population's demise. In contrast, a lack of recolonization over a 25-year period would provide evidence against the hypothesis that researcher intervention alone was responsible for the decline and disappearance of the Serengeti wild dogs. Researchers have a permanent presence in and around the formerly inhabited area which, in addition to the large number of tourists in this area, would have detected the reestablishment of wild dogs. We additionally used data from an extensive camera-trap survey, which covered 1,125 km 2 of the area, to evaluate whether wild dogs were detected and therefore potentially recolonized the area.
## | re sults
## | prediction 1: effects of handling-induced stress on survival
Of the 121 animals immobilized between 2006 and 2016, 87.6% (n = 106) survived at least 12 months post-handling, while 91.7% (n = 111) and 95.9% (n = 116) survived more than 6 and 3 months postimmobilization, respectively. The high survival rate does not support the hypothesis that handling negatively impacts wild dog survival.
## | prediction 2: effects of short-term and longterm stress on survival
Between 2012 and 2016, 67 wild dogs from six different packs were captured and translocated to SNP, after a mean period of 313 days in enclosures (range: 76 to 499 days). Survival of these individuals was high: 95.5% (n = 64 of 67) of wild dogs survived more than 12 months post-handling. Furthermore, all six translocated packs survived more than 12 months post-handling. In contrast, survival to 12 months post-handling for wild dogs that were immobilized, but not captured and translocated, was 77.8% (n = 42 of 54). Thus, despite the longer-term handling and stress resulting from the capture and translocation process, mortality was higher in non-translocated packs that were exposed to short-term handling and stress, indicating that factors other than handling had a greater effect on survival.
Therefore, longer-term stressful interventions did not evoke disease outbreaks, and the high survival rate does not support Burrows' hypothesis.
## | prediction 3: recolonization
Monitoring of wild dogs in the ecosystem has continued since 2005.
Over the past decade, the population has increased, with as many as 13 known packs within the immediate area at times. Although these packs were in close proximity to SNP and the former study area, no recolonization of the Serengeti plains has occurred. This area is frequently visited by tourists and the TAWIRI researchers have a permanent presence within the park. Consequently, recolonization by these conspicuous carnivores would almost certainly have been rapidly detected. The wild dog's failure to recolonize is also evident from a large-scale camera trapping survey ("Snapshot Serengeti") that covered 1,125 km 2 of the former study area. Between 2010 and 2013, a total of 225 cameras accumulated 1.2 million images over 99,241 camera-trap days. This large number of images resulted in 334,671 speciesspecific capture events, documenting the presence of 40 mammalian species, but not wild dogs (table 1 in. In comparison, these capture events included 1,272 images of cheetah (Acinonyx jubatus), another carnivore that occurs at a low density, as well as 4,266 of lion (Panthera leo), and 5,303 of spotted hyaena (Crocuta crocuta).
Dispersal and pack formation did occur outside SNP with packs establishing both within the LGCA and NCA as well as further away in Kenya . However, no new or established packs returned to occupy the Serengeti plains region where they were studied between 1964 and 1991. Transient dispersal groups were recorded within the former study area soon after the disappearance and sporadically in the years thereafter [bib_ref] Population-dynamics, intervention and survival in African wild dogs (Lycaon pictus), Burrows [/bib_ref] , confirming the area's accessibility to wild dogs. Recolonization did not occur despite cessation of wild dog research (that Burrows' hypothesis attributed as causal for the extinction) in the area. Moreover, there was adequate prey availability [bib_ref] Local extinction in a small and declining population -Wild dogs in the..., Ginsberg [/bib_ref] , and a territorial void would have been created by the lack of resident wild dog packs. The wild dogs' failure to recolonize SNP prompted the initiation of the wild dog reintroduction program in 2012. Despite being released in SNP and within relatively close proximity to the plains, none of the six reintroduced wild dog packs occupied the previously inhabited habitat, further highlighting the wild dog's avoidance thereof. Within the first 12 months, three of the reintroduced packs had established territories outside SNP, two packs along the north-western boundary and only one entirely inside SNP, in rugged terrain to the west of the former (plains) study area.
## | d iscuss i on
Burrows' hypothesis postulates that the decline and disappearance of the African wild dog population on the Serengeti plains was a direct result of researcher-induced disease outbreaks. Despite the resistance of the scientific community at large to accept this hypothesis, the proponents thereof have consistently defended it [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] [bib_ref] Population-dynamics, intervention and survival in African wild dogs (Lycaon pictus), Burrows [/bib_ref] [bib_ref] Wild dogs in the Serengeti ecosystem: What really happened, East [/bib_ref] [bib_ref] Stress hormones and radiocollaring of African wild dogs, East [/bib_ref]. Using a multifaceted approach and data from the same wild dog population, where disease is still prevalent, we found no support for Burrows' hypothesis. Survival following both short-and longterm human interventions was high and no decline in the number of resident packs occurred. Furthermore, recolonization of the former study area has not occurred during the past 25 years, despite active reintroduction attempts and the cessation of wild dog research, strongly suggesting that researcher intervention alone was not responsible for the wild dog decline and eventual disappearance from SNP in 1991. Our results are supported by earlier work showing that the survival of adult and yearling wild dogs in reintroduced packs (all individuals captured and handled) and freeranging packs (majority of individuals unhandled; maximum of two adults per pack handled for radio collar deployment) did not differ.
Both rabies and canine distemper virus can be fatal to wild dog individuals and packs [bib_ref] Fatal canine distemper infection in a pack of African wild dogs in..., Goller [/bib_ref] [bib_ref] Rabies in African wild dogs (Lycaon pictus) in the Madikwe Game Reserve, Hofmeyr [/bib_ref] [bib_ref] Rabies and African wild dogs in Kenya, Kat [/bib_ref] and both diseases were present within the Serengeti Ecosystem at the time of the wild dogs' disappearance [bib_ref] African wild dogs (Lycaon pictus) endangered by a canine distemper epizootic among..., Alexander [/bib_ref] [bib_ref] Maintenance of a microparasite infecting several host species: Rabies in the Serengeti, Cleaveland [/bib_ref] [bib_ref] Aspects of rabies infection and control in the conservation of the African..., Gascoyne [/bib_ref]. It is likely that one of these diseases caused the final disappearance of the study packs during 1990-1991, but the lack of biological samples prevented the determination thereof.
Burrows' hypothesis was entirely focused on rabies, yet canine distemper virus was another potential cause of mortality and a disease to which the wild dogs had not been vaccinated [bib_ref] Local extinction in a small and declining population -Wild dogs in the..., Ginsberg [/bib_ref] [bib_ref] Cause of wild dog deaths, Macdonald [/bib_ref]. Indeed, less than three years later, canine distemper virus was responsible for the death of a third of SNP's lion population [bib_ref] A canine distemper virus epidemic in Serengeti lions (Panthera leo), Roelke-Parker [/bib_ref].
More concerning, however, was the assumption that all wild dog individuals and packs had died. Little data on the actual number of dead wild dogs and packs were presented, and a large number of "deaths" were entirely circumstantial. The two Serengeti packs (n = 34 individuals), that were vaccinated in September 1990, had disappeared five to ten months after vaccination [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref]. Four of seven radio collars were recovered, while three were unaccounted for [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref]. Instead of verified mortality data, [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] assumed that collared and uncollared wild dogs had the same mortality rate, and by means of extrapolation reasoned that at least 57% (4 of 7) of all wild dogs had died. These authors proceeded even further and suggested that the entire population had died based on a lack of resightings. While four individuals may have died, due to unknown reasons, this cannot be used as evidence for the death of 34 individuals. This is particularly pertinent given that during the five months post-vaccination, the two wild dog packs spilt up and formed five packs [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref]. Such dynamics have been observed in the Serengeti Ecosystem to result in dispersing pack members covering hundreds of kilometers . Lacking satellite GPS telemetry, such movements would most likely have gone undetected, and the study animals would not have been sighted again. Failure to observe individuals or packs in their former range can therefore not be equated with pack mortality.
Moreover, pack fission and new pack formation occurred during a period when emigration from and immigration to the study area was confirmed [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] , increasing the probability of disease transmission between distant parts of the ecosystem.
Post-dispersal animals that had immigrated into packs survived significantly shorter than pre-dispersers [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref]. While it was argued that post-dispersal individuals were exposed to increased social stress in their new packs, making them more susceptible to stress-induced disease reactivation [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] , it is perhaps more likely that these individuals had been exposed to disease during their dispersal movements.
Previous studies found no evidence of handling-induced mortality using data from wild dog populations in five different ecosystems [bib_ref] Handling and survivorship of african wild dog (Lycaon-pictus) in 5 ecosystems, Ginsberg [/bib_ref]. However, [bib_ref] Population-dynamics, intervention and survival in African wild dogs (Lycaon pictus), Burrows [/bib_ref] argued that this finding was not relevant to the Serengeti wild dog extinction because the prevalence of and wild dog exposure to pathogens in those ecosystems was unknown. Instead, these authors argued that the potential absence of disease would prevent wild dog exposure and thus the development of a latent virus from researcher-induced stress. By working in the same ecosystem, with the same wild dog population that is still vulnerable to disease, we circumvented this problem, yet found no evidence of researcher-induced mortality in the Serengeti region. Ironically,invoked the handling-induced mortality hypothesis to explain declines in the Moremi Game Reserve (Botswana) and
Kruger National Park (South Africa) wild dog populations, despite specifically mentioning these populations previously when refuting [bib_ref] Handling and survivorship of african wild dog (Lycaon-pictus) in 5 ecosystems, Ginsberg [/bib_ref]. Such broad-scale application of the handling-induced mortality hypothesis without clear evidence highlights the necessity of determining the true cause of the Serengeti wild dog disappearance before unnecessary restrictions on wild dog, and possibly other endangered species, research is implemented elsewhere.
We found that wild dogs that were immobilized, captured, transported, and held for several months (thereby exposed to prolonged stress) survived longer than those that were only briefly immobilized and thereafter released. This opposes the idea of fatal stress-induced reactivation of rabies. Furthermore, at least three wild dogs were collared in SNP in 1975, yet no deaths or disease outbreaks were reported, whereas an unhandled wild dog pack of seven individuals died during the extinction period [bib_ref] Response to burrows, Gascoyne [/bib_ref]. Therefore, Burrows' hypothesis does not hold true in the Serengeti ecosystem before, during, or after the disappearance of the Serengeti wild dogs.
According to the data presented by [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] [bib_ref] Population-dynamics, intervention and survival in African wild dogs (Lycaon pictus), Burrows [/bib_ref] "the question about why they had long been in decline is arguably the more important" [bib_ref] Serengeti wild dogs: What really happened?, Dye [/bib_ref]. More than two decades later, the wild dog's failure to recolonize the Serengeti plains, despite their occurrence immediately alongside, may be equally important, as well as revealing, as to the cause of their disappearance.
What then could have caused the wild dog population decline and disappearance, and still hinder recovery decades later? Evidence from several ecosystems indicates that wild dogs are vulnerable to competition from lions and hyaenas, both through direct mortality [bib_ref] The impact of lions on the demography and ecology of endangered African..., Groom [/bib_ref] [bib_ref] Conserving the African wild dog Lycaon pictus. I. Diagnosing and treating causes..., Woodroffe [/bib_ref] and kleptoparasitism [bib_ref] Feeding success of African wild dogs (Lycaon pictus) in the Serengeti: The..., Carbone [/bib_ref]. The high risk posed by lions results in wild dogs avoiding them at all times [bib_ref] African wild dogs as a fugitive species: Playback experiments investigate how wild..., Webster [/bib_ref] and, at the landscape scale, wild dog densities are inversely correlated with lion and spotted hyaena densities [bib_ref] Factors affecting the density and distribution of wild dogs in the Kruger..., Mills [/bib_ref]. During the period of the Serengeti wild dog population decline, the spotted hyaena population increased by 150% (from 2,200 to 5,500) and similarly large increases were recorded in the lion population [bib_ref] Demography, extinction and intervention in a small population -The case of the..., Burrows [/bib_ref] , with concomitant decreases in wild dog pup survival and adult longevity [bib_ref] Local extinction in a small and declining population -Wild dogs in the..., Ginsberg [/bib_ref]. During the 1960s and 1970s, fewer than half of Serengeti wild dog kills were attended by hyaenas, increasing to 85% during the 1980s, and likely preventing wild dogs meeting their energy requirements [bib_ref] Feeding success of African wild dogs (Lycaon pictus) in the Serengeti: The..., Carbone [/bib_ref]. Such effects were already apparent during the 1970s, when competition with hyaenas for food and disease resulted in no wild dog pups surviving to 12 months of age between July 1974 and January 1976 [bib_ref] Social organization of African wild dogs (Lycaon pictus) on the Serengeti Plains, Frame [/bib_ref].
The wild dog population persisted in the eastern part of the ecosystem and mostly in LGCA.
LGCA is comprised of heterogeneous savanna habitat that differs largely from the homogenous, flat Serengeti grasslands from which the wild dogs disappeared. Heterogeneous habitats provide variability in resource abundance and distribution, affecting species interactions [bib_ref] Habitat heterogeneity and mammalian predator-prey interactions, Gorini [/bib_ref]. Lions and hyaenas respond to this resource variability and, as the dominant apex carnivores, select the most resource-rich habitats, avoiding rugged areas, for instance, in favor of prey-rich flatter terrain [bib_ref] Factors affecting the density and distribution of wild dogs in the Kruger..., Mills [/bib_ref]. The resulting spatial variability in lion and hyaena densities facilitates avoidance by subordinate wild dogs [bib_ref] Factors affecting the density and distribution of wild dogs in the Kruger..., Mills [/bib_ref] and the development of a landscape of fear where high-risk habitats are avoided and competition refuges are actively selected, particularly during the vulnerable denning season when rugged and densely vegetated areas, with fewer lions, are selected by wild dogs [bib_ref] Den site selection, pack composition, and reproductive success in endangered African wild..., Davies [/bib_ref] [bib_ref] Heading for the hills: Risk avoidance drives den site selection in African..., Jackson [/bib_ref]. Indeed, fluctuations in lion densities have been shown to elicit changes in wild dog behavior and habitat selection . In the absence of strong top-down regulation, behavioral flexibility would likely see profitable habitats, such as the plains, being exploited. Lower interspecific competition thereby likely played a significant role in the wild dog population's persistence on the Serengeti plains prior to 1991 [bib_ref] Cheetahs and wild dogs show contrasting patterns of suppression by lions, Swanson [/bib_ref] , while higher densities of lions and hyaenas today inhibits recolonization of these habitats [bib_ref] Local extinction in a small and declining population -Wild dogs in the..., Ginsberg [/bib_ref]. We postulate that the disappearance from the Serengeti plains was instead merely a range contraction driven by increasing competitor densities with an outbreak of disease dealing the final blow to the remaining individuals and had little to do with researcher-induced mortality.
Many threatened species are difficult to study due to their cryptic habits and/or low densities [bib_ref] Conserving wild dogs, Creel [/bib_ref]. Advocates of hypothesis argue that, due to the supposed negative effects, research and monitoring of such species should be conducted using entirely non-invasive techniques. While animal welfare and ethical considerations should remain paramount, much of the information pertinent to management and conservation of threatened species would be impossible to attain in the absence of radio telemetry and other techniques that require researcher intervention [bib_ref] Conserving wild dogs, Creel [/bib_ref]. Conserving wide-ranging carnivores is particularly challenging in an increasingly human-dominated world, and the probability of conservation initiatives being successful are greatly increased by a thorough understanding of the nuances of species' behavior and ecology. Consequently, in the case of the African wild dog, information gained through research involving radio telemetry and other interventions has most likely contributed to the conservation of the species as a whole, rather than compromised it.
## Ack n owled g m ents
We are grateful to two anonymous referees who provided constructive comments. We thank TAWIRI for granting permission to conduct the study. Wild dog research was funded by several donors, includ- No. 641918 (AfricanBioServices). We extend our sincere thanks to the Serengeti Wild Dog Conservation Project's field team for their tireless efforts during the data collection period.
## Co n fli c t o f i nte r e s t s
We have no competing interests.
## Auth o r co ntr i b uti o n s
CRJ conceived the study, analyzed the data, and drafted the manuscript. EHM, EEM, RDF conducted wild dog immobilization, collected wild dog survival data, and contributed to the writing of the manuscript. ABD, FF, ER, and RFM contributed to the study design and helped draft the manuscript. All authors read and approved the manuscript for publication.
## Data acce ss i b i lit y
The dataset will uploaded as supplementary material upon acceptance of the manuscript.
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Nitric Oxide Signaling in the Auditory Pathway
Nitric oxide (NO) is of fundamental importance in regulating immune, cardiovascular, reproductive, neuromuscular, and nervous system function. It is rapidly synthesized and cannot be confined, it is highly reactive, so its lifetime is measured in seconds. These distinctive properties (contrasting with classical neurotransmitters and neuromodulators) give rise to the concept of NO as a "volume transmitter," where it is generated from an active source, diffuses to interact with proteins and receptors within a sphere of influence or volume, but limited in distance and time by its short half-life. In the auditory system, the neuronal NO-synthetizing enzyme, nNOS, is highly expressed and tightly coupled to postsynaptic calcium influx at excitatory synapses. This provides a powerful activity-dependent control of postsynaptic intrinsic excitability via cGMP generation, protein kinase G activation and modulation of voltage-gated conductances. NO may also regulate vesicle mobility via retrograde signaling. This Mini Review focuses on the auditory system, but highlights general mechanisms by which NO mediates neuronal intrinsic plasticity and synaptic transmission. The dependence of NO generation on synaptic and sound-evoked activity has important local modulatory actions and NO serves as a "volume transmitter" in the auditory brainstem. It also has potentially destructive consequences during intense activity or on spill-over from other NO sources during pathological conditions, when aberrant signaling may interfere with the precisely timed and tonotopically organized auditory system.Keywords: auditory processing, neuronal excitability and ion channel regulation, hearing loss, neuronal nitric oxide synthase (nNOS), volume transmission, synaptic plasticity Abbreviations: cGMP, cyclic guanosine monophosphate; EPSC, excitatory postsynaptic current; GABA, gammaaminobutyric acid; GABA A R, gamma-aminobutyric acid ionotropic receptor; GTP, guanosine triphosphate; HCN, hyperpolarization-activated cyclic nucleotide gated cation channel; HCN1, hyperpolarization-activated cyclic nucleotide gated cation channel type 1; HCN2, hyperpolarization-activated cyclic nucleotide gated cation channel type 2; KCC2, potassium-chloride cotransporter type 2; LSO, lateral superior olive; MNTB, medial nucleus of the trapezoid body; MSO, medial superior olive; N, number of synaptic release sites; NADPH, nicotinamide adenine dinucleotide phosphate; NMDAR, N-methyl-D-aspartic acid or N-methyl-D-aspartate receptor; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; P, release probability; PKG, protein kinase G; PSD95, postsynaptic density 95; sGC, soluble guanylate cyclase; SPN, superior paraolivary nucleus; SR, spontaneous rate.
# Introduction
Nitric oxide (NO) is a small molecule, highly mobile, highly reactive and soluble in water and lipid membranes, so that once synthesized it cannot be contained. While its lifetime in biological tissues may be short, its mobility permits unimpeded diffusion over significant cellular distances. The discovery of the action of "Endothelium-Derived Relaxing Factor" on vascular smooth muscle and its identification as nitric oxide earned Furchgott, . NO action in the brain was first linked with NMDAR-mediated increases in cGMP in the cerebellumand its general signaling mechanisms in the brain have been widely reviewed.
Even the NO "receptor" is unconventional, in being a cytoplasmic hemoprotein ("soluble" guanylyl cyclase, sGC) generating cGMP from GTP. Although a misnomer, we have stuck with the term "soluble" and use of "sGC" to abbreviate guanylyl cyclase. It has been shown elsewhere in the brain, including in the inferior colliculus, that the GC is actually not soluble, but anchored to PSD-95 at the synapse. Indeed, the signaling cascade exhibits extreme amplification, so that physiological signaling is thought to be achieved by NO in the nanomolar concentrations.
Nitric oxide is synthetized from L-arginine and oxygen using NADPH and co-factors. This reaction is mediated by neuronal nitric oxide synthase (nNOS) in the brain. In the postsynaptic density of glutamatergic synapses, nNOS is activitydependent and coupled through calmodulin to calcium influx at NMDARs. The canonical nNOS signaling pathway is shown in , with examples of pharmacological agents . The concentration of cGMP in any one cellular compartment is not only determined by the rate of production, but also by degradation through local phosphodiesterases, which further modulate signaling . Although cGMP may exert direct action on cyclic nucleotide-gated channelsthe majority of the signaling is via activation of protein kinase G (PKG) extending NO signaling capabilities, with different sGC isoforms providing important tissue-specific control. Facilitation of this signaling pathway is achieved by spatial proximity using cytoskeletal scaffolding proteins to bind sequential enzymes in the pathway, so nNOS is located in the postsynaptic density through PSD-95, which also binds NMDAR.
Beyond the proven link to calcium influx through NMDAR, nNOS can be activated by calcium influx through calciumpermeable AMPA receptorsand L-type voltage-gated calcium channels; see . NO signaling also modulates neuronal intrinsic excitability by acting on voltage-gated calcium, sodium, and potassium channels.
Nitric oxide modulates neuronal excitability very broadly and yet nNOS knockout mice survive, as if NO is "part" of a massively redundant system (and perhaps compensated by the remaining eNOS and iNOS genes). NO signaling is highly ubiquitous in the animal kingdomand its breadth and diversity means we have yet to build consensus about its physiological roles in the nervous system. The literature has myriad observations (including those of the authors) that have yet to be consolidated into their full physiological context. The hypothesis of retrograde NO transmission has particularly fascinated neuroscientists, for which the evidence is reviewed elsewhere. However, a presynaptic focus may have biased investigations away from other NO signaling roles: consequently, less attention has focused on NO-mediated cGMP signaling beyond the synapse, on kinase regulation of ion channels, and non-cGMP signaling via nitrosylation, control of gene expression or as a free radical. The auditory pathway provides a system in which many of these issues can be explored. In fact, the generation of cGMP, NO-induced intrinsic plasticity, synaptic plasticity and changes in in vivo firing rates have been clearly demonstrated in the auditory brainstem: cochlear nucleus:, Superior Olivary Complex:, and Inferior Colliculus:and in an animal model of tinnitus.
## Nitric oxide signaling pathways in auditory neurons
There are multiple elements to understanding NO signaling in the auditory system: evidence for the presence of key signaling molecules in the pathway (nNOS/sGC/NADPH, see, identification of the target proteins and ion channels modulated, and observation of physiological/behavioral change on pharmacological intervention or genetic manipulation. This evidence must be weighed against physiological data and normal behavior since there is the potential for spill-over from other NOgenerating systems and pathology, for example associated with iNOS activation during inflammatory processes. An important caveat in studying NO signaling is the extent to which an in vitro experimental system supports NO signaling (e.g., possessing an arginine source, NO donor validation, etc.) and whether an in vivo system is achieving NO activation (or inactivation) within a physiological or pathological context.
Adenosine 5 -triphosphate (ATP) is a major neurotransmitter and neuromodulator in the cochlea causing an increase in intracellular calcium. NO inhibits this ATP-induced calcium response via a negative feedback mechanism in inner hair cells, while at the same time enhancing the ATP-induced calcium response in outer hair cells and spiral ganglion neurons. Noise exposure increases nNOS expression in cochlear nucleus neuronsand in spiral ganglion neurons, causing the NO concentration in the cochlea to rise from about 300 to 600 nM. The interaction of nNOS with activity-dependent calcium increases might be a component of the feedback in protecting inner hair cells from noise overexposure. Application of nNOS inhibitors or NO donors in vivo, differentially affected spontaneous and sound-evoked firing rates in different cell types, which may contribute to increased gain during tinnitus.
There have been many studies of short-term plasticity at the giant calyx of Held synapse in the auditory brainstem, but activity-dependent FIGURE 1 | Pharmacology of nitric oxide signaling. NO is generated by glutamatergic stimulation of NMDARs, but other sources of calcium from Calcium permeable AMPAR or L-type calcium channels are also recognized. Calcium influx activates nNOS (via calmodulin) which catalyzes the conversion of the amino-acid arginine to citrulline, releasing NO. nNOS activity may be blocked by competitive antagonists such as L-NAME (NG-Nitro-L-arginine methyl ester HCl), absorbed by chelating agents, or generated independently of nNOS by perfusion of NO donors. NO diffuses across cytoplasm, membranes and between cells to bind to its intracellular receptor -soluble guanylyl cyclase (sGC) which catalyzes GTP to cGMP -a cyclic nucleotide which activates protein kinase G (PKG). ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) is a competitive blocker of sGC, while BAY 41-2272 is a positive modulator. KT 5873 is an antagonist of PKG. cGMP signaling may in turn be suppressed with phosphodiesterases, such as PDE5, which can be blocked by sildenafil. Blockers or antagonists are shown in red, chelating agents in orange, and positive modulators in green. The canonical pathway is indicated by the thick black arrows, with links from other sources by fine arrows, and the spectrum of PKG actions via dashed arrows.
long-term plasticity has never been reported at this giant synapse. However, it is not always appreciated that NO reduces EPSC amplitudes at the calyx of Held through postsynaptic AMPAR modulation rather than a presynaptic mechanism. Such a postsynaptic NO-action is corroborated by the lack of NO-modulation of presynaptic potassium currents, which would have changed transmitter release via the action potential. Nevertheless, other studies have demonstrated PKG-mediated modulation of synaptic vesicle endocytosis using capacitance measurements, although no change in transmitter release was reported. It is important to recognize that the probability of transmitter release, the number of release sites and rates of exocytosis and vesicle recycling are in a complex equilibrium. Increased release probability (P) is "offset" by a reduced number of release sites (N) possessing fusion competent vesicles; hence after modulation the synapse may be in a different state (higher P, lower N; or lower P, higher N) even though there may be little evidence of a change in EPSC amplitude. Nevertheless, NO-signaling does cause an increase in spontaneous EPSCs in VCN T-stellate cells.
Direct effects of NO on evoked transmitter release have yet to be reported in the auditory pathway, so it is reasonable to postulate that NO-modulation of postsynaptic neuronal excitability (rather than synaptic mechanisms) is its primary mechanism of action. These actions may be mediated by the canonical cGMP second messenger and/or PKG-mediated phosphorylation of ion channels, for which there is direct evidence; or NO actions could be mediated by peroxynitrite formation or protein modification, such as nitrosylation .
In neurons of the medial nucleus of the trapezoid body (MNTB), synaptic stimulation of the calyx of Held synapse (or perfusion of NO donors) raised cGMP and increased action potential duration, due to modulation of postsynaptic Kv3 and Kv2 potassium channels. This is due to local activity-dependent generation of NO, and reciprocal modulation of potassium channel activity: so that Kv3 takes a lesser role and Kv2 takes a greater role in postsynaptic action potential repolarization, following NO signaling. This shift in intrinsic excitability reveals the hallmark of volume transmission, in that active synapses influence local quiescent neurons (having no synaptic input). This has implications for ion channel expression that follows a tonotopic gradient, such as HCN or Kv3 channels, which might be opposed (or amplified) by gradients of NO signaling, and hence ion channel activity will reflect the sum of channel expression and channel modulation.Auditory cortex
## Primary auditory cortex (au1)
Histology/physiologyNitric oxide also modulates HCN1 and HCN2 channels, which are differentially expressed across the superior olivary complex. The MNTB expresses HCN2, which has slow kinetics, while in the medial and lateral superior olive (MSO, LSO) and in the superior paraolivary nucleus (SPN), HCN channels are dominated by HCN1 subunits, which have fast kinetics. NO had distinct actions on these two channels: it facilitated HCN2 in a cGMP-dependent manner and inhibited and slowed HCN1 kinetics in a cGMP-independent manner. Regulation of HCN currents is a key means of setting and regulating resting membrane potentials and the neuron membrane time-constant, since the higher Na + permeability of HCN channels will drive the equilibrium to more positive potentials. In turn, a higher resting conductance generates a faster membrane time-constant, thereby modulating integration of synaptic inputs.
Another important homeostatic process is the control of intracellular chloride concentrations. A developmental shift in the chloride equilibrium potential in young animals is documented across many areas of the CNS, including the auditory brainstem. "Inhibitory" neurotransmitters such as GABA and glycine mediate depolarizing synaptic responses in neonatal animals, which become hyperpolarizing around the time of hearing onset, due to an upregulation of the potassiumchloride cotransporter 2 (KCC2;. Very high levels of KCC2 (driving the chloride equilibrium to around −100 mV) are expressed in the SPN and in combination with large glycinergic inputs (from the MNTB) and high levels of HCN1 currents, enable the ionic computation of the end of a sound . Activitydependent regulation of KCC2 has been widely documented in the hippocampus and neocortex where changes in chloride gradients impact the strength of GABA A R-mediated inhibition. In the SPN the strength of glycinergic inhibition is suppressed via a cGMP-dependent NO signaling at KCC2; creating a shift in the chloride equilibrium by +15 mV. This action is specific to those neurons that are expressing KCC2, which allows differential modulation of chloride reversal potentials in different neuronal populations, all of which may be receiving the same inhibitory projection (for example from the MNTB).
## Discussion and open questions
Nitric oxide signaling is widespread, with diverse sites and convoluted actions in the nervous system. Consequently, it is often difficult to identify the source of NO signaling for a specific physiological or behavioral output, and difficult to separate physiological roles from pathological consequences, with the potential for spill-over from one synthase into the signaling system of another, e.g., iNOS to nNOS. NO is an important mediator of inflammation and pathology via up-regulation of iNOS in microglia (generating micromolar concentrations of NO). Microglia are present in the auditory brainstem, where they are involved in developmental pruning of the calyx of Held synapseand in regulating inflammation. Inflammation is associated with noise-induced hearing lossand mediated by pro-inflammatory cytokines. Hearing loss and inflammation can also be caused by severe hyperbilirubinemia, where subsequent degeneration of the calyx of Held synapse is mitigated by blocking NO signaling. It is worth speculating that these links between hearing loss, inflammation and NO signaling could be associated with pathological actions of microglia. The wide actions of nitric oxide, nitrosylation, nitrergic stress, and inflammation are associated with multiple neurodegenerative disease mechanismsand perhaps underlies broader NO mediated pathology .
Nitric Oxide has a broad impact on auditory neurons and signaling. It increases evoked firing rates by enhancing intrinsic excitability, by reducing inhibitory strength and by potentiating excitatory inputs via positive feedback. An interesting facet of auditory signaling are high rates of spontaneous AP firing; these spontaneous rates (SRs) arise from a combination of transmitter release at inner hair cells and the intrinsic excitability of all neurons along the pathway. There is a progressive decrease in SRs from the cochlea to the cortex, that seems to be mirrored by higher nNOS expression in the brainstem and midbrain compared to lower nNOS expression in MGB and cortex. High SRs are advantageous for temporal processing tasks in the brainstem, but are less important at higher auditory centers (such as the MGB and cortex) where auditory processing has evolved from a temporal code toward a rate code. The idea that auditory brainstem SRs carry information has been comprehensively discussed elsewhere. While synchronization and phase-locking of AP firing are important properties of sound-evoked activity, non-sound-evoked, spontaneous firing is synchronized only during developmentor possibly during pathological auditory signaling. SRs in the healthy, mature auditory system are not synchronized. This is important because incoming soundevoked activity defines a time window within which an action potential could be generated, intrinsic excitability permitting. So when SR is high, there is a high probability that a neuron is refractory when a sound-evoked stimulus arrives, but the stochastic distribution and desynchronization of SR between neurons maximizes the number of short latency action potentials across the population. NO-mediated modulation of SR could maintain a desynchronized SR, ensuring temporally precise and faithful transmission of responses to sound. The lower SR in higher auditory brain areas would render NO-mediated desynchronization of SR redundant, in contrast to the developing auditory system. An open question for the future is the extent to which activitydependent NO signaling controls basal activity rates: a low SR before hearing onset requires little NO, and high SR on maturation needs more NO, while a stressed auditory system following noise exposure would demand even higher NO concentrations. Recruitment of NO has been shown following noise exposureand could be involved in the development of tinnitus. The question of whether NO signaling is a cause of tinnitus or a response to correct aberrant excitability and desynchronized SR, will require future studies.
The proposed role in desynchronizing SR might explain why NO-volume transmission does not necessarily interfere with the precise tonotopically dominated sound evoked processing. A common theme of NO action in the auditory system is the homeostatic control of excitability, be that synaptic excitation/inhibition, spontaneous firing rates or neuronal intrinsic excitability. The contribution of NO to synaptic plasticity and memory formation is widely accepted in higher brain centers. Recent studies in the fruit fly have proposed that NO is more associated with active forgetting and updating of memories. Such mechanisms might underlie auditory re-mapping following temporary hearing loss. Failure to update memories in the absence of NO might also explain impaired auditory fear conditioning in nNOS knockout mice.
There is strong evidence for the presence of NO signaling within the auditory brainstem. There are also broad observations of NO-mediated modulation of neuronal excitability and synaptic transmission. However, a consensus on the roles of NO in the auditory pathway has yet to be reached. Elsewhere there is ample evidence for NO involvement in synaptic plasticity, but less agreement about common downstream mechanisms. This no doubt reflects the broad signaling capabilities of cGMP and PKG (and alternate signaling by direct reactions of NO with proteins). Perhaps we need to integrate our investigations of NO signaling over a much broader range of targets (genetic, ion channel, cell signaling, metabolism/growth) in homeostasis, synaptic transmission and intrinsic excitability, and include (or control for) the potential for spill-over from pathological to physiological signaling. The superior olivary complex may lack the complexity of higher centers, but it has a well-characterized anatomy and physiology in which these complex interacting systems can be carefully explored.
## Key concepts
- NO generation is activity-dependent and through NMDAR activation at excitatory synapses. - Signaling involves both cGMP -dependent andindependent signaling cascades. - NO acts by diffusion through a process of Volume Transmission to regulate excitability of neurons (including those that are active and inactive within a sphere of influence). - NO modulates postsynaptic neuronal excitability via modulation of voltage-gated ion channels. - Aberrant signaling underlies impaired auditory processing via changes in excitability and spontaneous firing rates.
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Against the stream: drugs policy needs to be turned on its head
2019
Baroness Molly Meacher BJPsych Bulletin
43201910.1192/bjb.2018.98First received 25 Oct 2018, accepted 1 Nov 2018Against the stream: drugs policy needs to be turned on its head Former Chair, East London NHS Foundation Trust, UK Correspondence to Baroness Molly Meacher ([email protected])
Human beings have taken mind altering drugs since the Stone Age, but the current global 'war on drugs' dates only from 1961. At that time, the addictive qualities of drugs like heroin and cocaine led the United Nations Member States to conclude that drastic action had to be taken as they were 'concerned with the health and welfare of mankind' 1the objective of the United Nations Single Convention on Narcotic Drugs. The assumption at the time was that a drugfree world could be created if those who produce, sell, possess or use certain addictive drugs were severely punished. Currently, in the UK, those arrested for possession of a controlled drug (e.g. heroin, cocaine, ecstasy or cannabis) can have a maximum prison sentence of 7 years under the UK Misuse of Drugs Act 1971. Producers and suppliers can be put behind bars for a maximum of 14 years.
A policy objective to advance the health and welfare of mankind is fine if the policy makers know the consequences of their proposed policies. In fact, instead of advancing the health and welfare of mankind, the drug laws that followed the United Nations Convention have led to untold violence and corruption in the producer countries, drug-related deaths, the accumulation of wealth worth billions of dollars by terrorists and violent criminals, the non-availability of essential pain-relieving medicines in many developing countries and the emergence of an extremely dangerous online market for synthetic psychoactive drugs. Of course, none of these consequences was predicted in 1961. It is not that the policy makers at that time were bad, they were simply ignorant of the consequences of their policies.
For political reasons, two of the most dangerous drugs widely used across the globealcohol and tobaccowere excluded from the Convention. Although rated as less dangerous than heroin and cocaine on a carefully devised scale of harm, both these drugs have been rated well above cannabis and ecstasy in their potential danger to the individual. [bib_ref] Development of a rational scale to assess the harm of drugs of..., Nutt [/bib_ref] A Royal College of Psychiatrists Working Party report 3 concluded that, 'In the long run, society will only be at ease with its drug control policies if they are based on a rational assessment of the risks associated with the different psychoactive substances and an objective appraisal of the consequences of previous policy changes, rather than on moral postures, the mistaken assumptions of the past and the accidents of history' (p. 259).
This suggests we need an entirely new approach to controlled drugs. The starting point must be a clear definition of the objectives of drug policy. The All Parliamentary Group for Drug Policy Reform 4 proposed the following objectives to the United Nations:
(a) to ensure the adequate availability of essential controlled medicines to those who need them (relevant to the many developing nations who have minimal or no access to morphine); (b) in production and supply countries, to prioritise education, community development, infrastructure development and employment in vulnerable communities; (c) in user countries, to minimise addiction and the harms associated with drug use.
In 1961 there was widespread consensus that a criminalising approach to the sale and use of heroin, cocaine and cannabis was appropriate, but this is no longer the case today. Now, even the Global Commission on Drugs Policy reports that the prohibitionist approach has failed.Arguments for and against drug prohibition in relation to heroin and cocaine may be more finely balanced, but there has been a major swing both among scientists and politicians toward the view that the illegal status of less harmful drugs, especially cannabis, does more harm than good.
Considerable concern has been raised concerning the decriminalisation of cannabis as a result of studies showing links between 'skunk' (high-potency cannabis) and the onset of psychosis. An influential study has shown that people who use skunk daily are five times more likely to develop psychosis than those who do not. [bib_ref] Proportion of patients in south London with first-episode psychosis attributable to use..., Forti [/bib_ref] However, the same study showed that, when the effects of low-potency cannabis were examined, hash users did 'not have any increase in risk of psychotic disorders compared with non-users, irrespective of their frequency of use'. Further, although it is now widely accepted that there is a causal relationship between regular high-potency cannabis users and psychosis, the possible importance of the effect of confounding factors makes the significance of even this finding for drugs policy unclear. It has been estimated, for example, that 98% of regular cannabis users will not develop a psychotic disorder. [bib_ref] Association between cannabis and psychosis: epidemiological evidence, Gage [/bib_ref] Further, decriminalisation would allow much more effective control, especially of high-potency cannabis, than is the case at the present time.
The uncertainty regarding the effects of decriminalisation can only be resolved by examining the effect of decriminalising legislation where it is occurring elsewhere in the world. There are now a number of studies examining the effects of drug law liberalisation, especially, but not only, in relation to cannabis. A recent review suggested that liberalisation of cannabis laws is associated with a slight increase in use of cannabis among the young. 8 A cross-national study of 38 countries confirmed this finding, noting that the increase was only detectable after 5 years and then mainly in girls. [bib_ref] Cannabis liberalization and adolescence cannabis use: a cross-national study in 38 countries, Shi [/bib_ref] Further, although adolescent use remains criminalised in US states where marijuana use has been legalised for adults, decriminalisation has led to decreases in possession and felony arrests among adolescents as well as reduction of associated juvenile-justice involvement. [bib_ref] Child and adolescent psychiatry, marijuana and psychosis. Policy implications for treatment providers, Bagot [/bib_ref] It has also been shown in a 20-country comparison that cannabis law liberalisation leads to increased help-seeking behaviour for people with drug problems, an encouraging finding suggesting that if some of the savings made as a result of the discontinuation of prohibition policies were put into increasing and improving drug services, any negative effects might be significantly reduced. [bib_ref] The impact of drug policy liberalisation on willingness to seek help for..., Benfer [/bib_ref] It has recently been suggested that positive experience from cannabis law liberalisation might lead to some countries looking more critically at their laws relating to other potentially more dangerous drugs. [bib_ref] Could cannabis liberalisation lead to wider changes in drug policies and outcomes?, Hughes [/bib_ref] There is already some evidence to suggest this might have beneficial effects. In 2001, Portugal changed its approach to the possession of all drugs. The drugs remained illegal, so the policy did not resolve the problem of illegal drug dealers enriching themselves by selling contaminated drugs. However, children and young people who go through a drug-taking phase do not end up with a criminal record and can much more easily give up the habit and progress with their education and employmentthe best protections from addiction.
This policy is not 'soft' on drug users. If a police officer finds a young person with drugs, they will be taken to the police station and required to hand over the drugs, they are then referred to a Commission for the Dissuasion of Drug Addiction or tribunal including a legal, health and a social work professional. The tribunal will determine whether the drug possessor is addicted to drugs. If so, they will be referred for treatment. The treatment becomes the basis of a contractual agreement between the drug user and the tribunal. If the drug user breaks the contract, they could receive an administrative penalty, although this rarely happens. Importantly this has no implications for their future employment. A casual user is sent on their way by the tribunal and strongly told not to continue using the drug. Portugal invested heavily in prevention, treatment, harm reduction and social integration services. The combination of decriminalisation with improved health and social care services probably account for the good results.
Importantly the policy has been extensively evaluated. [bib_ref] What can we learn from the Portuguese decriminalization of illicit drugs?, Stevens [/bib_ref] Portugal now has levels of drug use well below the national European average. The numbers sent to the criminal courts in Portugal fell from more than 14 000 to 5000-6000 a year after the policy was introduced. The proportion of offenders for drug-related offences fell from 44 to 21% between 1999 and 2012. The numbers of addicted children and young people has decreased. All the same, critical analysis of studies of those who claim that the Portuguese drug policy has been a resounding success or, in contrast, a disastrous failure suggest that the evidence does not support either extreme view. [bib_ref] A resounding success or a disastrous failure: re-examining the interpretation of evidence..., Stevens [/bib_ref] Switzerland has shown how to replace drug dealers with heroin treatment services. The services largely cater for poly drug users. The service has three parts: the drug consumption room (DCR), the heroin clinic and the methadone clinic. The service providers have an agreement with the police that anyone approaching the DCR will not be arrested for drug possession. The DCR is a vital part of the service. A doctor spends time there each week, treating ulcers and other health problems, and a social worker is available to help with housing, financial and other social issues. Addicted clients who come in off the street with their illegal drugs are welcomed and cared for. Over about 3 weeks these two professionals encourage the street drug users to come along to the clinic and have clean heroin in exchange for agreeing to a demanding contract. These chaotic individuals are required to hand over their benefits in the early stages, to make sure their rent and bills are paid. They are given back the money they need for food or other essentials, but not enough for them to buy drugs.
The constraints are worth it in return for the clean heroin as well as the psychological and social care. The Swiss heroin treatment programme has been rigorously evaluated. [bib_ref] Heroin-assisted treatment in Switzerland: a case study in policy change, Uchtenhagen [/bib_ref] The results are impressive. Until they arrived at the clinic these individuals were committing an average of 80 crimes a month to feed their addiction. After 18 months in treatment, one third are entirely drug-free and leading normal lives; a further third are leading their lives within the law, but still taking some heroin or methadone. The last third need more time to achieve their objectives. The savings to the tax payer and the benefits to the community from reduced crime levels are huge. The estimate is that for every franc spent on this service, two francs are saved for the taxpayer. The cost of the service per person is 15 000 euros. Not cheap but well worth it.
In the meantime, in England, the Durham Police are beginning to use the Swiss route for users of all narcotic drugs and even for low-level drug dealers and traffickers.Their Check Point programme recognises that many who are arrested for theft motivated by drugs and other less serious crimes have underlying mental health and social problems. The programme offers drug-related offenders and others a 4-month contract. This requires them to engage with treatment and not to reoffend. If they succeed on their contract then no further criminal justice action is taken. If successful in rehabilitating drug users and cutting reoffending, this will surely be an important policy across the country. The government will be funding 10 pilots of Checkpoint and 25 police forces are wanting to apply to be involved.
To conclude, an independent review of UK drug policies is urgently needed. Each drug needs to be individually considered. Regulation of heroin, for example, needs to be very different from the regulation of cannabis or ecstasy. The objectives must be to reduce addiction and limit as far as possible the harms associated with drug use. Drug policy reform would also dramatically reduce the ill-gotten gains from the drugs trade of terrorists and violent criminals.
In fact, we need to turn, not just policy about cannabis, but our whole drugs policy in its head. Opponents of the legalisation of cannabis, who suggest that this might well represent a slippery slope leading to the legalisation of other, currently proscribed drugs are right. But that is exactly what needs to happen.
About the authorBaroness Molly Meacher is formerly Chair of the East London and City Mental Health Trust, UK.
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10.1155/2016/1564257
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CCBY
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5027300
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27688825
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s2orc_pubmed_articles
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Rosmarinic Acid and Melissa officinalis Extracts Differently Affect Glioblastoma Cells
Lemon balm (Melissa officinalis L.) has many biological effects but especially important is its neuroprotective activity. The aim of the study is to produce different extracts of Melissa officinalis and analyse their chemical composition and biological properties on rat glioblastoma C6 cells. Results revealed that rosmarinic acid (RA) is the predominant compound of lemon balm extracts. RA has cytotoxic effect on glioblastoma cells (LC 50 290.5 M after the incubation of 24 h and LC 50 171.3 M after 48 h). RA at concentration 80-130 M suppresses the cell proliferation and has an antioxidant effect. 200 M and higher concentrations of RA have a prooxidant effect and initiate cell death through necrosis. The aqueous extract of lemon balm is also enriched in phenolic compounds: protocatechuic, caftaric, caffeic, ferulic, and cichoric acids and flavonoid luteolin-7-glucoside. This extract at concentrations 50 M-200 M RA has cytotoxic activity and initiates cell death through apoptosis. Extracts prepared with 70% ethanol contain the biggest amount of active compounds. These extracts have the highest cytotoxic activity on glioblastoma cells. They initiate generation of intracellular ROS and cell death through apoptosis and necrosis. Our data suggest that differently prepared lemon balm extracts differently affect glioblastoma cells and can be used as neuroprotective agents in several therapeutic strategies.
# Introduction
Glioblastoma multiforme (GBM), the most common and the most lethal CNS cancer, causes approximately 50% of all brain tumors. Chemotherapy and radiotherapy are used for treatment. Temozolomide is a first-line medicament for treatment of GBM; however this drug has many adverse effects and average lifespan of patients after treatment is about 1 year. Therefore, nowadays the fact that a "single" targeted therapy might not be the most effective approach and multitargeting would be the rational approach for killing a heterogeneous population of cancer cells in a tumor is often discussed. The relevance of natural agents of dietary origin in human cancer is appreciated, because it is a part of the normal diets in various cultures, these agents are nontoxic to humans and are able to modulate multiple signalling pathways [bib_ref] Polyphenols as modulator of oxidative stress in cancer disease: new therapeutic strategies, Mileo [/bib_ref]. It is important to determine if natural bioactive compounds (extracts of Melissa officinalis and rosmarinic acid), the neuroprotective effect of which is proven, could be potential candidates for additional therapy in brain cancer cases.
Lemon balm (Melissa officinalis) and its main active substance, rosmarinic acid (RA), have multiple neuroprotective effects. Scientific data show that rosmarinic acid could decrease level of intracellular reactive species and the level of DNA damage induced by ethanol in mice [bib_ref] Rosmarinic acid as a protective agent against genotoxicity of ethanol in mice, Oliveira [/bib_ref]. RA produces a significant neuroprotective potential in rats with ischemia and reperfusion: it reduces apoptosis and necrosis, increases cell survival, and decreases LDH leakage rate in cultured SH-SY5Y cells [bib_ref] Rosmarinic acid protects against experimental diabetes with cerebral ischemia: relation to inflammation..., Luan [/bib_ref]. Pre-and posttreatment with RA decrease ciguatoxin-mediated neurotoxicity diminishes the extracellular LDH activity and DNA damage in primary human neurons [bib_ref] Neuroprotective effects of rosmarinic acid on ciguatoxin in primary human neurons, Braidy [/bib_ref]. RA exhibits neuroprotective effects in the neurotoxicity of amyloid -(A -) induced cognitive dysfunction and has an antidepressant-like property in animal models of depression [bib_ref] A natural scavenger of peroxynitrites, rosmarinic acid, protects against impairment of memory..., Alkam [/bib_ref]. It is now widely studied RA anticancer activity. RA was applied to various human cancer cell lines like NCI-H82, DU-145, Hep-3B, K-562, MCF-7, PC-3, MDA-MB-231, and it was shown that RA may inhibit cell proliferation, induce apoptosis, and decrease viability of investigated cells in dose-dependent manner [bib_ref] Inhibitory effects of rosemary extracts, carnosic acid and rosmarinic acid on the..., Yesil-Celiktas [/bib_ref] [bib_ref] Salvia fruticosa, Salvia officinalis, and rosmarinic acid induce apoptosis and inhibit proliferation..., Xavier [/bib_ref] Since the number of brain cancer cases has increased in the past decades and the effects of RA on tumor cells are not clearly identified, the aim of this work is to identify the effects of this substance on the most aggressive type of brain tumors, glioblastoma cells.
A lot of research is being performed in order to prove the effect of extracts, made from Melissa officinalis, in treatment of different forms of brain diseases [bib_ref] Bioassay-guided fractionation of lemon balm (Melissa officinalis L.) using an in vitro..., Awad [/bib_ref] [bib_ref] Pilot trial of Melissa officinalis L. leaf extract in the treatment of..., Cases [/bib_ref] [bib_ref] Protective effect of Melissa officinalis aqueous extract against Mn-induced oxidative stress in..., Martins [/bib_ref] [bib_ref] Inhibitory activity of Melissa officinalis L. extract on Herpes simplex virus type..., Mazzanti [/bib_ref] [bib_ref] Inhibitory effects of Lemon balm (Melissa officinalis, L.) extract on the formation..., Miroliaei [/bib_ref] [bib_ref] Chemical composition analysis of the essential oil of Melissa officinalis L. from..., Taherpour [/bib_ref]. Investigations on rats have been performed, which established that the aqueous or methanolic extract of lemon balm affects the GABA transaminase in the brain as an inhibitor in anxiety, epilepsy, and so forth, [bib_ref] Bioassay-guided fractionation of lemon balm (Melissa officinalis L.) using an in vitro..., Awad [/bib_ref] [bib_ref] Pilot trial of Melissa officinalis L. leaf extract in the treatment of..., Cases [/bib_ref]. According to Akhondzadeh et al. the effect is achieved by stimulating the activity of acetylcholine receptors in the central nervous system. Scientific data has proven that Melissa officinalis extracts may have anticancer activity. Encalada with coauthors demonstrated cytotoxic effect of the 50% ethanolic and aqueous extract against human colon cancer cells [bib_ref] Antiproliferative effect of Melissa officinalis on human colon cancer cell line, Encalada [/bib_ref]. Weidner with coauthors evaluated the effect of ethanolic lemon balm extract on HT-29 and T84 human colon carcinoma cells. Experimental data showed that investigated extract inhibits the proliferation of colon carcinoma cells and induces apoptosis through formation of ROS [bib_ref] Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells..., Weidner [/bib_ref].
Most studies have shown that the biological effect of Melissa officinalis extracts, as well as other plants from the Lamiaceae family, mainly depends on RA concentration [bib_ref] Protective effect of Melissa officinalis aqueous extract against Mn-induced oxidative stress in..., Martins [/bib_ref] [bib_ref] Inhibitory activity of Melissa officinalis L. extract on Herpes simplex virus type..., Mazzanti [/bib_ref] [bib_ref] Inhibitory effects of Lemon balm (Melissa officinalis, L.) extract on the formation..., Miroliaei [/bib_ref]. Differently prepared extracts are enriched with other biologically active compounds, which, acting synergistically with RA, may increase biological effect of the extracts. Extracts are produced by using various extraction solvents. Water as solvent is always used in preparation of extract (tea) at home. However, most of biologically active compounds have low solubility in water. Ethanol is the most popular solvent in industry for producing liquid extracts intended for oral use. Additionally, ethanol is capable of dissolving most of biologically active compounds nonsoluble in water. In summary, the amount of active compounds in differently prepared extracts depends on solvent and production conditions.
The aim of this study was to produce different liquid extracts of lemon balm (Melissa officinalis L.) and analyse the chemical composition, investigate their antiproliferative, antioxidant, and cytotoxic effects on rat glioblastoma C6 cells, and compare them with effects of rosmarinic acid.
# Materials and methods
## Chemicals and reagents.
Raw lemon balm (Melissae folium) was obtained from JSC "Acorus calamus" (Vilnius, Lithuania), and dry lemon balm extract (sicc. Extractum Melissae officinalis) was obtained from Naturex (France). All the reagents and standards were of analytical grade. Luteolin-7-glucoside, caffeic acid, and rosmarinic acid were obtained from Extrasynthese (Genay, France), protocatechuic acid, caftaric acid, ferulic acid, and cichoric acid from Fluka (Buchs, Switzerland). Dulbecco's modified Eagle's medium (DMEM), Ampliflu6 Red, and 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH 2 -DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), HPLCgrade acetonitrile, and trifluoroacetic acid (TFA) obtained from Sigma-Aldrich GmbH (Buchs, Switzerland). Deionized water was acquired from a Milli-Q purification system (Bedford, USA).
## Preparation of lemon balm
Extracts. The lemon balm dry extract was dissolved in purified water at the ratio 1 : 100. Manufactured extract (N1) is filtered through paper filter.
Extracts of lemon balm (N2, N3) are produced using 40% and 70% ethanol solutions as extract solvents. Raw material and extract solvent ratio is 1 : 1. Crushed herbal of lemon balm is soaked in an appropriate amount of solvent and left for maceration for seven days [bib_ref] Chemical composition analysis of the essential oil of Melissa officinalis L. from..., Taherpour [/bib_ref]. After extraction, extracts of lemon balm were filtered through paper filter.
## Analysis of extracts by high-performance liquid chromatography.
Chromatographic analysis was carried out using Waters Alliance e2695 Separations Module equipped with a Waters 2998 PDA Detector (Milford, USA). The separation was performed on an ACE Excel 3 SuperC18 analytical column (Aberdeen, Scotland) (250 × 4.6 mm, 3 m) at 25 ∘ C. The mobile phase consisted of 0.1% TFA in deionized water (A) and acetonitrile (B). The gradient elution was as follows: 0-30 min, 15%-30% B; 30-50 min, 30%-60% B; 50-55 min, 60%-90% B; and 55-60 min, 90%-15% B. The flow rate was 0.5 mL min −1 , and the injection volume was 10 L. The detector was set in the 200-400 nm range. The chromatographic data were acquired and processed with Empower 3 software (Milford, USA).
## Cell line and cell
Culture. The C6 rat glioma model has been widely used in experimental neuro-oncology to evaluate the therapeutic efficacy of a variety of modalities, including chemotherapy [bib_ref] Rat brain tumor models in experimental neuro-oncology: the C6, 9L, T9, RG2,..., Barth [/bib_ref]. A unique feature of C6 culture is that, exactly like a GBM tumor, it contains a subculture of cancer stem cells that express CD133 and nestin, which are widely used markers for brain CSCs [bib_ref] Detection of cancer stem cells from the C6 glioma cell line, Zhou [/bib_ref] ; therefore we choose this cell line for our investigations.
Rat glioblastoma C6 cells were purchased from the Cell Lines Service GmbH (Germany). C6 cells of convenient concentration were seeded in culture flasks containing DMEM with 10% of fetal bovine serum, 100 U/mL penicillin and 100 g/mL streptomycin. The cultures were then incubated at 37 ∘ C, with 5% CO 2 and saturated humidity; culture transfer was performed once every 3-4 days. treated without (control) or with different concentrations of investigated solutions for 24 or 48 h. After treatment DMEM medium was removed from wells, and then cells were washed twice with 100 mL/well Phosphate Buffered Saline (PBS). After washing, 180 L/well PBS was added along with 20 L/well of 5 mg/mL MTT dye dissolved in PBS to each well. The cells were incubated with MTT for 2 h. Blue Formosan crystals formed in the intact cells were dissolved in DMSO (100 L/well). The absorption was measured at 570 nm and 620 nm as reference with a microplate spectrophotometer (Sunrise, Tecan Group Ltd., Switzerland). The results were expressed as percentages of MTT reduction, with the absorbance exhibited by the control cells being as 100%.
Concentration of ethanol used for control varied depending on the amount of extracts used for investigation, the maximal concentration was 2.5% of ethanol.
The LC 50 concentration was calculated by nonlinear regression analysis, fitting the data to equations, using the software package SigmaPlot 12.0 version (Systat Software Inc.).
## Assessment of cell count and cell death by hoechst
and Propidium Iodide Staining. Cell count was assessed using Hoechst 33258 and propidium iodide staining. At first, C6 cell suspension was dispersed to 24-well plates (2500 cells/well). After 24 h different concentrations of analysed preparations were added to the medium for 24 h, 48 h, and 72 h. After treatment, 15 min before investigation 5 g/mL of Hoechst 33258 and 2 g/mL of propidium iodide were directly added to the culture medium. After incubation viable, apoptotic, and necrotic cells were counted under fluorescence microscope.
## Measurement of intracellular rs generation.
The production of RS was assessed using the 2 ,7 -dichlorofluorescein diacetate (DCFH-DA). After incubation of C6 cells in 96well plates (20000 cells/well) for 24 h, they were incubated with DCFH-DA (10 M) in HBSS at 37 ∘ C for 30 min. During this time a part of DCFH is diffused into the cells. The dye, which was not diffused into the cells during incubation and remained in the outside medium, was washed twice with PBS. Wells were filled with a HBSS medium, enriched with investigated solutions of different concentrations. In the presence of cellular oxidizing agents, DCFH is oxidized to the highly fluorescent compound dichlorofluorescein (DCF); thus, the fluorescence intensity is proportional to the amount of RS produced in the cells. The fluorescence of DCF was detected by fluorometer at excitation and emission wavelengths of 488 and 525 nm, respectively.
# Statistical analysis.
Results are presented as means ± standard error. Statistical analysis was by one-way analysis of variance (ANOVA), followed by Dunnett's posttest using the software package SigmaPlot 12.0 version (Systat Software Inc.). A value of < 0.05 was taken as the level of significance.
# Results
## Chemical composition of differently prepared extracts.
The chemical composition of the lemon balm extracts as obtained by HPLC method is shown in [fig_ref] Table 1: Quantity of identified active compounds in differently prepared lemon balm extracts [/fig_ref]. Results revealed that RA is the predominant active ingredient of lemon balm extracts. Since RA is soluble in water, its concentration in aqueous extracts is relatively high, although the concentration of other identified substances is higher in ethanol solutions. Extracts prepared with 40% ethanol contain significantly smaller amount of all identified compounds in comparison with 70% ethanol. Results of our study demonstrated that 70% ethanol is the best solvent for extracting biologically active compounds from lemon balm. 40% and 70% ethanolic extracts have affected cell viability significantly stronger [fig_ref] Figure 3: Effects of different concentrations of ethanolic extracts [/fig_ref] if compared with N1 or RA alone. In both cases when the amount of investigated extracts was small (10 M of RA), cell viability has increased after 24 h of incubation , extracts with 40-100 M RA have statistically significantly decreased cell viability by 25-98% after 24 h of incubation. After 48 h of incubation the viability decreased by 50% with extracts containing 40 M RA and more. Different results were found by analysing the antiproliferative and cytotoxic activity of N 1-3 extracts.
## Effect of analysed preparations on cell
## Assessment of antiproliferative and cytotoxic
The aqueous extract N1, used in concentrations 50 M-100 M RA [fig_ref] Figure 4: Effect of different concentrations of [/fig_ref] ), has increased or has not changed the number of cells after 24 h incubation and decreased the number after 48 and 72 h. This extract caused death of C6 cells mainly by apoptosis (89±4%) and only a small number of cells died due to necrosis (12 ± 3%) [fig_ref] 2. 5: Assessment of Cell Viability 2 [/fig_ref].
The number of cells has increased in extracts N2 and N3 with 5-10 M RA concentration, whereas higher concentrations resulted in dead cells. Cell death by apoptosis (61 ± 7%) and necrosis (34 ± 5%) has been determined [fig_ref] 2. 5: Assessment of Cell Viability 2 [/fig_ref].
## Assessment of antioxidant/prooxidant activity of analysed preparations.
Since the extracts in low concentrations increased the proliferation of cells we have hypothesised that this effect can be initiated by an increased amount of intracellular reactive species (RS) and our further experiments were aimed to assess effect of the analysed preparations on the concentration of RS.
At first we analysed the effect of RA on the amount of RS. The results show [fig_ref] Figure 6: The effect of [/fig_ref] All concentrations of aqueous extract increased the amount of RS [fig_ref] Figure 6: The effect of [/fig_ref] ; a statistically significant difference was achieved at concentrations of 150 M and 200 M, which after incubation of 2 h increased the amount of RS by 16% and 27%.
All investigated concentrations of N2 and N3 extracts (
# Discussion
Studies have suggested that diets rich in RA and RA rich extracts together with other phenolic compounds may exert Oxidative Medicine and Cellular Longevity neuroprotective effects in neuroinflammation, and neurodegeneration, as well as chemical-induced neurotoxicity and oxidative stress. Earlier studies revealed that preparations from Melissa officinalis may act as a modulator of mood and cognitive function and has antidepressant effect [bib_ref] Rosmarinic acid-rich extracts of summer savory (Satureja hortensis L.) protect jurkat T..., Chkhikvishvili [/bib_ref] [bib_ref] The cellular protective effects of rosmarinic acid: from bench to bedside, Nabavi [/bib_ref] [bib_ref] Therapeutic and nutraceutical potential of rosmarinic acid-cytoprotective properties and pharmacokinetic profile, Nunes [/bib_ref] [bib_ref] Inhibition of amyloid fibrillation and cytotoxicity of lysozyme fibrillation products by polyphenols, Shariatizi [/bib_ref]. There is an increasing interest to identify plant-derived natural products with antitumor activities [bib_ref] Bioactive flavonoids, antioxidant behaviour, and cytoprotective effects of dried grapefruit peels (Citrus..., Castro-Vazquez [/bib_ref] ; therefore we seek to investigate the effect of RA alone and its rich extracts, made of lemon balm, on glioblastoma cells.
Our experiments have shown that RA alone, depending on its concentration, has a proliferation-inhibiting effect. Results showed that, after an incubation of 48 and 72 hours, RA at concentrations 80-130 M efficiently inhibited the cell proliferation. Makino with coworkers demonstrated that DNA synthesis, stimulated by PDGE and TNF-was significantly decreased by RA (IC 50 1.4 g/mL and 3.8 g/mL) and in process of cell proliferation RA might regulate DNA synthesis both in early and in late signal transduction [bib_ref] Inhibitory effects of rosmarinic acid on the proliferation of cultured murine mesangial..., Makino [/bib_ref].
However, there are experiments demonstrating that RA showed proliferative effects rather than cytotoxic activity in almost all cell lines tested with the highest effect in K-562 cells, exhibiting a cell viability of 205% at 139 M [bib_ref] Inhibitory effects of rosemary extracts, carnosic acid and rosmarinic acid on the..., Yesil-Celiktas [/bib_ref]. Our experiments revealed that RA reduces C6 cell viability; however, rather very high RA concentrations are needed to achieve this effect (LC 50 after 24 h and 48 h incubation 290.51 M and 171.3 M, resp.) and only necrotic cells were found [fig_ref] 2. 5: Assessment of Cell Viability 2 [/fig_ref] and 5(f)). However, Hur with his coworkers found that after 24 h treatment of RA (3-30 M) Jurkat cells displayed apoptosis, and at least 48 h of incubation with RA was required to induce apoptosis in almost 80-100% of the cells [bib_ref] Rosmarinic acid induces p56lck-dependent apoptosis in jurkat and peripheral T cells via..., Hur [/bib_ref]. Moon and coworkers reported that RA alone exhibited little effect on the cell viability in human leukemia cells. However, a combination of TNF-and RA induced apoptosis [bib_ref] Rosmarinic acid sensitizes cell death through suppression of TNF--induced NF-B activation and..., Moon [/bib_ref]. Even though there are studies revealing that RA induces apoptosis, our experiments, in which RA alone was studied, induced only necrosis in C6 glioblastoma cells. What is more, relatively high concentrations of RA are needed to reduce their viability.
There are many studies showing that RA has antioxidant properties attenuating oxidative stress and neuronal cell death [bib_ref] Rosmarinic acid protects human dopaminergic neuronal cells against hydrogen peroxide-induced apoptosis, Lee [/bib_ref] [bib_ref] Rosmarinic acid inhibits the formation of reactive oxygen and nitrogen species in..., Qiao [/bib_ref]. Ghaffari with coworkers show that H 2 O 2induced cytotoxicity in N2A cells was suppressed by treatment with RA. Moreover, RA is very effective in attenuating the disruption of lactate dehydrogenase, mitochondrial membrane potential, and intracellular ROS [bib_ref] Rosmarinic acid mediated neuroprotective effects against H 2 O 2 -induced neuronal..., Ghaffari [/bib_ref]. The results of our experiments show that the amount of RS in C6 cells depends on the concentration of RA and smaller studied concentrations (50-150 M) reduced the amount of intracellular radicals. Therefore, these results confirm that, depending on the concentration, RA has an antioxidant effect and reduced the amount of intracellular radicals; thus, it can be used as a neuroprotective agent against inflammatory, neurodegenerative diseases (Alzheimer's disease and Parkinson's disease) Extracts, prepared from lemon balm, not only contain rosmarinic acid, but also are enriched in other phenolic compounds. Biologically active substances identified in our extracts are protocatechuic, caftaric, caffeic, ferulic, and cichoric acids and flavonoid luteolin-7-glucoside. These our data confirm results of other investigators which found that extracts of Melissa officinalis are rich in phenolic compounds [bib_ref] Extraction of phenolic compounds from melissa using microwave and ultrasound, Ince [/bib_ref]. Naturally occurring flavonoids and phenolic acids are the hydrophilic and lipophilic nature. Investigations of chemical composition of our prepared extracts showed that the amount of identified compounds increases in the order N3 (solvent 70% ethanol) > N2 (solvent 40% ethanol) > N1 (aqueous extract).
The extract prepared in aqueous method contains amount of RA bigger than in extract N2, but amount of other biologically active substances is 2-10-fold lower than in ethanolic extracts N2 and N3. Chemical composition determined biological action: N1 reduced the viability of C6 cells significantly weaker than ethanol extracts, but ∼30% stronger than RA alone. Upon studying the cell proliferation, it was found that the amount of cells after 24 hours, compared to control, does not decrease, but after 48 and 72 hours it decreases significantly. It is important to note that this extract initiates the death of cells mainly through apoptosis. The studies of intracellular RS showed that this extract, depending on the concentration, increased the amount of RS; however this increase is significantly lower, compared to the increase of RS caused by ethanol extracts. Hence, it could be true that the death of cells is initiated not only by the increased amount of intracellular RS, but also by other mechanisms.
Ethanolic (40% and 70%) extracts initiated the death of cells 4-5 times stronger than that of RA alone or aqueous extract N1. Investigations of intracellular RS concentrations demonstrated that N2 and N3 extracts have a prooxidative effect; that is, the cells treated with these extracts contained significantly more RS, compared to control [fig_ref] Table 2: increased the amount of intracellular RS and after 2 h it was... [/fig_ref]. The beneficial health effects of medicinal plants rich in polyphenols are often attributed to their potent antioxidant activities. However, it is known that medicinal plants may also exert prooxidant effects [bib_ref] Medicinal plants and antioxidants: what do we learn from cell culture and..., Tang [/bib_ref]. Prooxidant effects are not necessarily bad because RS may act as signalling molecules of intracellular pathways. It is known that mild prooxidant effect promotes cell proliferation and higher amounts of RS induce cell death through apoptosis or necrosis, depending on additional conditions. Our investigations with ethanolic extracts revealed that lowest concentrations of extracts investigated in our experiments (10 M RA) increased concentration of intracellular RS and proliferation of C6 cells. In cases of bigger amount of investigated extracts (40-75 M RA) higher concentrations of RS were detected, and cells dying through apoptosis and necrosis were found. Weidner with coworkers have also found that ethanolic lemon balm extract inhibits the proliferation and induces apoptosis in HT-29 and T84 human colon carcinoma cell through formation of ROS [bib_ref] Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells..., Weidner [/bib_ref]. Our results as well as other authors results demonstrate that ethanolic extracts exert strong in vitro antitumor activity.
Taking everything into account, RA and other polyphenols identified in extracts are known as (1) radical scavengers and they could directly neutralize ROS; (2) they could also regulate intracellular antioxidant system capacity. Kim with coauthors demonstrated that RA reversed the downregulations of GSH, SOD, and Bcl-2 [bib_ref] Inhibitory effects of rosmarinic acid on adriamycininduced apoptosis in H9c2 cardiac muscle..., Kim [/bib_ref]. Therefore, it would be useful to assess and compare the effects of investigated preparations on activity of intracellular antioxidant systems. Higher concentrations of RA and extracts (investigated concentrations) increase intracellular RS levels. Although results of our and other investigators show that RS play a significant role in cell death, it remains unclear which RS (e.g., hydrogen peroxide, superoxide, or others) are responsible for the cytotoxicity of investigated preparations. Identification of RS could be useful to hypothesise about mechanisms of action.
It is proven that RS that are generated intracellularly can induce mitochondrial depolarization and release of cytochrome c into the cytosol and thus participate in the activation of the caspase-3 cascade. So, one of regulators of cell's life/death in apoptosis and necrosis are mitochondria [bib_ref] Role of the mitochondrion in programmed necrosis, Baines [/bib_ref]. Several literature sources revealed that RA induced apoptosis through mitochondrial pathway [bib_ref] Rosmarinic acid induces p56lck-dependent apoptosis in jurkat and peripheral T cells via..., Hur [/bib_ref]. Other biologically active compounds presented in extracts also may modulate mitochondrial functions, and total effect of extracts on mitochondria depends on the characteristics of complex of biologically active compounds [bib_ref] Caffeic acid induces apoptosis in human cervical cancer cells through the mitochondrial..., Chang [/bib_ref] [bib_ref] Effect of Perilla Frutescens extracts and rosmarinic acid on rat heart mitochondrial..., Raudone [/bib_ref]. Therefore evaluation of effect of investigated preparations on mitochondrial functions could be beneficial to reveal detailed mechanisms of action in the future. Extracts prepared with 70% ethanol contain the biggest amount of active compounds; their concentration is lower in extract, prepared with 40% ethanol. Despite different concentrations of biologically active compounds, they have a high cytotoxic activity on glioblastoma C6 cells. These extracts initiate the generation of intracellular RS and cell death through apoptosis and necrosis.
# Conclusions
[fig] 2. 5: Assessment of Cell Viability 2.5.1. MTT Assay. Cell viability was assessed by measuring the ability of cells to metabolize MTT. After incubation of C6 cells in 96-well plates (20000 cells/well) for 24 h, they were [/fig]
[fig] Figure 1, Figure 2: Effects of different concentrations of RA on the viability of C6 cells. C6 cells were treated with different concentrations (50-400 M) of RA for 24 and 48 hours. Cell viability was assessed using MTT method. Data are presented as means of percentage of the untreated control cells ± SE ( = 5). * < 0.05 versus control 24 h, # < 0.05 versus control 48 h. Effects of different concentrations of aqueous extract N1 on the viability of C6 cells. C6 cells were treated with different concentrations of aqueous extract (50-200 M of RA) for 24 and 48 hours. Cell viability was assessed using MTT method. Data are presented as means of percentage of the untreated control cells ± SE ( = 4-5). * < 0.05 versus control 24 h, # < 0.05 versus control 48 h. [/fig]
[fig] Figure 3: Effects of different concentrations of ethanolic extracts (a) N2 and (b) N3 on the viability of C6 cells. C6 cells were treated with different concentrations of ethanolic extracts (10-100 M of RA) for 24 and 48 hours. Cell viability was assessed using MTT method. Data are presented as means of percentage of the untreated control cells ± SE ( = 3). * < 0.05 versus control 24 h, # < 0.05 versus control 48 h. [/fig]
[fig] Figure 4: Effect of different concentrations of (a) RA and (b) N1 on C6 cell proliferation. Cell count was performed by adding Hoechst 33258 and propidium iodide dyes and counting the cells under fluorescence microscope. Data are presented as means of cell counts ± SE ( = 4-5). * < 0.05 versus control.and other biologic processes associated with enhanced ROS level. Higher examined RA concentrations (200-400 M) increased the amount of intracellular radicals in C6 cells. It is established that polyphenolic compounds often initiate cell death mechanisms by increasing the amount of ROS. Since, by studying the effect of RA on the viability of C6 cells, we have found that after the 24-hour incubation it statistically significantly reduces in the presence of 200 M concentration and higher; it could be stated that one of the mechanisms by which RA causes the death of glioblastoma C6 cells depends on ROS-sensitive pathways. [/fig]
[fig] Figure 5: Typical images of glioblastoma C6 cells after incubation with different concentrations of RA and extracts. (a) Control after 24 h incubation; (b) N1 200 M RA; (c) N3 50 M RA; (d) control after 48 h incubation; (e) 170 M RA; (f) 200 M RA. [/fig]
[fig] Figure 6: The effect of (a) RA and (b) N1 extract on concentration of intracellular RS. C6 cells were pretreated with 10 M DCFH-DA and then treated with different concentrations of RA and aqueous extract N1 for 0; 0.5; 1; 1.5; and 2 hours. Fluorescence intensity, which is proportional to intracellular RS concentration, was detected by using a fluorometer at excitation and emission wavelengths of 488 and 525 nm, respectively. Data is presented by fluorescence intensity ± SE ( = 3). [/fig]
[table] Table 1: Quantity of identified active compounds in differently prepared lemon balm extracts. [/table]
[table] Table 2: increased the amount of intracellular RS and after 2 h it was ×1.86-2.23 higher at RA concentration of 75 M. [/table]
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10.3390/foods9091184
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CCBY
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7554819
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32867028
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s2orc_pubmed_articles
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Changes in the Bacterial Diversity of Human Milk during Late Lactation Period (Weeks 21 to 48)
Breast milk from a single mother was collected during a 28-week lactation period. Bacterial diversity was studied by amplicon sequencing analysis of the V3-V4 variable region of the 16S rRNA gene. Firmicutes and Proteobacteria were the main phyla detected in the milk samples, followed by Actinobacteria and Bacteroidetes. The proportion of Firmicutes to Proteobacteria changed considerably depending on the sampling week. A total of 411 genera or higher taxons were detected in the set of samples. Genus Streptococcus was detected during the 28-week sampling period, at relative abundances between 2.0% and 68.8%, and it was the most abundant group in 14 of the samples. Carnobacterium and Lactobacillus had low relative abundances. At the genus level, bacterial diversity changed considerably at certain weeks within the studied period. The weeks or periods with lowest relative abundance of Streptococcus had more diverse bacterial compositions including genera belonging to Proteobacteria that were poorly represented in the rest of the samples.
# Introduction
Human milk is considered to be an important source of bacteria for the newborn. Many of these bacteria may be human commensals or have potential probiotic effects [bib_ref] Inhibition of Staphylococcus aureus by the commensal bacteria of human milk, Heikkila [/bib_ref]. Lactic acid bacteria, such as Lactobacillus fermentum, L. gasseri, L. rhamnosus, isolated from human breast milk, can be regarded as potential probiotic bacteria [bib_ref] Lactobacillus fermentum strains from human breast milk with probiotic properties and cholesterol-lowering..., Asan-Ozusaglam [/bib_ref] [bib_ref] Impact of mode of delivery on the milk microbiota composition of healthy..., Cabrera-Rubio [/bib_ref] [bib_ref] Impact of human milk bacteria and oligosaccharides on neonatal gut microbiota establishment..., Jost [/bib_ref]. Previous studies have suggested that commensal coagulase-negative staphylococci and viridans streptococci found in breast milk can reduce the acquisition of undesired pathogens by infants exposed to hospital environments [bib_ref] The human milk microbiota: Origin and potential roles in health and disease, Fernández [/bib_ref]. In addition, some of the bacterial strains found in human milk may have a large potential to improve the mother's health [bib_ref] Human milk microbiota: Origin and potential uses, Fernández [/bib_ref]. Furthermore, bacteria ingested during breastfeeding contribute to the development of the infant gut microbiome [bib_ref] Association between breast milk bacterial communities and establishment and development of the..., Pannaraj [/bib_ref]. The benefits of breastfeeding also extend to a reduction of respiratory and gastrointestinal tract infections and to a correct education of the immune system, with a concomitant reduction of the risks to develop several diseases such as obesity, diabetes, or inflammatory bowel diseases [bib_ref] Association between breast milk bacterial communities and establishment and development of the..., Pannaraj [/bib_ref] [bib_ref] Dynamics and stabilization of the human gut microbiome during the first year..., Bäckhed [/bib_ref] [bib_ref] Lactobacillus rhamnosus from human breast milk shows therapeutic function against foodborne infection..., Li [/bib_ref].
In addition to classical studies based on isolation and identification of bacteria from human milk [bib_ref] The human milk microbiota: Origin and potential roles in health and disease, Fernández [/bib_ref] , culture-independent studies have provided a large amount of information on the human milk microbiota. The culture-independent approaches based on amplification and sequencing of variable regions within the 16S rDNA gene, allow the detection in a single step of both aerobic and anaerobic bacteria as well as bacteria that, in spite of being in a low proportion in the population, may play significant roles. Recent review papers have summarized the major findings of previous studies on the microbiota from human milk samples based on culture-independent approaches [bib_ref] Systematic review of the human milk microbiota, Fitzstevens [/bib_ref] [bib_ref] Breast milk microbiota: A review of the factors that influence composition, Zimmermann [/bib_ref]. Many of the studies have focused on the influence of different factors such as the mother's diet and health status, maternal age, child delivery method, probiotic use, HIV infection, administration of antibiotics or collection/feeding method, and involve samples from several subjects. Such studies led to the proposal of a core microbiota for the human milk or at least a list of the bacterial genera most frequently found [bib_ref] Breast milk microbiota: A review of the factors that influence composition, Zimmermann [/bib_ref]. Nevertheless, the influence of the lactation stage has been studied to a much less extent. Cabrera-Rubio et al. [bib_ref] The human milk microbiome changes over lactation and is shaped by maternal..., Cabrera-Rubio [/bib_ref] analyzed the milk samples from 18 women at three sampling points, e.g., within 2 days after mothers gave birth in the maternity hospital (colostrum) and at 1 and 6 months after delivery at home. The main results obtained after 16S rRNA gene amplification and pyrosequencing indicated that the human milk microbiome changes over lactation: Weissella, Leuconostoc, Staphylococcus, Streptococcus, and Lactococcus were predominant in colostrum samples, whereas in 1-and 6-month milk samples, the typical inhabitants of the oral cavity (e.g., Veillonella, Leptotrichia, and Prevotella) increased significantly. Khodayar-Pardo et al. [bib_ref] Impact of lactation stage, gestational age and mode of delivery on breast..., Khodayar-Pardo [/bib_ref] applied quantitative polymerase chain reaction (PCR) to study the microbial composition of milk samples collected from 322 mothers within the first month of exclusive breastfeeding and reported that total bacteria, Bifidobacterium and Enterococcus spp. counts, increased throughout the lactation period. Most of these studies, however, do not report individual variations in the microbiota during the lactation period. Nevertheless, it is suspected that different changes may occur at the individual level due to different factors. The aim of the present study was to investigate the microbiota during the mid-to-late lactation period in breast milk from a single mother and to analyze possible changes in bacterial diversity during the period.
# Materials and methods
## Sample collection
Written informed consent was obtained, in accordance with the Declaration of Helsinki. Samples were taken from a single 38-year-old Latin American female donor, suffering from asthma and overweight, during a lactating period between weeks 21 and 48, inclusive, after cesarean delivery. Samples were taken three times in the day before baby lactation. The hands of the volunteer were cleaned with soap and covered with sterile gloves. Then, the nipples and surrounding areola were cleaned with cotton soaked with 70% ethanol. The first drops of milk were discarded, and then 5-7 mL of milk was extracted from each breast manually using a sterile manual pump (Philips Avent SCF330/20; Philips Ibérica, Madrid, Spain). The milk was transferred to sterile falcon test tubes, stored at 4 - C, and transported on ice to the laboratory within the next 12 h, where it was stored at −20 - C until analysis.
## Dna extraction
Thawed milk samples from the same day were mixed thoroughly and centrifuged at 16,000× g for 7 min in a refrigerated centrifuge 5424 R (Eppendorf, Corp., Hamburg, Germany). After removal of the supernatants, total DNA from the remaining pellets was extracted with a QIAamp Stool DNA Fast Mini Kit (Qiagen, Madrid, Spain) following the manufacturer instructions. The quality and quantity of the extracted DNA was determined by QuantiFluor ® ONE dsDNA system (Promega, Madison, WI, USA). The DNA was stored at −20 - C until analysis.
## Dna sequencing and analysis
The 16S rDNA V3-V4 regions were amplified following the Illumina Metagenomics Sequencing Library Preparation protocol (Illumina, Inc., San Diego, CA, USA). Illumina adapter overhang nucleotide sequences were added to the gene-specific sequences. The following 16S rDNA gene amplicon PCR primer sequences were used: forward primer: 5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG; reverse primer: 5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC [bib_ref] Evaluation of general 16S ribosomal RNA gene PCR primers for classical and..., Klindworth [/bib_ref]. Microbial genomic DNA (5 ng/µL in 10 mM Tris pH 8.5) was used to initiate the protocol. After 16S rDNA gene amplification, the multiplexing step was performed using Nextera XT Index Kit (Illumina). Then, 1 µL of the PCR product was run on a Bioanalyzer DNA 1000 chip to verify the size (expected size~550 bp). After size verification, the libraries were sequenced using a 2 × 300 pb paired-end run on a MiSeq Sequencer according to the manufacturer's instructions (Illumina). Quality assessment was performed by the use of prinseq-lite program [bib_ref] Quality control and preprocessing of metagenomic datasets, Schmieder [/bib_ref]. The sequence data were analyzed using qiime2 pipeline [bib_ref] Global patterns of 16S rRNA diversity at a depth of millions of..., Caporaso [/bib_ref]. Denoising, paired-ends joining, and chimera depletion were performed starting from paired ends data using DADA2 pipeline [bib_ref] DADA2: High-resolution sample inference from Illumina amplicon data, Callahan [/bib_ref]. Taxonomic affiliations were assigned using the Naive Bayesian classifier integrated in quiime2 plugins and the SILVA_release_132 database. Statistical analysis was carried out with SPSS software version 24 (IBM Corp., Foster City, CA, USA).
# Results
## Characteristics of sequence reads
The numbers of reads assigned to operational taxonomic units (OTUs) and the alpha diversity indicators are shown in [fig_ref] Table 1: N • of reads and alpha diversity indexes at genus level of... [/fig_ref]. The number of assigned reads ranged from 140 to 1,375,641. A few samples yielded very low numbers of reads (e.g., samples corresponding to weeks [bib_ref] Breast milk microbiota: A review of the factors that influence composition, Zimmermann [/bib_ref] [bib_ref] Quality control and preprocessing of metagenomic datasets, Schmieder [/bib_ref] [bib_ref] Mother-to-infant transmission of intestinal bifidobacterial strains has an impact on the early..., Makino [/bib_ref] , and 26 of the sampling period) and yielded a very low number of observations (between 3 and 9). In addition, samples 11 and 26 showed an abnormal bacterial composition. Therefore, these samples were excluded from the analysis.
## Bacterial diversity in breast milk samples
Firmicutes and Proteobacteria were the main phyla detected in the milk samples [fig_ref] Figure 1: Bacterial diversity of breast milk samples at Phylum [/fig_ref]. The proportion of Firmicutes to Proteobacteria changed considerably depending on the sampling week. Firmicutes were most abundant in samples from weeks 1, 3, 7, 14-17, 20-25, and 27-28 of the sampling period. However, Proteobacteria were predominant in the rest of the samples. Actinobacteria were the third most important group in most of the samples, followed by Bacteroidetes. Bacteroidetes had higher relative abundances than Actinobacteria in samples corresponding to weeks 6 and 10-13. The abovementioned phyla represented between 98.0% and 99.8% of the OTUs. detected in samples from weeks 5, 8-10, 12-13, 18, and 20. Box-plot representation of the relative abundances of the main genera across the samples [fig_ref] Figure 2: Box-plot representation of the relative abundances of the 38 main bacterial genera... [/fig_ref] indicated that Streptococcus was the main genus in the milk [fig_ref] Figure 2: Box-plot representation of the relative abundances of the 38 main bacterial genera... [/fig_ref]. Data on the relative abundance of Streptococcus were clustered in three groups of high relative abundances with two intercalated periods of low relative abundances [fig_ref] Figure 2: Box-plot representation of the relative abundances of the 38 main bacterial genera... [/fig_ref] , insert) and then analyzed by univariate statistical analysis (ANOVA, Tukey's test, Kruskal-Wallis, Dunn's post hoc). The results revealed that the three main groups of samples (G1A, G1B, G1C) had significantly higher relative abundances (p < 0.05) than the low-abundance samples. Apparently, groups G1B and G1C had higher relative abundances than group G1A (which would suggest an increase in the relative abundance of Streptococcus by the end of the sampling period). However, the differences between the three groups were not statistically significant (p > 0.05) for any of the univariate analyses carried out. with relative abundances between 1.0% and 1.2%. Enterobacteriaceae were highly represented in most of the samples, ranking sometimes in first position in relative abundance. Members of Pseudomonadaceae, Moraxellaceae, and Xanthomonadaceae were also relevant groups among the Proteobacteria. Microccaceae and Weeksellaceae were the main representatives among Actinobacteria and Bacteroidetes, respectively.
The 38 genera with relative abundances ≥ 2.5% (representing between 81.7 and 97.5 of OTUs) are shown in [fig_ref] Figure 1: Bacterial diversity of breast milk samples at Phylum [/fig_ref]. Genus Streptococcus was detected in all the samples (being the most abundant OTU in 14 samples), although there were large differences in its relative abundances between samples. For example, genus Streptococcus had relative abundances that were above 50% in samples from weeks 3, 14, 17, and 21-23. By contrast, the lowest relative abundances for this genus were detected in samples from weeks 5, 8-10, 12-13, 18, and 20. Box-plot representation of the relative abundances of the main genera across the samples [fig_ref] Figure 2: Box-plot representation of the relative abundances of the 38 main bacterial genera... [/fig_ref] indicated that Streptococcus was the main genus in the milk [fig_ref] Figure 2: Box-plot representation of the relative abundances of the 38 main bacterial genera... [/fig_ref]. Data on the relative abundance of Streptococcus were clustered in three groups of high relative abundances with two intercalated periods of low relative abundances [fig_ref] Figure 2: Box-plot representation of the relative abundances of the 38 main bacterial genera... [/fig_ref] , insert) and then analyzed by univariate statistical analysis (ANOVA, Tukey's test, Kruskal-Wallis, Dunn's post hoc). The results revealed that the three main groups of samples (G1A, G1B, G1C) had significantly higher relative abundances (p < 0.05) than the low-abundance samples. Apparently, groups G1B and G1C had higher relative abundances than group G1A (which would suggest an increase in the relative abundance of Streptococcus by the end of the sampling period). However, the differences between the three groups were not statistically significant (p > 0.05) for any of the univariate analyses carried out. Another genus represented in all breast milk samples was Staphylococcus [fig_ref] Figure 1: Bacterial diversity of breast milk samples at Phylum [/fig_ref] , with relative abundances from 0.3% to 14%. The lactic acid bacteria Carnobacterium and Lactobacillus had very low relative abundances. Aerobic endospore formers (Paenibacillus, Brevibacillus, and Bacillus) were detected Foods 2020, 9, 1184 6 of 10 in many of the samples, in some cases with high relative abundances. Gemella was also detected in many samples, and Listeria was detected in three samples (reaching 6.8% in one sample).
Most of the Enterobacteriales belonged to Fam. Enterobacteriaceae (Others). Members of the genera Pantoea, Serratia, and Enterobacter were also relevant in some samples [fig_ref] Figure 1: Bacterial diversity of breast milk samples at Phylum [/fig_ref]. Members of the genera Acinetobacter, Haemophilus, and Neisseria were detected in some samples. Pseudomonas had low relative abundances, except for three samples (weeks . Several samples showed high relative abundances of members of family Burkholderiaceae (Cupriavidus), with remarkably high values at week 5. All samples with a high relative abundance of Cupriavidus also had higher relative abundances of Vulcaniibacterium (Xanthomonadaceae). Among Actinobacteria, the main representative was Rothia (with relative abundances between 3% and 10% in many samples).
PCoA [fig_ref] Figure 3: Principal coordinates analysis of breast milk samples taken at different weeks [/fig_ref] revealed a main cluster of samples (all of them having a mid-to high relative abundance of OTUs belonging to genus Staphylococcus), with at least two minor clusters (samples W8, W12, W13) characterized by a very low relative abundance of Streptococcus and high relative abundances of Pseudomonas/Acinetobacter/Bacillus, and samples W5, W9, W10, W18 (also having very low relative abundances of Streptococcus and higher relative abundances of Cupriavidus/Vulcaniibacterium together with other bacterial groups).
28). Groups with low relative abundance: G2A (weeks 5, 8-10, 12-13), G2B (weeks 18, 20). a statistically significant differences (p < 0.05) with G2A.
Another genus represented in all breast milk samples was Staphylococcus [fig_ref] Figure 1: Bacterial diversity of breast milk samples at Phylum [/fig_ref] , with relative abundances from 0.3% to 14%. The lactic acid bacteria Carnobacterium and Lactobacillus had very low relative abundances. Aerobic endospore formers (Paenibacillus, Brevibacillus, and Bacillus) were detected in many of the samples, in some cases with high relative abundances. Gemella was also detected in many samples, and Listeria was detected in three samples (reaching 6.8% in one sample).
Most of the Enterobacteriales belonged to Fam. Enterobacteriaceae (Others). Members of the genera Pantoea, Serratia, and Enterobacter were also relevant in some samples [fig_ref] Figure 1: Bacterial diversity of breast milk samples at Phylum [/fig_ref]. Members of the genera Acinetobacter, Haemophilus, and Neisseria were detected in some samples. Pseudomonas had low relative abundances, except for three samples [fig_ref] Figure 1: Bacterial diversity of breast milk samples at Phylum [/fig_ref]. Several samples showed high relative abundances of members of family Burkholderiaceae (Cupriavidus), with remarkably high values at week 5. All samples with a high relative abundance of Cupriavidus also had higher relative abundances of Vulcaniibacterium (Xanthomonadaceae). Among Actinobacteria, the main representative was Rothia (with relative abundances between 3% and 10% in many samples).
PCoA [fig_ref] Figure 3: Principal coordinates analysis of breast milk samples taken at different weeks [/fig_ref] revealed a main cluster of samples (all of them having a mid-to high relative abundance of OTUs belonging to genus Staphylococcus), with at least two minor clusters (samples W8, W12, W13) characterized by a very low relative abundance of Streptococcus and high relative abundances of Pseudomonas/Acinetobacter/Bacillus, and samples W5, W9, W10, W18 (also having very low relative abundances of Streptococcus and higher relative abundances of Cupriavidus/Vulcaniibacterium together with other bacterial groups).
# Discussion
Results from the present study provided information on the changes in bacterial diversity in human milk from a single individual during the late lactating period. By focusing on a single donor, the present study avoided the confounding effect of data from different subjects and allowed to follow this mother continuously. Although the data may be more difficult to analyze, the study revealed a sample variability that otherwise may be unnoticed in studies involving a compendium of samples. Many previous studies have addressed the microbial composition of human milk, but usually from a compendium of samples taken from different individuals and taken at early to mid lactating period. Zimmermann and Curtis [bib_ref] Breast milk microbiota: A review of the factors that influence composition, Zimmermann [/bib_ref] identified 44 studies investigating 3105 breast milk samples from 2655 women, and reported that the most frequently found genera were Staphylococcus, Streptococcus, Lactobacillus, Pseudomonas, Bifidobacterium, Corynebacterium, Enterococcus, Acinetobacter, Rothia, Cutibacterium, Veillonella, and Bacteroides. Another review paper reported that Streptococcus and Staphylococcus appear to be widely predominant in human milk without regard to differences in geographic location or analytic methods [bib_ref] Systematic review of the human milk microbiota, Fitzstevens [/bib_ref].
Results from the present study also identified main genera reported in previous studies (Streptococcus, Staphylococcus, Pseudomonas, Acinetobacter, Rothia, Cutibacterium, and Veillonella). Furthermore, the microbial composition of several samples from the present study resembled, in a certain way, that of the children salivary microbiome reported by other authors [bib_ref] Exploring the salivary microbiome of children stratified by the oral hygiene index, Mashima [/bib_ref] (with the following common genera: Streptococcus, Veillonella, Rothia, Leptotrichia, Haemophilus, and Neisseria). These results would suggest colonization of mammary glands by bacteria from the baby's mouth. As a matter of fact, human milk is considered to be colonized by bacteria from the mother's gut and skin or the infant's mouth [bib_ref] Mother-to-infant transmission of intestinal bifidobacterial strains has an impact on the early..., Makino [/bib_ref] [bib_ref] The origin of human milk bacteria: Is there a bacterial entero-mammary pathway..., Rodríguez [/bib_ref].
Streptococcus was the predominant OTU in 14 of the samples. Remarkably, those samples with lower relative abundances of Streptococcus had different microbial compositions. While the sequence reads obtained in the present study only allowed identification at the genus level, a previous study reported the presence of the following species of Streptococcus in human milk: S. mitis, S. infantis, S. cristatus, S. salivarius, S. mutans, S. sanguinis, S. gordonii, and S. sanguinosus [bib_ref] Relationship between milk microbiota, bacterial load, macronutrients, and human cells during lactation, Boix-Amoros [/bib_ref]. Streptococci may produce different types of antimicrobial substances including hydrogen peroxide, organic acids, and bacteriocins [bib_ref] Streptococcal bacteriocins and the case for Streptococcus salivarius as model oral probiotics, Wescombe [/bib_ref] [bib_ref] Antimicrobial effects of commensal oral species are regulated by environmental factors, Herrero [/bib_ref]. The obtained results would suggest an ecological role of Streptococcus in the control of microbial populations in breast milk. Staphylococcus was also detected in all breast milk samples, with relative abundances reaching up to 14% in one sample. It has been suggested that commensal coagulase-negative staphylococci and viridans streptococci from breast milk could reduce the acquisition of undesired pathogens by infants, especially when exposed to hospital environments [bib_ref] The human milk microbiota: Origin and potential roles in health and disease, Fernández [/bib_ref] [bib_ref] Intranasal application of S. epidermidis prevents colonization by methicillinresistant Staphylococcus aureus in..., Park [/bib_ref].
Contrary to other studies, Lactobacillus had low relative abundance in the studied milk samples, and other related genera (Enterococcus, Weissella, Leuconostoc) represented less than 2%. This could be related to the late lactation period. Carnobacterium was represented in two samples (2.1-3.8%). Carnobacterium has seldom been reported in human milk, although this bacterium could be a new, largely unexplored candidate for novel probiotic bacteria from human milk. One study reported that the presence of Carnobacterium in milk was associated with cesarean delivery [bib_ref] The human milk microbiome changes over lactation and is shaped by maternal..., Cabrera-Rubio [/bib_ref] , which is in agreement with the delivery procedure in the present study. It is believed that cesarean delivery influences the milk microbiota because of the differential exposure of the newborn to skin and environmental bacteria instead of vaginal microbiota and because the skin and oral cavity of newborns act as sources of colonization of the bacteria in breast milk [bib_ref] The human milk microbiome changes over lactation and is shaped by maternal..., Cabrera-Rubio [/bib_ref].
Aerobic endosporeformers of the genera Bacillus and Paenibacillus also had high relative abundances in several milk samples and contributed (together with Streptococcus) to the increase of Firmicutes during late lactation. Remarkably, genus Bacillus reached ca. 25% relative abundance in two samples (weeks . The presence of Bacillus in breast milk samples has also been reported in several previous studies [bib_ref] Relationship between milk microbiota, bacterial load, macronutrients, and human cells during lactation, Boix-Amoros [/bib_ref] [bib_ref] Geographical location specific composition of cultured microbiota and Lactobacillus occurrence in human..., Ding [/bib_ref] [bib_ref] Bacteria in expressed breastmilk from mothers of premature infants and maternal hygienic..., Dahaban [/bib_ref]. Patel et al.reported both the presence of Bacillus and Paenibacillus in human milk, but Bacillus was associated with subacute or acute mastitis. However, during the sampling period of the present study, the mother did not report any signs of mastitis. Members of genus Bacillus are ubiquitous, sporulating, saprophytic microorganisms that can readily contaminate human milk during its collection or storage [bib_ref] Recent actuality about Bacillus cereus and human milk bank: A new sensitive..., Rigourd [/bib_ref]. Among them, B. cereus is a matter of concern in stored breast milk (specially in breast milk banks) since it can produce food poisoning toxins and may also cause severe illness in neonates [bib_ref] Recent actuality about Bacillus cereus and human milk bank: A new sensitive..., Rigourd [/bib_ref] [bib_ref] Banked human milk and quantitative risk assessment of Bacillus cereus infection in..., Lewin [/bib_ref]. On the other hand, selected strains of Bacillus species are commercialized as human probiotics [bib_ref] Bacillus strains as human probiotics: Characterization, safety, microbiome, and probiotic carrier, Lee [/bib_ref] and strains of Bacillus coaulans and Bacillus clausii are being investigated for pediatric and infant formula applications [bib_ref] Probiotics for the prevention of pediatric antibiotic-associated diarrhea, Guo [/bib_ref] [bib_ref] Probiotics for preterm infants: A strain-specific systematic review and network meta-analysis, Van Den Akker [/bib_ref].
Results from the present study indicate large variations in the microbiota of the breast milk from a single woman during the sampling period. Tentatively, some of the changes could be explained by environmental contamination of the milk. The baby was already in the weaning period and crawling. Therefore, transfer of microbiota from the baby to the mother's breast could account for some of the contaminants observed, including Enterobacteria. Previous work has suggested that the environment, utensils, and water could contribute considerably to the microbiota detected in breast milk [bib_ref] Human milk microbiota: Origin and potential uses, Fernández [/bib_ref]. A second way of contamination could be during milk expression by the breast pump. It has been reported that breast pumps may be difficult to decontaminate and increase the bacterial contamination of Foods 2020, [bib_ref] Lactobacillus rhamnosus from human breast milk shows therapeutic function against foodborne infection..., Li [/bib_ref] 8 of 10 the milk [bib_ref] Contamination of breast milk obtained by manual expression and breast pumps in..., Boo [/bib_ref]. And, finally, contamination could also occur during sample processing and analysis operations. Additionally, previous studies reported that contaminant DNA (sample collection and preparation, laboratory environment and reagents, personnel . . . ) and cross contamination (e.g., during sequencing runs) can influence the results of next-generation sequencing approaches [bib_ref] Contamination in low microbial biomass microbiome studies: Issues and recommendations, Eisenhofer [/bib_ref] [bib_ref] Reagent and laboratory contamination can critically impact sequence-based microbiome analyses, Salter [/bib_ref]. However, none of the variations reported in the present study were observed in other studies carried out in the same lab and using the same reagents and therefore it seems unlikely that they may be due to lab contamination. In addition, the negative controls included in the Illumina amplification and sequencing protocol clearly ruled out the possibility of contamination at this step.
# Conclusions
In conclusion, results from the present study reveal major changes in the microbiota of breast milk from a single mother during the late breastfeeding period. The main changes include a decrease in the relative abundance of Staphylococcus and an increase of other microbial groups belonging to phylum Proteobacteria. Factors such as environmental contamination and changes in the baby habits (weaning and crawling) could account for the observed differences during the sampling period.
[fig] Figure 1: Bacterial diversity of breast milk samples at Phylum (a), Family (b) and Genus (c) levels. The different sampling weeks (W) are represented. The 30 families that had relative abundances ≥ 2.5% are shown in Figure 1b. These covered between 88.1 and 98.2 of OTUs. Firmicutes were represented mainly by Fam. Streptococcaceae. This group was found at relative abundances in a range from 2.0% to 68.8%. The following families were represented in many of the samples: Staphylococcaceae, Bacillaceae, Paenibacillaceae, and Veillonellaceae. Members of O. Clostridiales Family XI were detected only in a few of the samples. Unidentified members of O. Lactobacillales and Carnobacteriaceae were also relevant in some samples, with relative abundances ranging from ca. 1.5% to ca. 4.5%. Lactobacillaceae only were represented in two samples (W2, W10), [/fig]
[fig] Figure 2: Box-plot representation of the relative abundances of the 38 main bacterial genera detected in the milk samples. Insert: Box-plot representation of the relative abundances of gen. Streptococcus during the sampling period. Data were grouped by weeks according to relative abundance. Groups with high relative abundance: G1A (weeks 1-4, 6-7), G1B (weeks 14, 16-17), G1C (weeks 21-25, 27-28). Groups with low relative abundance: G2A (weeks 5, 8-10, 12-13), G2B (weeks 18, 20). a statistically significant differences (p < 0.05) with G2A. [/fig]
[fig] Figure 3: Principal coordinates analysis of breast milk samples taken at different weeks (W) during the sampling period. [/fig]
[table] Table 1: N • of reads and alpha diversity indexes at genus level of breast milk samples at different weeks (W) of the sampling period. [/table]
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10.1371/journal.pone.0242951
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CCBY
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s2orc_pubmed_articles
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Correction: Alpha-crystallin mutations alter lens metabolites in mouse models of human cataracts
[fig] Reference 1 32833997 Fig 5 g001 Fig 6: Frankfater C, Bozeman SL, Hsu F-F, Andley UP (2020) Alpha-crystallin mutations alter lens metabolites in mouse models of human cataracts. PLoS ONE 15(8): e0238081. https://doi.org/10.1371/journal. pone.0238081 PMID: Major and minor sterols in Cryab-R120G mouse lenses are compared to WT lenses. Four or six lenses from mice of each genotype were individually analyzed, and the average percentage was determined. Data are presented as the means ± S.D. ( � P < 0.05).https://doi.org/10.1371/journal.pone.0242951.Amino acid content in Cryab-R120G mouse lenses are compared to WT mouse lenses. The lenses were first derivatized with TMS, and the amino acids were detected as single-or double-derivatized species. Four or six lenses from mice of each genotype were individually analyzed, and the average percent area of each amino acid was determined. Data are presented as the means ± S.D. ( � P < 0.05). [/fig]
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A five-safes approach to a secure and scalable genomics data repository
# Introduction
Data footprint for genomics projects has increased rapidly. The UK Biobank, for example, holds about 11 petabytes of genomic data, which are projected to grow above 40 petabytes by 2025. To effectively store and process data at petabyte scale requires significant information technology (IT) capacity, operated by experienced specialists. These capacities are often out of reach for small-to-medium size companies and academic labs. There is also the added issue of effectively sharing large-scale data with collaborators. To transfer 1 petabyte of data across a network, which allows actual sustained throughput (not line speed) of 1024Mbps, will take more than 90 days. If data are moved through the public internet, the transfer time is estimated to be at least 5 times longer.Public cloud computing platforms, such as Amazon Web Services (AWS), Google Cloud Platform (GCP), and Microsoft Azure, provide feasible ways around these constraints. Commercial clouds provide elastic and scalable IT resources (i.e., servers and storage) allowing users to grow their IT infrastructure in tandem with data generation without loss in reliability, availability, or performance. Operators can also adjust the scale and subsequent costs of cloud-based IT operations on demand, to match different project phases. In contrast, operators building on-premise data centers must build in sufficient capacity to take on the peak load of the project and not the most common load level. Therefore, an on-premise system operator will have to bear the cost of maintaining the entire system designed for peak usage, even during lull periods when most of the servers are idle. In addition, once data are available on the cloud, collaboration and sharing can be achieved by having users run analytics within the same ''cloud region'' where the data reside, effectively side-stepping the challenge of moving large data across networks. The National Human Genome Research Institute (NHGRI) Analysis Visualization and Informatics Lab-space (AnVIL) project, for example, leverages GCP to host and share more than 3 petabytes of genomic data. [bib_ref] Inverting the model of genomics data sharing with the NHGRI genomic data..., Schatz [/bib_ref] For analysis, AnVIL provides its users with the ability to work directly on cloud by integrating with various analytics platforms, including Galaxy, Juypter, and Dockstore. These platforms enable users to bring computation to the data repository, effectively ''inverting the model of data sharing''. [bib_ref] Inverting the model of genomics data sharing with the NHGRI genomic data..., Schatz [/bib_ref] However, unrestricted use of cloud computing can introduce significant security risks. Without well-designed access controls, data placed on cloud can potentially be accessed from anywhere and by anyone with internet access. Active data on cloud may spend considerable time moving through storage and servers shared with many other users, allowing data to be silently replicated many times over while in flight. Furthermore, cloud data sharing requires setting up an endpoint which is accessible from the internet. As evidenced by remote attacks through OpenSSH with GNU Bash, this may create opportunities for malicious actors who can either break in using forged credentials or vulnerabilities in the software used to host the data.There have been several initiatives to address these security concerns of genomic data. The Global Alliance for Genomics and Health (GA4GH) has developed protocols, tools, and policies for responsible sharing of genomics data. [bib_ref] The international data governance landscape, Bernier [/bib_ref] In particular, the GA4GH Data Security workstream has delivered a data security infrastructure policy, which outlines recommendations for securing IT infrastructure used for genomic and clinical data.The Authentication and Authorization Infrastructure guide additionally provides a comprehensive framework for cloud users to safely authenticate users and assign authorizations for data use.The Ministry of Health of Singapore has also issued a HealthTech Instruction Manual (HIM), providing instructions for IT and data governance.These include standard procedures and algorithms for data encryption, configuration of network partition and data access points, and management and securing user accounts.Major cloud service providers (CSPs) already supply many tools needed to build a secure platform following best practices and compliance to regulations.Groups such as the National COVID Cohort (N3C) by the National Center for Advancing Translational Sciences [bib_ref] The national COVID cohort collaborative (N3C): rationale, design, infrastructure, and deployment, Haendel [/bib_ref] have built virtual data enclaves that leverage scale and elasticity of CSP while enforcing strict access controls, such as blocking all data egress from the enclave. Such data enclave implementations may be too restrictive for facilitating other genomic research and collaborations. For example, researchers may prefer to use a customized analytics environment that comes with a wide range of tools and reference data from the internet. On an enclave such as N3C, users will have to work with analytics environment that comes with the platform. Other platforms such as AnVIL, while providing extensibility and ease of integration with other platforms and excellent flexibility for researchers, may not be aligned with specific national regulations on how genomic data should be protected. For example, human whole-genome data from public hospitals have been classified as ''Restricted Sensitive'' under the Singapore Government's data classification framework. As such, building a large data platform on CSP remains a complex task requiring deep IT expertise and knowledge of relevant regulations that may not be available in an academic genomics lab. [bib_ref] Considerations for genomic data privacy and security when working in the cloud, Carter [/bib_ref] The Research Assets Provisioning and Tracking Online Repository (RAPTOR) was developed to fill this niche. RAPTOR is a serverless, cloud-native, genomics data repository and analytics platform that strives to balance flexibility for data use with IT security and regulatory compliance [fig_ref] Figure 1: RAPTOR Overview and the modes of data analysis supported RAPTOR provides access... [/fig_ref]. Our key compliance measures include enforcing encryption at rest and in flight with key separation (using pre-approved algorithms only), stringent egress controls, and data that must be hosted within Singapore's jurisdiction (applicable to CSP). RAPTOR is hosted on AWS, Singapore region. Through the use of a ''5-safes'' framework, RAPTOR provides researchers the ability to build their own analytics environment to leverage elasticity and scalability of cloud computing for large-scale data analysis, without having to worry about IT security and regulatory compliance. With data-in-place analytics, RAPTOR also circumvents the challenge of transferring large datasets across public networks and allows strong accountability of data usage.
# Results
RAPTOR is designed to be foundationally secure and embedded with all elements essential for data governance. Security and data governance strategies and procedures are considered and designed into the platform using a ''5-Safes'' framework-safe purpose, safe people, safe settings, safe data, and safe output.
## Safe purpose
Safe purpose refers to measures adopted to ensure data contributors' control of data deposited with RAPTOR. Users who wish to access a dataset must submit an access request on the platform. Mandatory Singapore, Republic of SingaporeIt is mandatory to provide at least one data access committee (DAC) contact when depositing data onto RAPTOR, and RAPTOR will forward any access request to the relevant DAC. After evaluation, the DAC has the option to approve or deny the access request using RAPTOR's data management console. The DAC may also choose to grant access to specific subsets of files or allow access with modified parameters. For instance, a DAC may choose to modify certain data access expiry dates for specific sub-datasets on the same console.
RAPTOR hosts data on AWS S3. The hosting buckets are configured to block all access except those coming through a specific S3 endpoint. An S3 endpoint is analogous to a proxy for a webserver, providing an interface for the bucket to interact with the outside world without any direct connection. Access policies can be applied to the endpoint to govern traffic to the bucket. For example, in the case of allowing read access from a specific Elastic Computing Cloud (EC2) instance, when data users request to work on a dataset, a customized policy is generated on the fly based on permissions granted by the DAC.
Once access has been granted, users can analyze the dataset using RAPTOR's Analytics Workspace. This is an on-demand, dedicated virtual network where all interactions by a user with the chosen dataset can occur. The Analytics Workspace comes in three flavors: single-node AWS EC2 virtual machine instance, elastic spark cluster, and high-performance computing cluster. Through this design, RAPTOR automates the provisioning of selected data, creation and configuration of virtual machines, and the enforcement of security policies, on one computational resource.
Within the EC2 instance, users have full administrator rights by default and can install tools directly from the internet. To save operating costs, workspaces can be shut down when not in use, with the virtual server retaining its mount points, tools, and environment. Only the terminate command will destroy the workspace completely. Users have the option of exporting their virtual machine, the Amazon Machine Image (AMI), to be shared with collaborators. AMIs flagged for export will be reviewed by RAPTOR administrators to ensure the image is safe and does not contain malware data. Administrators also will work with relevant DAC to ensure the custom image does not violate terms of use. Second, RAPTOR's elastic spark cluster invokes AWS Elastic MapReduce Service to provision an Apache SPARK cluster (https://spark.apache.org/) with a Zeppelin notebook (https://zeppelin.apache.org/) serving as the front end. This workspace also comes with common genomic analysis tools, such as Hail (https://hail.is/) pre-installed to facilitate data analytics. Third, within the high-performance computing cluster, users can create a dedicated Linux computing iScience Article cluster complete with SLURM scheduler (https://www.schedmd.com/) and a share storage for processing data requiring heavy computational demands.
Workspaces do not create local copies of datasets. The operating system mount points read data directly from where selected datasets are housed on RAPTOR, thereby allowing users to sidestep the issue of moving large data files across the network. RAPTOR uses AWS FSX to provision a Lustre file system (https:// www.lustre.org/) for both runtime scratch and analysis outputs. Outputs and results are flushed into S3 for persistent storage when the workspace is shut down. AWS service endpoints are used to route function calls to native AWS services. This allows the workspace to function even if all external network access is disabled. Similar to data on S3, data on scratch and output staging are encrypted with advanced encryption standard, 256 bits (AES256) with key separation.
Importantly, the DAC can modify the conditions of data being shared even after RAPTOR has granted user access. For instance, DACs may revoke a user's permission to modify the original dataset while still allowing the user to access data for specific analysis. Additionally, the DAC may stop all access immediately with a kill switch, i.e., the mounted drive containing the selected dataset will immediately disconnect from the analytics workspace. Under safe usage, data contributors are thus able to have effective and direct control of datasets housed on RAPTOR.
Mechanisms are also in place to protect against ''insider-attacks'' from malicious users with valid RAPTOR credentials. Access to resources within RAPTOR, including data on S3, are restricted using policies for both network addresses and identity and access management (IAM) role of the calling application programming interface (API). Upon a user starting a workspace, RAPTOR creates a new IAM role unique to the user and the request. This newly created role will be attached to all API calls from its attached workspace. Policies guarding data access will allow role access only to data, which 1) were specifically requested during workspace creation and 2) has had access granted by the relevant DAC. Attempts by a user to access any other data (assuming the user has gained insider information on paths on S3) will be rejected by RAPTOR's service endpoints. This created role will be destroyed upon workspace termination. Additionally, all analytic workspaces are segregated by subnets, and traffic between workspace subnets is blocked. This prevents malicious users attempting to steal session credentials by running metadata queries on another concurrently running workspace. [fig_ref] Table 1: Numbers of total, common, and rare SNPs obtained after imputation of the... [/fig_ref] provides key security differentiators between RAPTOR, the National Center for Advancing Translational Sciences (NCATS) N3C data enclave, and NHGRI AnVIL. Notably, RAPTOR administrators are out of loop regarding data access requests. Rather, representatives from the appropriate DACs will approve or reject requests using the RAPTOR console, and RAPTOR adminstrators only provide technical support to DAC representatives.
## Safe user
RAPTOR ensures all users within the system are properly validated and does not allow anonymized access. As part of the user registration process, RAPTOR administrators verify the identity of the applicant. This typically involves running checks with the applicant's institute or collaborator.
User management and authentication controls in RAPTOR are handled by AWS Cognito due to its compliance with key security standards including ISO 27001, HIPPA BAA, and Multi-Tiered Cloud Security (MTCS). 18,19 RAPTOR does not store user credentials. All user records are encrypted both at rest and in transit. A 2-factor authentication is mandatory for all users for better protection, which also discourages users from sharing accounts. Additionally, Cognito allows integration with major identity providers, including active directory, and supports protocols including OAuth2, security assertion markup language (SAML), and OpenID Connect, 20 facilitating future integration with systems that conform to GA4GH authentication and authorization infrastructure.RAPTOR's safe user protocols can also extend to the platform's core administration and development team. For example, in the case of Singapore, developers and system administrators working on RAPTOR must receive security clearance from Singapore's Ministry of Home Affairs to work with restricted data. These measures ensure that data deposited in RAPTOR will only be accessed by approved individuals. iScience Article Safe settings RAPTOR employs a multi-layer strategy to protect hosted data against unauthorized access, starting with the host infrastructure. RAPTOR is a serverless, cloud-native application that leverages CSP with the Platform-as-a-Service (PaaS) model. RAPTOR's features are constructed from scripts and functions by hosting CSP's native services [fig_ref] Figure 2: As a serverless application, RAPTOR is composed of native AWS services integrated... [/fig_ref]. For example, RAPTOR's graphical user interface is built from a set of Java scripts hosted on AWS CloudFront, meaning that RAPTOR does not manage or operate any servers. Notably, AWS is among CSP adopted to host public services for the Singaporean governmentand was one of the first to attain MTCS level 3 (highest level) under SS584:2020, a security standard designed by Infocomm Media Development Authority (IMDA) Singapore,which is required for CSP to host data and services for the Singapore government. Thus, adopting AWS in RAPTOR for application and data security allows us to provide stringent operating procedures for infrastructure security.
Data encryption provides an added layer of data protection. RAPTOR encrypts all data with symmetric AES256. This extends to data stored on interim server stores within the analytics workspace, dataset metadata, and search indexes. To guard against accidental leakage and to provide additional protection against malicious actors, data encryption keys are stored in a separate system, the AWS key management system (KMS). RAPTOR leverages AWS Parameters Store to encrypt key IDs to prevent accidental leakage of key identifiers when invoking routines. All connections between the user's computer and RAPTOR are encrypted with SSH2, rivest cipher 4 (RC4), or trasport layer security (TLS) version no older than 1.2. iScience Article RAPTOR further provides enhanced data protection for data requiring egress restrictions with the Secure Analytics Workspace (Secure Sandbox). This is a locked-down version of a regular analytics workspace. It provides complete network isolation (no internet), blocking all data egress. A specially provisioned bastion node using Remote Desktop Protocol (RDP) with copy and print redirection disabled is the only way for users to access a Secure Analytics Workspace. Bastion nodes can further restrict its access to whitelisted IPs and subnets.
To validate the effectiveness of RAPTOR's security measures, RAPTOR undergoes penetration tests and vulnerability assessment by Council of Registered Ethical Security Testers (CREST)-certified assessors at least once every twelve months, to coincide with our platform revision cycle. During the annual assessment, RAPTOR will be assessed against well-known exploits (published Common Vulnerabilities and Exposure [CVEs]) and potential weakness in any of RAPTOR's system dependencies. Our current release cycle deploys a major revision every 10 months, and we engage a CREST assessor to ensure that there has been no inadvertent weakening of our security posture in the new release. In addition, between a major release, the team will run open worldwide application security project (OSWAP) vulnerability and dependency scanners every three months to ensure RAPTOR stays current with respect to security patches. Critical security issues from Computer Emergency Response Team (CERT) alerts are addressed as soon as possible (for example, zero-day exploits).
Beyond the direct security measures, RAPTOR has an extensive event logging system, which tracks all actions performed on datasets, including each time it comes up during a search, when a user submits an access request, and every instance the dataset is provisioned to a workspace. RAPTOR's logs provide network (transmission control protocol/internet protocol [TCP/IP])-level granularity and are encrypted to protect against tampering. Logs are kept for at least 12 months and can be made available for evaluations upon request.
Taken collectively, RAPTOR ensures that there are adequate mechanisms to protect hosted data, all system vulnerabilities will be promptly patched, and extensive logs are available to allow reconstruction of events to identify issues or support audits.
## Safe data and safe output
Safe data and safe output refers to RAPTOR's data protection mechanism for ingress and egress. RAPTOR's ingress procedure requires the data contributors to deposit their data into a pre-set staging S3 bucket. Data going into RAPTOR are both screened for malware and checked for authorization from the project's DAC before they become available for use on RAPTOR. Data hosted on RAPTOR cannot be modified without authorization from the data contributor. Data on RAPTOR are transparently spread across multiple AWS S3 storage tiers to optimize between cost and access efficiency. The use of AWS S3 also provides data with 11 9s in data durability and 2 9s in data availability.For data contributors who desire higher levels of assurance, options such as data immutability, file-level versioning, and crossregion backups are available.
To enable egress protection, RAPTOR allows data contributors to mandate the use of Secure Analytics Workspace for users working on their data. To copy files out of a Secure Analytics Workspace, users must submit an egress request on RAPTOR. A copy of the data for egress will be made in a bucket. The requestors will not have any access to this bucket, and requestors cannot change the contents of the files to be egressed after submitting a request. RAPTOR will notify the data contributor of the egress request.
The data contributor will then have full file-level access to the files RAPTOR had duplicated. The contributor can spin up a ''Review workspace'' to run content filtering or validation routines on the files submitted. The contributor can subsequently approve the egress request from their data management console, and the requestors can export these approved files. If the egress request consists of several datasets that have been combined, all contributors of the respective datasets are required to approve the egress request before the requestor will be allowed to perform data egress.
## Case study: secure imputation analysis on raptor
Utilizing population-specific reference panels during imputation can significantly improve the accuracy of detecting low-frequency variants (minor-allele frequency [MAF] <1%) for the relevant study population. [bib_ref] Rare variant genotype imputation with thousands of study-specific whole-genome sequences: implications for..., Pistis [/bib_ref] [bib_ref] Decoding asian genomic diversity-Singapore's national precision medicine strategy, Wong [/bib_ref] Additional imputation on the same SCHS dataset was performed using Trans-Omics in Precision Medicine (TOPMed) [bib_ref] Sequencing of 53,831 diverse genomes from the NHLBI TOPMed program, Taliun [/bib_ref] imputation reference panel (version r2) that includes data from 97,256 reference samples (https://imputation.biodatacatalyst.nhlbi.nih.gov). The quality of imputed SNPs from both analyses was determined by impute r 2 values; high-quality common SNPs (MAF R1%) were those with an impute r 2 > 0.3, and high-quality rare SNPs (MAF <1%) were those with an impute r 2 > 0.6. [fig_ref] Figure 1: RAPTOR Overview and the modes of data analysis supported RAPTOR provides access... [/fig_ref] provides a flowchart detailing sequence of action, from the user's perspective, including logging into RAPTOR, selecting appropriate datasets, performing analyses, and egressing data. The SG10K data deposited in RAPTOR were flagged as sensitive, and thus the SCHS genotyped data were linked to the SG10K reference panels in the Secure Sandbox. Access to this instance was restricted to a Windows bastion node, and users could only connect to the bastion node with the Windows RDP that has print and clipboard function disabled. Users working on Windows or MacOS will connect to the bastion node with Microsoft's freely available RDP client, while those working on Linux are recommended to use Remmina, an open-source client for Microsoft RDP. Within the bastion node, users will find Secure Shell (ssh) credentials to access the imputation server, with both the SCHS study and the SG10K panels data available as mount points. After the end of imputation, we submitted an egress request for the folder containing imputed dosage data.
## Ancestry-matched reference panels improve imputation of rare variants
We compared the imputation performance in the SCHS after imputing for additional variants using the Trans-Omics in Precision Medicine (TOPMed) [bib_ref] Sequencing of 53,831 diverse genomes from the NHLBI TOPMed program, Taliun [/bib_ref] and local SG10K reference panels using the RAPTOR platform. Expectedly, high-quality common variants (MAF R1%) obtained after imputation on the TOPMed and SG10K reference panels were similar (7,236,027 and 7,263,376 biallelic SNPs obtained after TOPMed and SG10K imputations, respectively, [fig_ref] Table 1: Numbers of total, common, and rare SNPs obtained after imputation of the... [/fig_ref]. However, a substantially higher number of high-quality rare variants (MAF <1%) were obtained in the SCHS study through imputation with local SG10K reference panels as compared to the TOPMed imputed data (1,271,426 additional rare SNPs from SG10K imputation procedures, [fig_ref] Table 1: Numbers of total, common, and rare SNPs obtained after imputation of the... [/fig_ref].
Five-safes framework enabled efficient usage of consortia level data in RAPTOR BCAC data consisted of South Asian and South-East Asian ancestry subjects genotyped on the Infinium OncoArray (N = 27, 501) and the illumina collaborative oncological gene-environment study (iCOGS) array (N = 12,500). Data were highlighted as sensitive and similarly required mandatory use of the Secure Sandbox, as well as requiring limited usage for only imputation protocols. The BCAC study folder was linked to the SG10K reference panels in the Secure Sandbox, and access to this instance was similarly restricted to a Windows bastion node paired exclusively to the instance running imputation service. Of note, even on a single AWS EC2 instance (r5.8xLarge), this entire work for imputation of the relatively large BCAC dataset (N = 40,001) in a secure setting took only around 20 days (end-to-end) and incurred modest costs of about USD 3,000 worth of AWS utilization, highlighting that the RAPTOR platform enables effective and secure usage of large-scale consortia-level data. iScience Article Remote data retrieval using GA4GH standards Data integration with other data platforms, such as combining RAPTOR's genomic data with another repository holding phenotype information, is a key feature of RAPTOR. GA4GH workstreams have defined several standards and APIs to facilitate data federation across different genomic repositories. The first task in any data exchange is to discover and list datasets available in the remote host. This is most effective when performed before initializing user authentication and authorization. Hence, common standards are crucial in enabling two different data sources to exchange ''data catalogs'' securely without authentication. The GA4GH Discovery workstream's Beacon V2 standard [bib_ref] Beacon v2 Reference Implementation: a toolkit to enable federated sharing of genomic..., Rueda [/bib_ref] provides an efficient mechanism for such activity. We do not anticipate major roadblocks adding this functionality to RAPTOR as the Discovery workstream has provided a working reference for implementation of the Beacon v2 standard. Furthermore, as RAPTOR is serverless, every conceptual ''layer'' of the application is exposed via APIs and a thin integration layer between the Beacon v2 reference implementation and RAPTOR's hosting services can be readily incorporated. The integration layer will serve to translate functions and calls from the Beacon v2 implementation into native service calls for RAPTOR, allowing RAPTOR to reuse the Beacon v2 reference implementation with minimal modifications. The GA4GH Data Repository Service (DRS)is a set of APIs providing consumers (both users and workflows) with direct access to data in a repository. A key feature of DRS is the provision of a Universal Resource Identifier (URI) to provide an exclusive identification for a single file or group of files within a repository. This will allow data consumers to access resources without prior knowledge of repository's data organization or file hierarchy. RAPTOR also enables data contributors to define groups of files or directories termed a Collection. RAPTOR Collections can be shared and referenced by data consumers independently of Collection's parent dataset. Therefore, it may be possible to extend RAPTOR Collections to a DRS-compliant resource. DAC approval can be integrated into authentication and authorization workflows before retrieving data using DRS. Destination endpoint IP address for remote retrieval can also be whitelisted.
## Federated analysis with data in place
Even with DRS approval and IP whitelisting, remote data retrieval may inevitably weaken RAPTOR's safe purpose assurance. In addition, when working with datasets on the scale of multiple terabytes or more, cost and latency for replicating large data volume across networks can quickly become prohibitive. A more feasible approach would be to send the compute job to the data and return outputs of the job. GA4GH has defined three standards for sending compute jobs to be executed on remote sites, including the Tools Registry Service (TRS), Workflow Execution Service (WES), and Task Execution Service (TES).TRS provides standardization for tools discovery, providing standardized descriptions of docker-based tools and popular workflow engines. 37 Dockstore (https://dockstore.org/) is an example of a TRS-compliant container repository. WES then builds on top of TRS to provide a common interface to interact with TRS tools and workflows. TES is similar to WES in that it also defines an interface defining and running compute tasks.TES is differentiated from WES in that a TES task can be modeled as a single job execution such as executing a single script or a command, while a WES is designed for executing a pre-composed workflow. It is possible for a TES to be ''nested'' within a WES. For example, a Nextflow workflow can serve as a TES client submitting tasks to a TES server.These three services, however, are insufficient for RAPTOR to provide federation services to third-party repositories. As a serverless application, RAPTOR does not have any ready virtual servers to execute remote workflows or tasks requests. The Analytics Workspace is also not suitable as it is designed for direct interactions with end-users and would be unwieldy to automate. In addition, these standards do not provide an easy way to inform RAPTOR of the type of virtual machines required for execution. As an example, while TES allows for resource specification within a task request, the resource request is defined in the form of cores, random access memory (ram), disk size, and name or URI of a docker container. These parameters alone are not sufficient to identify an instance type. Specifically, on AWS, just specifying ''2 cores and 8 GB ram'' maps to at least 7 different instance types, each with a unique hardware profile, optimized for different use-case. There is also no means of specifying a type of platform (x86 or ARM, Memory-optimized, or graphical process unitenabled), and loading containers or images from third-party repository is not compatible with RAPTOR's operations. To preserve RAPTOR's safe purpose assurance, RAPTOR administrators would have to manually clear an AMI or container before allowing it for use, which would require the container or image to be hosted within a restricted repository trusted by RAPTOR administrators. The remote user would thus have to perform an ''out-of-band'' communication with the data contributor on RAPTOR to learn the identifier of the AMI or container to be referenced with TES. This may complicate workflow scripting or automation. iScience Article To overcome these limitations, RAPTOR's development team evaluated an early concept where we could predefine and associate an AMI with a dataset. AMIs allow users to define key features for a virtual server, such as platform-configured tools (x86 or ARM, ROCm or CUDA) and even reference files. At the same time, AMIs provide some flexibility for users to determine the appropriate sizing (core counts, amount of ram, volume of attached disks) during runtime. The exchange only involves the universal identifier of the AMI, not the actual image file. When a remote compute request has been received, RAPTOR will initialize a virtual machine using AWS Batch, with core count and ram size matching the values from TES. Batch will initialize the virtual machine, execute the command, write back the output to requestor, and terminate the machine.
Case study: Prototype of federated analysis on RAPTOR As a case study, the RAPTOR development team recognized the challenge of performing these activities within the defined set of GA4GH APIs. We thus implemented an early-stage proof of concept where a RAPTOR instance received a remote computation request together with an AMI and docker identifier, with task execution and termination of the virtual server after the results were returned.
In the prototype, we evaluated a federated imputation workflow in RAPTOR by performing imputation on a Primary Open Angle Glaucoma (POAG) dataset using Minimac4 (version 1.0.0) with the same SG10K health reference panels. However, in this scenario, two RAPTOR instances were created, one hosting POAG and the other SG10K health panels. Each customized instance had added functions, providing a minimal implementation of TES and task invocation via the API Gateway and Batch. The team limited the proof of concept's scope to TES since the initial DRS exchange only involved inserting additional data content under ''alias'' section of response to a DRS ''Get'' call. Hence, we configured the RAPTOR instances assuming that information can be packed within the single DRS get call. Specifically, these include the AMI id, mini-mac4 imputation commands, mount paths and customized AWS IAM role (role created for this workflow only, will be destroyed after the end of workflow), and egress IP address. The IAM role and IP were used to configure POAG RAPTOR to allow network filesystem (NFS) read-write mounts from the SG10K RAPTOR. This allowed SG10K RAPTOR to read and write data to POAG RAPTOR.
The complete imputation workflow could then be broken down into four main TES tasks: 1) creating unphased variant call format (VCF) files, 2) conversion to phased VCFs, 3) addition of prefix ''chr'' to entries in phased VCFs, and 4) imputation. During the evaluation, the POAG RAPTOR submitted the four TES tasks creation calls in sequence. Each task was invoked after the previous one had been completed successfully (validated via TES ''Get task'' call). With every task received, the SG10K RAPTOR will submit a job to AWS Batch service using its account with a predefined Virtual Private Network (VPC) using parameters received from TES calls. To allow reading both datasets during analysis and writing of outputs to either (or both) of RAPTOR instances, a virtual machine was set up to run filesystem in userspace (FUSE) mounts to both POAG RAPTOR and SG10K RAPTOR. AWS Batch service will run the TES tasks and write outputs back into POAG RAPTOR. After the end of an AWS Batch job, AWS Batch Service will automatically stop and delete all resources associated with the task (except files written to output directories). Users on POAG RAPTOR can monitor the task status by issuing TES ''Get task'' calls to SG10K RAPTOR. Once a task was completed, the user will submit the subsequent TES task until all 4 tasks are completed .
Using the POC system, we completed the imputation of POAG chr21 with SG10K Health. Results from the POC system were consistent with results from work done on the production RAPTOR platform (wholegenome imputation of POAG with SG10K Health panel). With this approach, the ''host'' site controls the tools, security policies, and the virtual machines, while the remote user directs where the outputs are written. The use of Batch ensures all interim data in scratch will be deleted. Hence, we believe this protype approach can provide both the remote user and the host strong protections against data leaks.
# Discussion
## Value of raptor-like platforms for genomics research
A cornerstone to precision and personalized medicine is the ability to effectively access and thoroughly evaluate a multitude of genomic datasets to improve our molecular understanding of health and disease processes. Managing large-scale data analysis across multiple datasets, however, remains challenging. Hyper-scale computational capabilities provided by CSP such as AWS provide a full suite of computational resources to enable concurrent in-data-analysis data on an AWS S3 Bucket. Nevertheless, harnessing these resources requires knowledge on programming and cloud computing expertise. Additionally, the sensitive ll OPEN ACCESS iScience 26, 106546, April 21, 2023 9
iScience Article nature of genomics data and increased emphasis on data governance and security limit capabilities of individual labs to setup a cloud platform for effective large-scale genomic collaboration studies. Data contributors from public sectors also routinely request for specific controls to be put in place, such as AES256 symmetric for data encryption at rest and the version of TLS/SSH protocols for data encryption during transit. Data hosting facilities may also require certifications such as International Organization for Standardization (ISO) 27001 or multi-tier cloud security (MCTS) Level 3.
RAPTOR provides users with a platform, which addresses these computation and security concerns. Through RAPTOR, users seamlessly run their analysis as they would have on an on-premise system or their private cloud machine while still staying compliant to overarching governance and security regulations. Our case example on performing imputation on large-scale and consortia-level datasets demonstrates these capabilities in RAPTOR and highlights the potential of integrating various genetic resources to improve genetic studies, while, at the same time, remaining compliant to required restrictions of sensitive genetic data.
Beyond security, datasets hosted on RAPTOR become FAIR (Findable, Accessible, Interoperable, and Repeatable). [bib_ref] The FAIR Guiding Principles for scientific data management and stewardship, Wilkinson [/bib_ref] RAPTOR allows contributors to add metadata as tags to their datasets, allowing for quick way to filter out relevant datasets using RAPTOR's simple search form (example: Show all datasets where ''Country = Singapore and ethnicity = Malay"). RAPTOR users can submit request to access any dataset hosted on RAPTOR, with levels of access determined by the respective DACs. As RAPTOR manages all data provisioning tasks, users can start working on the requested datasets immediately upon receiving approval. RAPTOR's AMI-sharing feature further allows users to save and share their intermediate data and work environment (i.e., Juypter-style Notebook or even the whole Linux machine) with other RAPTOR users, enabling interoperability and repeatability.
RAPTOR went live in 2021 and has uniquely built a collection of Asian genetic SNP array-based genetic datasets that are primarily from the local Singapore ethnic population groups (Singapore Chinese, Malay, and Indian datasets). As of October 2022, RAPTOR is hosting close to 100,000 samples with more than half from Singapore [fig_ref] Table 2: Datasets hosted on RAPTOR [/fig_ref].
## Challenges with scaling up of hosted data
The footprint for next generation sequencing (NGS) data is easily an order of magnitude higher than array data. While we do not foresee major issues to scale up data footprints for RAPTOR's data management (DynamoDB and Lambda) and bulk storage (S3) services, larger data may pose challenges in staging latency and operating cost.
Several RAPTOR operations require data to be staged across buckets or data stores. In particular, data within a workspace have to be staged from S3 into FSX Luster. FSX only creates file metadata when provisioned, relying on lazy loading as a mechanism for moving data into FSX Luster. Load time is usually limited . Using existing GA4GH DRS and TES for federated computation on RAPTOR (i) A client invokes Get to describe an existing data collection 'C' on RAPTOR. In addition to content and access descriptions, RAPTOR also sends identity of the AMI associated with C under 'alias'. The provided information includes AMI id, pre-installed tools, mount points for accessing data, and the customized IAM role and endpoint IP; these are used by RAPTOR to read and write data from the remote client. (ii) The remote client invokes a task using TES. In the call, inputs are used for the client to inform RAPTOR which URI is to be mapped to which mount point. Under executors, the remote client will inform RAPTOR which AMI is to be used. (iii) Batch instantiates an EC2 machine AMI and parameters provided by TES task. The machine will mount paths from POAG RAPTOR and SG10K RAPTOR within the same machine using the customized IAM role. iScience Article by S3 read throughput (sustained throughput usually slightly over 100 MBs -1 ), and load time scales linearly with file size. Hence a user working on a sizable collection of whole-genome sequencing data may experience considerably longer delay when performing initial file reads. With larger data footprints, users also likely require a larger scratch (FSX Luster) which has to stay ''alive'' for a longer duration. Processing large NGS files (such as VCF manipulation, variant calling) would often require machines with high runtime memory (RAM). Such machines would cost more leading to higher runtime costs.
As RAPTOR scales in both number of datasets and its footprint, cost recovery becomes an increasingly important aspect for RAPTOR operations. RAPTOR recovers most of its cost from data users. Data owners pay the minimum amount to partially cover platform overheads, for example, constant security scans running in the background. Conversely, data users will pay for the full cost of resources used, plus a small markup to ensure platform sustainability. This model encourages data owners to host their datasets on RAPTOR as the majority of costs are derived from data usage and not through storage of data.
RAPTOR provides a few tools to help data users manage their costs. A cost dashboard on the user's landing zone displays user's cost incurred. Additionally, users have the choice to suspend their workspace when there is a pause in their work. Unlike ''terminate'', suspend does not delete the working environment, but the user will not incur cost on virtual machines in suspend mode. In addition, the team will be introducing features to allow users to set email alert once their incurred costs go over defined threshold.
Beyond cost and data latency, onboarding of more datasets may also add to the workload of the operations team. RAPTOR employs a ''man-in-the-middle'' approach for data security. Activities such as data ingress and AMI import require review and approval by RAPTOR's administrators. This is one of the contributing factors to RAPTOR adopting a shared responsibility model for data management. The RAPTOR administrators focus on enforcing platform and IT security, while the DAC controls safe use of data. This approach allows RAPTOR administrators to focus on a very defined aspect of data security and compliance and not become overburdened with workload as we scale up the datasets hosted within RAPTOR.
## Risk management
RAPTOR's internal risk assessment has highlighted two main risks: 1) security breach from malware introduced during data ingress or AMI import and 2) security vulnerabilities or failures within AWS's native infrastructure. RAPTOR takes two approaches to address the first risk. Firstly, RAPTOR administrators work closely with DAC representatives to ensure they understand how RAPTOR procedures provide data safety. iScience Article RAPTOR administrators will ensure DAC representatives are trained and are comfortable with using RAPTOR's controls. Secondly, RAPTOR does not allow direct data ingress or egress. Data must be isolated on a different storage bucket while it is being cleared, thus creating a barrier to prevent malicious actors leveraging egress or ingress reviews to launch attacks. This isolated review mechanism also applies to AMI imports.
To mitigate the second risk, we also adopted a zero-trust system design for RAPTOR. Segregration is applied judiciously with system zero trust between all. RAPTOR is hosted on a dedicated AWS account, with all development and testing occurring in a separate account. All AWS roles for operating RAPTOR are segregated. For instance, a dedicated role is used to deploy RAPTOR services and another for regular operations. All accounts are protected with multi-factor authentication with 3 months password rotation. There are no ''super user'' accounts that can manage all resources in this AWS account (root account disabled). RAPTOR services are segregated into different virtual networks. All services are guarded via policy, network, and keys. There is no assumed trust between any resource within RAPTOR. All network activity within this account is monitored and logged. Our rationale for the high level of segregation is the underlying assumption that if there is an unknown vulnerability in a key native service, processes must be implemented to limit damage and ''blast radius''. In addition, ''break-glass'' protocols are in place to where administrators can activate kill switches to immediately lock down all assets within RAPTOR.
## Future objectivies for raptor
In the long term, platforms such as RAPTOR are likely to represent options as trusted custodians for national-scale genomic data. RAPTOR's next phase will also focus on expanding the types of data hosted on the platform. Various transcriptomic datasets from local population datasets are expected to be housed in RAPTOR, facilitating larger-scale transcriptomic studies and combinatorial expression quantitative trail loci studies. To do so, RAPTOR will continuously update security measures and operating procedures to ensure compliance with relevant data and IT governance policies. While we have not extensively reviewed RAPTOR's alignment to similar policies from other jurisdictions, we note that RAPTOR's core features facilitate alignment to 5 of the 7 core principles of European Union's data protection law (GDPR).These include lawfulness, fairness, and transparency and purpose limitation by providing the DAC with control over how data are used and who can access data, data minimization where data contributors may create cub-collections of data for sharing, integrity and confidentiality through RAPTOR's security capabilities, and encryption adoptions as well as accountability through the storage of logs of every activity on RAPTOR. Under the shared responsibility model of RAPTOR, data validation and ensuring proper use of data are deferred to the DAC. The DAC reviews all access requests and approves all data ingress and egress. Therefore, principles on accuracy and storage limitations are usually beyond the scope for a platform such as RAPTOR and best specified by the data contributor and DAC.
## Federated analysis and data sharing using ga4gh protocols
For RAPTOR, the ability to integrate with other repositories (either via data movement or federated analysis) will be essential for the next phase of development. GA4GH protocols will be the key interface between RAPTOR and other repositories and platforms. The key challenge, as highlighted, is ensuring this implementation continues while retaining the 5 safes assurances. One current limitation in our study was that, for our DRS implementation, there required be a mandatory, out-of-band approval seeking with dataset's DAC before a dataset can be retrieved to remote site. Notably, while DRS's standards require the use of OAuth2 tokens for authentication, the standard is silent on the authentication and authorization procedures. RAPTOR is already utilizing OAuth2 for user authentication. RAPTOR's authentication mechanism can be extended to applications and scripts (i.e., automated flow triggered by user or software), with IP whitelisting as replacement for 2FA. However, once data have egressed, there would be no effective way for DAC to track subsequent usage or enforce additional compliances. It should, however, be noted that RAPTOR data marked as sensitive (mandatory use of secure analysis workspace) will not be valid for retrieval using DRS.
We propose that RAPTOR's federated analysis will provide stronger adherence to the 5 safes. The federated analysis environment and tools are pre-determined by the data contributors (i.e., the remote data site) and are immutable. All endpoints are IP-locked, with the pre-determined write out process. The data owners therefore have full control of how the data are to be used and the outputs which will be shared with the remote user. Data access expiry can be automatically enforced by RAPTOR. iScience Article RAPTOR's federated analysis is implemented with GA4GH DRS and TES API interfaces. RAPTOR utilizes free text sections within the DRS and TEST schemas to exchange essential integration information, including the data paths and tools available for use. The goal of using DRS and TES APIs is not to allow ''ad-hoc'' invocations from remote users. The goal of using GA4GH APIs is mostly to reduce the implementation and customization overheads.
## Raptor's technical advantage and roadmap
A key advantage from building RAPTOR as a ''serverless'' application is that RAPTOR is completely abstracted from both the system hardware and software, including the operating system. Instead, core RAPTOR services including the user interface and user management are plugged directly into AWS's hyper-scale compute and storage fabric. All button clicks and function calls are distributed to a managed cluster of servers that ensures consistent performance that scales with load while keeping the cost of upkeep low since RAPTOR is billed only for the resources for the functions executed, not for the number of servers it ran on. Apart from cost efficiency and performance scaling, a serverless resource also provides an easier pathway for integration with existing applications and services. The reason is that major CSP such as AWS reduces entire system infrastructure as a service to a series of functions calls that are by design consistent with cloud-native service calls utilized for core RAPTOR functions. This means it is programmatically similar for us to deploy a complete system of applications as to implement a button on the user interface, hence appreciably reducing the effort and time required to integrate third-party applications. For example, we have plans to enhance hosted data's findability by providing improvements on metadata capture and adopting GA4GH's Data Use Ontology (DUO).However, instead of building more native services, we are evaluating ways to integrate with third-party tool suites such as those from Center for Expanded Data Annotation and Retrieval (CEDAR).We will package the entire system into a softwaredefined infrastructure that will be provisioned on demand.
Based from learnings from this prototype, the next developmental goal for RAPTOR is put to implement a feature of complete data exchange with third-party data sources (data federation) using combinations of community defined standards, including GA4GH Beacon v2, DRS, and WES.
In conclusion, we propose that the RAPTOR computational platform provides researchers a significant resource that enables power of cloud-based computing while ensuring a safe and secure environment to meet regulatory requirements for genomic data. Additionally, RAPTOR enables flexibility in data federation and offers potentials to integrate multiple data repositories that would enable for more effective analysis of large-scale genomic data.
## Limitations of the study
One limitation of this study is that it reviews approaches to security but not implementation of controls in details which is also important for scaling up and user acceptance. A second limitation is that we did not explore effectiveness of alternate emerging security approaches in complying with relevant security policies, such as privacy-preserving analytics.
# Star+methods
Detailed methods are provided in the online version of this paper and include the following:
[formula] d KEY RESOURCES [/formula]
[fig] Figure 1: RAPTOR Overview and the modes of data analysis supported RAPTOR provides access via Standalone Linux machines with sudo access, Juypter Notebooks, and EMR Cluster with pre-configured Hail tools. [/fig]
[fig] Figure 2: As a serverless application, RAPTOR is composed of native AWS services integrated together with Lambda functions User Interfaces are composed of CloudFront hosting graphical user interfaces made with Java scripts. User authentications are managed with Cognito. Hosted datasets sit on S3 (with automated tiering) while all metadata are stored on DynamoDB. Data-staging activities are managed using S3 Batch. Data ingress and egress are managed through TransferFamily. The Analytics workspace relies on FSX to provide scratch storage, and depending on the mode of compute, either EC2, Elastic Map Reduce or Parallel Cluster will provide computing power. Data access from the nodes is regulated by Service Endpoints. All permissions and authorisations are managed using IAM. Encryption keys used by S3, EBS, and DynamoDB are stored within KMS. All RAPTOR activities are written into AWS QLDB. [/fig]
[table] Table 1: Numbers of total, common, and rare SNPs obtained after imputation of the SCHS dataset with TOPMed and SG10K imputation panels [/table]
[table] Table 2: Datasets hosted on RAPTOR. # denotes studies with samples from Singapore [/table]
[table] TABLE d RESOURCE: AVAILABILITY B Lead contact B Materials availability SUPPLEMENTAL INFORMATION Supplemental information can be found online at https://doi.org/10.1016/j.isci.2023.106546. We would like to thank Charissa Chang of AWS Singapore, Kenny Max, and Smit Callum of AWS Envision Engineering. RAPTOR grew out of an early prototype Kenny and Smit built on top of AWS Service [/table]
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Born to sweet delight: Using natural models of malaria protection to understand and neutralize P. falciparum pathogenesis
# Introduction
Across millennia of coevolution, malaria parasites have mediated their human hosts' patterns of settlement, authored their historical milestones, and shaped their genome. The human genome harbors archived responses to the pressure of severe, life-threatening malaria, and chief among these are innate variants of erythrocytes, which serve as principal cellular hosts for the parasite. Since JBS Haldane first speculated that red blood cell (RBC) variants may confer some resistance to malaria, numerous studies using diverse approaches have identified an array of innate variants, which can be grouped as disorders of the erythrocyte cytoskeleton (e.g., Southeast Asian ovalocytosis), variation in erythrocyte surface antigens (e.g., ABO and Duffy), enzymatic aberrations (e.g., glucose-6-phosphate dehydrogenase deficiency), and mutants of α-or β-globin proteins, either as deletions (e.g., α-thalassemia) or point mutations (e.g., hemoglobin [Hb] S or C). To varying degrees, each of these variants of erythrocytes have been selected over generations owing to enhanced fitness when human populations are exposed to intense malaria transmission.
These variants can provide striking protection: as one example, in African children, heterozygosity for sickle Hb reduces the risk of severe, life-threatening malaria by over 90%. This degree of protection against severe malaria exceeds any other preventive medical intervention, including the 34% protection conferred by the most advanced malaria vaccine, RTS,S/AS01 [bib_ref] Efficacy and safety of the RTS,S/AS01 malaria vaccine during 18 months after..., Rts S Clinical Trials [/bib_ref]. Because of this, these models of naturally occurring protection need to be exploited to better understand the mechanisms of parasite pathogenesis and how these can be neutralized. Now, nearly 7 decades following Haldane's hypothesis, there exists a pressing need to translate these observations into strategies to interrupt malaria and render fewer children born to the endless night of immiserating malaria.
## Historical perspectives
Malaria is transmitted between humans by Anopheles spp. mosquitos and is caused by five species of Plasmodium spp. parasites-Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. P. falciparum and P. vivax are distributed most widely throughout the tropics and cause most human disease. P. vivax is very rare in most of Africa because most Africans lack expression on their erythrocytes of the Duffy antigen receptor for chemokines (DARC), which enables the efficient invasion of red cells by P. vivax. Globally, P. falciparum is the most common and the deadliest species, and it kills over 400,000 people annually, mostly children in sub-Saharan Africa. After a short stage in hepatocytes, the principal target in humans is the erythrocyte, which serves as the primary platform for parasite propagation. Consequently, variants of erythrocytes are the principal known means by which humans have developed protection, most notably through mutations in Hb [fig_ref] Table 1: Common Hb variants and their phenotypes [/fig_ref]. Hb is formed by a tetramer of two α-and two β-globin proteins; mutations of Hb result from either deletion of globin gene copies (in the thalassemias) or in coding point mutations in β-globin. A wide variety of point mutations in β-globin have been described, and most important for malaria protection are the substitutions of glutamate at position 6 for either valine (to produce HbS) or lysine (HbC). The inheritance of two mutated β-globins (as either HbSS or HbSC) produces sickle cell anemia and significant multisystem morbidity; in contrast, the inheritance of either sickle-cell trait (HbAS) or homozygous HbC (HbCC) produces little morbidity but substantial protection.
## Clinical epidemiology
Interestingly, both HbAS and HbCC specifically protect children from severe disease, suggesting that they attenuate the specific pathogenic mechanisms of either the parasite or host while allowing children to be infected with P. falciparum. This conclusion is supported by contrasts between the degrees of protection conferred by HbAS against severe malaria (91%), uncomplicated malaria (31%), and asymptomatic parasitization (none). The risk of severe malaria is also reduced by homozygous (37%) or heterozygous (17%) α-thalassemia, but these variants confer no protection from uncomplicated malaria. In addition to their individual effects, epistasis has been repeatedly reported between HbAS and α-thalassemia, in which the coinheritance of both mutations negates the protective effects of either against severe malaria [bib_ref] Negative epistasis between the malaria-protective effects of alpha+-thalassemia and the sickle cell..., Williams [/bib_ref]. This clear biological and clinical interaction between the two variants suggests that candidate molecular mechanisms of protection should follow a similar pattern.
## In vitro effects
Numerous in vitro studies have reported the effects of Hb variants on parasite cellular phenotypes. Generally, in vitro studies have rejected the hypothesis that parasites are unable to invade hemoglobinopathic RBCs: invasion has been reported as normal for HbAS [bib_ref] The interaction between sickle haemoglobin and the malarial parasite Plasmodium falciparum, Pasvol [/bib_ref] , HbCC [bib_ref] Synchronized cultures of P falciparum in abnormal red cells: the mechanism of..., Olson [/bib_ref] , HbAC [bib_ref] Synchronized cultures of P falciparum in abnormal red cells: the mechanism of..., Olson [/bib_ref] , HbAE, and HbEE cells [bib_ref] Reduced deformability of thalassemic erythrocytes and erythrocytes with abnormal hemoglobins and relation..., Bunyaratvej [/bib_ref] , as well as for α-thalassemic RBCsand those containing HbF [bib_ref] A role for fetal hemoglobin and maternal immune IgG in infant resistance..., Amaratunga [/bib_ref]. Studies of parasite growth have been conflicting: early studies [bib_ref] The interaction between sickle haemoglobin and the malarial parasite Plasmodium falciparum, Pasvol [/bib_ref] [bib_ref] Erythrocytic mechanism of sickle cell resistance to malaria, Friedman [/bib_ref] [bib_ref] Cellular mechanism for the protective effect of haemoglobin S against P. falciparum..., Pasvol [/bib_ref] reported that, compared with HbAA RBCs, growth of parasites was attenuated specifically at low oxygen tension (approximately 5%) in HbAS or HbCC RBCs, but more recent studies have reported equivalent growth in HbAA, HbAS, and HbAC RBCs [bib_ref] Oxidative insult can induce malaria-protective trait of sickle and fetal erythrocytes, Cyrklaff [/bib_ref] [bib_ref] Differential time-dependent volumetric and surface area changes and delayed induction of new..., Waldecker [/bib_ref] in similarly hypoxic environments. However, a recent, more detailed investigation of the stage-specific growth effects of hypoxia in HbAS cells reported that hypoxia attenuates parasite maturation specifically in the latter stages of parasite development, during which infected erythrocytes typically sequester in hypoxic deep vascular beds [bib_ref] Resistance to Plasmodium falciparum in sickle cell trait erythrocytes is driven by..., Archer [/bib_ref]. How this phenomenon may operate in vivo or be present in other Hb variants remain open questions.
## Cellular pathogenesis
Cellular mechanisms of pathogenesis are principally governed by interactions between infected RBCs (iRBCs) and extracellular ligands on host cells, including endothelium, leukocytes, and uninfected RBCs; these interactions are enabled by the expression of parasitederived proteins on the RBC surface, including the hypervariable protein families P. falciparum erythrocyte membrane protein 1 (PfEMP1), repetitive interspersed families of polypeptides (RIFIN), and subtelomeric variable open reading frame (STEVOR). This cytoadherence allows iRBCs to sequester in the microvasculature and avoid splenic clearance, attenuate immune activation [bib_ref] Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors, Saito [/bib_ref] , and activate endothelial cells [bib_ref] Direct activation of human endothelial cells by Plasmodium falciparum-infected erythrocytes, Viebig [/bib_ref]. In vitro studies reveal that HbAS and HbCC reduce the cytoadherence of iRBCs to human microvascular endothelial cells and produce aberrant expression of PfEMP1 on the iRBC surface compared with HbAA [bib_ref] Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle hemoglobin, Cholera [/bib_ref] [bib_ref] Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against..., Fairhurst [/bib_ref]. The reduction in PfEMP1 expression is important because PfEMP1 variants enable the interaction of iRBCs with endothelial receptors including endothelial protein C receptor (ePCR), intercellular adhesion molecule 1 (ICAM-1), and CD36 that are increasingly recognized as important in the pathogenesis of severe malaria [bib_ref] Severe malaria is associated with parasite binding to endothelial protein C receptor, Turner [/bib_ref]. Intriguingly, a more recent study reported that whereas HbAS reduced PfEMP1 expression, adherence to recombinant ICAM-1 and CD36, and adherence to uninfected RBCs, these phenotypic effects were reversed by coinheritance with α-thalassemia, mirroring the clinical epidemiology of HbAS, α-thalassemia, and malaria risk [bib_ref] Negative epistasis between the malaria-protective effects of alpha+-thalassemia and the sickle cell..., Williams [/bib_ref]. These attenuated interactions have functional consequences: in a recent study, HbAS reduced the ability of iRBCs to activate endothelium, possibly as a result of the reduced quality of adhesion of the iRBC to endothelium [bib_ref] The sickle cell trait affects contact dynamics and endothelial cell activation in..., Lansche [/bib_ref]. Taken together, these data suggest that the ability of HbCC and HbAS to disrupt P. falciparum's ability to interact with extracellular ligands contributes significantly to their protection from severe malaria.
## Molecular pathogenesis
As noted above, P. falciparum exports a large number of proteins to the iRBC surface, and a principal component of the export machinery is Maurer's clefts, the parasite-derived Golgilike membranous structures that serve as transport hubs for PfEMP1 and other exported proteins. As the parasite matures in the RBC, these Maurer's clefts are conveyed along a scaffold of host-cell actin that the parasite has repurposed to assemble a transport network; in HbCC and HbSC RBCs, Maurer's clefts are dysmorphic, the actin assemblage is disorganized [bib_ref] Hemoglobins S and C interfere with actin remodeling in Plasmodium falciparum-infected erythrocytes, Cyrklaff [/bib_ref] , Maurer's cleft motion is impaired [bib_ref] Haemoglobin S and C affect the motion of Maurer's clefts in Plasmodium..., Kilian [/bib_ref] , and protein export to the RBC surface is delayed [bib_ref] Hemoglobin S and C affect protein export in Plasmodium falciparum-infected erythrocytes, Kilian [/bib_ref]. Some of these phenotypes have been induced by the exposure of HbAA RBCs to oxidation prior to infection, suggesting that oxidative insult from β-globin variants is an important contributor [bib_ref] Oxidative insult can induce malaria-protective trait of sickle and fetal erythrocytes, Cyrklaff [/bib_ref]. In a separate line of inquiry, host microRNAs (miRNAs) harbored within HbAS RBCs attenuated parasite growth and perturbed parasite protein translation owing to the production of chimeric mRNAs formed by the fusion of host miRNA and parasite mRNA transcripts [bib_ref] Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation..., Lamonte [/bib_ref] , suggesting a novel mechanism of interactions between host RBC and parasite.
Collectively, these studies suggest that HbAS exerts pervasive effects on parasite mechanisms and underscores the efficiency of this system for exploring fundamental mechanisms of parasite pathogenesis (Box 1).
## A pathway to progress
Just as pathogen drug-resistance phenotypes furnish a model by which to better understand drug mechanisms of action, human disease resistance enables us to more efficiently explore pathogen mechanisms of disease and thereby identify targets for intervention. There are two models for this approach. The first model relates to P. vivax malaria. As noted above, many Africans are protected from P. vivax because the absence of DARC on the erythrocyte surface prevents RBC invasion by P. vivax. This model of protection enabled the identification of the parasite-derived binding partner for DARC: P. vivax Duffy binding protein (PvDBP). In populations exposed to P. vivax, naturally occurring antibodies to PvDBP are associated with protection from vivax malaria (in DARC-positive people), and this apparent functional protection has prioritized PvDBP as an advanced candidate antigen for vivax-specific vaccination strategies [bib_ref] Human vaccination against Plasmodium vivax Duffy-binding protein induces strain-transcending antibodies, Payne [/bib_ref]. The second model relates to pregnancy-associated P. falciparum malaria. Epidemiologically, pregnant women are at high risk in endemic areas for falciparum malaria but develop over successive pregnancies acquired immunity to its consequences, including placental malaria. Placental malaria results from interactions between chondroitin sulfate A in the syncytiotrophoblast and a parasite-derived surface protein VAR2CSA; this essential interaction, and the model of acquired immunity to it, enabled the identification of functional antibody responses that attenuate placental malaria. Currently, two early-phase vaccines are being developed to generate responses directed against VAR2CSA and protect women and their offspring from placental malaria. Adaptive evolution in human populations has furnished to us natural strategies by which to neutralize malaria parasites in the form of erythrocyte variants. By using the protective phenotypes to infer essential mechanisms of disease, we can rationally develop new approaches to preventing and treating this ancient and immiserating disease.
[table] Table 1: Common Hb variants and their phenotypes. [/table]
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Contemporaneous SARS-CoV-2-Neutralizing Antibodies Mediated by N-glycan Shields
Mutations and the glycosylation of epitopes can convert immunogenic epitopes into nonimmunogenic ones via natural selection or evolutionary pressure, thereby decreasing their sensitivity to neutralizing antibodies.Based on Thomas Francis's theory, memory B and T cells induced during primary infections or vaccination will freeze the new mutated epitopes specific to naïve B and T cells from the repertoire.On this basis, some researchers argue that the current vaccines derived from the previous strains of the SARS-CoV-2 virus do not increase immunity and may also prevent the immune response against new epitopes.However, evidence shows that even if the binding affinity is reduced, the previous antibodies or T cell receptors (TCRs) can still bind to this new epitope of the Beta, Gamma, and Delta variant if their concentration is high enough (from a booster injection) and neutralize the virus.This paper presents some convincing immunological reasons that may challenge this theory and argue for the continuation of universal vaccination to prevent further mutations of the SARS-CoV-2 virus.Simultaneously, the information presented can be used to develop vaccines that target novel epitopes or create new recombinant drugs that do not lose their effectiveness when the virus mutates.
One crucial aspect of SARS-CoV-2 is the ability of the virus to rapidly mutate and create antigenically distinct strains.Changes in the amino acids and glycosylation or deglycosylation of sites create new epitopes by changing the previous epitopes.These established new epitopes form novel N-glycan shields that can mediate other contemporaneous SARS-CoV-2-neutralizing antibodies.
## Introduction 2.1. contextual framework
The Original antigenic sin (OAS) theory, described in 1960 by Thomas Francis, states that the immune system preferentially uses immunological memory based on a previous infection when encountering a second, slightly different version of that foreign pathogen.This leaves the immune system "trapped" by its first response to each antigen and unable to mount potentially more effective responses during subsequent infections.Based on this theory, memory B and T cells induced during infections or vaccinations with the primary variant of the pathogen will freeze the new mutated epitopes' specific naïve B and T cells (cross-reactive memory against specific naïve B or T cells) from the repertoire [bib_ref] Previous Humoral Immunity to the Endemic Seasonal Alphacoronaviruses NL63 and 229E Is..., Focosi [/bib_ref].Some researchers argue that if a booster dose of the SARS-CoV-2 vaccine from the primary variant is administered, even when there are common epitopes between the two variants, the immune response against the new uncommon epitopes will be prevented, thus failing to enhance immunity [bib_ref] Impact of antigenic evolution and original antigenic sin on SARS-CoV-2 immunity, Aguilar-Bretones [/bib_ref].Garrity et al. [bib_ref] Refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope, Garrity [/bib_ref] introduced this process in relation to the human immunodeficiency virus (HIV) and coined it decotope, or immune decoy epitopes.This process was defined as a shift from immunodominant epitopes to a limited pool of neutralizing antibodies providing low protection [bib_ref] Refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope, Garrity [/bib_ref] [bib_ref] Glycan masking in vaccine design: Targets, immunogens and applications, Martina [/bib_ref].Here, we argue against this notion and present several ways in which the immune system can still mount a response against mutated variants.
## Perspective
Suppose a primary antigen with several epitopes enters the body.The epitopes are divided into several categories: exposed immunogenic epitopes, hidden immunogenic epitopes, exposed non-immunogenic epitopes, and hidden non-immunogenic epitopes.The exposed immunogenic epitopes (linear and conformational) are expected to naturally stimulate the cellular and humoral immune system and induce T and B cell memories.Similarly, the hidden immunogenic linear epitopes will stimulate the humoral and cellular immune systems by producing antigen-specific antibodies (Ab1) during the first immune reaction following exposure to the antigen.In the case of SARS-CoV-2, mutations might have several consequences: the exposed immunogenic epitopes may change so that the antibodies produced against these epitopes no longer can neutralize the virus, and the virus will remain pathogenic.
Furthermore, the production of anti-idiotype antibodies (Ab2) can be induced, specifically targeting Ab1 to inhibit its action by forming immune complexes.Paratopes of Ab2 can mimic antigens by binding to Ab1.Moreover, their structural similarities enable them to bind to the specific receptor of the original antigen and induce agonist or antagonist cell signaling in the cells targeted by the virus.This mechanism mimics the pathological reaction and triggers a long-term response after the first contact.Characterizing the action of Ab2 would help us to understand several adverse effects during SARS-CoV-2 infection or vaccination [bib_ref] A Possible Role for Anti-idiotype Antibodies in SARS-CoV-2 Infection and Vaccination, Murphy [/bib_ref].
## Immunologic reasons challenging oas theory
After mutation due to changes in amino acids or glycosylation (or deglycosylation), new epitopes are created by changing the previous epitopes.Even if the binding affinity is reduced, the previous antibodies or T cell receptors (TCRs) can still bind to this new epitope if their concentration is high enough (from a booster injection) to neutralize the virus.This has been demonstrated based on the neutralization of new variants of the SARS-CoV-2 virus (Beta, Gamma, and Delta) through convalescent and post-vaccination sera with high titers [bib_ref] The Omicron variant is highly resistant against antibody-mediated neutralization: Implications for control..., Hoffmann [/bib_ref].On the other hand, if the binding affinity is too low and the mutated epitopes constitute critical residues, or new glycosylation/deglycosylation occurs on the residues, improving access to the ACE2 receptor, the affinity of the antibodies will drop significantly [bib_ref] A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2, Shi [/bib_ref] [bib_ref] N-Linked Glycans and K147 Residue on Hemagglutinin Synergize To Elicit Broadly Reactive..., Huang [/bib_ref] [bib_ref] The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity, Li [/bib_ref].The more specific naive B and T cells in the repertoire will also gain access, bind to the mutated epitopes, and be stimulated.Evidence shows that changes in the glycan components of the SARS-CoV-2 S protein can profoundly influence the epitopes targeted by neutralizing antibodies [bib_ref] Profiling and characterization of SARS-CoV-2 mutants' infectivity and antigenicity, Wang [/bib_ref] [bib_ref] Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent..., Cao [/bib_ref] [bib_ref] Site-specific glycan analysis of the SARS-CoV-2 spike, Watanabe [/bib_ref] [bib_ref] Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein, Walls [/bib_ref] (Figure [fig_ref] Figure 1: Figure 1 [/fig_ref].Another argument is that mutations and glycosylation can convert immunogenic epitopes into non-immunogenic ones via natural selection or evolutionary pressure, decreasing their sensitivity to neutralizing antibodies [bib_ref] Glycan shield and epitope masking of a coronavirus spike protein observed by..., Walls [/bib_ref].Unlike bacteria, in which glycans are encoded by the bacterial genome and treated as "nonself" epitopes by their corresponding hosts, viruses take advantage of the host cell machinery for glycosylation.Another argument is that mutations and glycosylation can convert immunogenic epitopes into non-immunogenic ones via natural selection or evolutionary pressure, decreasing their sensitivity to neutralizing antibodies [bib_ref] Glycan shield and epitope masking of a coronavirus spike protein observed by..., Walls [/bib_ref].Unlike bacteria, in which glycans are encoded by the bacterial genome and treated as "nonself" epitopes by their correspond-ing hosts, viruses take advantage of the host cell machinery for glycosylation.Generally, they are decorated with the "self"-glycans.These "self"-glycans are generally considered as a strategy for escaping the host immune response [bib_ref] Coronaviruses' sugar shields as vaccine candidates, Wang [/bib_ref] [bib_ref] Glycans of SARS-CoV-2 Spike Protein in Virus Infection and Antibody Production, Zhao [/bib_ref].Even though the previous antibodies or TCRs do not bind to the converted epitopes, the new epitopes do not stimulate more specific naive B cells and T cells from the repertoire.However, we cannot rely on this antigenic sin for previous B or T cell memories.
Glycans disrupt the immune response and promote immune escape by inhibiting recognition by antibodies and immune cells.In influenza, the severity of the disease depends on the glycosylation profile of surface proteins such as hemagglutinin (HA) from past exposure.Infection with a highly glycosylated variant induces a deficient adaptive response with a lack of neutralizing antibodies and a robust T response.In the event of reinfection with a low-glycosylated variant, antibody-mediated neutralization is significantly reduced, and the disease is more severe.This explains why hosts previously infected with highly glycosylated seasonal H1N1 viruses develop severe lower respiratory tract disease due to the pandemic H1N1 strain [bib_ref] Glycan shielding of the influenza virus hemagglutinin contributes to immunopathology in mice, Wanzeck [/bib_ref].
Furthermore, some evidence indicates that some new glycosylation on the SARS-CoV-2 spike glycoprotein not only shields the immunogenic epitope but also, at the same time, may change the S binding affinity to ACE2 (Figure [fig_ref] Figure 1: Figure 1 [/fig_ref].Zhao et al. suggested the essential roles of glycosylation in mediating receptor binding, antigenic shielding, and potentially, the evolution/divergence of these glycoproteins.This process could be necessary for vaccine stabilization via glycosylation and could be considered when manufacturing future SARS-CoV-2 vaccines [bib_ref] Vulnerabilities in coronavirus glycan shields despite extensive glycosylation, Watanabe [/bib_ref].
The professional antigen-presenting cells (APC), B lymphocytes, also present an opportunity to refute the OAS theory.Any antigen it recognizes can present its different epitopes and the epitope that specifically binds to its receptor, can bind to T helper lymphocytes in the MHC class II cleft (Figure [fig_ref] Figure 1: Figure 1 [/fig_ref].The glycan-specific B lymphocytes can be helped by the previous memory T cells specific to epitopes other than the mutated (glycosylated or deglycosylated) epitopes of the SARS-CoV-2 spike.Here, it can be concluded that using the boosters of existing vaccines can at least induce memory T cells specific to epitopes other than the mutated epitopes of the SARS-CoV-2 spike (common epitopes of the primary and the mutated variants).These non-mutated T-dependent epitopes can help B lymphocytes to mount a specific response to new SARS-CoV-2 variants.Therefore, not only do the previous B lymphocytes fail to prevent the stimulation of B lymphocytes specific to new epitopes, but they also involve T helper cells in order to induce the response of new B lymphocytes specific to the mutated epitopes [bib_ref] SARS-CoV-2 vaccination induces immunological T cell memory able to cross-recognize variants from..., Tarke [/bib_ref].
After mutation, the change in the epitopes mentioned above may lead to a situation where the previous vaccine-induced antibody's binding to their adjacent epitopes increases their binding affinity to ACE2.As Liu et al. reported, several antibodies bind to parts of the SARS-CoV-2 N-terminal domain (NTD), increasing its binding to the ACE2 receptor [bib_ref] An infectivityenhancing site on the SARS-CoV-2 spike protein targeted by antibodies, Liu [/bib_ref].This is an FcR-independent antibody-dependent enhancement (ADE) of the infection induced by the previous SARS-CoV-2 variants.Other evidence indicates that individuals vaccinated with antigens against Wuhan-Hu-1 and then infected with the Alpha or Delta variants have a relatively decreased response to variant-specific epitopes compared to unvaccinated individuals who become naturally infected [bib_ref] Immune imprinting, breadth of variant recognition, and germinal center response in human..., Röltgen [/bib_ref].Unvaccinated individuals were found to have immunity against SARS-CoV-2 only if they had a prior infection.Unvaccinated individuals who were naturally infected had an 85% lower risk of contracting COVID-19 than those who were not naturally infected [bib_ref] Rates of COVID-19 Among Unvaccinated Adults with Prior COVID-19, Ridgway [/bib_ref].This was seen in both mild and severe disease cases.This could be due to the Fc-independent ADE, as there is no evidence that previous memory B lymphocytes prevent the stimulation of B lymphocytes specific to new epitopes [bib_ref] Immune imprinting, breadth of variant recognition, and germinal center response in human..., Röltgen [/bib_ref] [bib_ref] Immune imprinting and SARS-CoV-2 vaccine design, Wheatley [/bib_ref].Although these Fc-dependent and -independent ADE antibodies were produced through the previous virus variants' stimulation (i.e., very few vaccine platforms or natural infection), the leading cause of this problem was the mutation of the virus.So, the mutated antigen is the culprit, not the original antigen (Figure [fig_ref] Figure 1: Figure 1 [/fig_ref].conducted a review of the analyses of COVID-19 vaccine efficacy in older persons who received the second booster compared to unvaccinated people and those who received only a single COVID-19 vaccine booster.The second vaccine booster maintained high effectiveness against adverse COVID-19 outcomes such as hospitalization, intensive care unit admission, and death (i.e., between 77 and 86%) and also showed an efficacy around 10% higher than the single booster.The efficacy of the second vaccine booster declined over time, decreasing by 33-46% when assessed at >120 days from administration.The results of these ad interim analyses of the ongoing Italian nationwide COVID-19 vaccination campaign suggested that regular boosting with COVID-19 vaccines may be advisable for older persons.
## Mattiuzzi and lippi
The SARS-CoV-2 S glycoprotein (SGP) is a trimeric class I fusion protein that comprises two subunits, including the S1, the receptor-binding subunit attached to the S2, the fusionmediating subunit, such that the complete entity is a trimer of S1-S2 heterodimers.The S2 subunit is anchored to the virus via a membrane-spanning domain.Once assembled and processed by a protease within the cell, the fusion protein is maintained in a metastable state known as the prefusion conformation.Usually, the receptor-binding subunit (S1) overlays its fusion-mediating counterpart (S2) and temporarily locks it into an energetically unfavorable conformation.When the receptor-binding subunit engages ACE2, its structure alters so that it releases the S2 subunit to undergo profound conformational changes, revealing a hydrophobic region at the N-terminus of the S2 subunit.This region can be inserted into the cell membrane, creating a linkage protein between the cell membranes and the virus.The release of these energies is sufficient to pull the two membranes together so as to allow them to fuse.The S2 subunit is now in its post-fusion conformation.As a vaccine candidate, the mutated SARS-CoV-2 SGP can produce epitopes for virus-neutralizing antibodies (NAbs).When used as an immunogen, it induces NAbs that, in turn, will bind to the same protein on the virus surface, impairing its functions and neutralizing its infectivity.The relevant NAb epitopes are optimally displayed or observed only when the trimer is in its conformational perfusion.
Conversely, post-fusion or other aberrant protein conformations induce primarily nonneutralizing antibodies (non-NAbs) with no or limited protective capacity.In other cases, non-NAbs can even be harmful.When expressed as a recombinant protein, the SARS-CoV-2 SGP trimer can undergo spontaneous conformational changes and decay into its post-fusion form, inducing non-NAbs.These non-NAbs can be categorized as Fc-antibody-dependent infection enhancement antibodies produced by the previous vaccination, and the induction of such non-NAbs results in immune complex (IC) deposition and complement activation.These IC deposits have been linked to COVID-19 disease severity and many of the longterm consequences of SARS-CoV-2 infection such as arthritis and vasculitis [bib_ref] Immune complexes as culprits of immunopathology in severe COVID-19, Kolb [/bib_ref].This viral persistence based on the enhanced interference of non-NAbs has also been noted in HIV.The destruction of CD4+ T cells, downregulation of MHC molecules, and the establishment of latent viral genomes are often linked to immune evasion, disease progression, and non-NAbs [bib_ref] Multiantibody strategies for HIV, Hiatt [/bib_ref].Overcoming this problem requires the application of protein-engineering stabilizing methods to create mimics stabling the prefusion trimer that presents critical NAb epitopes with appropriate fidelity [bib_ref] Immunogenicity of novel mRNA COVID-19 vaccine MRT5500 in mice and non-human primates, Kalnin [/bib_ref].Multi-mAb therapy is a promising approach to treating HIV infection and may be a candidate for SARS-CoV-2 [bib_ref] Multiantibody strategies for HIV, Hiatt [/bib_ref].It would allow for the bypassing of viral escape strategies and restoration of an adapted endogenous immune response.These antibodies inhibit the more effective, broadly cross-neutralizing antibodies (bnAbs), preventing the immune reaction from progressing.
## Designing future vaccines with glycosylation in mind
The most utilized and internationally accepted SARS-CoV-2 vaccines are nucleic acid vaccines such as the Pfizer-BioNTech and the Moderna Spikevax.For RNA-based mRNA vaccines, the mRNA is transcribed in vitro from DNA before the RNA is encapsulated in the nanoparticles prior to vaccine administration, so no DNA is present, and no transcription is needed in the nucleus [bib_ref] Glycosylation as a key parameter in the design of nucleic acid vaccines, Ozdilek [/bib_ref] [bib_ref] Developing mRNA-vaccine technologies, Schlake [/bib_ref] [bib_ref] Current prospects for mRNA gene delivery, Yamamoto [/bib_ref].
While these vaccines are some of the most efficacious available, there is potential for host glycosylation to negatively impact their success.Previous studies have found that host protein glycosylation impairs immune responses against bacteria even after using nucleic acid vaccines [bib_ref] Host protein glycosylation in nucleic acid vaccines as a potential hurdle in..., Ozdilek [/bib_ref].Similar findings are seen in viruses where glycosylation changes the structure and efficacy of the mRNA-derived viral antigens through post-translational modifications [bib_ref] Glycosylation as a key parameter in the design of nucleic acid vaccines, Ozdilek [/bib_ref].One proposed theory for this phenomenon is that glycans shield the protein surface so that the protein folding deviates from its native form [bib_ref] Glycosylation as a key parameter in the design of nucleic acid vaccines, Ozdilek [/bib_ref] (Figure [fig_ref] Figure 1: Figure 1 [/fig_ref].The receptor binding domain (RBD) of the SARS-CoV-2 S protein, which is responsible for recognizing and binding to the ACE2 receptor, is found in a closed or inaccessible receptor conformation, preventing it from being recognized by immune cells [bib_ref] Glycan masking of a non-neutralising epitope enhances neutralising antibodies targeting the RBD..., Carnell [/bib_ref].However, the receptor binding motif (RBM), the most crucial component for attachment to the ACE2 receptor, remains untouched and stable [bib_ref] Glycan masking of a non-neutralising epitope enhances neutralising antibodies targeting the RBD..., Carnell [/bib_ref].Glycan masking is also implicated in this immune shifting, or a shift in the immune response away from specific epitopes while focusing on other epitopes to decrease the extent of off-target antibody production.This also has been linked with reduced ADE activity and increased B cell regulation and testing [bib_ref] Glycan masking in vaccine design: Targets, immunogens and applications, Martina [/bib_ref].However, there are some downsides to glycan masking.The process is complicated and requires extensive rounds of glycosylation in the correct sites, or it may lead to misfolding and off-target downstream effects [bib_ref] Glycan masking in vaccine design: Targets, immunogens and applications, Martina [/bib_ref].
A new approach introduces specific glycosylation sites onto the RBD to counteract this issue and thus elicit more robust immune responses to both previous and mutated variants.A novel study by Carnell et al. proposed a modified RBD spike subunit that triggers more broad neutralizing immune responses as a potentially improved booster vaccine [bib_ref] Glycan masking of a non-neutralising epitope enhances neutralising antibodies targeting the RBD..., Carnell [/bib_ref].Their methods were based on studies conducted with MERS and influenza viruses and involved removing and adding new glycan sites.In preliminary results, they demonstrated that this method could produce more neutralizing antibodies when compared to wild-type SARS-CoV-2 RBD.
Moreover, data suggests that the N-glycosylation and O-glycosylation of proteins can occur [bib_ref] Glycosylation as a key parameter in the design of nucleic acid vaccines, Ozdilek [/bib_ref].This glycosylation has been shown to impact NAb epitopes by creating steric hindrance that prevents proper antibody responses and has also been implicated in the recruitment of T cells [bib_ref] Glycosylation as a key parameter in the design of nucleic acid vaccines, Ozdilek [/bib_ref].Targeting these N-glycosylation sites may prove to be beneficial in creating more efficient vaccines that can withstand several mutations; however, in practice, it is more complex.There are 22 potential N-glycoSites on the S protein of SARS-CoV-2, which, as Huang et al. highlighted, make the process of analysis and vaccine design more difficult [bib_ref] In-depth characterization of protein N-glycosylation for a COVID-19 variant-design vaccine spike protein, Huang [/bib_ref].The role of N-glycan epitopes extends beyond the recruitment of immune cells; they are crucial for viral entry by mediating membrane fusion.This is seen in the HIV-1 virus, where the densely packed N-glycans on the viral envelope are responsible for pathogenesis.This concept has also been studied in relation to other pathogens, such as influenza and Ebola [bib_ref] Glycosylation as a key parameter in the design of nucleic acid vaccines, Ozdilek [/bib_ref].
From the SARS-CoV-2 pandemic, we can learn a lot about vaccine design and efficacy.It is evident that glycosylation plays an essential and contrasting role in immunogenicity; therefore, it has been suggested that removing the glycosylation sites around vulnerable NAb epitopes may be an effective strategy.The ACE2 receptor binding domain of the SARS-CoV-2 spike protein, which is required for viral anchoring to enable cell entry, is not glycosylated to increase the fitness of the virus [bib_ref] SARS-CoV-2 Glycosylation Suggests That Vaccines Should Have Adopted the S1 Subunit as..., Fernández [/bib_ref].However, the other epitopes in the S protein are glycosylated, which has been suggested to divert the vaccine-induced immune response.
Another method that may improve the efficacy of these nucleic acid vaccines is to create proper glycosylation matches.This can be accomplished by ensuring that the same cell types infected during viral infection express the nucleic acid vaccines.The rationale for this approach is based on the fact that the surface glycoproteins of enveloped viruses use the host glycosylation machinery, and the glycosylation of the viral proteins must be highly similar to the glycosylation of the mRNA vaccines [bib_ref] SARS-CoV-2 Glycosylation Suggests That Vaccines Should Have Adopted the S1 Subunit as..., Fernández [/bib_ref].Adopting the S1 antigen instead of the whole S protein is another potential suggestion for improving these nucleic acid vaccines.In this way, the immune response to these vaccines is targeted not only in the virion phase but also in the activation phase.This activation phase frees the ACE2-anchoring S1 subunit, providing more opportunities for the vaccine to expose the NAb epitopes to the immune system and prevent immune evasion [bib_ref] SARS-CoV-2 Glycosylation Suggests That Vaccines Should Have Adopted the S1 Subunit as..., Fernández [/bib_ref].While these suggestions may improve the immune response to the SARS-CoV-2 virus, other factors should be considered.Namely, host inflammation has been shown to alter the glycosylation process and must be considered when designing new vaccines [bib_ref] Glycosylation in health and disease, Reily [/bib_ref].This can be a detrimental factor in vaccine design, as a key feature of COVID-19 is a cytokine release syndrome (CRS) that leads to a prolonged inflammatory phase.Some of the pro-inflammatory cytokines upregulated in COVID-19 patients include TNF-α, IL-6, IL-8, IL-17, and IL-1β, which are responsible for the increased ability of cells to respond to the infection by increasing the recruitment of neutrophils, macrophages, and T lymphocytes and increasing vascular permeability .The downside of CRS is acute injury to the lung, microbiota alteration, and other damage to cellular and organ functions seen in many patients who have previously experienced severe COVID-19 symptoms .As this is a concern for most patients, identifying glycosylation sites for vaccine design may prove even more difficult than anticipated.In other non-SARS-CoV-2 diseases, the induction of pro-inflammatory cytokines leads to conformational changes in the N-glycosylation of endothelial cells [bib_ref] Glycosylation in health and disease, Reily [/bib_ref].In reverse, glycosylation can also impact inflammatory responses by regulating the recruitment of leukocytes [bib_ref] Glycosylation in health and disease, Reily [/bib_ref] , demonstrating that the relationship between glycosylation and inflammation is a directly correlation.There is a gap in the literature discussing this relationship in terms of SARS-CoV-2 infection; however, our knowledge of the exact impact of inflammation and glycosylation on each other will be useful when designing vaccines for future COVID-19 variants and may even be potentially translatable to future pandemics.
# Conclusions
While the COVID-19 pandemic may seem to be a part of history, it presents a great learning opportunity for preparing for a potential future pandemic of the exact same nature.If vaccines are produced and used for the new Omicron variants, how will we know that future mutated variants will not cause the same problem again?Since the probability of such a problem occurring is very low, evidence based on the existing data indicates that the cross-reactivity of antibodies is a phenomenal concept that can either be advantageous or detrimental to the host [bib_ref] Computer Modeling of Clonal Dominance: Memory-Anti-Naïve and Its Curbing by Attrition, Castiglione [/bib_ref].For example, it is possible that the antibodies produced against the recent subvariants of Omicron may, for the future subvariant, have a helpful cross-reactive reaction on the N-terminal domain epitopes of the S trimer, which constitute the two primary neutralizing targets for the neutralizing antibody.This reaction would decrease the RBD binding affinity of SARS-CoV-2 to ACE2.So, it is logical to continue universal vaccination in order to prevent the cycle of multiplication and further mutation of the SARS-CoV-2 virus on the population level.At the same time, efforts should be made to develop new vaccines that target novel epitopes absent from the primary variant or to create new recombinant drugs that do not lose their effectiveness when the virus changes [bib_ref] An immunotherapeutic method for COVID-19 patients: A soluble ACE2-Anti-CD16 VHH to block..., Sheikhi [/bib_ref].This knowledge can help to ensure that we are more prepared for the next pandemic with efficacious vaccines that may be developed quickly.
[fig] Figure 1: Figure 1.The various ways in which the immune system can still mount a response against mutated variants.(A) Despite the single mutation in the epitope of the second variant, the epitope-specific antibodies from the first variant can still bind and neutralize the new variant if the amount of antibodies is high enough from a booster injection.(B) The SARS-CoV-2 spike epitope may be converted into a non-immunogenic form after mutation, allowing the binding of the S protein to the ACE2 receptor.(C) The new epitope-specific B cell presents the prior epitope to the helper T cell with the aid of a co-stimulatory signal from the prior epitope-specific memory helper T cell.(D) Mutated variants may have increased affinity to bind to ACE2 due to conformational changes in the SPG through FcR-independent antibody-dependent enhancement (Fc-independent ADE). [/fig]
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10.5414/CNCS109759
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s2orc_pubmed_articles
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Systemic p-ANCA vasculitis with fatal outcome, arising in the setting of methimazole use
Here we report a fatal case of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) due to methimazole use in a 64-year-old woman. She was initially hospitalized for abdominal pain and possible colitis, and subsequently developed hematuria, renal failure, and hemoptysis. The serologic work-up revealed positive antinuclear antibody (ANA) and perinuclear-antineutrophilic cytoplasm antibodies (p-ANCA), with positive antimyeloperoxidase. Three weeks following admission, the patient was found to be pulseless, and expired. At autopsy, microscopic review included widespread transmural necrotizing vasculitis and crescentic glomerulonephritis in the kidney, and diffuse pulmonary alveolar hemorrhage; focal coronary artery intimal vasculitis and necrotizing pericarditis were also noted. Several drugs have been associated with the development of ANCApositive diseases, including propylthiouracil, hydralazine, allopurinol, penicillamine, and levamisole in cocaine. Association of ANCA vasculitis with methimazole exposure is less known, and severe presentation with fatal outcome, as seen in our patient, is exceedingly rare. We reviewed clinical and histopathologic features of drug-induced ANCA vasculitis associated with methimazole to raise awareness of this potentially life-threatening complication associated with this agent.
## Case presentation
A 64-year-old female with a history of hypertension, asthma, pulmonary fibrosis, and hyperthyroidism secondary to multinodular goiter presented with abdominal pain and diarrhea and was admitted for possible colitis. On admission, she was noted to have acute kidney injury (AKI) with serum creatinine (Scr) of 2.8 mg/dL (baseline Scr was 1.2 mg/dL). She denied use of nonste-roidal anti-inflammatory drugs, proton pump inhibitors, or herbal medications; however, she reported having completed a course of clarithromycin for a respiratory infection 2 weeks prior. Her only home medication at the time of presentation was methimazole, which she had been taking for ~ 2.5 years (5 mg/p.o). She denied smoking or the use of illicit drugs, and her social history was otherwise nonrevealing. Subsequently, she developed gross hematuria with worsening AKI.
Laboratory data revealed an elevated serum C-reactive protein, white blood cell (WBC) leukocytosis of 29.1 × 10 3 /µL, blood urea nitrogen was 50 mg/dL, Scr increased to 5.67 mg/dL, and potassium was elevated to 7.2 mg/dL. Serological work-up was positive for antinuclear antibody (ANA, 1:640), perinuclear antineutrophilic cytoplasmic antibodies (p-ANCA, 1:320), and myeloperoxidase antibody (MPO, 109.4), and negative for human immunodeficiency virus, hepatitis B, hepatitis C, rheumatoid factor, ribonucleoprotein antibody, double stranded (ds)-DNA antibody, Sjogren SSA and SSB antibodies, and antiglomerular basement membrane (GBM) antibody. Serum complement levels were within normal limits; C3 = 99 mg/dL (reference range 81 -157) and C4 = 34 mg/dL (reference range 13 -39). Serum free light chain ratio was not elevated, and serum immunofixation did not show any monoclonal gammopathy. Urinalysis showed 173 red blood cells, 13 WBCs, and spot urine protein to creatinine ratio was elevated at 1.2. Additionally, a chest X-ray showed bilateral pleural effusions. Kidney ultrasound revealed increased bilateral cortical echogenicity with bilateral hydronephrosis. The patient was started on empirical therapy for Clostridium difficile due to her diarrhea, but the test for C. diff toxin was negative. CT of abdomen was performed that was negative for infectious processes. She also underwent urine culture, stool culture, and blood cultures testing multiple times, however, they were all negative. Stool was also negative for ova, parasites, and protozoa.
Given the negative anti-GBM and ds-DNA antibodies, normal complement levels, and positive ANCA serologic test with anti-MPO specificity in this patient presenting with pulmonary-renal syndrome, ANCA-driven vasculitis and pauci-immune crescentic glomerulonephritis was at the top of our differential diagnosis. Foley catheter was placed, and she was initiated on pulse dose corticosteroids due to clinical suspicion of ANCA)-associated vasculitis (AAV). She subsequently underwent kidney biopsy, which showed severe necrotizing small vessel vasculitis and crescentic glomerulonephritis, consistent with AAV. Methimazole was discontinued. Three days after the kidney biopsy, the patient developed hemoptysis and was initiated on plasmapheresis for concern of pulmonary alveolar hemorrhage. She eventually became oliguric, requiring hemodialysis. Few days later, she underwent change in mental status and eventually coded. cardiopulmonary resuscitation was unsuccessful, and the patient expired.
## Pathology evaluation
Following the patient's death, an autopsy was performed. Microscopic evaluation of the kidney parenchyma was compatible with the findings of the recent prior kidney biopsy; there was widespread necrotizing leukocytoclastic vasculitis, with extensive transmural necrosis and polymorphonuclear cell infiltration [fig_ref] Figure 1: Histopathologic findings include [/fig_ref]. The light microscopy sample contained 30 glomeruli, 2 of which were globally sclerosed. Approximately 20% of the glomeruli revealed cellular crescents. Uninvolved glomeruli did not show significant changes; the capillaries were of normal thickness and texture, the mesangium was only segmentally prominent, and no significant hypercellularity was seen in endocapillary or mesangial spaces. Immunofluorescence studies performed on sections of paraffin-embedded tissue revealed nonspecific reactivity. No significant immune type glomerular deposits were seen on electron microscopy. Chronic changes were mild.
Autopsy examination also revealed widespread pulmonary hemorrhage and focal subintimal vasculitis of a coronary artery [fig_ref] Figure 1: Histopathologic findings include [/fig_ref]. There was also focal necrotizing pericarditis adjacent to an involved artery. Other organs did not show significant changes that could explain the clinical course.
# Discussion
AAV is a group of small-vessel vasculitides, encompassing granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA) [bib_ref] The clinical presentation and therapy of diseases related to anti-neutrophil cytoplasmic antibodies..., Weiner [/bib_ref]. There are two major ANCA autoantibodies -the cytoplasmic (c-ANCA), which confers antigen specificity for proteinase 3, and perinuclear (p-ANCA), with specificity for MPO; the cytoplasmic and perinuclear forms refer to the pattern of reactivity seen by indirect immunofluorescence test on alcohol-fixed test cells exposed to patients' serum-carrying ANCA antibodies. ANCArelated vasculitides are often idiopathic, however, infections and drugs are the most common triggers for onset of this disease.
In the retrospective analysis of ANCArelated vasculitis, patients with the highest anti-MPO antibody titers were reviewed for the use of commonly implicated offending drugs; 60% (18 of 30) of patients had been exposed for a minimum of 9 months (and in some cases for many years) to hydralazine, propylthiouracil (PTU), penicillamine, allopurinol, or sulfasalazine, frequently with renal involvement, and sometimes with biopsy-proven crescentic glomerulonephritis [bib_ref] Drugassociated antineutrophil cytoplasmic antibodypositive vasculitis: prevalence among patients with high titers of..., Choi [/bib_ref]. Apart from variability in dose and length of drug exposure, there is no good correlation of antibody titer with severity of presentation and the number of organs involved; in addition, the appearance of ANCA antibodies in serum and onset of ANCA-associated vasculitis are not necessarily correlated [bib_ref] Clinical characteristics of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis caused by antithyroid drugs, Noh [/bib_ref].
Methimazole is less commonly associated with the ANCA-related vasculitis than PTU. In a large study of ANCA-related complications associated with these 2 drugs, the adverse reactions were commonly characterized as severe, with 1 fatal outcome among 23 patients exposed to methimazole [bib_ref] Clinical characteristics of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis caused by antithyroid drugs, Noh [/bib_ref]. The lack of awareness of the association of methimazole use with ANCA-related disease may have resulted in lower recognition rates of the milder forms. The use of methimazole has been increasing with the growing prevalence of thyroid disease, raising the risk for methimazole complications. Other than vasculitis, commonly cited complications include arthritis, agranulocytosis, autoimmune hepatitis, cholestasis, hypoglycemia, pancreatitis, and hypoprothrombinemia [bib_ref] Clinical characteristics of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis caused by antithyroid drugs, Noh [/bib_ref]. The vasculitic process often involves kidneys and lungs [bib_ref] Methimazole-induced ANCAassociated vasculitis with diffuse alveolar haemorrhage, Arai [/bib_ref] [bib_ref] Methimazole-induced antineutrophil cytoplasmic antibody-associated diffuse alveolar haemorrhage in a Chinese woman with..., Lau [/bib_ref]. Other target organs include joints, muscles, nerves, or skin, with variable clinical presentation [bib_ref] Clinical characteristics of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis caused by antithyroid drugs, Noh [/bib_ref] [bib_ref] Cutaneous leukocytoclastic vasculitis in the presence of methimazole therapy, Ribeiro [/bib_ref].
The pathogenesis of methimazole-induced AAV is still not fully understood. The mechanism of PTU-induced ANCA disease has been studied with more interest, and over the years, several hypotheses have been entertained. Some of them include the notion that PTU accumulates in neutrophils, binds to MPO, and causes its alteration to a more antigenic form [bib_ref] Antibodies to myeloperoxidase in propylthiouracil-induced autoimmune disease in the cat, Waldhauser [/bib_ref]. Another theory suggests that sera from patients that develop AAV in association with PTU have high reactivity towards specific MPO fragments when compared to those without AAV [bib_ref] Epitope analysis of anti-myeloperoxidase antibodies in propylthiouracil-induced antineutrophil cytoplasmic antibody-associated vasculitis, Wang [/bib_ref]. More recent research suggests that excessive activation of neutrophils by ANCAs induces formation of neutrophil extracellular traps (NETs), which are involved not only in ANCA-mediated vascular injury but also in the production of ANCAs themselves [bib_ref] Pathogenesis and therapeutic interventions for ANCA-associated vasculitis, Nakazawa [/bib_ref]. Nakazawa et al. [bib_ref] Abnormal conformation and impaired degradation of propylthiouracil-induced neutrophil extracellular traps: implications of..., Nakazawa [/bib_ref] showed that PTU can induce abnormal conformation and degradation of the NETs, suggesting that PTU plays a direct role in AAV pathogenesis. A similar process may exist with methimazole exposure, although further studies are needed to confirm this mechanism of pathogenesis.
Early recognition of this complication is critical, since discontinuation of the offending drug can not only lead to improvement in kidney function, but results in overall clinical recovery [bib_ref] Clinical and histological features of antineutrophil cytoplasmic antibody-associated vasculitis related to antithyroid..., Hasegawa [/bib_ref]. In patients where major organ injury is present, immunosuppression may be needed to suppress the inflammation and prevent permanent damage. Drugassociated ANCA vasculitis should be considered in patients with a history of culprit drug exposure, high-titer anti-MPO antibodies, and the presence of other autoantibodies including antinuclear, antihistone, antiproteinase 3, antielastase, antilactoferrin, or antiphospholipid antibody [bib_ref] Drugassociated antineutrophil cytoplasmic antibodypositive vasculitis: prevalence among patients with high titers of..., Choi [/bib_ref]. The presence of these antibodies is not a feature of idiopathic ANCA-associated disease [bib_ref] Drugassociated antineutrophil cytoplasmic antibodypositive vasculitis: prevalence among patients with high titers of..., Choi [/bib_ref]. ANCA-related glomerular disease is typically pauci-immune, however, it can be associated with immune complex deposits; the presence of an immune complex-mediated component on a kidney biopsy, in what otherwise appears as an ANCA-driven process, should prompt further investigation of drug exposures. Patients treated with methimazole should be monitored for serological evidence of ANCA antibodies and clinical manifestations of AAV, regardless of the dose and length of drug exposure. Patient survival and the risk of end-stage kidney disease are closely associated with degree of renal dysfunction at time of presentation. Physicians, including endocrinologists, pulmonologists, and nephrologists, should be aware of this potentially life-threatening, severe adverse event related to methimazole use.
# Funding
No funding was provided for this manuscript.
[fig] Figure 1: Histopathologic findings include: A: Necrotizing leukocytoclastic vasculitis with crescent formation in the glomerulus to the right, on a kidney biopsy performed days prior to the patient's death (PAS stain, × 100). B: focal intimal coronary artery vasculitis (arrow; H & E-stained section, × 40). C: Diffuse pulmonary alveolar hemorrhage (H & E-stained section, × 40). [/fig]
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10.3390/membranes5030369
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CCBY
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26266426
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s2orc_pubmed_articles
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Bio-Inspired Aquaporinz Containing Double-Skinned Forward Osmosis Membrane Synthesized through Layer-by-Layer Assembly
We demonstrated a novel AquaporinZ (AqpZ)-incorporated double-skinned forward osmosis (FO) membrane by layer-by-layer (LbL) assembly strategy. Positively charged poly(ethyleneimine) (PEI) and negatively charged poly(sodium 4-styrenesulfonate) (PSS) were alternately deposited on both the top and bottom surfaces of a hydrolyzed polyacrylonitrile (H-PAN) substrate. Subsequently, an AqpZ-embedded 1,2-dioleloyl-snglycero-3-phosphocholine (DOPC)/1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) supported lipid bilayer (SLB) was formed on PSS-terminated (T-PSS) membrane via vesicle rupture method. The morphology and structure of the biomimetic membranes were characterized by in situ atomic force microscopy (AFM), scanning electron microscope (SEM), Fourier transform infrared spectrometer using the attenuated total reflection technique (ATR-FTIR), and contact angle. Moreover, the FO performance of the resultant membrane was measured by using 2 M MgCl2 solution as draw solution and deionized (DI) water as feed solution, respectively. The membrane with a protein-to-lipid weight ratio (P/L) of 1/50 exhibits 13.2 L/m 2 h water flux and 3.2 g/m 2 h reversed flux by using FO mode, as well as 15.6 L/m 2 h water flux and 3.4 L/m 2 h reversed flux for PRO mode (the draw solution is placed against the active layer). It was also shown that the SLB layer of the double-skinnedOPEN ACCESSMembranes 2015, 5 370 FO membrane can increase the surface hydrophilicity and reduce the surface roughness, which leads to an improved anti-fouling performance against humic acid foulant. The current work introduced a new method of fabricating high performance biomimetic FO membrane by combining AqpZ and a double-skinned structure based on LbL assembly.
# Introduction
Aquaporins (AQPs) are a kind of transmembrane water channel protein that selectively allow the transport of water molecules because of their narrow channels with a dimensional structure and distinct charge characteristics [bib_ref] The aquaporin-Z water channel gene of Escherichia coli: Structure, organization and phylogeny, Calamita [/bib_ref]. It has been reported that the osmotic permeability of a single AQP is between 6 × 10 −14 to 24 × 10 −14 m 3 ·s −1 [bib_ref] Water transport in aquaporins: Osmotic permeability matrix analysis of molecular dynamics simulations, Hashido [/bib_ref]. AQP-containing biomimetic membranes hold great potential in water purification and seawater desalination due to the excellent permeability and the selectivity of AQP. AqpZ, the simplest member of the AQP family, is of particular interest for water treatment and desalination because of its lack of N-linked glycan, its resistance to proteolysis, and its high osmotic water permeability [bib_ref] Developing cell-free biology for industrial applications, Swartz [/bib_ref].
To date, several methods have been applied to fabricate AQP-containing biomimetic membranes, most of which are based on depositing AQP-incorporated vesicles onto porous substrates. Many efforts have been made to improve the performance of the AQP-based biomimetic membranes, including direct deposition of vesicles onto the substrates, pressure-assisted vesicle fusion, electrostatic force-induced vesicle adsorption, magnetic-enhanced vesicle deposition, and chemical crosslinking between functionalized lipid and the substrate [bib_ref] Layer-by-Layer Assembly of Aquaporin Z-Incorporated Biomimetic Membranes for Water Purification, Wang [/bib_ref] [bib_ref] Preparation of supported lipid membranes for aquaporin Z incorporation, Li [/bib_ref] [bib_ref] Highly permeable aquaporin-embedded biomimetic membranes featuring a magnetic-aided approach, Sun [/bib_ref] [bib_ref] Study on water transport through a mechanically robust Aquaporin Z biomimetic membrane, Wang [/bib_ref]. Wang et al. reported on an AqpZ-embedded supported lipid bilayer (SLB) by using an electrostatic layer-by-layer (LbL) deposition method to prepare a robust biomimetic membrane, which demonstrated an excellent nanofiltration (NF) performance [bib_ref] Layer-by-Layer Assembly of Aquaporin Z-Incorporated Biomimetic Membranes for Water Purification, Wang [/bib_ref]. fabricated SLB biomimetic membranes by spin-coating positively charged proteoliposomes on a modified NF-270 membrane via vesicle fusion [bib_ref] Preparation of supported lipid membranes for aquaporin Z incorporation, Li [/bib_ref]. Sun et al. encapsulated magnetic nanoparticles into the vesicles and utilized magnetic force to enhance the adsorption amount of AqpZ-incorporated vesicles on the polyelectrolyte film [bib_ref] Highly permeable aquaporin-embedded biomimetic membranes featuring a magnetic-aided approach, Sun [/bib_ref]. Wang et al. reported a method of synthesizing a FO biomimetic membrane [bib_ref] Study on water transport through a mechanically robust Aquaporin Z biomimetic membrane, Wang [/bib_ref]. In their process, the UV-crosslinked AqpZ-ABA block copolymer proteopolymersomes were immobilized on the membrane support, followed by stabilization through a layer-by-layer polydopamine-histidine coating process [bib_ref] Study on water transport through a mechanically robust Aquaporin Z biomimetic membrane, Wang [/bib_ref].
Forward osmosis (FO) utilizes an osmotic pressure gradient across the semipermeable membrane to separate clean water from feed solution (FS) [bib_ref] Forward osmosis: Principles, applications, and recent developments, Cath [/bib_ref]. Advantages of FO over pressure-driven membrane processes include lower energy consumption, less fouling propensity, and easy cleaning [bib_ref] Forward osmosis: Principles, applications, and recent developments, Cath [/bib_ref] [bib_ref] Chemical and physical aspects of organic fouling of forward osmosis membranes, Mi [/bib_ref]. There are more and more fields where FO can be applied, such as seawater desalination, wastewater treatment, FO-based membrane bioreactors, food processing, and power generation, etc. [bib_ref] Chemical and physical aspects of organic fouling of forward osmosis membranes, Mi [/bib_ref]. However, due to the asymmetric structure of the traditional FO membranes, the phenomenon called "internal concentration polarization" (ICP) is created severely, and it can lower the effective osmotic driving force for water transport and limit the water flux of the FO process to a large extent. When the feed solution is placed against the active layer (FO mode), dilutive ICP occurs within the membrane support layer as water permeates across the membrane from FS to draw solution (DS). On the contrary, concentrative ICP occurs as the solute in the feed solution accumulates within the membrane support layer, when the draw solution is placed against the active layer (PRO mode) [bib_ref] Recent developments in forward osmosis: Opportunities and challenges, Zhao [/bib_ref] [bib_ref] Influence of concentrative and dilutive internal concentration polarization on flux behavior in..., Mccutcheon [/bib_ref]. In the FO process, ICP usually causes a severe decline in water flux in the FO mode [bib_ref] Network modeling for studying the effect of support structure on internal concentration..., Li [/bib_ref] [bib_ref] Coupled effects of internal concentration polarization and fouling on flux behavior of..., Tang [/bib_ref]. However, foulants can hardly enter the pores of the porous support. For PRO mode, ICP is drastically reduced, but foulants tend to go into the porous support easily [bib_ref] Relating solution physicochemical properties to internal concentration polarization in forward osmosis, Zhao [/bib_ref]. In order to make full use of the advantages of the two different modes, double-skinned FO membranes have been designed and fabricated [bib_ref] Double-skinned forward osmosis membranes based on layer-by-layer assembly-FO performance and fouling behavior, Qi [/bib_ref] [bib_ref] Modeling double-skinned FO membranes, Tang [/bib_ref]. Compared with traditional single-skinned FO membranes, a dense rejection skin is formed on the bottom surface of the membrane. This additional skin can prevent solute reverse diffusion and reduce ICP effectively [bib_ref] Highly permeable double-skinned forward osmosis membranes for anti-fouling in the emulsified oil-water..., Duong [/bib_ref].
In our work, we combined AqpZ with double-skinned FO membranes to further improve the performances of the biomimetic FO membranes. The electrostatic LbL deposition method, which is based on the alternating adsorption of oppositely charged components [bib_ref] Stimuli-responsive LbL capsules and nanoshells for drug delivery, Delcea [/bib_ref] [bib_ref] Lipid Coating on Polyelectrolyte Surface Modified Colloidal Particles and Polyelectrolyte Capsules, Moya [/bib_ref] , has been demonstrated to form a robust biomimetic membrane [bib_ref] Layer-by-Layer Assembly of Aquaporin Z-Incorporated Biomimetic Membranes for Water Purification, Wang [/bib_ref]. Thus, we used this method to fabricate T-PSS membranes on the basis of porous substrates. AqpZ-embedded SLB was formed via vesicle rupture to obtain the bio-inspired AqpZ containing the double-skinned FO membrane. As shown in [fig_ref] Figure 1: Schematic diagram of the formation procedures for AqpZ-incorporated double-skinned FO membrane [/fig_ref] , on the top surface of a hydrolyzed polyacrylonitrile (H-PAN), poly(ethyleneimine) (PEI) was deposited to form the polycation layer, then polystyrene sulfonate (PSS) was deposited to form the polyanion layer. The process was repeated on the bottom surface of H-PAN. The positively charged 1,2-dioleloyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-3-trimethylammo-nium-propane (chloride salt) (DOTAP) mixture proteoliposomes were deposited onto the top surface of the LbL membrane to form a SLB. In the current work, we prepared three kinds of membranes: PEI-terminated double-skinned (T-PEI) FO membranes, PSS-terminated double-skinned (T-PSS) FO membranes, and AqpZ-incorporated double-skinned (T-AqpZ) FO membranes. FO performances and fouling tests of AqpZ-incorporated double-skinned FO membranes were investigated and compared with the single-skinned FO membranes.
## Experimental
# Materials and chemicals
Polyacrylonitrile (PAN, Mw ~50,000) was kindly supplied by the Shanghai Jingshan Petrochemical Company. N,N-dimethylformamide (DMF) and lithium chloride were purchased from Sinopharm (Shanghai, China). Polyethyleneimine (PEI, Mw ~750,000) and poly(sodium 4-styrene-sulfonate) (PSS, Mw ~70,000), 1-n-octyl-β-d-glucopyranoside (OG, purity > 99%) and glycerol (purity > 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1,2-dioleloyl-sn-glycero-3-phosphocholine (DOPC, purity > 99%) and 1,2-dioleoyl-3-trimethylammo-nium-propane (chloride salt) (DOTAP) were obtained from Avanti Polar Lipids (USA). AqpZ was expressed and purified using previously described procedures [bib_ref] Enhanced functional expression of aquaporin Z via fusion of in situ cleavable..., Zhang [/bib_ref]. Deionized water (Millipore) with a resistivity of 18.2 MΩ·cm was used to prepare buffer solutions and for FO measurement. Buffer solution used through the experiment was phosphate buffer solution (pH = 7.5) prepared with 10 mM KH2PO4, 10 mM K2HPO4 3H2O, and 150 mM NaCl.
## Preparation of liposomes and proteoliposomes
Liposome samples were prepared by the film rehydration method as previously described [bib_ref] Studies of supported phospholipid bilayers formed on nanofiltration membranes surface, Wang [/bib_ref]. Briefly, a N2 stream was used to evaporate chloroform from a chloroform solution of DOPC/DOTAP mixture with a molar ratio of DOPC: DOTAP = 4:1 to obtain a lipid film. The dried lipid film was left standing overnight in a vacuum to remove the residual organic solvent, and then rehydrated in phosphate buffer through vortexing, followed by five freeze-thaw cycles. The final solution was extruded 11 times with 100 nm polycarbonate membranes through an extruder system (Avanti Polar Lipids, Alabaster, AL, USA) to get the uniform small unilamellar vesicles with a final concentration of 0.1 mg/mL.
A certain amount of AqpZ solution was added into 0.1 mg/mL liposome solution containing 1 wt % OG to prepared samples with different protein-to-lipid ratios (P/L = 1/200, 1/100, 1/50). The solution was dialyzed in a 6-8 KDa molecular weight cut-off (MWCO) dialysis bag (Spectra Por) against 1 L phosphate buffer for three days at room temperature. The buffer was refreshed every day. After dialysis, the proteoliposome solution was extruded through a polycarbonate membrane with a mean pore size of 100 nm as stated above for further use.
## Preparation of pan substrates
PAN substrate was fabricated by using the phase inversion method. The PAN casting solution consisted of 18 wt % PAN and 2 wt % LiCl in DMF. The solution was stirred for 24 h at 60 °C to obtain a homogeneous solution and then cooled to room temperature for 24 h to remove the residual bubbles. A casting knife was used to cast the homogeneous solution on a clean glass plate, and then the glass plate was immediately immersed into a distilled water coagulation bath to form PAN substrates. Subsequently, the PAN substrates were soaked in a 1.5 M NaOH solution at 45 °C for 1.5 h and then rinsed by DI water until the pH of the rinse water became neutral [bib_ref] Influence of the properties of layer-by-layer active layers on forward osmosis performance, Qi [/bib_ref]. The alkali solution-treated PAN membrane was denoted as H-PAN.
## Fabrication of t-pss membrane
The LbL membrane was prepared by immersing the H-PAN substrate in 1 wt % PEI and 1 wt % PSS solution alternatively. Each deposition step lasted 30 min followed by a rinse of DI water for 5 min to remove the redundant polyelectrolytes. The procedure above could be repeated so that multilayer polyelectrolyte membranes with a specific bilayer number were fabricated. In our work, we adopted one PEI/PSS layer for the top rejection skin and then a similar method was used for the preparation of the bottom rejection layer. In that way, T-PSS membranes were fabricated.
## Aqpz-incorporated slb formation on t-pss membrane
The prepared liposome or proteoliposome solutions were spread on the top layer of the T-PSS membrane and incubated at room temperature for 4 h. Then the biomimetic membrane was rinsed by DI water for 5 min to remove the residual vesicles.
## Vesicle characterizations
Zetasizer (NanoZS 90, Malvern Instruments Limited, Worcestershire, UK) was used to characterize the size and zeta potential of liposomes and proteoliposomes. A 633 nm laser source, with a fixed detector angle of 90°, was used in the dynamic mode. We used a stopped-flow apparatus (Applied Photophysics Ltd., Surrey, UK), with light source at emission wavelength of 577 nm, to characterize the permeability of the liposomes and proteoliposomes. Samples were rapidly mixed with a high-osmotic solution (0.5 mol/L sucrose). The osmolarity difference (∆osm = 290 mosm/L, Osmomat 030, Gonotec, Germany) impelled the water effluent from the vesicles. By fitting the data to an exponential increase equation, the initial rate of vesicle shrinkage (k) was determined. Thereafter, the osmotic water permeability (Pf) was calculated by using the following equation:
[formula] Pf = k / ((S/V0) × VW × Δosm)(1) [/formula]
where S/V0 is the vesicle's initial surface-to-volume ratio, VW is the molar volume of water (0.018 L/mol), and ∆osm is the osmolarity difference after mixing. All of the measurements were carried out at 25 ± 0.1 °C.
## Membrane characterizations
The functionalized groups of the resultant membrane were detected by the Fourier transform infrared (FTIR) spectrometer (Tensor 27, Bruker, Germany), using the attenuated total reflection (ATR) technique.
The hydrophilicity of the prepared membrane was characterized by an automatic contact angle meter (DSA100, Kruss, Germany). Then 0.5 mL DI water was dropped onto the membrane surface using a microsyringe under ambient conditions and contact angle was measured until no further change. At least six random locations were measured for each sample in order to minimize the experimental error and then obtain the average value. Both the top surfaces and bottom surfaces of the double-skinned LbL FO membranes were measured.
The surface morphology and roughness of the membranes were investigated via atomic force microscopy (AFM, Nanoscope V MultiMode, Veeco, Plainview, NY, USA). Tapping mode measurements in liquid were performed by using NP-S cantilevers (short lever, nominal spring constant 0.06 N/m). Surface roughness values were obtained from AFM images with the help of the instrument software.
The morphology and structure of original and modified membranes were analyzed by scanning electron microscope (SEM, S-4800, Hitachi, Japan). Dry membrane samples were frozen in liquid nitrogen and subsequently cracked in order to obtain the cross sections.
## Fo performance
FO performances of the fabricated membranes were conducted in a lab-scale cross-flow FO filtration unit with an effective membrane area of 36.0 cm 2 . The linear velocity was 6.4 cm/s. We denoted the AqpZ-embedded SLB active layer facing FS as the FO mode. Accordingly, the active layer facing DS was denoted as pressure retarded osmosis (PRO) mode. The FO performances of double-skinned membranes were evaluated under both FO and PRO mode. As a control, single-skinned membranes, which were prepared by depositing only the top layer of H-PAN with polyelectrolytes and AqpZ-embedded SLB, were also measured.
Then, 2 mol/L MgCl2 solution was used as DS and DI water was used as FS, respectively. The water flux was calculated by measuring the weight change of DS every 2 min. The solute reverse flux of the membrane was calculated from the conductivity change.
The water flux (Jv in L/m 2 ·h) and solute reverse flux (Js in g/m 2 ·h) were calculated by the following equations [bib_ref] Highly crosslinked layer-by-layer polyelectrolyte FO membranes: Understanding effects of salt concentration and..., Duong [/bib_ref] :
[formula] = ∆ ×∆ (2) [/formula]
where ΔV is the volume changes of DI water (density is 1000 g/L); Am is the effective membrane area; Δt is the measuring time interval.
[formula] = ×∆(3) [/formula]
where V0 and Vt are the initial and final volumes of feed solution, respectively; C0 and Ct are the initial and final solute concentrations of feed solution, respectively.
The anti-fouling tests of double-skinned membranes were performed in the FO mode. Prior to the fouling test, the FO membrane was first tested using 2 mol/L MgCl2 DS and DI water FS for 1 h to obtain the initial water flux. Anti-fouling tests were performed with 5 mg/L humic acid (HA) as the FS for 1 h. Subsequently, both the FS and DS were changed into DI water to rinse the membrane for 0.5 h, which was the cleaning process. Finally, 2 mol/L MgCl2 DS and DI water FS were used for 1 h to measure the recovered water flux. The fouling and cleaning cycles were performed three times, alternatively. [fig_ref] Table 1: Characterization of DOPC, DOPC/DOTAP liposomes, and proteoliposomes [/fig_ref] summarizes the size and zeta potential of DOPC, DOPC/DOTAP liposomes, and proteoliposomes at different P/L (w/w) ratios. The vesicles exhibited a narrow size range from 114 to 122 nm and their polydispersity index (PDI) was consistently smaller than 0.2, indicating a narrow size distribution of the vesicles [bib_ref] Interaction of cationic surfactant and anionic polyelectrolytes in mixed aqueous solutions, Petzold [/bib_ref]. Moreover, proteoliposomes showed decreased zeta potential after incorporation of AqpZ because the incorporated proteins were negatively charged in phosphate buffer saline PBS (pH = 7.5) [bib_ref] Fusion behaviour of aquaporin Z incorporated proteoliposomes investigated by quartz crystal microbalance..., Li [/bib_ref] [bib_ref] Preparation of high performance nanofiltration (NF) membranes incorporated with aquaporin Z, Li [/bib_ref]. Apparently, the incorporation of AqpZ had a major influence on the physicochemical properties of the vesicles. [fig_ref] Table 1: Characterization of DOPC, DOPC/DOTAP liposomes, and proteoliposomes [/fig_ref] also summarized the water permeability data. The permeability of AqpZ-incorporated proteoliposomes was measured by the stopped-flow apparatus. Here, proteoliposomes showed a much higher shrinkage rate (k) than liposomes, which could be attributed to the higher water permeation of AqpZ. Moreover, the permeability of proteoliposomes increased as the P/L ratio increased because more incorporated AqpZ enhanced the permeability of the vesicles. Based on the data of the proteoliposomes with the protein-to-lipid molar ratio of 1:50, the permeability of each AqpZ was calculated to be 32.4 × 10 −14 cm 3 ·s −1 , which was comparable to the previously reported results [bib_ref] Effects of Proteoliposome Composition and Draw Solution Types on Separation Performance of..., Zhao [/bib_ref] [bib_ref] Functional Reconstitution and Characterization of AqpZ, the E. coli Water Channel Protein, Borgnia [/bib_ref].
# Results and discussion
## Characteristics of liposomes and proteoliposomes
## Atr-ftir characterization
ATR-FTIR analyses were performed to verify that the AqpZ-incorporated double-skinned FO membranes were successfully fabricated. As shown in [fig_ref] Figure 2: ATR-FTIR spectra of various membranes [/fig_ref] , the peak at 2245 cm −1 was due to stretching vibration of the C≡N moiety of the PAN substrate. After being treated by 1.5 M NaOH solution, most of the CN groups convert to COO − groups as previously reported [bib_ref] Layer-by-Layer Assembly of Aquaporin Z-Incorporated Biomimetic Membranes for Water Purification, Wang [/bib_ref]. As a result, the H-PAN membrane exhibited a peak at approximately 1677 cm −1 , representing the C=O bond in COO − groups. After PEI deposition, the intensity of the N-H bend and C-N stretch of the CONH group increased at around 1553 cm −1 , which suggested that the nucleophilic amine groups were formed on the membrane. For the T-PSS membrane, two new peaks appeared at 1068 cm −1 and 1042 cm −1 , which corresponded to the S=O bond stretching vibration. When AqpZ-incorporated DOPC/DOTAP SLB was formed on the polyelectrolyte membrane (the membrane we denoted as T-AqpZ), the characteristic peak attributed to C-C-N + groups appeared at around 947 cm −1 . As well, the peak at 715 cm −1 was attributed to the vibration of P-O-C groups. The spectra of the membrane bottom surfaces with different outermost layers were shown in [fig_ref] Figure 2: ATR-FTIR spectra of various membranes [/fig_ref]. The characteristic peaks belonging to H-PAN, PEI, and PSS could be found, which demonstrated that the AqpZ-incorporated FO membrane with a double-skinned structure was successfully fabricated.
## Hydrophilicity of membranes
The hydrophilicity of the membrane surface is usually evaluated by the water contact angle. In general, a higher contact angle of the membrane surface indicates higher hydrophobicity. The water contact angles of the FO membranes are illustrated in [fig_ref] Figure 3: Contact angles of PAN, H-PAN, T-PEI [/fig_ref]. The water contact angle of PAN was 56.6°. After deposition of PEI/PSS, the water contact angle dropped to 31.6°. This can be understood by the fact that acidic COO − groups and some amine groups on the surface of the T-PSS membrane have a strong interaction with water molecules. Finally, after covering with proteoliposomes, the water contact angle made a further decrease to 20.1°. This can be understood by the fact that the SLB with high hydrophilicity covered the surface of the membrane and greatly improved the surface hydrophilicity [bib_ref] Towards supported bolaamphiphile membranes for water filtration: Roles of lipid and substrate, Kaufman [/bib_ref].
## Morphologies of the fo membrane
The SEM images of fabricated membranes are shown in [fig_ref] Figure 4: SEM images of various membranes [/fig_ref]. After the deposition of AqpZ-embedded SLB, the gaps in the top surface were filled so that the membrane appeared to be smoother and less defective. As for the cross-section images, finger-like pores were formed in the asymmetric membranes [bib_ref] Fabrication and characterization of forward osmosis hollow fiber membranes with antifouling NF-like..., Setiawan [/bib_ref]. Both the top surface and the bottom surface had a dense selective layer and this layer on the bottom surface was looser than that on the top surface, which was attributed to the difference of roughness and pore size of the top surface and the bottom surface. This was due to the fact that the large pores on the bottom surface led to an uneven deposition of polyelectrolytes and proteoliposomes. The cross-section images proved the fabrication of the double-skinned membrane structure [bib_ref] Double-skinned forward osmosis membranes based on layer-by-layer assembly-FO performance and fouling behavior, Qi [/bib_ref].
## Afm characterization
AFM was used to characterize the roughness of various membranes. As shown in [fig_ref] Figure 5: AFM images [/fig_ref] , top surfaces of the membranes were flatter than bottom surfaces, correlating well with the SEM images. The roughness of the top surface ranged from 4.131 to 10.638 mm, which was much smaller than the roughness of the bottom surface, ranging from 12.215 to 20.641 mm. This might be induced by more large pores existing at the bottom surface. After LbL deposition, the roughness of both the top and the bottom surface of membranes became higher than the H-PAN substrate because of the random adsorption of polyelectrolytes on the membranes [bib_ref] Characteristics of polyelectrolyte multilayers: Effect of PEI anchoring layer and posttreatment after..., Kolasinska [/bib_ref] [bib_ref] Surface engineerings of polyacrylonitrile-based asymmetric membranes towards biomedical applications: An overview, Wang [/bib_ref]. Moreover, the PSS layer led to the decrease of roughness due to the shorter and more inflexible hydrocarbon chains of PSS than PEI. When the SLB was formed on the T-PSS membrane, the roughness decreased accordingly, and was even lower than the H-PAN substrate [bib_ref] Characteristics of polyelectrolyte multilayers: Effect of PEI anchoring layer and posttreatment after..., Kolasinska [/bib_ref]. This can be understood by the fact that AqpZ-embedded SLB filled up the gaps on the substrates, which in turn proved the successful formation of AqpZ-embedded SLB.
## Fo performances of double-skinned membranes
The FO performance of AqpZ-incorporated double-skinned FO membranes was evaluated by using a 2M MgCl2 solution as the DS and DI water as the FS. Both the FO and PRO modes (the draw solution is placed against the active layer) were measured and shown in [fig_ref] Figure 6: FO performances of double-skinned membranes by using 2 mol/L MgCl2 as the... [/fig_ref]. The Jv of AqpZ-absent SLB-covered double-skinned FO membrane was 3.5-4.8 L/m 2 ·h and the Js/Jv was about 0.2 g/L. The low water flux could be explained by the fact that the lipid bilayer was impermeable to water. However, due to the existence of defects on the SLB, the membrane exhibited a low water flux and reversed salt solution flux [bib_ref] Preparation of supported lipid membranes for aquaporin Z incorporation, Li [/bib_ref]. After the embedding of AqpZ with different protein-to-lipid ratios (P/L from 1/200 to 1/50), Jv increased from 13.7 to 15.6 L/m 2 ·h by using the PRO mode, while the Js/Jv slightly increased from 0.21 to 0.24 g/L. This can be understood by considering the fact that plenty of water channels were provided by AqpZ for water molecules to pass through the biomimetic membranes. However, the zeta potential of proteoliposomes decreased with the increase of the P/L ratio (as shown in [fig_ref] Table 1: Characterization of DOPC, DOPC/DOTAP liposomes, and proteoliposomes [/fig_ref]. Therefore, the interaction between the polyelectrolyte layer and the SLB layer weakened, and then more defects appeared and induced the increase of reversed solute flux. A similar phenomenon was also observed by Wang et al. [bib_ref] Highly permeable and selective pore-spanning biomimetic membrane embedded with aquaporin, Wang [/bib_ref]. It was also worth noting that the membranes exhibited higher Jv and lower Js/Jv at the PRO mode than at the FO mode. This was mainly because of the denser top layer at the PRO mode, which prevented more draw solutes from entering into the membrane and reduced the ICP level as a result [bib_ref] Double-skinned forward osmosis membranes based on layer-by-layer assembly-FO performance and fouling behavior, Qi [/bib_ref].
## Fo performances of single-skinned and double-skinned fo membranes
As shown in [fig_ref] Figure 7: FO performances of AqpZ-incorporated single-skinned and double-skinned FO membranes in the FO... [/fig_ref] , AqpZ-incorporated single-skinned FO membranes were also measured for comparison. Both the double-skinned membranes and single-skinned membranes were performed in the FO mode, where the active layer faced the FS. In the FO mode, dilutive ICP occurs within the membrane support layer as water permeates across the membrane from the FS to the draw solution, which usually causes a severe decline in water flux, so the flux experiments were performed in the FO mode to confirm that the double-skinned FO membrane could reduce the ICP level. The single-skinned FO membranes incorporated with AqpZ (P/L = 1/50) had the highest Jv at 14.5 L/m 2 ·h. The reason for this result could be related to two aspects. Firstly, single-skinned FO membranes possessed less resistance than double-skinned FO membranes because of the lack of a secondary selective layer. Secondly, membranes with P/L = 1/50 provided more water channels than membranes with P/L = 1/100 and P/L = 1/200, which allowed more water molecules to pass through the membrane. Although single-skinned membranes showed higher Jv, the double-skinned structure significantly reduced Js/Jv, indicating the higher solute rejection of the double-skinned FO membranes. Both kinds of membranes showed excellent abilities of water separation compared to membranes without AqpZ (P/L = 0), which confirmed the fact that AqpZ was beneficial to FO biomimetic membranes.
## Fo fouling tests
The FO fouling tests were performed with AqpZ (P/L = 1/50)-incorporated membranes in the PRO mode. In the PRO mode, foulants could go into the porous support easily. Therefore, the PRO mode was used to test the anti-fouling capacity of the double-skinned FO membrane. [fig_ref] Figure 8: Normalized fluxes of AqpZ-incorporated single-skinned and double-skinned FO membranes during three cycles... [/fig_ref] shows the comparison of double-skinned FO membranes and single-skinned FO membranes with normalized fluxes. Before fouling, the initial water fluxes of both membranes were measured by using 2M MgCl2 as the DS and DI water as the FS for 1 h. The water flux slightly dropped because of the decrease of the driven pressure induced by the dilutive ICP [bib_ref] Influence of concentrative and dilutive internal concentration polarization on flux behavior in..., Mccutcheon [/bib_ref]. Remarkably, the double-skinned membrane had more advantages in the anti-fouling test. The single-skinned membrane suffereda severe decline in water flux (decreased to ~70.5%) within a few minutes after the fouling test. Similar results were reported for several types of single-skinned FO membranes [bib_ref] Chemical and physical aspects of organic fouling of forward osmosis membranes, Mi [/bib_ref] [bib_ref] Double-skinned forward osmosis membranes based on layer-by-layer assembly-FO performance and fouling behavior, Qi [/bib_ref]. This can be understood by the fact that HA in FS could easily enter into the pores of the PAN substrates and cause increased membrane resistance and reduced water molecule transport, which in turn causes severe flux loss [bib_ref] Double-skinned forward osmosis membranes based on layer-by-layer assembly-FO performance and fouling behavior, Qi [/bib_ref]. However, the water flux of double-skinned membranes just decreased slightly (decreased to ~85.5%), which demonstrated that the double-skinned FO membrane could effectively mitigate fouling. This may be caused by the fact that the added secondary selective layer was dense enough to prevent the foulant from clogging the water molecule channels in the membrane [bib_ref] Double-skinned forward osmosis membranes based on layer-by-layer assembly-FO performance and fouling behavior, Qi [/bib_ref]. Moreover, the double-skinned FO membranes exhibited higher normalized recovery flux at 85.1%, whereas, the normalized recovery flux of single-skinned FO membranes was 71.8% after three cycles of fouling and cleaning. It indicated that the double-skinned FO membranes possessed huge potential in anti-fouling capacity. Our work provided a novel structure of AqpZ-incorporated FO membranes, which enhanced the anti-fouling stability and offers a clear advantage for FO applications.
# Conclusions
We successfully fabricated and characterized the AqpZ-incorporated double-skinned FO membranes based on layer-by-layer assembly in the current work. To the best knowledge of the authors, this is the first study reporting the combination of AqpZ and double-skinned FO membranes. The addition of AqpZ has made a great contribution to the water flux and salt rejection. The changes were obvious in the water flux. The double-skinned FO membranes possessed a relatively loose secondary selective layer, which decreased ICP and ensured the water flux. Through fouling tests, the double-skinned FO membranes demonstrated a much better anti-fouling capacity compared to the single-skinned FO membranes.
# Acknowledgments
This work was supported by the National Natural Science Foundation of China (21106139, 21476219, and 21276226), the Science and Technology Development Plan of Shandong Province (2014GSF116006), and the Science and Technology Program for Fundamental Research of Qingdao (13-1-4-251-jch). This is MCTL contribution No. 78.
# Author contributions
Zhining Wang designed and planned this research; Shuzheng Wang fabricated the membranes and tested the performance of the membranes; Wande Ding assisted in fabrication and test; Zhinan Xu and Jin Cai prepared and purified the AqpZ. Shuzheng Wang wrote the body of this paper under the guidance of Zhining Wang; Zhinan Xu, Wande Ding and Jin Cai modified this paper.
[fig] Figure 1: Schematic diagram of the formation procedures for AqpZ-incorporated double-skinned FO membrane. [/fig]
[fig] Figure 2: ATR-FTIR spectra of various membranes. (A) Top surfaces of membranes; (B) Bottom surfaces of membranes. [/fig]
[fig] Figure 3: Contact angles of PAN, H-PAN, T-PEI (top surface), T-PEI (bottom surface), T-PSS (top surface), T-PSS (bottom surface), and T-AqpZ membrane. PAN-OH T-PEI(Top)T-PEI(Bottom)T-PSS(Top)T-PSS(Bottom) T- [/fig]
[fig] Figure 4: SEM images of various membranes. (A,B) top surface of T-PSS membrane; (C,D) bottom surface of T-PSS membrane; (E,F) top surface of T-AqpZ membrane; (G) cross-section of top surface of T-PSS membrane; (H) cross-section of bottom surface of T-PSS membrane. [/fig]
[fig] Figure 5: AFM images (scan size 2 × 2 μm 2 ，z-scale = 100 nm) of the various membranes. (A) Top surface of H-PAN; (B) Top surface of T-PEI; (C) Top surface of T-PSS; (D) Top surface of T-AqpZ; (E) Bottom surface of H-PAN; (F) Bottom surface of T-PEI; (G) Bottom surface of T-PSS. [/fig]
[fig] Figure 6: FO performances of double-skinned membranes by using 2 mol/L MgCl2 as the DS and DI water as the FS. (A) Jv of double-skinned membranes; (B) Js/Jv of double-skinned membranes. [/fig]
[fig] Figure 7: FO performances of AqpZ-incorporated single-skinned and double-skinned FO membranes in the FO mode. (A) Jv of AqpZ-incorporated single-skinned and double-skinned FO membranes; (B) Js/Jv of AqpZ-incorporated single-skinned and double-skinned FO membranes. [/fig]
[fig] Figure 8: Normalized fluxes of AqpZ-incorporated single-skinned and double-skinned FO membranes during three cycles of humic acid (HA) solution and DI water FO process. [/fig]
[table] Table 1: Characterization of DOPC, DOPC/DOTAP liposomes, and proteoliposomes. [/table]
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Implications of Elevated Fibrosis-4 Index in Patients Receiving Trans-Catheter Aortic Valve Replacement
Citation: Imamura, T.; Narang, N.; Onoda, H.; Tanaka, S.; Ushijima, R.; Sobajima, M.; Fukuda, N.; Ueno, H.; Kinugawa, K. Implications of Elevated Fibrosis-4 Index in Patients Receiving Trans-Catheter Aortic Valve Replacement.
# Background
Survival following trans-catheter aortic valve replacement (TAVR) has improved considerably partly due to improvements in peri-procedural management and patient selection optimization [bib_ref] Transcatheter or Surgical Aortic-Valve Replacement in Intermediate-Risk Patients, Leon [/bib_ref] [bib_ref] Transcatheter Aortic-Valve Replacement with a Balloon-Expandable Valve in Low-Risk Patients, Mack [/bib_ref]. Nevertheless, adverse events after TAVR can occur, including decompensated heart failure [bib_ref] Predictors and Risk Calculator of Early Unplanned Hospital Readmission Following Contemporary Self-Expanding..., Sanchez [/bib_ref] [bib_ref] Determinants and Impact of Heart Failure Readmission Following Transcatheter Aortic Valve Replacement, Auffret [/bib_ref]. It is clinically challenging to ascertain predictive factors that are associated with heart failure recurrence following TAVR given the complex interplay of valvular and intrinsic myocardial dysfunction.
The fibrosis-4 index is a calculated metric used originally to assess the risk of hepatic fibrosis progression in patients with hepatitis [bib_ref] Comparison of diagnostic accuracy of aspartate aminotransferase to platelet ratio index and..., Xiao [/bib_ref]. This index is calculated using four standard clinical parameters associated with hepatic function: {[glutamic oxaloacetic transaminase (IU/L)] × [age (years)]}/{[platelet counts (×10 9 /L) × [glutamic pyruvic transaminase (IU/L)] 1/2 } [bib_ref] Comparison of diagnostic accuracy of aspartate aminotransferase to platelet ratio index and..., Xiao [/bib_ref]. Recently, the fibrosis-4 index has been used in patients with chronic heart failure [bib_ref] Impact of predictive value of Fibrosis-4 index in patients hospitalized for acute..., Shibata [/bib_ref] , where an elevated fibrosis-4 index may be associated with systemic congestion and worse clinical prognosis [bib_ref] Prognostic significance of liver stiffness assessed by fibrosis-4 index in patients with..., Takae [/bib_ref].
This study aimed to investigate the association between the fibrosis-4 index and risk of heart failure recurrence following TAVR.
# Methods
## Patient selection
Patients who underwent TAVR at our institute between 2015 and 2020 were included in this retrospective study. All patients were followed from index discharge for 2 years or until Jun 2021. Written informed consent was obtained from all participants on admission. The institutional review board approved the study protocol.
## Tavr procedure
Patients with severe aortic stenosis with max velocity > 4.0 m/s, mean pressure gradient > 40 mmHg, or aortic valve area < 1.0 cm 2 were considered for TAVR in a multidisciplinary heart-valve team conference.
All patients received TAVR according to the standard procedure. Patients received self-expandable valves (Corevalve, Evolut R, Evlolut PRO, or Evolut PRO+; Medtronic plc., Minneapolis, Minnesota) or balloon-expandable valves (Sapien XT or Sapien 3; Edwards Lifesciences Inc., Irvine, CA, USA) via trans-femoral, trans-aorta, trans-subclavian, or direct aorta approach under general or local anesthesia support.
## Clinical variables
Demographic, laboratory, echocardiographic, hemodynamics, and medication data within one week before TAVR were collected. Laboratory, echocardiographic, and medication data following TAVR were also collected.
The baseline fibrosis-4 index was calculated as an independent variable according to the following formula: {[glutamic oxaloacetic transaminase (IU/L)] × [age (years)]}/{[platelet counts (×10 9 /L) × [glutamic pyruvic transaminase (IU/L)] 1/2 } [bib_ref] Comparison of diagnostic accuracy of aspartate aminotransferase to platelet ratio index and..., Xiao [/bib_ref]. The fibrosis-4 index was calculated also following TAVR.
## Clinical outcomes
All patients were followed up at our institute or affiliated institutes. Heart failure readmissions that required IV diuretics or any other intensive therapies under in-hospital observation were defined as the primary endpoint. All-cause death was also counted. The plasma B-type natriuretic peptide level at one-year follow-up was collected.
# Statistical analysis
Continuous variables were presented as a median and an interquartile range and compared using the Mann-Whitney U test. Categorical variables were presented as numbers and percentages and compared using Fisher's exact test. A two-tailed p < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS Statistics 22 (SPSS Inc, Armonk, IL, USA) and ESR (Ver 1.54; Saitama Medical Center Jichi Medical University, Saitama, JPN).
The primary endpoint was two-year heart failure readmissions. A time-dependent receiver operating characteristics analysis was conducted to calculate the cutoff for the baseline fibrosis-4 index at the primary endpoint using a Youden index. The patient cohort was stratified into two groups using this cutoff.
Cumulative incidence comparisons with a Gray test between the two groups were performed by accounting for death as a competing risk. Negative binomial regression analyses were performed to compare event rates between the two groups. Fine-Gray proportional hazard ratio regression analyses were performed to investigate the impact of the baseline fibrosis-4 index on the primary endpoint by adjusting for potential baseline characteristics that were significantly different in the comparison study (age, body surface area, hemoglobin, plasma B-type natriuretic peptide, and peak velocity at aortic valve).
# Results
## Baseline characteristics
A total of 272 patients were included [fig_ref] Table 1: Pre-procedural baseline characteristics [/fig_ref]. Median age was 85 (82, 88) years old, and 28% were men. The median plasma B-type natriuretic peptide level was 271 (123, 556) pg/mL. The median left ventricular ejection fraction was 64% (54%, 70%). The median peak velocity at aortic valve was 4.5 (4.0, 4.9) m/s. The median STS score was 5.3 (4.2, 7.7), and the median EURO II score was 3.5 . The median fibrosis-4 index was 2.8 (2.2, 3.7). The distribution of the fibrosis-4 index at baseline is displayed in [fig_ref] Figure 1: Distribution of the baseline fibrosis-4 index [/fig_ref].
## Post-procedural data
Following TAVR, the median plasma B-type natriuretic peptide was 127 (86, 271) pg/mL. The median peak velocity at aortic valve was 2.1 (1.8, 2.4) m/s. All other clinical parameters are displayed in [fig_ref] Table 2: Post-procedural clinical data [/fig_ref].
## Baseline fibrosis-4 index and heart failure readmissions
The cutoff of the baseline fibrosis-4 index associated with the primary endpoint was calculated as 3.79 [fig_ref] Figure 2: Cutoff of fibrosis-4 index for the primary endpoint [/fig_ref]. Patients with a high fibrosis-4 index (>3.79) were older, had lower hemoglobin levels, and had higher plasma B-type natriuretic peptide levels than the low fibrosis-4 index group (p < 0.05 for all; [fig_ref] Table 1: Pre-procedural baseline characteristics [/fig_ref]. Following TAVR, those with a high fibrosis-4 index had a lower estimated glomerular filtration ratio and higher plasma B-type natriuretic peptide (p < 0.05 for both; [fig_ref] Table 2: Post-procedural clinical data [/fig_ref]. During the observational period following the index discharge for a median of 730 (704, 730) days, there were 11 and 8 heart failure readmissions in the high and low fibrosis-4 index groups, respectively. Most of them (15/19) were preserved ejection fraction, and others were mildly reduced ejection fraction (3/19) or reduced ejection fraction (1/19). A 2-year cumulative incidence of the primary endpoint was higher in the high fibrosis-4 index group (18% versus 4%, p < 0.001; [fig_ref] Figure 3: Cont. [/fig_ref]. The event rates of the primary endpoint were higher in the high fibrosis-4 index group (0.1041 versus 0.0222 events/year), with an incidence rate ratio of 5.99 (95% confidence interval 5.65-6.33, p < 0.001; [fig_ref] Figure 3: Cont. [/fig_ref]. The impact of baseline fibrosis-4 index on the incidence of primary endpoint was calculated as a hazard ratio 1.27 (95% confidence interval 1.04-1.54, p = 0.019), which was adjusted for five potential confounders that were significantly different in the comparison analysis (age, body surface area, hemoglobin, plasma B-type natriuretic peptide, and peak velocity at aortic valve) [fig_ref] Table 3: Impact of the baseline fibrosis-4 index on the primary endpoint [/fig_ref].
## Other clinical outcomes
At one-year follow-up, median plasma B-type natriuretic peptide did not significantly differ between the two groups (100 (71, 165) pg/mL versus 87 (42, 164) pg/mL, p = 0.15). There were 4 and 16 deaths in the high and low fibrosis-4 index groups, respectively. The 2-year cumulative mortality rate did not significantly differ between the two groups (4% versus 5%, p = 0.66) [fig_ref] Figure 3: Cont. [/fig_ref].
## Changes in fibrosis-4 index following tavr
Among the 64 patients with high baseline fibrosis-4 indexes, 58 had fibrosis-4 indexes that were also obtained at index discharge. Of them, 18 patients (31%) achieved decreases in their fibrosis-4 index below the cutoff level (<3.79) at index discharge. There were no significant differences in the baseline characteristics between the two groups (decreased and persistently high groups), except for a higher max velocity at the aortic valve in the group with decreases in their fibrosis-4 index (p = 0.010; [fig_ref] Table 4: Baseline characteristics among those with a high baseline fibrosis-4 index [/fig_ref]. The group with a decreased fibrosis-4 indexes tended to be associated with a lower cumulative incidence of the primary endpoint (6% versus 26%, p = 0.079; [fig_ref] Figure 4: Cumulative incidence of the primary endpoint accounting for death as a competing... [/fig_ref]. Mortality was not significantly different between the two groups (5% versus 3%, p = 0.55). Abbreviations are the same as in [fig_ref] Table 1: Pre-procedural baseline characteristics [/fig_ref]. Continuous variables are presented as median and interquartile and compared using the Mann-Whitney U test. Categorical variables are presented as numbers and percentages and compared using the Fischer's exact test. * p < 0.05. Among the 64 patients with high baseline fibrosis-4 indexes, 6 were excluded due to a lack of data.
# Discussion
In this study, we investigated the impact of the baseline fibrosis-4 index on heart failure readmissions following TAVR. We observed that (1) the baseline fibrosis-4 index was elevated in patients with severe aortic stenosis and was associated with more advanced heart failure; (2) the baseline fibrosis-4 index was associated with an increased incidence of heart failure readmissions following TAVR, even after adjustment for other potential confounders; and 3) the peri-procedural improvement in fibrosis-4 index tended to be associated with a lower incidence of heart failure readmissions.
## Fibrosis-4 index and aortic stenosis
The fibrosis-4 index was originally introduced to predict the progression of hepatic fibrosis in patients with hepatic diseases [bib_ref] Comparison of diagnostic accuracy of aspartate aminotransferase to platelet ratio index and..., Xiao [/bib_ref]. The use of this index has recently expanded to assess the degree of right-sided volume overload and to predict prognosis in patients with acute/chronic heart failure [bib_ref] Impact of predictive value of Fibrosis-4 index in patients hospitalized for acute..., Shibata [/bib_ref] [bib_ref] Prognostic significance of liver stiffness assessed by fibrosis-4 index in patients with..., Takae [/bib_ref] [bib_ref] Clinical Significance of the Fibrosis-4 Index in Patients with Acute Heart Failure..., Shirakabe [/bib_ref] , pulmonary hypertension due to left heart disease [bib_ref] The Fibrosis-4 Index Is Useful for Predicting Mortality in Patients with Pulmonary..., Yokokawa [/bib_ref] , and atrial fibrillation [bib_ref] Impact of the Fibrosis-4 Index on Risk Stratification of Cardiovascular Events and..., Saito [/bib_ref]. The fibrosis-4 index would reflect hepatic injury via systemic congestion, mostly due to hepatic congestion, and hypo-perfusion due to low cardiac output. This is the first study to investigate the fibrosis-4 index in patients with aortic stenosis. The median value was 2.8, which is almost comparable with other previous studies including heart failure cohorts [bib_ref] Impact of predictive value of Fibrosis-4 index in patients hospitalized for acute..., Shibata [/bib_ref] [bib_ref] Prognostic significance of liver stiffness assessed by fibrosis-4 index in patients with..., Takae [/bib_ref]. Of note, in a study by Takae and colleagues, a higher fibrosis-4 index was associated with clinical outcomes in patients with diastolic heart failure but not in patients with systolic heart failure [bib_ref] Prognostic significance of liver stiffness assessed by fibrosis-4 index in patients with..., Takae [/bib_ref]. They hypothesized that the underlying mechanisms that explained their findings would be systemic tissue fibrosis with a similar pathophysiology between the heart and the liver. A similar pathophysiology might exist in our cohort with aortic stenosis, which often accompanies diastolic heart failure with myocardial fibrosis.
In our study, those with a higher fibrosis-4 index had incremental plasma levels of Btype natriuretic peptide and dilutional anemia, both of which may suggest greater systemic volume overload and more advanced heart failure. Greater impairment of renal function might reflect glomerular congestion due to cardio-renal syndrome in patients with a higher fibrosis-4 index after TAVR. Given these findings, we strongly encourage clinicians to calculate the fibrosis-4 index in all patients with aortic stenosis to assess the refractory systemic congestion and the degree of heart failure progression.
## Prognostic implication of fibrosis-4 index
Recurrent heart failure following TAVR has negative prognostic implications [bib_ref] Determinants and Impact of Heart Failure Readmission Following Transcatheter Aortic Valve Replacement, Auffret [/bib_ref]. Elevations in intra-cardiac filling pressures and the existence of refractory pulmonary hypertension are associated with poor outcomes following TAVR [bib_ref] Decoupling Between Pulmonary Artery Diastolic and Wedge Pressure Following Transcatheter Aortic Valve..., Imamura [/bib_ref]. The existence of advanced tricuspid regurgitation is also independently associated with poor outcomes after TAVR [bib_ref] Association of Tricuspid Regurgitation With Transcatheter Aortic Valve Replacement Outcomes: A Report..., Mccarthy [/bib_ref]. Collectively, it is not surprising that a baseline elevated fibrosis-4 index, probably indicating these clinical conditions, was associated with recurrent heart failure following TAVR. Beyond the assessment of heart failure severity, we highly encourage clinicians to calculate the fibrosis-4 index for risk stratification and appropriate shared decision making for all TAVR candidates, particularly the elderly cohort. Some patients who are older with advanced frail and multiple comorbidities might not select TAVR following repeat shared decision making if their calculated fibrosis-4 index is high.
A post-procedural improvement in fibrosis-4 index tended to be associated with a lower incidence of heart failure recurrence. We also showed this important finding in the shared decision-making process. Although further studies are needed to establish how to improve the fibrosis-4 index, aggressive therapeutic approaches using neuro-hormonal blockers and diuretics might be promising tools.
# Limitations
This is a retrospective study consisting of a moderate sample size. We observed the association between baseline fibrosis-4 index levels and heart failure recurrence. Detailed mechanisms that explain the causality remains uncertain. We cannot draw any conclusions between fibrosis-4 index levels and mortality due to the low total number of deaths following TAVR. We performed multivariate analyses but other unadjusted potential confounders may also have impacted the risk of the primary endpoint. Patients with liver cirrhosis did not receive TAVR and were not included in this study. Sarajlic and colleagues demonstrated that the sex-specific gene expression was associated with the valvular phenotypes [bib_ref] Artificial Intelligence Models Reveal Sex-Specific Gene Expression in Aortic Valve Calcification, Sarajlic [/bib_ref]. The uneven sex proportion in our study might have also biased our findings.
# Conclusions
The baseline elevated fibrosis-4 index, which is often associated with systemic congestion, was associated with increased heart failure incidence following TAVR. We highly encourage clinicians to calculate the fibrosis-4 index for all TAVR candidates for risk stratification and shared decision making. An aggressive intervention to decrease the score, including neuro-hormonal blockers and diuretics, might improve the clinical outcomes following TAVR. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Data are available from the corresponding author upon reasonable requests.
## Conflicts of interest:
The authors declare no conflict of interest.
[fig] Figure 1: Distribution of the baseline fibrosis-4 index. [/fig]
[fig] Figure 2: Cutoff of fibrosis-4 index for the primary endpoint. HF, heart failure. Cutoff was calculated as 3.79 of the fibrosis-4 index. [/fig]
[fig] Figure 3: Cont. [/fig]
[fig] Figure 4: Cumulative incidence of the primary endpoint accounting for death as a competing risk among those with a high fibrosis-4 index at baseline. [/fig]
[fig] Author: Contributions: Conceptualization, T.I.; methodology, T.I.; software, T.I.; validation, N.N.; formal analysis, T.I.; investigation, H.O., S.T. and R.U.; resources, K.K.; data curation, M.S. and N.F.; writing-original draft preparation, T.I.; writing-review and editing, N.N. and H.U.; visualization, T.I.; supervision, K.K.; project administration, T.I.; funding acquisition, K.K. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Ethics Committee of University of Toyama (R2015154, 11 April 2016). [/fig]
[table] Table 1: Pre-procedural baseline characteristics. eGFR, estimated glomerular filtration ratio; MR, mitral regurgitation; TR, tricuspid regurgitation. Continuous variables are presented as median and interquartile and compared using the Mann-Whitney U test. Categorical variables are presented as numbers and percentages and compared using the Fischer's exact test. * p < 0.05. [/table]
[table] Table 2: Post-procedural clinical data. [/table]
[table] Table 3: Impact of the baseline fibrosis-4 index on the primary endpoint. * p < 0.05 by Fine-Gray hazard ratio regression analysis. For the adjustment, potential confounders that were significantly different inTable 1were used: age, body surface area, hemoglobin, plasma B-type natriuretic peptide, and max velocity at aortic valve. [/table]
[table] Table 4: Baseline characteristics among those with a high baseline fibrosis-4 index. [/table]
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Ex vivo peripheral nerve detection of rats by spontaneous Raman spectroscopy
Nerve-sparing surgery is increasingly being applied to avoid functional deficits of the limbs and organs following surgery. Peripheral nerves that should be preserved are, however, sometimes misidentified due to similarity of shape and color to non-nerve tissues. To avoid misidentification of peripheral nerves, development of an in situ nerve detection method is desired. In this study, we report the label-free detection of ex vivo peripheral nerves of Wistar rats by using Raman spectroscopy. We obtained Raman spectra of peripheral nerves (myelinated and unmyelinated nerves) and their adjacent tissues of Wistar rats without any treatment such as fixation and/or staining. For the identification of tissue species and further analysis of spectral features, we proposed a principal component regression-based discriminant analysis with representative Raman spectra of peripheral nerves and their adjacent tissues. Our prediction model selectively detected myelinated nerves and unmyelinated nerves of Wistar rats with respective sensitivities of 95.5% and 88.3% and specificities of 99.4% and 93.5%. Furthermore, important spectral features for the identification of tissue species were revealed by detailed analysis of principal components of representative Raman spectra of tissues. Our proposed approach may provide a unique and powerful tool for peripheral nerve detection for nerve-sparing surgery in the future.Nerve-sparing surgery, which aims to preserve peripheral nerves near the tissue being removed, is now recognized as a highly effective therapeutic procedure that preserves functions of the limbs and organs following surgery and improves the patients' quality of life. Nerve-sparing surgery has widely been applied in such fields as urological surgery, gynecological surgery, head and neck surgery, and gastrointestinal surgery 1-13 . In general, assessment by the surgeon's own eye and/or white-light imaging is applied to identify peripheral nerves. Peripheral nerves that should be preserved are, however, sometimes misidentified due to similarity of shape and color to non-nerve tissues. Electrical stimulation of peripheral nerves can be employed for motor nerve identification 14,15 . Electrical stimulation, however, cannot be applied to sensory nerves and autonomic nerves. Dye-based visualization methods are also proposed 16,17 . Although dye labeling enhances the visualization contrast of nerves against background tissues, toxicity and nerve specificity are matters of concern for human application. Optical coherence tomography (OCT) and multi-photon microscopy (MPM) were also proposed for peripheral nerve detection[18][19][20][21][22]. OCT and MPM detect peripheral nerves by three-dimensional visualization of peripheral nerves based on light interferometry or inherent autofluorescence of peripheral nerves. These techniques, however, provide only morphological information on peripheral nerves due to low molecular sensitivity of these techniques, and as a result final identification of peripheral nerves should be made by a surgeon.To realize the direct detection of the peripheral nerves on the basis of molecular constituents of peripheral nerves, we have recently proposed a nerve detection technique employing spontaneous Raman
## Ex vivo peripheral nerve detection of rats by spontaneous raman spectroscopy takeo minamikawa 1 , yoshinori harada 1 & tetsuro takamatsu 2
Nerve-sparing surgery is increasingly being applied to avoid functional deficits of the limbs and organs following surgery. Peripheral nerves that should be preserved are, however, sometimes misidentified due to similarity of shape and color to non-nerve tissues. To avoid misidentification of peripheral nerves, development of an in situ nerve detection method is desired. In this study, we report the label-free detection of ex vivo peripheral nerves of Wistar rats by using Raman spectroscopy. We obtained Raman spectra of peripheral nerves (myelinated and unmyelinated nerves) and their adjacent tissues of Wistar rats without any treatment such as fixation and/or staining. For the identification of tissue species and further analysis of spectral features, we proposed a principal component regression-based discriminant analysis with representative Raman spectra of peripheral nerves and their adjacent tissues. Our prediction model selectively detected myelinated nerves and unmyelinated nerves of Wistar rats with respective sensitivities of 95.5% and 88.3% and specificities of 99.4% and 93.5%. Furthermore, important spectral features for the identification of tissue species were revealed by detailed analysis of principal components of representative Raman spectra of tissues. Our proposed approach may provide a unique and powerful tool for peripheral nerve detection for nerve-sparing surgery in the future.
Nerve-sparing surgery, which aims to preserve peripheral nerves near the tissue being removed, is now recognized as a highly effective therapeutic procedure that preserves functions of the limbs and organs following surgery and improves the patients' quality of life. Nerve-sparing surgery has widely been applied in such fields as urological surgery, gynecological surgery, head and neck surgery, and gastrointestinal surgery [bib_ref] A nerve-sparing radical hysterectomy: guidelines and feasibility in Western patients, Trimbos [/bib_ref] [bib_ref] Nerve-sparing radical prostatectomy: evaluation of results after 250 patients, Catalona [/bib_ref] [bib_ref] Avoiding long-term disturbance to bladder and sexual function in pelvic surgery, particularly..., Havenga [/bib_ref] [bib_ref] Nerve-sparing surgery in rectal cancer: feasibility and functional results, Mancini [/bib_ref] [bib_ref] Pain, quality of life, and spinal accessory nerve status after neck dissection, Terrell [/bib_ref] [bib_ref] Facial nerve morbidity following parotid surgery for benign disease: the Cleveland Clinic..., Mehle [/bib_ref] [bib_ref] Prostate cancer: Nerve-sparing surgery and risk of positive surgical margins, Boehm [/bib_ref] [bib_ref] Risk factors associated with positive surgical margins following radical prostatectomy for clinically..., Roder [/bib_ref] [bib_ref] Clinical efficacy and safety of nerve-sparing radical hysterectomy for cervical cancer: a..., Long [/bib_ref] [bib_ref] Nerve-sparing radical abdominal trachelectomy versus nerve-sparing radical hysterectomy in early-stage (FIGO IA2-IB)..., Van Gent [/bib_ref] [bib_ref] Facial nerve preservation surgery for koos grade 3 and 4 vestibular schwannomas, Anaizi [/bib_ref] [bib_ref] Quality of life, shoulder range of motion, and spinal accessory nerve status..., Eickmeyer [/bib_ref] [bib_ref] Impact of robotic surgery on sexual and urinary functions after fully robotic..., Luca [/bib_ref]. In general, assessment by the surgeon's own eye and/or white-light imaging is applied to identify peripheral nerves. Peripheral nerves that should be preserved are, however, sometimes misidentified due to similarity of shape and color to non-nerve tissues. Electrical stimulation of peripheral nerves can be employed for motor nerve identification [bib_ref] Facial nerve monitoring during acoustic neuroma removal, Benecke [/bib_ref] [bib_ref] Risk factors of paralysis and functional outcome after recurrent laryngeal nerve monitoring..., Dralle [/bib_ref]. Electrical stimulation, however, cannot be applied to sensory nerves and autonomic nerves. Dye-based visualization methods are also proposed [bib_ref] Nerve mapping for prostatectomies: novel technologies under development, Ponnusamy [/bib_ref] [bib_ref] Fluorescent peptides highlight peripheral nerves during surgery in mice, Whitney [/bib_ref]. Although dye labeling enhances the visualization contrast of nerves against background tissues, toxicity and nerve specificity are matters of concern for human application. Optical coherence tomography (OCT) and multi-photon microscopy (MPM) were also proposed for peripheral nerve detection [bib_ref] Optical coherence tomography: technology and applications for neuroimaging, Boppart [/bib_ref] [bib_ref] Extracting structural features of rat sciatic nerve using polarization-sensitive spectral domain optical..., Islam [/bib_ref] [bib_ref] Imaging the cavernous nerves in the rat prostate using optical coherence tomography, Fried [/bib_ref] [bib_ref] Multiphoton microscopy of prostate and periprostatic neural tissue: a promising imaging technique..., Yadav [/bib_ref] [bib_ref] Multiphoton microscopy for structure identification in human prostate and periprostatic tissue: implications..., Tewari [/bib_ref]. OCT and MPM detect peripheral nerves by three-dimensional visualization of peripheral nerves based on light interferometry or inherent autofluorescence of peripheral nerves. These techniques, however, provide only morphological information on peripheral nerves due to low molecular sensitivity of these techniques, and as a result final identification of peripheral nerves should be made by a surgeon.
To realize the direct detection of the peripheral nerves on the basis of molecular constituents of peripheral nerves, we have recently proposed a nerve detection technique employing spontaneous Raman spectroscopy [bib_ref] Label-free detection of peripheral nerve tissues against adjacent tissues by spontaneous Raman..., Minamikawa [/bib_ref]. Spontaneous Raman spectroscopy provides information about molecular vibrations that appear in inelastic scattered light of monochromatic excitation light. Since molecular vibration is strongly related to the molecular species and structures of tissues, spectral analysis of inelastic scattered light allows us to identify tissue species via molecular vibrations [bib_ref] Determination of human coronary artery composition by Raman spectroscopy, Brennan [/bib_ref] [bib_ref] Tissue characterization using high wave number Raman spectroscopy, Koljenovic [/bib_ref] [bib_ref] Diagnosing breast cancer by using Raman spectroscopy, Haka [/bib_ref] [bib_ref] Emerging concepts in deep Raman spectroscopy of biological tissue, Matousek [/bib_ref] [bib_ref] Raman molecular imaging of cells and tissues: towards functional diagnostic imaging without..., Harada [/bib_ref]. As Raman spectroscopy does not require any treatment such as staining and/or fixation, ex vivo and in vivo observation of living tissues can be realized noninvasively [bib_ref] Subsurface sensing of biomedical tissues using a miniaturized Raman probe: study of..., Yamamoto [/bib_ref] [bib_ref] Label-free biochemical imaging of heart tissue with high-speed spontaneous Raman microscopy, Ogawa [/bib_ref] [bib_ref] Ex vivo and in vivo diagnosis of C6 glioblastoma development by Raman..., Beljebbar [/bib_ref] [bib_ref] Fiberoptic confocal raman spectroscopy for real-time in vivo diagnosis of dysplasia in..., Bergholt [/bib_ref] [bib_ref] Fiber-optic Raman spectroscopy probes gastric carcinogenesis in vivo at endoscopy, Bergholt [/bib_ref] [bib_ref] In vivo blood glucose quantification using Raman spectroscopy, Shao [/bib_ref] [bib_ref] In vivo detection of dysplastic tissue by Raman spectroscopy, Bakker Schut [/bib_ref]. Spontaneous Raman spectroscopy and its alternative Raman spectroscopy, coherent anti-Stokes Raman spectroscopy, have been applied to myelinated nerve detection in both the central and the peripheral nervous systems [bib_ref] Vibrational spectroscopic imaging and multiphoton microscopy of spinal cord injury, Galli [/bib_ref] [bib_ref] Label-free high-resolution imaging of prostate glands and cavernous nerves using coherent anti-Stokes..., Gao [/bib_ref] [bib_ref] Application of Raman spectroscopy for visualizing biochemical changes during peripheral nerve injury..., Morisaki [/bib_ref] [bib_ref] In vivo coherent anti-Stokes Raman scattering imaging of sciatic nerve tissue, Huff [/bib_ref]. Unmyelinated nerves that are also important to maintain functions of organs were not visualized. In our previous report, we have provided a proof-of-principle demonstration of selective detection of peripheral nerves including myelinated and unmyelinated nerves against adjacent tissues by using dried microsection samples after a frozen-thawed procedure [bib_ref] Label-free detection of peripheral nerve tissues against adjacent tissues by spontaneous Raman..., Minamikawa [/bib_ref]. In such dried microsection samples, cross-sections of nerve fibers were directly examined by means of Raman spectroscopy. However, in situ macroscopic detection must be approached across the surrounding tissues such as the perineurium. The feasibility of our peripheral nerve detection method is still unclear.
In this study, we investigated the feasibility of peripheral nerve detection with Raman spectroscopy by using fresh ex vivo macrosamples from Wistar rats. We examined myelinated nerve, unmyelinated nerve, adipose tissue, collagenous tissue, and skeletal muscle without any treatments such as fixation, staining, or microsectioning. A principal component regression (PCR)-based discriminant analysis was performed for the selective detection and further spectral analysis of peripheral nerves against adjacent tissues, and the detection power of peripheral nerves was examined.
# Results
Raman spectra of nerves and adjacent tissue in fresh ex vivo samples. For selective peripheral nerve detection, we measured Raman spectra of the peripheral nerves and the predominant contents around peripheral nerves as follows: myelinated nerve, unmyelinated nerve, adipose tissue, collagenous tissue, and skeletal or smooth muscle. We excised representative tissues of these tissue species, i.e., sciatic nerves as myelinated nerve, vagus nerves as unmyelinated nerve, abdominal adipose tissues as adipose tissue, Achilles' tendon as collagenous tissue, and femoral muscle as skeletal muscle. In our previous study, we found that the Raman spectra of peripheral nerves had almost no site-specific dependency, thus we chose sciatic nerves and vagus nerves as representative peripheral nerves [bib_ref] Label-free detection of peripheral nerve tissues against adjacent tissues by spontaneous Raman..., Minamikawa [/bib_ref]. Our previous study also revealed that the representative Raman spectrum of smooth muscle in blood vessels was very similar to that of skeletal muscle. We thus investigated only the skeletal muscle was investigated in this study. Areaaveraged representative Raman spectra of these tissues were obtained ex vivo, and are shown in [fig_ref] Figure 1: Representative ex vivo Raman spectra of myelinated nerve [/fig_ref]. Since there were no significant Raman bands related to tissues in the Raman spectra between 1800 and 2750 cm −1 , we showed only the Raman spectra from 725 to 1800 and from 2750 to 3090 cm −1 in [fig_ref] Figure 1: Representative ex vivo Raman spectra of myelinated nerve [/fig_ref]. We did not find any spectral differences of Raman spectra of respective tissue species among animals. Tissue species of the observed region were confirmed by histological analysis with hematoxylin-eosin staining after Raman observation.
Myelinated nerves and unmyelinated nerves are both important for maintaining organ functions. Myelinated nerves have a myelin sheath that is composed of abundant lipids, whereas unmyelinated nerves have no myelin sheath. This molecular difference mainly appeared at 2850 cm −1 , assigned to CH 2 stretching vibration mode. The spectral features of both myelinated and unmyelinated nerves in fresh ex vivo macrosamples were similar to those reported in our previous study using dried microsection samples, with Raman bands at 1000, 1126, 1168, 1297, 1336, 1445, 1585, 1654, 2885 and 2932 cm −1 23 . In fresh ex vivo peripheral nerves, relatively broad Raman bands of amide I appearing at 1654 cm −1 as indicated by the arrow in [fig_ref] Figure 1: Representative ex vivo Raman spectra of myelinated nerve [/fig_ref] were obtained. Furthermore, the relative intensity of CH 3 stretching vibration mode appearing at 2932 cm −1 as indicated by the arrow in [fig_ref] Figure 1: Representative ex vivo Raman spectra of myelinated nerve [/fig_ref] was relatively strong comparing with that observed in our previous study using dried macrosection samples. These results were different from those seen in Raman spectra of dried microsection samples, and thus indicated that the protein contributions in fresh ex vivo peripheral nerves were relatively large. In our previous study with dried microsection samples, we directly observed peripheral nerves [bib_ref] Label-free detection of peripheral nerve tissues against adjacent tissues by spontaneous Raman..., Minamikawa [/bib_ref]. In this study with fresh ex vivo peripheral nerves, we observed the peripheral nerves across the perineurium that mainly comprises collagens, which might result in the Raman features of the protein contributions that appeared in the fresh ex vivo samples.
Adipose tissue, collagenous tissue, and skeletal muscle are predominant components around peripheral nerves. In adipose tissue, spectral features appeared at 1080, 1260, 1299, 1441, 1655, 1747, 2855, 2899 and 2930 cm −1 . The adipose tissue was composed of fat cells with large lipid droplets that occupied most regions of the fat cells. The lipid droplets were predominantly composed of triglycerides, and thus the Raman bands of adipose tissue are mainly assigned to lipid-related bands such as CH 2 bending mode (1441 cm −1 ), C = C double bond stretching mode (1655 cm −1 ), C = O double bond stretching mode (1747 cm −1 ), CH 2 stretching modes (2855 and 2899 cm −1 ), and CH 3 stretching mode (2930 cm −1 ) as indicated by the arrows in [fig_ref] Figure 1: Representative ex vivo Raman spectra of myelinated nerve [/fig_ref] ,b. In skeletal muscle, the main Raman bands appeared at 746, 1000, 1124, 1309, 1333, 1450, 1550, 1581, 1650, 2883 and 2934 cm −1 . Especially relatively strong Raman bands at 746, 1124, 1309 and 1581 cm −1 as indicated by the arrows in [fig_ref] Figure 1: Representative ex vivo Raman spectra of myelinated nerve [/fig_ref] might be assigned to reduced cytochromes present in mitochondria in skeletal muscle cells [bib_ref] Label-free Raman observation of cytochrome c dynamics during apoptosis, Okada [/bib_ref] [bib_ref] Mapping of redox state of mitochondrial cytochromes in live cardiomyocytes using Raman..., Brazhe [/bib_ref]. The Raman bands that related to cellular components such as phenylalanine (1000 cm −1 ), CH 2 bending mode (1450 cm −1 ), Amide I (1650 cm −1 ), and CH 3 stretching modes (2934 cm −1 ) as indicated by the arrowheads in [fig_ref] Figure 1: Representative ex vivo Raman spectra of myelinated nerve [/fig_ref] ,b were also appeared. For investigation of collagenous tissue, we obtained Raman spectra of Achilles' tendon. Collagenous tissue is generally present around peripheral nerves, e.g., perineurium and fibrous connective tissue; complete surgical isolation of perineurium and fibrous connective tissue in macrosamples was difficult due to their small size. Our previous study revealed that fibrous connective tissue surrounding peripheral nerves is mainly composed of collagen type-I 23 . Therefore, we used tendon that is predominantly composed of collagen type-I as a model tissue of collagenous tissue. The main Raman bands of collagenous tissue appeared at 812, 853, 935, 1000, 1030, 1241, 1448, 1662, 2883 and 2943 cm −1 . Especially, − PO 4 3 (1000 cm −1 ), Amide III (1241 cm −1 ), CH 2 bending mode (1448 cm −1 ), Amide I (1662 cm −1 ), CH 3 symmetric stretching mode (2883 cm −1 ), and CH 3 symmetric and CH 2 asymmetric stretching modes (2943 cm −1 ) as indicated by the arrows in [fig_ref] Figure 1: Representative ex vivo Raman spectra of myelinated nerve [/fig_ref] ,b were representative Raman bands related to collagen type-I.
## Principal component analysis of raman spectral data set.
We constructed a prediction model based on PCR-based discriminant analysis. We first performed principal component analysis using the representative Raman spectra of myelinated nerve, unmyelinated nerve, adipose tissue, collagenous tissue, and skeletal muscle as shown in Eq. 2 (see Materials and Methods). We employed both Raman spectra of fingerprint region and high wavenumber region to realize reliable prediction as discussed in our previous study [bib_ref] Label-free detection of peripheral nerve tissues against adjacent tissues by spontaneous Raman..., Minamikawa [/bib_ref]. To simply elucidate the differences of Raman spectra among tissue species, we performed principal component analysis with the representative Raman spectra of tissue species. Loading and score plots of the representative Raman spectral data set are shown in. In our model, we used 5 Raman spectra derived from 5 tissue species in S base , and thus a total of 5 principal components (PCs) were calculated. Contribution ratios of 1st PC (denoted as PC1; similar notations for other PCs), PC2, PC3, PC4, and PC5 were 90.7%, 8.38%, 0.655%, 0.171%, and 0.0701%, respectively.
In the score plot related to the Raman spectrum of myelinated nerve, PC1 has the largest score, where the absolute score values of the other PCs were less than 10% of PC1. This result indicated that the loading plot of PC1 predominantly had the spectral features of the Raman spectrum of myelinated nerve, e.g. 1000, 1122, 1168, 1297, 1334, 1445, 1583, 1653, 2854, 2894 and 2932 cm −1 . The relative intensity ratio in the CH region (2800-3090 cm −1 ) was slightly different from the representative Raman spectrum of myelinated nerve shown in [fig_ref] Figure 1: Representative ex vivo Raman spectra of myelinated nerve [/fig_ref]. To reconstruct the Raman spectrum of myelinated nerve with proper intensity ratio in the CH region, other PCs were needed, especially PC3 and PC4.
In the score plot related to the Raman spectrum of unmyelinated nerve, PC1 had the highest score in the positive direction, and PC2 was the predominant component in the negative direction. This result indicated that the positive peaks appearing in the loading plot of PC2 did not contain or had small contributions in comparison with those of PC1 in the Raman spectrum of unmyelinated nerve. In contrast, the negative peaks appearing in the loading plot of PC2 were emphasized or appeared in the Raman spectrum of unmyelinated nerve. In the comparison between the score plot of the myelinated nerve and that of unmyelinated nerve, PC2 showed the major difference. This result indicated that the loading plots of PC2 might contain features of spectral difference between the Raman spectrum of myelinated nerve and that of unmyelinated nerve. The loading plot of PC2 contained positive peaks at 2853 cm −1 and 2890 cm −1 , indicating the difference of CH 2 vibration modes between the Raman spectra of myelinated nerve and unmyelinated nerve. The negative peaks at 2948 and 2984 cm −1 and the positive peak at 3012 cm −1 might reflect in detail the difference of CH 3 vibration modes between them. Furthermore, there was a peak at 1655 cm −1 in the positive direction. This result might indicate that the C = C double bond stretching mode (1655 cm −1 ) of lipids in the myelin sheath was contained in the Raman spectrum of myelinated nerve. This difference was not apparent in the representative Raman spectra due to the broad spectrum of Amide I.
In the score plot related to the Raman spectrum of adipose tissue, PC1 and PC2 were the predominant components. Several peaks assigned to triglycerides of lipid droplets appeared in the positive direction of the loading plot of PC2, including 1655 (C = C double bond stretching mode), 1747 (C = O band carbonyl stretching mode), and 2853 cm −1 (CH 2 symmetric stretching mode) [bib_ref] Fast and minimally invasive determination of the unsaturation index of white fat..., Giarola [/bib_ref]. Furthermore, the peak at 1437 cm −1 in the loading plot of PC2 indicated a spectral feature of peak shift of CH 2 bending mode appearing around 1450 cm −1 to lower wavenumber in the Raman spectrum of adipose tissue compared with those of the other tissues. Actually, the score plots of PC2 of myelinated nerve and adipose tissue had positive value, while those in the other tissues had negative value. These results indicated that the spectral features of lipid-containing tissues, such as myelinated nerve and adipose tissue, appeared in the positive peaks of PC2.
In the score plot related to the Raman spectrum of collagenous tissue, PC1 was the predominant component in the positive direction, and PC2 was that in the negative direction, similar to the score plot related to the Raman spectrum of unmyelinated nerve. The score plots related to the Raman spectra of unmyelinated nerve and of the collagenous tissue differed in that the relative values of the score plots of PC2 and PC3 in the collagenous tissue were slightly larger than those in the unmyelinated nerve. Furthermore, the score plots of PC4 and PC5 had negative values in the collagenous tissue, while the score plots of PC4 and PC5 respectively were almost zero and had positive value in the unmyelinated nerve.
In the score plot related to the Raman spectrum of skeletal muscle, PC1 was the predominant component in the positive direction, and PC2 and PC3 were the predominant components in the negative direction. The score plot of PC3 for the skeletal muscle was negative, while the score plots of PC3 in the other tissues were positive or almost zero. This result suggests that the negative peaks on the loading plot of PC3 indicated spectral features of skeletal muscle, such as characteristic Raman bands of cytochromes (746, 1124, 1309, and 1581 cm −1 ).
## Regression coefficient matrix for pcr-based prediction.
For the prediction of tissue species, we calculated regression coefficient matrix C by Eq. 3 (see Materials and Methods). Mean regression coefficients calculated with 1000 observation points (one pixel obtained from a line shaped spectral-image was defined as an observation point) are shown in [fig_ref] Figure 3: Regression coefficient plots for [/fig_ref]. The regression coefficient matrices were calculated without the dimensional reduction of PCs. For the myelinated nerve and unmyelinated nerve, the regression coefficient corresponding to the Raman spectrum of each type of nerve exhibited the highest value. The regression coefficient corresponding to the Raman spectrum of collagenous tissue had also relatively large value in the myelinated nerve and the unmyelinated nerve. The value of the regression coefficient indicated the relative contribution of the base spectral data set S base in the observed spectrum used in Eq. 3. In our experiments, we obtained Raman spectrum of fresh ex vivo peripheral nerve by illuminating from the lateral side, and thus the contribution of the perineurium must be included. The perineurium is predominantly composed of collagenous tissue, and therefore the contribution of collagenous tissue appeared in the regression coefficients for the myelinated nerve and the unmyelinated nerve. The other tissues, such as adipose tissue, collagenous tissue, and skeletal muscle, predominantly had the regression coefficients of their own tissue, respectively. Thus, the regression coefficients corresponding to each tissue type exhibited the highest value, and the other coefficients were much smaller than the regression coefficients of corresponding tissues.
Prediction capability of the PCR-based prediction model. By using the regression coefficient matrix, we obtained a prediction model by using quadratic discriminant analysis. Cross-validated sensitivities and specificities of the prediction model without dimensional reduction of PCs are shown in . The PCR-based prediction model realized sensitivities of 95.5%, 88.3%, 96.5%, 89.2%, and 88.2% and specificities of 99.4%, 93.5%, 100%, 98.0%, and 98.6%, respectively, for the prediction of myelinated nerve, unmyelinated nerve, adipose tissue, collagenous tissue, and skeletal muscle.
We also estimated cross-validated sensitivities and specificities with dimensional reduction of each PC . In the dimensional reduction of PC1, in which PC2, PC3, PC4, and PC5 were employed for the estimation of regression coefficient in Eq. 3 (see Materials and Methods), notable decrease of sensitivity and specificity compared with the sensitivity and specificity obtained without dimensional reduction of PCs was not obtained, indicating that PC1 was not critical for the prediction of tissue species in this model.
In the dimensional reduction of PC2, we obtained notable decrease of the sensitivity for adipose tissue, but not for the other tissues. This result indicated that the peaks appearing in the loading plot of PC2, such as lipid-related peaks at 1437, 1655, 1747, and 2855 cm −1 , are important for the prediction of adipose tissue, and had insignificant effect on the prediction of the other tissues.
In the dimensional reduction of PC3, notable decrease of sensitivity was obtained for the sensitivities for myelinated nerve, unmyelinated nerve, and skeletal muscle. Especially, the sensitivity for skeletal muscle was decreased to almost half of that in the case without dimensional reduction. This result indicated that the most important components of the Raman spectrum for skeletal muscle for selective prediction appeared in the loading plot of PC3, such as characteristic Raman bands of cytochrome c (746, 1124, 1309, and 1581 cm −1 ). According to the result that the sensitivities for myelinated nerve and unmyelinated nerve were also decreased, the loading plot of PC3 also comprised spectral features for selective prediction of myelinated nerve and unmyelinated nerve.
In the dimensional reduction of PC4, notable decrease was obtained for the sensitivities for myelinated nerve and unmyelinated nerve, while no such decrease was noted for the sensitivity for peripheral nerve, when both myelinated and unmyelinated nerves were considered to be peripheral nerves. In contrast, in the dimensional reduction of PC3, the sensitivity for peripheral nerve was decreased following the decrease of the sensitivities for myelinated nerve and unmyelinated nerve. These results indicated that the loading plots of PC3 and PC4 respectively included spectral features for the prediction of peripheral nerves against the other tissues and for that of each peripheral nerve type.
In the dimensional reduction of PC5, notable decrease was obtained for the sensitivity for unmyelinated nerve. The sensitivity for peripheral nerve was also decreased, indicating that the loading plot of PC5 is comprised of the important spectral features for the prediction for unmyelinated nerve against other non-peripheral nerve tissue species. Actually, most of the mispredictions for unmyelinated nerve were collagenous tissue (data not shown). Although the contribution ratio of PC5 was only 0.0701%, PC5 might be comprised of important spectral features for the prediction of unmyelinated nerve against collagenous tissues.
# Discussion
Our recent study demonstrated the proof-of-principle of Raman spectroscopy for the selective detection of peripheral nerves by using dried microsections after a frozen-thawed procedure [bib_ref] Label-free detection of peripheral nerve tissues against adjacent tissues by spontaneous Raman..., Minamikawa [/bib_ref]. Here, we extended the detection to ex vivo spectral analysis of fresh tissue macrosamples, and performed selective detection of peripheral nerves against adjacent tissues and further spectral analysis of fresh ex vivo tissues by means of a PCR-based prediction model. Although surgically excised fresh ex vivo tissues were used for the experiments in the present study, sample conditions were almost identical to in vivo conditions. The result presented in this study suggests that the Raman spectroscopic prediction of peripheral nerves has potential for application in intraoperative use. Raman spectroscopy offers the advantages of fast, in situ, non-destructive, and molecular vibration-based observation, enabling the selective detection of peripheral nerves against adjacent tissues without any treatment such as fixation or staining.
It is considered that the most appropriate cases for application of this method might be the detection of small peripheral nerves ranging from 100 μ m to 1 mm in diameter. In general, peripheral nerves larger than 1 mm innervate a well-known course with relatively small individual variability, and most of the peripheral nerves can be identified by the human eye or under white-light imaging. In contrast, in the case of peripheral nerves smaller than 100 μ m, organ functions generally recover by regeneration of peripheral nerves even if these nerves are disrupted [bib_ref] Histologic assessment of nerve regeneration in the rat, Mackinnon [/bib_ref]. However, the actual size at which peripheral nerves should be preserved is still unknown. This is because there is no quantitative and intraoperative identification method of small peripheral nerves in current surgery, and surgical damage of peripheral nerves is estimated based on only post-operative recovery of the patient's nerve function without the evaluation of resection rate of small peripheral nerves. Use of our proposed method offers the potential of revealing actual peripheral nerves that should be preserved by quantitative evaluation of the resection rate of peripheral nerves. There are some limitations of this study. Firstly, the estimated size of peripheral nerves used in this study was limited. As myelinated nerve, we examined the sciatic nerve of Wistar rats, which ranges from a few tens of micrometers to millimeters in diameter. However, we used vagus nerve for unmyelinated nerve, which is only less than 100 μ m in diameter. The other peripheral unmyelinated nerves are narrower than the vagus nerve in the Wistar rat. For further confirmation of this method by using larger peripheral unmyelinated nerves ranging from 100 μ m to 1 mm in diameter, larger animals such as the rabbit and pig, and also humans should thus be used. We should also evaluate the effect of the thickness of perineurium depending on the size of the peripheral nerves. Secondly, the toxicity of light irradiation to peripheral nerves is still unclear. In this study, we observed no serious damage of the peripheral nerves and other tissues, such as charring, burning, or structural distortion. For the determination of the adequate irradiation condition including laser power, irradiation area, and exposure time in the observation of Raman spectra of peripheral nerves, further studies are required that include follow-up evaluation of organ functions after surgical use in vivo. Thirdly, the optimum excitation wavelength for the peripheral nerve detection with Raman spectroscopy is still unclear. High signal intensity of Raman scattering could be obtained by using short excitation light such as the 532-nm light that we employed in this study due to high Raman scattering coefficient of tissues and the availability of a high quantum efficiency detector, although near-infrared light is better for decreasing autofluorescence, absorption, and Rayleigh scattering of tissues. Furthermore, potential phototoxicity of excitation light to samples is also a matter of concern.
For clinical use, we should investigate the effective excitation light for peripheral nerve detection with Raman spectroscopy by using actual peripheral nerves of patients in future studies. Fourthly, the penetration depth of Raman spectroscopy using 532 nm excitation light was limited to several tens to a few hundreds of a micrometer due to the scattering of light in turbid media [bib_ref] Going deeper than microscopy: the optical imaging frontier in biology, Ntziachristos [/bib_ref]. The penetration depth might be sufficient because the applicable diameter of peripheral nerve using this method ranges from 100 μ m to 1 mm, of which the perineurium might range from a few tens to a few hundreds of a micrometer, as discussed above. If required, we can apply the following methods for further enhancement of penetration depth: use of longer wavelength of excitation such as 780 nm, and application of spatially offset Raman spectroscopy [bib_ref] Prospects for in vivo Raman spectroscopy, Hanlon [/bib_ref] [bib_ref] Subsurface probing in diffusely scattering media using spatially offset Raman spectroscopy, Matousek [/bib_ref] [bib_ref] Near-infrared Raman spectroscopy for optical diagnosis of lung cancer, Huang [/bib_ref]. These methods can enhance the penetration depth of Raman spectroscopy to a few times to tens of times.
# Conclusions
In the present study, we demonstrated label-free and selective detection of fresh ex vivo peripheral nerves of Wistar rats by using Raman spectroscopy with PCR-based discriminant analysis. By using PCR-based discriminant analysis with representative Raman spectra of peripheral nerves and their adjacent tissues, we revealed important spectral features for the prediction of tissue species. We also successfully and selectively detected peripheral nerves including myelinated nerves and unmyelinated nerves with high sensitivity and specificity. Although for the application of our proposed method to clinical use, further development of an intraoperative Raman spectroscopy system is required, our proposed approach may provide a unique and powerful tool for peripheral nerve detection for nerve-sparing surgery in the future.
# Materials and methods
Raman spectroscopy. A slit-scanning confocal Raman microscope (RAMAN-11; Nanophoton, Osaka, Japan) was used for acquiring Raman spectra and Raman spectral images, as described in our previous studies [bib_ref] Label-free detection of peripheral nerve tissues against adjacent tissues by spontaneous Raman..., Minamikawa [/bib_ref] [bib_ref] Label-free biochemical imaging of heart tissue with high-speed spontaneous Raman microscopy, Ogawa [/bib_ref] [bib_ref] Intracellular dynamics of topoisomerase I inhibitor, CPT-11, by slit-scanning confocal Raman microscopy, Harada [/bib_ref] [bib_ref] Label-free evaluation of myocardial infarction and its repair by spontaneous Raman spectroscopy, Nishiki-Muranishi [/bib_ref]. A frequency doubled Nd:YAG laser (532 nm) was employed for excitation. The excitation laser beam was focused into a line (735 μ m in length and approximately diffraction limit in width) on a sample through a cylindrical lens and a long working distance objective lens (UPlanFL N, ×10, NA = 0.3; Olympus, Tokyo, Japan). Raman scattering was collected with the same objective lens, focused onto the input slit of the spectrometer with a 600 grooves/mm grating, and detected with a two-dimensional image sensor (Pixis 400BR, − 70 °C, 1340 × 400 pixels; Princeton Instruments, Trenton, NJ, USA). Owing to the line shaped focus of the excitation beam, a one-dimensional spectral image from the line-illuminated site was collected with the same objective lens (one pixel in space corresponds to 1.8 μ m in length and approximately 0.89 μ m in width). The excitation laser power was 235 μ W/μ m 2 on the sample plane. The exposure time for each line and the slit width of the spectrometer were respectively set at 10 s and 70 μ m (corresponding spectral resolution was 7 cm −1 ) for all experiments.
## Animals.
All animal experiments were conducted with the approval of and in accordance with guidelines from the Committee for Animal Research, Kyoto Prefectural University of Medicine. Sciatic nerves (1 cm in length), vagus nerves (1 cm in length), abdominal adipose tissues (approximately 1 × 1 × 1 cm 3 ), Achilles' tendon (0.5-1 cm in length), and femoral muscle (approximately 1 × 1 × 1 cm 3 ) were excised from male Wistar rats (8-10 weeks old) after euthanasia. A total of 5 rats were used for analysis. One sample of respective tissue species from each rat was studied. The excised fresh tissues were put on a cover slip (No.1; Matsunami Glass Ind., Ltd., Osaka, Japan), and were kept wet throughout the experiment with physiological saline. A schematic diagram of ex vivo Raman spectroscopy is shown in [fig_ref] Figure 4: Experimental conditions for ex vivo Raman spectroscopy [/fig_ref]. Spectral preprocessing. Nanophoton's software was utilized for preprocessing individual Raman spectra. Wavenumbers of all Raman spectra were calibrated by using the known Raman bands of ethanol. To extract the Raman spectrum from broad fluorescence background, we applied modified polynomial curve fitting method [bib_ref] Automated Method for Subtraction of Fluorescence from Biological Raman Spectra, Lieber [/bib_ref]. We estimated the autofluorescence component superposed on the Raman spectrum by calculating a modified least-squares fifth-order polynomial curve with 10 iterations, and then subtracted this polynomial from the raw spectra.
## Construction of prediction model using principal component regression-based discriminant
analysis. We employed PCR for the construction of a prediction model of tissues. The PCR is one of the common statistical methods, which provides linearly independent spectral datasets called principle components (PCs). In spectral analysis, linearly independent spectra are useful for extracting spectral features owing to its independency of each spectral component. In this study, PCR was performed by using Matlab (Mathworks, Natick, MA, USA). Firstly, we assumed that the observed Raman spectrum S tissue could be expressed by user-defined Raman spectra of tissue species S base with regression coefficient matrix C:
[formula] = , ( ) S CS 1 tissue b ase [/formula]
where S tissue and S base were normalized with the square root summation of their Raman spectra ranging from 725 cm −1 to 3090 cm −1 . In this study, we assumed that S base was the spectral data set of representative Raman spectra of myelinated nerve, unmyelinated nerve, collagenous tissue, adipose tissue, and skeletal muscle, which were normalized with the total intensity of the wavenumber region ranging from 725 cm −1 to 3090 cm −1 . The spectral data set of S base was decomposed to loading matrix P and score matrix T with principal component analysis:
[formula] = , ( ) S TP 2 t base [/formula]
where P was orthonormal basis. Then, we calculated the regression coefficient matrix with Moore-Penrose pseudo inverse:
[formula] = , ( ) + C S PT 3 tissue [/formula]
where T + indicated the Moore-Penrose pseudo inverse of T matrix. Since data points of discrete Raman spectra obtained with the CCD camera in the spectrometer (1300 pix) were much larger than the number of assumed tissue species in S base (5 tissue species), Eq. 3 reflects the most reliable regression coefficient matrix with the square root sum of the prediction error. To obatain a reliable PT + , we used area-averaged representative Raman spectra with high signal-to-noise ratio for S base (400-3000 pixels averaging). By using the regression coefficient matrix C, we constructed a prediction model with discriminant analysis. Quadratic discriminant analysis was used for the prediction. A total of 1000 Raman spectra in each tissue species was obtained from 5 rats as training data sets for prediction. Detection sensitivity and specificity were calculated by leave-one-out cross-validation with these data sets.
[fig] Figure 1: Representative ex vivo Raman spectra of myelinated nerve (MN), unmyelinated nerve (UMN), adipose tissue (A), collagenous tissue (C), and skeletal muscle (SM). (a) Raman spectra in fingerprint region, and (b) in high wavenumber region. a.u., arbitrary units. Scientific RepoRts | 5:17165 | DOI: 10.1038/srep17165 [/fig]
[fig] Figure 2: (a) Loading and (b) score plots of Raman spectra of tissue species Sbase calculated by principal component analysis. [/fig]
[fig] Figure 3: Regression coefficient plots for (a) myelinated nerve, (b) unmyelinated nerve, (c) adipose tissue, (d) collagenous tissue, and (e) skeletal muscle. Each error bar indicates standard deviation. (c) indicates each coefficient. MN, myelinated nerve; UMN, unmyelinated nerve; A, adipose tissue; C, collagenous tissue; and SM, skeletal muscle. [/fig]
[fig] Scientific: RepoRts | 5:17165 | DOI: 10.1038/srep17165 Histological examination. For the confirmation of histology of excised tissues, we performed hematoxylin-eosin staining. The excised tissues were embedded in frozen section compound (FSC 22; Leica, Wetzlar, Germany) after Raman observation. The embedded samples were snapfrozen in dry ice-acetone, and then stored at − 80 °C. The frozen samples were sliced into 5-μ m-thick sections with a cryostat microtome (CM1900; Leica, Wetzlar, Germany). The sections were fixed with 10% formalin, and stained with hematoxylin-eosin. [/fig]
[fig] Figure 4: Experimental conditions for ex vivo Raman spectroscopy. [/fig]
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Sex Difference Impacts on the Relationship between Paraoxonase-1 (PON1) and Type 2 Diabetes
Type-2 diabetes (T2D) and its cardiovascular complications are related to sex. Increasing evidence suggests that paraoxonase 1 (PON1) activity, an antioxidant enzyme bound to high-density lipoproteins (HDL), is implicated in the onset and clinical progression of T2D. Since we previously showed that PON1 is a sexual dimorphic protein, we now investigated whether sex might impact the relationship between PON1 and this chronic disease. To address this aim, we assessed PON1 activity in the sera of 778 patients, including controls (women, n = 383; men, n = 198) and diabetics (women, n = 79; men = 118). PON1 activity decreased in both women and men with T2D compared with controls (p < 0.05 and p > 0.001, respectively), but the change was 50% larger in the female cohort. In line with this result, the enzyme activity was associated with serum glucose level only in women (r = −0.160, p = 0.002). Notably, only within this gender category, lower PON1 activity was independently associated with increased odds of being diabetic (odds ratio (95% Confidence interval: 2.162 (1.075-5.678)). In conclusion, our study suggests that PON1-deficiency in T2D is a gender-specific phenomenon, with women being more affected than men. This could contribute to the partial loss of female cardiovascular advantage associated with T2D.
# Introduction
Increasing evidence suggests that type-2 diabetes (T2D) and its macro-and micro-vascular complications are influenced by gender [bib_ref] Diabetes abrogates sex differences and aggravates cardiometabolic risk in postmenopausal women, Mascarenhas-Melo [/bib_ref] [bib_ref] Diabetes and gender, Gale [/bib_ref]. In particular, although women without T2D present a lower risk of cardiovascular diseases (CVDs) than men, the impairment in glucose metabolism seems to reverse this phenomenon [bib_ref] Diabetes abrogates sex differences and aggravates cardiometabolic risk in postmenopausal women, Mascarenhas-Melo [/bib_ref] [bib_ref] Sex and Gender Differences in Risk, Pathophysiology and Complications of Type 2..., Kautzky-Willer [/bib_ref] [bib_ref] Diabetes and cardiovascular disease. The Framingham study, Kannel [/bib_ref]. The impact of gender on cardiometabolic factors leading to T2D cardiovascular complications could account for this epidemiological evidence [bib_ref] Sex and Gender Differences in Risk, Pathophysiology and Complications of Type 2..., Kautzky-Willer [/bib_ref] [bib_ref] Sex-Gender Differences in Diabetes Vascular Complications and Treatment, Franconi [/bib_ref].
Lipid abnormalities are important features in patients with T2D, who typically present high triglycerides and low high-density lipoprotein cholesterol (HDL-c) [bib_ref] Diabetic dyslipidaemia, Durrington [/bib_ref] , thus contributing as main morbidity and mortality factors. Since it is well-known that this lipid pattern is similar in diabetic men and women, it cannot be the cause of the aforementioned sex-difference in T2D complications. Thus, other parameters should be considered to explain this epidemiological data.
One of the best candidates in this frame is high-density lipoprotein (HDL) functionality. Several lines of evidence show that the sole cholesterol content of HDL particles does not fully capture their atheroprotective functions [bib_ref] Inherited disorders of HDL metabolism and atherosclerosis, Hovingh [/bib_ref] [bib_ref] Dysfunctional HDL and atherosclerotic cardiovascular disease, Rosenson [/bib_ref]. HDL has been ascribed with many beneficial activities. For instance, it has been shown to possess antioxidant, anti-inflammatory, and endothelial cell maintenance functions as well as playing a role in mediating reverse cholesterol transport [bib_ref] Biological Consequences of Dysfunctional HDL, Pirillo [/bib_ref] [bib_ref] Modulation of oxidative stress, inflammation, and atherosclerosis by lipoprotein-associated phospholipase A2, Rosenson [/bib_ref] [bib_ref] Novel biological functions of high-density lipoprotein cholesterol, Mineo [/bib_ref]. These beneficial aspects of HDL are impaired in T2D, which significantly increases the risk of developing atherosclerosis and related diseases [bib_ref] The anti-inflammatory function of HDL is impaired in type 2 diabetes: Role..., Ebtehaj [/bib_ref].
Paraoxonase 1 (PON1) is an accessory protein of HDL which, in intimate and mutual coordination with Apolipoprotein A1 (APO A1), contributes to the lipoprotein functionality [bib_ref] Human paraoxonase-1 (PON1): Gene structure and expression, promiscuous activities and multiple physiological..., Mackness [/bib_ref] [bib_ref] Paraoxonase-1 activities in individuals with different HDL circulating levels: Implication in reverse..., Cervellati [/bib_ref]. In particular, it has been widely demonstrated that PON1 protects HDL, low-density lipoprotein (LDL), endothelial cells, and intimal macrophages from oxidative insult [bib_ref] Paraoxonases-1, -2 and -3: What are their functions?, Furlong [/bib_ref] [bib_ref] Paraoxonase 1 (PON1) is a more potent antioxidant and stimulant of macrophage..., Rosenblat [/bib_ref]. This protective action occurs via hydrolysis of the potential physiological substrate, lipo-lactones, which originate from the oxidative damage of phospholipids present on cell and lipoprotein surfaces [bib_ref] The catalytic histidine dyad of high density lipoprotein-associated serum paraoxonase-1 (PON1) is..., Rosenblat [/bib_ref] [bib_ref] Characterization of the PON1 active site using modeling simulation, in relation to..., Tavori [/bib_ref]. Lipoprotein oxidation is central to the development of macro-(atherosclerosis) and micro-vascular disease in diabetes, as highlighted by the toxicity of oxidized lipoproteins for endothelial cells and pericytes in retinal capillaries [bib_ref] Toxicity of mildly modified low-density lipoproteins to cultured retinal capillary endothelial cells..., Lyons [/bib_ref] [bib_ref] LOX-1: Regulation, Signaling and Its Role in, Kattoor [/bib_ref]. Some studies report that PON1 activity is greater in women than men, this could be ascribed to an influential effect of sex hormones on the expression of the protein [bib_ref] Sex difference: An important issue to consider in epidemiological and clinical studies..., Trentini [/bib_ref] [bib_ref] Modulation of paraoxonase (PON1) activity, Costa [/bib_ref]. Indeed, in vivo studies found that mice treated with a male-pattern growth hormone had decreased hepatic mRNA levels of PON1 [bib_ref] Hormonal and chemical regulation of paraoxonases in mice, Cheng [/bib_ref] , whereas estrogens increased the stabilization/regulation of PON1 without affecting its expression [bib_ref] Estradiol enhances cell-associated paraoxonase 1 (PON1) activity in vitro without altering PON1..., Ahmad [/bib_ref]. Nonetheless, the evidence of differential expression of PON1 in the context of T2D is still undisclosed. Indeed, the only known information about PON1 expression in T2D is related to its genetic polymorphisms, which have been suggested to be responsible for variations in serum PON1 activity of T2D patients [bib_ref] Association of SRB1 and PON1 gene polymorphisms with type 2 diabetes mellitus:..., Wamique [/bib_ref]. Consistently, the majority of population-based studies on the PON1 phenotype, found that low enzymatic activity is associated with T2D and related clinical complications, and this, in turn, could be due to the increased levels of pro-atherogenic oxidized LDL and HDL of these patients [bib_ref] Glycation of paraoxonase-1 inhibits its activity and impairs the ability of high-density..., Mastorikou [/bib_ref] [bib_ref] The paraoxonases: Role in human diseases and methodological difficulties in measurement, Camps [/bib_ref] [bib_ref] HDL 2 particles are associated with hyperglycaemia, lower PON1 activity and oxidative..., Stefanović [/bib_ref] [bib_ref] Protective Effect of Paraoxonase Activity in High-Density Lipoproteins against Erythrocyte Membranes Peroxidation:..., Ferretti [/bib_ref].
Recently, we have shown that sex significantly affects the interplay of PON1 with CVD risk factors, such as overall and central obesity [bib_ref] Sex difference: An important issue to consider in epidemiological and clinical studies..., Trentini [/bib_ref]. In this study, we investigated in a large population of men and women whether the effect of gender on PON1 might contribute to the sexual dimorphism that characterizes complicated T2D.
# Materials and methods
## Subjects
In the present study, we re-examined the data collected from three different cohorts: (1) subjects attending the metabolic outpatient clinic of Sant'Anna University Hospital (Ferrara, Italy); (2) outpatients undergoing bone densitometry testing at the Menopause and Osteoporosis Centre of the University of Ferrara; (3) outpatients referring to the Obesity Centre of Sant'Anna (more details in [bib_ref] 17β-estradiol levels and oxidative balance in a population of pre-, peri-, and..., Cervellati [/bib_ref] [bib_ref] arylesterase and lactonase activities of paraoxonase-1 (PON1) in obese and severely obese..., Cervellati [/bib_ref] [bib_ref] Decreased arylesterase activity of paraoxonase-1 (PON-1) might be a common denominator of..., Castellazzi [/bib_ref].
The research protocols conform to The Code of Ethics of the World Medical Association (Declaration of Helsinki) and were conducted accordingly to the guidelines for Good Clinical Practice (European Medicines Agency). The studies were approved by the Local Ethics Committee of the University of Ferrara, and written informed consent was obtained from each patient during the first office visit before the possible inclusion in the study. No personal information was available to the authors of the study to protect the anonymity of the patients.
Inclusion criteria were men/women older than 18 years. Exclusion criteria were infection, acute or chronic disease (affecting liver, kidney, lungs, etc.), cancer, dementia, pregnancy, and alcohol consumption >10 g daily.
The diagnosis of T2D was made by following the American Diabetes Association (ADA) criteria.
Accordingly, a total of 778 subjects were divided into unaffected Controls (n = 581) and T2D subjects (n = 197).
At study entry, clinical (a questionnaire about comorbidities, lifestyle, and blood pressure) and anthropometric data (i.e., body mass index, BMI) were collected from each patient (however, anthropometric data were available only for a part of the sample subjects). Glucose level was assayed in all samples by the Central laboratory of S. Anna University Hospital, Ferrara, whereas plasma lipid profile (including total cholesterol, triglycerides, HDL-c, and LDL-c) was retrieved only for a part of the whole population (total, n = 685; controls, n = 545; T2D, n = 140).
## Serum sampling and biochemical assays
Venous blood samples from patients were collected after overnight fasting and centrifuged at 1500g for 10 min. The serum was aliquoted and stored at −80 - C until analysis.
All assays were performed by UV-VIS spectrophotometric techniques in a 96-well plate format by using a Tecan Infinite M200 microplate reader (Tecan group Ltd.).
Arylesterase activity was assayed by adding 10 µL of serum to 240 µL of reaction mixture consisting of 1 mmol/L phenylacetate and 0.9 mmol/L CaCl 2 dissolved in 9 mmol/L Tris-HCl, pH 8, according to previously published methods [bib_ref] arylesterase and lactonase activities of paraoxonase-1 (PON1) in obese and severely obese..., Cervellati [/bib_ref]. A molar extinction coefficient of 1.3 × 10 3 L −1 mol −1 cm −1 was used for the calculation of enzyme activity, expressed in kilo unit per liter. One unit of arylesterase activity accounts for 1 µmol of phenol produced in a min under the conditions of the assay. The intra-assay Coefficient of Variation (CV) was 3.8% whereas the inter-assay CV was 9.7%.
Total cholesterol (Tc), HDL-c, triglycerides, and glucose were assayed by routine enzymatic-colorimetric methods and LDL-c was calculated according to the Friedewald formula.
# Statistical analysis
Continuous variables were first analyzed for normal distribution using the Kolmogorov-Smirnov tests. Since the variables were not normally distributed, group comparisons were performed using the Mann-Whitney U test or t-test on the square root transformed variable (to approach a normal distribution). A chi-squared test was used to compare categorical variables between groups. Bivariate correlation analyses were performed using Spearman's tests. Multiple regression analysis was used to determine the independence of the found associations involving arylesterase in men and women. For all the models, residuals were visually inspected and tested for normality. Multivariate logistic regression analysis was performed to evaluate the association between low levels of PON-arylesterase activity and the risk of T2D. To this end, PON-arylesterase activity values in the whole population were divided into quartiles based on the distribution data of the control group. The outcome variable was the absence/presence of diabetes, whereas PON-arylesterase organized into quartiles was the predictor, with the highest quartile considered as the reference category. To correct for confounding factors and test the independency of predictor, we included age centered on the mean (64.72 years), smoking habits, hypertension, and HDL-c as covariates. These two analyses were performed on men and women separately. The multinomial logistic regression was also performed on the PON-arylesterase continuous variable, after centering for the mean in the whole population (91.6004 kU/L) by evaluating the interaction term sex*PON-arylesterase activity as the main clue for the influence of both PON-arylesterase and sex on the outcome. Both uncorrected and corrected multivariable logistic regression (for age, smoking habits, hypertension, and HDL-c) were performed. In all the analyses, a p < 0.05 was considered statistically significant.
# Results
## Population characteristics
The demographic and clinical characteristics of the whole population and grouped according to sex and disease status are summarized in [fig_ref] Table 1: Main clinical and demographic characteristics of controls and diabetic subjects in the... [/fig_ref]. As expected, subjects with T2D were characterized by a general worst lipid profile (high triglycerides, and low HDL-c, p < 0.001) than controls, although they demonstrated slightly lower total cholesterol and LDL-C than controls (p < 0.001). On the contrary, comorbidities such as hypertension and CVDs where more frequent in diabetic subjects [fig_ref] Table 1: Main clinical and demographic characteristics of controls and diabetic subjects in the... [/fig_ref] , p < 0.001 for both comorbidities). The differences observed in the lipid profile and comorbidities prevalence were grossly conserved when men and women were considered separately (for the complete results, see [fig_ref] Table 1: Main clinical and demographic characteristics of controls and diabetic subjects in the... [/fig_ref]. PON-arylesterase activity measured in the whole population, or subjects grouped in women and men. As depicted, we found significantly lower levels of PON-arylesterase activity in subjects affected by T2D compared with controls (p < 0.001), the difference was maintained also within sexes (p < 0.001 and p < 0.05, for women and men, respectively). Mann-Whitney U test was used for comparisons. PON: paraoxonase-1; T2D: Type 2 diabetes. Diabetic subjects showed a decreased activity of PON1 when compared with controls (p < 0.001, effect size r = 0.24). The same difference, although with a different magnitude, was also observed in the two subsets of women and men (p < 0.001, effect size r = 0.25, and p < 0.05, effect size r = 0.11, respectively). Of note, these differences remained significant even by comparing the square root transformed variable in T2D subjects and controls with a t-test (whole population, p = 0.001, effect size r = 0.24; women, p = 0.001, effect size r = 0.25), with the exception of men which did not show any difference (p = 0.082, effect size r = 0.1).
## Pon-arylesterase in diabetic and non-diabetic women and men
In agreement with the apparent link between T2D and low PON1, we found an inverse correlation between serum glucose levels and PON1 activity in the whole population (r = −0.209, p < 0.01). Notably, this association was significant in women but not in men (r = −0.162, p < 0.01 and r = −0.099 p = 0.125, respectively) (see [fig_ref] Figure 1 displays: PON-arylesterase activity levels in controls and T2D in the whole population, and... [/fig_ref].
## Logistic regression analysis for the association of pon1 decrease and t2d in relation to sex
We further explored the possible influence of sex on the association between PON1 activity and T2D by employing a logistic regression analysis.
The analysis was performed on the whole population without grouping by sex and by treating PON-arylesterase activity as a continuous predictor, presence/absence of T2D as an outcome, and by evaluating both the main effect of sex and the sex* PON-arylesterase interaction as clues for the influence of sex on the outcome. As summarized in [fig_ref] Table 1: Main clinical and demographic characteristics of controls and diabetic subjects in the... [/fig_ref] (Model 1), PON-arylesterase activity was a significant predictor associated with T2D development, since an increase in one unit of its activity caused a decrease in the odds of being diagnosed with T2D (Odds Ratio, O.R., (95% Confidence Interval, 95%CI): 0.97 (0.96-0.98)). Sex was confirmed to be a strong risk factor for the disease, with men characterized by greater odds of being affected by T2D (O.R.: 2.55 (1.77-3.67)). In addition, PON-arylesterase and sex seem to interact given the significant interaction coefficient we observed in Model 1 (see [fig_ref] Table 1: Main clinical and demographic characteristics of controls and diabetic subjects in the... [/fig_ref]. Upon correction for possible confounders like age, smoking habits, hypertension, HDL-c (see [fig_ref] Table 1: Main clinical and demographic characteristics of controls and diabetic subjects in the... [/fig_ref] , Model 2), both the main effects (PON-arylesterase and sex) and the interaction (O.R.: 1.025 (1.002-1.045), p = 0.010) remained significant.
We further explored the association between sex and PON-arylesterase in T2D by dividing the activity into quartiles based on the distribution in the control group, and by comparing the odds of being diagnosed with T2D of subjects with activity lower than < 110.9 kU/L (the IV quartile cut-off) with those within the higher activity quartile (>110.9 kU/L). As displayed in [fig_ref] Figure 2: Odds ratios [/fig_ref]. Adding HDL-c to the covariate set caused a further decrease in the strength of the relationship, in particular in men, where low PON1 activity was no more related to high odds of being affected by T2D [fig_ref] Figure 2: Odds ratios [/fig_ref].
The observation that this effect was much more relevant in men than in women was likely due to the different strengths in the correlation between PON1 and HDL-c within these two groups [fig_ref] Figure 3: Correlations between PON-arylesterase activity and HDL-C levels in men [/fig_ref]. Indeed, we found that the association in the whole population sample (r = 0.317, p < 0.001) was mostly driven by men [fig_ref] Figure 3: Correlations between PON-arylesterase activity and HDL-C levels in men [/fig_ref] , men: r = 0.417, p < 0.0001, women: r = 0.117, p < 0.05). Of note, when subjects were divided according to diagnosis of diabetes, only T2D women (r = 0.387, p < 0.01) and men in both control and T2D groups demonstrated a significant positive association between PON-arylesterase activity and HDL-c (r = 0.368, p < 0.001 and r = 0.411, p < 0.001, respectively). and women with T2D (F). As displayed, the correlation we found was mainly driven by men in both control and diabetes groups.
# Discussion
The present study confirms and extends previous findings on the implication of PON1 in T2D, but also demonstrated for the first time that sex significantly affects the interplay between the antioxidant enzyme and this disease. Indeed, we found that PON1 was decreased in women and, to a lesser extent, in men, but only in women was the difference independent of confounders such as age, smoking status, hypertension, and HDL-c. On the contrary, the change in men appeared to merely reflect the decrease in the T2D-related HDL level. Of note, the calculated effect sizes, a measure of the magnitude of the association between two variables (in this case PON1 and T2D), suggest that the effect of sex on PON1 in the context of T2D is small/moderate but significant (r = 0.25 of women and r = 0.11 for men; according to the classical definition from Cohen, r = 0.1, 0.3, or 0.5 indicates a small, medium, or large effect size, respectively).
In line with other studies [bib_ref] Paraoxonase 1: A better atherosclerotic risk predictor than HDL in type 2..., Patra [/bib_ref] [bib_ref] Serum Paraoxonase Activity, Concentration, and Phenotype Distribution in Diabetes Mellitus and Its..., Abbott [/bib_ref] [bib_ref] Serum paraoxonase activity and its relationship to diabetic complications in patients with..., Ikeda [/bib_ref] , we found that PON1 activity is significantly lower in patients with T2D compared to controls. Many of the published investigations on this topic are characterized by a relatively small and medium sample size. A major disadvantage of these studies is that the confidence intervals of the observed relationships are very broad and therefore lack the precision to quantify involvement of PON1 in the disease development. Our study is, to the best of our knowledge, the largest one on this research topic.
Mounting evidence suggests a scenario where PON1 and T2D are reciprocally related [bib_ref] Glycation of paraoxonase-1 inhibits its activity and impairs the ability of high-density..., Mastorikou [/bib_ref] [bib_ref] The paraoxonases: Role in human diseases and methodological difficulties in measurement, Camps [/bib_ref] [bib_ref] HDL 2 particles are associated with hyperglycaemia, lower PON1 activity and oxidative..., Stefanović [/bib_ref] [bib_ref] Protective Effect of Paraoxonase Activity in High-Density Lipoproteins against Erythrocyte Membranes Peroxidation:..., Ferretti [/bib_ref] [bib_ref] 17β-estradiol levels and oxidative balance in a population of pre-, peri-, and..., Cervellati [/bib_ref] [bib_ref] arylesterase and lactonase activities of paraoxonase-1 (PON1) in obese and severely obese..., Cervellati [/bib_ref] [bib_ref] Decreased arylesterase activity of paraoxonase-1 (PON-1) might be a common denominator of..., Castellazzi [/bib_ref] [bib_ref] Paraoxonase 1: A better atherosclerotic risk predictor than HDL in type 2..., Patra [/bib_ref] [bib_ref] Serum Paraoxonase Activity, Concentration, and Phenotype Distribution in Diabetes Mellitus and Its..., Abbott [/bib_ref] [bib_ref] Serum paraoxonase activity and its relationship to diabetic complications in patients with..., Ikeda [/bib_ref]. Various factors contribute to the development of T2D, such as genetic, lifestyle, and environment [bib_ref] Modulation of paraoxonase (PON1) activity, Costa [/bib_ref] [bib_ref] Hormonal and chemical regulation of paraoxonases in mice, Cheng [/bib_ref] [bib_ref] Estradiol enhances cell-associated paraoxonase 1 (PON1) activity in vitro without altering PON1..., Ahmad [/bib_ref] [bib_ref] Association of SRB1 and PON1 gene polymorphisms with type 2 diabetes mellitus:..., Wamique [/bib_ref] [bib_ref] Glycation of paraoxonase-1 inhibits its activity and impairs the ability of high-density..., Mastorikou [/bib_ref] [bib_ref] The paraoxonases: Role in human diseases and methodological difficulties in measurement, Camps [/bib_ref] [bib_ref] HDL 2 particles are associated with hyperglycaemia, lower PON1 activity and oxidative..., Stefanović [/bib_ref] [bib_ref] Protective Effect of Paraoxonase Activity in High-Density Lipoproteins against Erythrocyte Membranes Peroxidation:..., Ferretti [/bib_ref] [bib_ref] 17β-estradiol levels and oxidative balance in a population of pre-, peri-, and..., Cervellati [/bib_ref] [bib_ref] arylesterase and lactonase activities of paraoxonase-1 (PON1) in obese and severely obese..., Cervellati [/bib_ref] [bib_ref] Decreased arylesterase activity of paraoxonase-1 (PON-1) might be a common denominator of..., Castellazzi [/bib_ref] [bib_ref] Paraoxonase 1: A better atherosclerotic risk predictor than HDL in type 2..., Patra [/bib_ref] [bib_ref] Serum Paraoxonase Activity, Concentration, and Phenotype Distribution in Diabetes Mellitus and Its..., Abbott [/bib_ref] [bib_ref] Serum paraoxonase activity and its relationship to diabetic complications in patients with..., Ikeda [/bib_ref] [bib_ref] Paraoxonase-1 and Early-Life Environmental Exposures, Marsillach [/bib_ref]. The genetic predisposition of having low PON1 expression and activity has been ascribed as one of these influential factors by several authors [bib_ref] Modulation of paraoxonase (PON1) activity, Costa [/bib_ref]. The most studied functional single-nucleotide polymorphisms (SNPs) in the PON1 gene are Q192R and L55M. A recent meta-analysis showed that these SNPs Q192R/L55M are significantly associated with the risk of T2D [bib_ref] European versus Asian differences for the associations between paraoxonase-1 genetic polymorphisms and..., Luo [/bib_ref]. In vitro studies suggest that PON1 could participate in T2D pathogenesis by protecting against glucose inflammation and oxidative stress (OxS) cytotoxicity and may play a role in the secretion of insulin from these cells [bib_ref] The antioxidant HDL-associated paraoxonase-1 (PON1) attenuates diabetes development and stimulates β-cell insulin..., Koren-Gluzer [/bib_ref] [bib_ref] Importance of paraoxonase 1 (PON1) as an antioxidant and antiatherogenic enzyme in..., Shokri [/bib_ref]. On the other hand, reduction in PON1 activity could also represent an effect of oxidative and glycation stress associated with T2D. Indeed, it has been shown that these challenges reduce the catalytic efficiency of the enzyme in a dose-dependent manner [bib_ref] Oxidative inactivation of lactonase activity of purified human paraoxonase 1 (PON1), Nguyen [/bib_ref]. However, the evidence of sex-influence of PON1 expression in the context of T2D is still lacking, and limited to animal studies evaluating only non-diabetic females and males [bib_ref] Hormonal and chemical regulation of paraoxonases in mice, Cheng [/bib_ref] [bib_ref] Estradiol enhances cell-associated paraoxonase 1 (PON1) activity in vitro without altering PON1..., Ahmad [/bib_ref].
Independently of the causality of PON1 in T2D, the published evidence is concordant on the fact that the contribution of the protein to the beneficial properties of HDL is compromised in diseased patients [bib_ref] Glycation of paraoxonase-1 inhibits its activity and impairs the ability of high-density..., Mastorikou [/bib_ref]. This PON1 deficiency reflects in a weaker anti-atherogenic action, with HDL from T2D patients being less effective in counteracting the inhibitory effect of oxidized LDL on vasorelaxation [bib_ref] Inability of HDL from type 2 diabetic patients to counteract the inhibitory..., Perségol [/bib_ref] and endothelial nitric oxide synthesis [bib_ref] Endothelial-vasoprotective effects of high-density lipoprotein are impaired in patients with type 2..., Sorrentino [/bib_ref]. In line with this reasoning, reduced PON1 activity has been linked to a high risk of T2D-related CVD complications in several studies [bib_ref] The paraoxonases: Role in human diseases and methodological difficulties in measurement, Camps [/bib_ref] [bib_ref] Paraoxonase 1: A better atherosclerotic risk predictor than HDL in type 2..., Patra [/bib_ref] [bib_ref] Association of the novel cardiovascular risk factors paraoxonase 1 and cystatin C..., Connelly [/bib_ref]. However, our data suggest that this decrease in enzyme activity is not homogeneously distributed in the diabetic population, but it might involve mostly the female part. Indeed, in women, but not in men, the presence of T2D appears to be associated with two independent adverse phenomena such as decreased HDL-c concentration (a typical feature of diabetic dyslipidemia) and function. This would suggest that T2D attenuates the reported "double" physiological female sex advantage represented by the high HDL-c concentration and PON1 activity level [bib_ref] Diabetes abrogates sex differences and aggravates cardiometabolic risk in postmenopausal women, Mascarenhas-Melo [/bib_ref] [bib_ref] Estrogen, hormonal replacement therapy and cardiovascular disease, Yang [/bib_ref] [bib_ref] Epidemiology of cardiovascular diseases in women in Europe, Panico [/bib_ref]. As previously postulated by us and others, high levels of this enzyme might contribute to the lower risk of CVD in women compared to men [bib_ref] Sex difference: An important issue to consider in epidemiological and clinical studies..., Trentini [/bib_ref] [bib_ref] Modulation of paraoxonase (PON1) activity, Costa [/bib_ref]. Thus, the sex-dependent decrease in PON1 could be one of many biological factors explaining why T2D increases the risk for CVD in females to a greater extent than in males (see comprehensive review on sex difference in T2D complications and comorbidities [bib_ref] Diabetes abrogates sex differences and aggravates cardiometabolic risk in postmenopausal women, Mascarenhas-Melo [/bib_ref] [bib_ref] Diabetes and gender, Gale [/bib_ref] [bib_ref] Sex and Gender Differences in Risk, Pathophysiology and Complications of Type 2..., Kautzky-Willer [/bib_ref]. Notably, the logistic regression outcomes suggest that only in women the decrease in PON1 activity due to T2D is independent of HDL-c. The strong confounding effect of HDL-c we observed is not surprising since this marker is a surrogate for HDL particle concentration which, in turn, is the main carrier of PON1 [bib_ref] Human paraoxonase-1 (PON1): Gene structure and expression, promiscuous activities and multiple physiological..., Mackness [/bib_ref] [bib_ref] The importance of high-density lipoproteins for paraoxonase-1 secretion, stability, and activity. Free..., James [/bib_ref].
However, the observational nature of our study makes challenging to speculate on the biological mechanisms and factors underlying the apparent sex-dependent change of PON1 activity in T2D patients. Nevertheless, some intriguing clues in this regard are provided by the analysis of the correlation between HDL-c (i.e., surrogate maker of HDL particle) and PON1. Indeed, we found that the correlation between the enzyme activity and the level of its main carrier in circulation is much stronger in men relative to women, and it disappeared when non-diabetic women were considered separately. This difference might be connected to the already demonstrated shift of the accessory protein from an HDL subtype to another when diabetes occurs [bib_ref] Distribution of Paraoxonase-1 (PON-1) and Lipoprotein Phospholipase A2 (Lp-PLA2) across Lipoprotein Subclasses..., Passaro [/bib_ref] , which could also impact the catalytic efficiency of the protein. HDL belongs to a highly heterogeneous family which is composed of particles differing to each other for size, density, lipid, and protein constituents. Contrary to men, in non-diabetic women, PON1 could be segregated in one subtype in healthy women (e.g., the small HDL 3 or the buoyant HDL 2 ), and this might explain the lack of association with the whole HDL-c level. Then, with the occurrence of diabetes and the related oxidation/glycation of the lipoprotein PON1 could redistribute in other subclasses.
This study was not without limitations. First, the cross-sectional design precluded the ability to determine with certainty any cause-effect relationship between PON1 activity and the increased susceptibility of women to T2D compared to men. Second, potential confounding factors not considered in the study and the unequal size of sample groups might have affected the reliability of the results. Despite these undeniable caveats, this is, to the best of our knowledge, the largest population-based study on this topic.
In conclusion, our study suggests that PON1-decreased activity in T2D is a gender-specific phenomenon, with women being more affected than men. This difference might, at least partially, explain why diabetic women are more prone to develop cardiovascular complications than diabetic men. The decrease in PON1, reflecting in a poorer HDL functional quality, could represent a target for nutraceutical or pharmacological treatment in both sexes, but in particular in women. More in general, our findings strengthen the concept that since many diseases can manifest differently in men and women, searching for biological factors underlying this diversity becomes of crucial importance for defining the medical strategy.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3921/9/8/683/s1, [fig_ref] Figure 1 displays: PON-arylesterase activity levels in controls and T2D in the whole population, and... [/fig_ref] : Correlations between glucose and PON1-arylesterase activity, [fig_ref] Table 1: Main clinical and demographic characteristics of controls and diabetic subjects in the... [/fig_ref] : Logistic regression analysis for the association between PON-arylesterase activity, T2D, and sex.
Author Contributions: Conceptualization, C.C. and A.T.; methodology, V.R. and C.B.; software, C.C. and A.T.; formal analysis, C.C. and A.T.; data curation, C.C., A.T., J.M.S., A.P., and C.B.; writing-original draft preparation, C.C., A.T., and V.R.; writing-review and editing, A.T., C.C., A.P., G.Z., G.B., and T.B.; supervision, C.C. All authors have read and agreed to the published version of the manuscript.
[fig] Figure 1 displays: PON-arylesterase activity levels in controls and T2D in the whole population, and separately in women and men. [/fig]
[fig] Figure 1: Figure 1. PON-arylesterase activity measured in the whole population, or subjects grouped in women and men. As depicted, we found significantly lower levels of PON-arylesterase activity in subjects affected by T2D compared with controls (p < 0.001), the difference was maintained also within sexes (p < 0.001 and p < 0.05, for women and men, respectively). Mann-Whitney U test was used for comparisons. PON: paraoxonase-1; T2D: Type 2 diabetes. [/fig]
[fig] Figure 2: Odds ratios (95% C.I.) for the diagnosis of T2D in women and men with low levels (lower than IV quartile) of serum PON-arylesterase activity. Women with low levels of PON-arylesterase activity demonstrated increased odds of being affected by T2D independently from all cofounding factors (left panel). On the contrary, while the uncorrected model and the model corrected for age, smoking and hypertension suggested increased odds for diabetes in men as well, the inclusion of HDL-c as covariate made the relationship non-significant. HDL-c: High Density Lipoprotein-cholesterol; T2D: Type 2 Diabetes. [/fig]
[fig] Figure 3: Correlations between PON-arylesterase activity and HDL-C levels in men (A) and women (B) and separated according to diagnosis in control men (C), men with T2D (E), control women (D) [/fig]
[table] Table 1: Main clinical and demographic characteristics of controls and diabetic subjects in the whole population and divided by sex. Glucose level was available for 634 subjects. § Information regarding the use of lipid-lowering drugs was available for 602 subjects. # Complete lipid profile was available for 685 subjects. [/table]
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10.1155/2018/9782419
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CCBY
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5889910
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29770145
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s2orc_pubmed_articles
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β-Carboline Silver Compound Binding Studies with Human Serum Albumin: A Comprehensive Multispectroscopic Analysis and Molecular Modeling Study
β-Carbolines (βCs) belong to the naturally occurring alkaloid family, derived from 9H-pyrido [3,4-b]indole, also known as norharmane (Hnor). Knowing the importance of the βCs alkaloid family in biological processes, a comprehensive binding study is reported of four Ag(I) compounds containing the ligand Hnor and having different counteranions, namely, NO 3 − , ClO 4 − , BF 4 − , and PF 6 − , with human serum albumin (HSA) as a model protein. Different approaches like UV-visible, fluorescence spectroscopy, circular dichroism (CD), and molecular docking studies have been used for this purpose. e fluorescence results establish that the phenomenon of binding of Ag(Hnor) complexes to HSA can be deduced from the static quenching mechanism. e results showed a significant binding propensity of the used Ag(I) compounds towards HSA. e role of the counteranion on the binding of Ag(I) compounds to HSA appeared to be remarkable. Compounds with (ClO 4 − ) and (NO 3 − ) were found to have the most efficient binding towards HSA as compared to BF 4 − and PF 6 − . Circular dichroism (CD) studies made clear that conformational changes in the secondary structure of HSA were induced by the presence of Ag(I) compounds. Also, the α-helical structure of HSA was found to get transformed into a β-sheeted structure. Interestingly, (ClO 4 − ) and (NO 3 − ) compounds were found to induce most substantial changes in the secondary structure of HSA. e outcome of this study may contribute to understanding the propensity of proteins involved in neurological diseases (such as Alzheimer's and Parkinson's diseases) to undergo a similar transition in the presence of Ag-β-carboline compounds.
# Introduction
e history of silver applications started from its use in coins and jewelry. Women loved to decorate themselves with various trinkets of silver, but it is less well known that this metal can be an excellent metallotherapeutic agent. In the human body, silver (Ag) is not acting as an endogenous metal and exhibits relatively low toxicity. Ag(I) coordination compounds with a variety of ligands having nitrogen, phosphorus, and/or sulfur donor atoms have large applications in medicinal and analytical chemistry [bib_ref] Supramolecular design of one-dimensional coordination polymers based on silver(I) complexes of aromatic..., Khlobystov [/bib_ref]. Specifically, the antibacterial and antifungal actions of Ag(I) compounds are well known [bib_ref] Give silver a shine, Fromm [/bib_ref] [bib_ref] Silver nanoparticles as a new generation of antimicrobials, Rai [/bib_ref] [bib_ref] Antibacterial silver, Clement [/bib_ref]. e properties of silver compounds, like aqueous solubility, light stability, and biological activity, can be modified by varying the number and types of organic ligands [bib_ref] Supramolecular design of one-dimensional coordination polymers based on silver(I) complexes of aromatic..., Khlobystov [/bib_ref] [bib_ref] Give silver a shine, Fromm [/bib_ref] [bib_ref] Silver nanoparticles as a new generation of antimicrobials, Rai [/bib_ref] [bib_ref] Antibacterial silver, Clement [/bib_ref]. Furthermore, in the more recent past, Ag(I) compounds have also been reported to display antitumor activity and have shown activities comparable to the clinical chemotherapeutic drug (cisplatin) [bib_ref] Anticancer and antimicrobial metallopharmaceutical agents based on palladium, gold, and silver N-Heterocyclic..., Ray [/bib_ref] [bib_ref] Noble metals in medicine: latest advance, Medici [/bib_ref] [bib_ref] Nanobio silver: its interactions with peptides and bacteria, and its uses in..., Eckhardt [/bib_ref] [bib_ref] Synthesis and in vitro antitumor activity of water soluble sulfonate-and esterfunctionalized silver(I)..., Gandin [/bib_ref].
Human serum albumin (HSA) is responsible for about 60% of the plasma protein in humans and is accountable for nearly 80% of the osmotic pressure of the blood, and it plays a prominent role in drug disposition and efficacy [bib_ref] Structure of serum albumin, Carter [/bib_ref]. Various drugs bind reversibly to albumin and other serum components, which thereby function as carriers [bib_ref] Chapter 33 plasma protein binding of drugs, Olson [/bib_ref]. Serum albumin is known to increase the solubility of many drugs in plasma and modulates their delivery to cells in vivo and in vitro [bib_ref] Serum albumin, Peters [/bib_ref] [bib_ref] Binding, unfolding and refolding dynamics of serum albumin, Anand [/bib_ref]. Hence, it is relevant to study the interactions of drug candidates with this protein.
Norharmane, 9H-pyrido indole, is a rather unconventional ligand, belonging to an alkaloid family called β-carbolines (βCs). e molecule can act as a coordinating ligand, an H-bonding donor/acceptor, and may also act on π-π stacking; all these properties may act in synergy. A schematic chemical structure of norharmane is included in [fig_ref] 3. 1: General Observations and Synthesis [/fig_ref].
β-Carbolines are present in many plants, arthropods, and insects. Endogenously, these alkaloids are synthesized by tryptophan or tryptophan-like indoleamines and also found in urine, plasma, platelets (∼0.1 nM), in the case of mammals. Interestingly, after alcohol intake and smoking, β-carboline concentrations are found to increase to ∼1.0 nM. It is also well known that some β-carboline derivates on photoexcitation induce chromosomal damages in mammal cells and may disarm viruses and bacteria [bib_ref] Mechanisms of DNA damage by photoexcited 9-methyl-β-carbolines, Vignoni [/bib_ref]. Moreover, substituted aromatic β-carbolines may enter into the brain by crossing the blood-brain barrier (BBB) and may then be converted into methyl derivatives by specific enzymes, like methyltransferases. Some β-carbolines like 2,9-dimethylβ-carbolines have exhibited mitochondrial damage leading to neurotoxicity, while 9-methyl-harmine has a neuroprotective effect. β-Carboline compounds are also known to reduce the expression of phosphorylated forms of the socalled tau protein, potently at multiple Alzheimer's diseaserelated sites [bib_ref] Mechanisms of DNA damage by photoexcited 9-methyl-β-carbolines, Vignoni [/bib_ref] [bib_ref] 9-Methyl-β-carboline up-regulates the appearance of differentiated dopaminergic neurones in primary mesencephalic culture, Hamann [/bib_ref] [bib_ref] β-Carboline compounds, including harmine, inhibit DYRK1A and tau phosphorylation at multiple Alzheimer's..., Frost [/bib_ref].
In a previous study, some − compound being even comparable to cisplatin in two different cancer cell lines [bib_ref] Light-stable bis(norharmane)silver(I) compounds: synthesis, characterization and antiproliferative effects in cancer cells, Khan [/bib_ref]. To explore the possible molecular mechanism of action, we have decided to undertake a study of protein binding of the four silver compounds [bib_ref] Supramolecular design of one-dimensional coordination polymers based on silver(I) complexes of aromatic..., Khlobystov [/bib_ref] with Hnor varying the ionic sphere by changing the counteranions, namely, using NO 3 − , ClO 4 − , BF 4 − , and PF [bib_ref] Silver nanoparticles as a new generation of antimicrobials, Rai [/bib_ref]. HSA has been selected as a model for the protein binding studies. As spectroscopic techniques, UV-visible, fluorescence spectroscopy, and circular dichroism (CD) techniques have been chosen. Finally, the interactions of the Ag(I) compounds with the protein were studied using molecular modeling.
## Experimental section
2.1. Starting Materials and Syntheses. AgNO 3 , AgClO 4 , AgBF 4 , AgPF 6 , DL-tryptophan, and formaldehyde were purchased from Sigma-Aldrich. Human serum albumin (HSA; ≥99%, Sigma, USA) was essentially fatty-acid free and globulin free, purchased from Sigma, and used as received.
We have synthesized the four Ag(I) compounds as described in before [bib_ref] Light-stable bis(norharmane)silver(I) compounds: synthesis, characterization and antiproliferative effects in cancer cells, Khan [/bib_ref]. Other standard laboratory chemicals were used as available. Studies of protein folding have mainly been carried out in buffered dilute aqueous solutions to avoid loss of protein to the aggregation phenomenon. Stock solutions of HSA (300 µM) and Ag(I) compounds were prepared in a mixture of dimethyl sulfoxide (5%) DMSO and 95% phosphate buffer (20 mM) of pH 7.4 (i.e., well above the isoelectric point of HSA, pH 4.7; hence, the protein possesses a net negative charge at this pH). Conductivity in solution was studied by using an Accumet AB30 Fisher Scientific conductometer at room temperature in MeOH solution of the compounds.
## Protein binding studies.
e samples of HSA were prepared in 20 mM phosphate buffer (pH 7.4), whereas silver complexes (1 mM) stock solution were prepared in DMSO and further diluted in 20 mM phosphate buffer to reach the desired concentration. In all the samples, the final concentration of DMSO was not more than 1%. e concentration of HSA was determined using the Beer-Lambert law with the molar extinction coefficient of 36500 M −1 ·cm −1 at 280 nm. To study structural changes in HSA by the addition of [Ag(I) (Hnor) 2 ](anion) compounds, the UV absorption spectra were measured, by variation of the concentration of the [Ag(I) (Hnor) 2 ](anion) compounds, while keeping the concentration of HSA constant. UV absorption spectra, from 240 nm to 320 nm, were recorded on a Perkin-Elmer Lambda 45 spectrophotometer at 25°C. Quartz cuvettes of 1 cm path length were used for the measurements. Fluorescence measurements were performed on a Hitachi spectrofluorometer (Model F 7000) equipped with a PC. e fluorescence spectra were collected at 25°C with a path length cell of 1 cm. e slit width used was 5 nm with a protein concentration of 5 µM. e used excitation wavelength for the protein was 295 nm. CD measurements were carried out on a Jasco spectropolarimeter (Model J-815) equipped with a microcomputer. e instrument was calibrated with D-10-camphorsulfonic acid. All the CD measurements were performed at 25°C with a thermostatically controlled cell holder, attached to a Neslab RTE-110 water bath with an accuracy of ±0.1°C. Spectra were collected with a scan speed of 0.2 nm/min and a response time of 1 s. Each spectrum was taken as the average of three scans. e far-UV CD spectra were measured at a protein concentration of 20 µM at a path length of 1 cm.
All the spectra were recorded after equilibration of the reaction mixture for 5 min.
## Molecular docking.
e rigid molecular docking studies were performed by using HEX 8.0.0 software [bib_ref] Docking essential dynamics eigenstructures, Mustard [/bib_ref] , which is an interactive molecular graphics program for calculating and displaying possible docking modes of protein. e Hex 8.0.0 performs protein docking using spherical polar Fourier correlations [bib_ref] Ultra-fast FFT protein docking on graphics processors, Ritchie [/bib_ref]. Hex 8.0.0 necessitates the ligand and the receptor as input in PDB format. e parameters used for docking include the following: correlation type: shape only; FFT mode: 3D; grid dimension: 0.6; receptor range: 180; ligand range: 180; twist range: 360; and distance range: 40. e coordinates of compounds 1 and 2 were taken from its crystal structure as a .cif file and were converted to the PDB format using Mercury software. The crystal structure of the human serum albumin (PDB ID: 1h9z) was downloaded from the protein data bank (http://www.rcsb.org./pdb). Visualization of minimum energy favorable docked poses has been performed using Discovery Studio 4.1and PyMOL [25]. , were done by dissolving the starting Ag salt and Hnor in acetonitrile (ratio 1 : 2); they were characterized according to the procedures reported by some of us previously [bib_ref] Light-stable bis(norharmane)silver(I) compounds: synthesis, characterization and antiproliferative effects in cancer cells, Khan [/bib_ref]. Conductivity studies in MeOH solution have shown that the compounds behave as 1 : 1 electrolytes. Given the known kinetic lability of Ag(I), it is assumed that the Hnor ligand may dissociate from and associate with the Ag ion, in solution, but on average, they are largely coordinated. e schematic structures of the ligand and the used Ag(I) compounds are depicted in [fig_ref] 3. 1: General Observations and Synthesis [/fig_ref].
# Results and discussion
## Hsa binding studies.
e interaction between the small bioactive molecules and protein receptors is a fundamental step in the drug discovery process. Obtaining a thorough idea of the interaction of the protein with chemical entities plays a vital role in the etiology of several diseases. e protein-drug intermediate products involved in governing various biochemical phenomena in both normal and diseased cells are known to play a significant role in metabolizing therapeutic compounds and their transport [bib_ref] Photoaffinity labeling in drug discovery and developments: chemical gateway for entering proteomic..., Hatanaka [/bib_ref] [bib_ref] Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural..., Robinette [/bib_ref].
## Uv absorption studies.
e binding propensity of the drug candidate with the biomolecule was first studied by using the UV technique. e cumulative absorption of three aromatic amino acid residues gives rise to an absorption peak at 280 nm for human serum albumins (HSA) [bib_ref] Ultraviolet absorption spectroscopy, Mach [/bib_ref]. With the concomitant increase in concentration of Ag(I) compounds, the absorbance of HSA increased, and shifts toward longer wavelengths were observed; the Ag(I) compounds give a definite pattern of the UV-Vis spectrum with weak absorbance at a higher concentration between 295 and 320 nm, ascribed to the ligand "Hnor." e profound enhancement of UV absorbance (hyperchromism) with a redshift (bathochromic effect) of 7 nm (1), 5 nm (2), 5 nm (3), and 6 nm (4) in the spectra is suggestive of the formation of adducts/intermediates between the Ag(I) compounds and HSA. HSA is known to act as an important extracellular antioxidant, and this antioxidant property resides in one free cysteine-derived redox-active thiol (-SH) group (i.e., Cys34), which can occur in either reduced or oxidized form. Ag(I) being a soft Lewis acid, its compounds are known to have a high affinity towards sulfur-ligand atoms and moderate affinity towards nitrogen donor atoms. However, binding of Ag(I) towards methionine (Met)/histidine(His) residues and disulfide bridges, and nitrogen atoms, such as deprotonated peptide nitrogen, imine, and indole nitrogen, also cannot be ruled out completely [bib_ref] Model peptide studies of Ag+ binding sites from the silver resistance protein..., Chabert [/bib_ref] [bib_ref] Electron transfer in peptides: on the formation of silver nanoparticles, Kracht [/bib_ref]. However, the compounds 1-4 are quite stable in the solution.
e difference spectra of HSA have confirmed that the conformational changes to HSA are due to binding of the Ag (I) compounds and also [fig_ref] Figure 2: displays the UV absorption spectra of HSA in the absence and presence... [/fig_ref] in Supplementary Materials). Nevertheless, the variation in binding extent/mode exhibited by the spectra of the compounds to HSA may be associated with the effect of the counteranions. e anions may have facilitated the microenvironmental changes of protein and exposed the targeted site of a subdomain of protein to assist or enhance the selectivity of the metal center, that is, Ag(I). e effect of anions has been studied with the help of molecular docking to get further insight into their role. e possible role of anions has been discussed in computational studies section. However, the dissociation of the Ag(I) compound is a matter of main concern that we have also studied and found quite stable complex association of Ag(I)-Hnor. Shen et al. [bib_ref] Studies on the interaction between Ag+ and human serum albumin, Shen [/bib_ref] have studied the interaction of Ag + alone with HSA. When we compared these results of Ag compounds with Ag + alone, silver compounds exhibited significantly worthy binding propensity compared to the Ag + alone. In the next stage, with the aim to gain more information about the mode of binding of the Ag(I) compounds with HSA, solution fluorescence studies were carried out.
## Luminescence studies.
e interaction between metal compounds and proteins has been widely investigated by using fluorescence spectroscopies [bib_ref] Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural..., Suryawanshi [/bib_ref] [bib_ref] Synthesis and crystal structure determination of copper(II)-complex: in vitro DNA and HSA..., Tabassum [/bib_ref] [bib_ref] Interaction and photo-induced cleavage studies of a copper based chemotherapeutic drug with..., Tabassum [/bib_ref]. e luminescence response of HSA, upon addition of different concentrations of Ag(I) compounds, was studied in 20 mM phosphate buffer by using an emission titration experiment. Preliminary luminescence studies of the four starting Ag compounds 1-4 have been reported earlier by some of us [bib_ref] Light-stable bis(norharmane)silver(I) compounds: synthesis, characterization and antiproliferative effects in cancer cells, Khan [/bib_ref]. e excitation at 290 nm for all four compound results in a strong luminescence band at around 370 nm. e fluorescence studies of the ligand "Hnor" as well as the free silver salt, "AgNO 3 ," were also studied under the same conditions to compare the effect of separate entities with a combination of the two. Upon excitation at 295 nm, fluorescence intensity around 340 nm is known to reflect the changes of the tryptophan residue microenvironment [bib_ref] Protein Stability, and Folding: eory and Practice, Royer [/bib_ref]. Encouragingly, the luminescence of HSA was found substantially decreased in the presence of increasing concentrations of [Ag(I)(Hnor) 2 ]ClO 4 , as depicted in . In , similar data are presented for compounds 2-4, with only marginal differences upon anion variation.
A gradual decrease in the luminescence of HSA was observed upon increasing concentration of Ag(I) compounds showed significant redshift at the maximal emission wavelength of tyrosine(Tyr) and tryptophan(Trp) residues of 7 nm (1), 5 nm (2), 4 nm (3), and 4 nm (4), which indicates the altered conformation of the HSA by decrease in the polarity around Tyr and Trp residues and increase in hydrophobicity. The continuous, gradual addition of Ag(I) compounds resulted in further decreases in the fluorescence intensity of HSA, which again is indicative of a strong interaction between the Ag(I) compounds and HSA. The factors linked to quenching of the fluorescence can be associated with, for example, excited-state reaction, energy transfer, molecular rearrangements, collisional quenching, and/or ground-state compound formation [bib_ref] Spectroscopy features of the binding of polyene antibiotics to human serum albumin, Romanini [/bib_ref]. erefore, the consequence of these molecular interactions enforcing towards the static quenching that comprises the establishment of a ground state adduct between the fluorophore and the quencher. e involvement of static quenching in the binding process can be determined with the help of analyzed values of bimolecular quenching constant (K q ). A value of K q higher than 2.0 × 10 10 M −1 ·s −1 reflects the quenching to be static [bib_ref] Oxygen quenching of fluorescence in solution: an experimental study of the diffusion..., Ware [/bib_ref] [bib_ref] Interaction of human serum albumin with sulfadiazine, Ali [/bib_ref]. e interaction of the Ag compounds with HSA was quantified; the Stern-Volmer equation has been employed [bib_ref] A fluorescence analysis of ANS bound to bovine serum albumin: binding properties..., Togashi [/bib_ref] :
[formula] F 0 F � 1 + K SV [Q] � 1 + K q τ 0 [Q],(1) [/formula]
where F 0 and F are the steady-state fluorescence intensities in the absence and presence of quencher at 340 nm, respectively. K sv is the Stern-Volmer quenching constant, K q stands for bimolecular quenching constant, τ 0 is the lifetime of the fluorophore in the absence of quencher, and [Q] is the concentration of quencher (i.e., the Ag compound). By calculating the quenching rate constants, K q , one can distinguish between the static quenching and the dynamic quenching, and this was evaluated by using the following equation:
[formula] K q � K SV τ 0 .(2) [/formula]
e value of tau(zero), τ 0 , for biopolymers is known to be 10 −8 s. [bib_ref] Synthesis and crystal structure determination of copper(II)-complex: in vitro DNA and HSA..., Tabassum [/bib_ref] e Stern-Volmer quenching constant (K sv ) for the fluorometric titration of the Ag(I) compounds into HSA solution was calculated from the linear relationship between F 0 /F and [Ag(I)(Hnor) 2 ]ClO 4 [fig_ref] Figure 5 1: [/fig_ref] and enlisted in [fig_ref] Table 1: Stern-Volmer quenching constants and bimolecular quenching rate constant for the interaction of... [/fig_ref]. In , similar data are presented for compounds 2-4, confirming the above observations. us, these data ascertain the static quenching in the interaction of Ag(I) compounds with HSA, by the calculated value of K q . e equilibrium between free and bound molecules, when small molecules bind independently to a set of similar sites on a macromolecule, was ascertained by a modified Stern-Volmer equation [bib_ref] Über die abklingzeit der fluoreszenz, Stern [/bib_ref] :
[formula] log F 0 − F F � logK + n log[Q],(3) [/formula]
where K and n are the binding constant and the number of binding sites, respectively. A plot of log(F 0 − F/F) versus log[Q] was used to determine the value of K and n (inset in [fig_ref] Figure 5 1: [/fig_ref]. Several forces, like electrostatic, hydrogen bonds, weak van der Waals, hydrophobic, and steric contacts, can be thought of being responsible for the interaction between the albumin and Ag compounds. e value of the binding constant, K, was used to calculate the standard free energy change ΔG°of the binding of the ligand to the HSA, by using the relationship [bib_ref] Über die abklingzeit der fluoreszenz, Stern [/bib_ref] :
[formula] ΔG � −2.303 RT logK.(4) [/formula]
e values of K, n, and ΔG (binding) are presented in [fig_ref] Table 1: Stern-Volmer quenching constants and bimolecular quenching rate constant for the interaction of... [/fig_ref] Since it is known that HSA is a monomeric, three-domain, allosteric protein with only one free cysteine, Cys34, the Ag(I) ion could selectively bind at this site because it has a strong preference for S-donor atoms. Also, it can be assumed that the counteranions have facilitated the microenvironmental changes and may have contributed to the exposure of Cys34 from the subdomain of HSA and facilitated that the sulfur atom of cysteine will coordinate to the Ag(I) center of the compounds. Nevertheless, the known affinity of Ag(I) for Met residues and disulfide bridges and nitrogen atoms of HSA coordination on such sites cannot be ignored completely. is hypothesis is supported by the observation that the extent of microenvironmental changes of the protein is much higher in the presence of additional ClO 4 − and NO 3 − anions (see below), and which agrees with the trend in their binding parameter [fig_ref] Table 1: Stern-Volmer quenching constants and bimolecular quenching rate constant for the interaction of... [/fig_ref] ; thus, a more profound effect on the Ag(I) compound and its HSA interaction is observed.
## Effects of addition of additional anions.
Given the fact that the highest anticancer activity and also the most substantial HSA interaction takes place in case of the perchlorate and nitrate salts, it was decided to add extra perchlorate (and also nitrate) for all cases and to study the effect on the binding affinity. So, we carried out two sets of experiments and studied the binding propensity of Ag(I) compounds with HSA. In one experiment, the extra ClO 4 − anion was added in a 1 : 4 ratio, compared to the Ag compound. In a second experiment, we used additional nitrate together with the Ag(I) compounds. Details are given in , and the quite similar details for the other 3 compounds are presented in [fig_ref] Figure 5 1: [/fig_ref] in Supplementary Materials.
To determine the role of only the anions (i.e., without Ag and Hnor), we also used KClO 4 and KNO 3 and studied their HSA binding by adding variable amounts of anions to the concentration maximum used in the experiments. We found that on the addition of KClO 4 and KNO 3 to HSA, only a negligible perturbance of the HSA structure has occurred. Hence, the effect of these anions (ClO 4 − and KNO 3 − ) on HSA conformation can be neglected in comparison to the effect of Ag compounds and Ag compounds + additional anions on HSA conformation. When the extra anions + compounds were titrated, the results obtained did show an exponential increase in binding affinity of the Ag(I) compounds.
ese findings can be attributed to the enhanced exposure of the cysteine sulfur atoms of HSA, most likely caused by the significant electrostatic effect of the additional anions. is behavior is indicative of the increased microenvironmental changes of the HSA, thereby allowing the more binding of the Ag(I) center to coordinate to the HSA binding site. e binding strength was evaluated by calculating the Stern-Volmer constant (K sv ), a number of the binding sites (n) and Gibbs free energy (ΔG); [fig_ref] Table 2: Parameters for the interaction of HSA with Ag [/fig_ref] In complexes 1 and 2, extra ClO 4 − showed significant increase in the binding affinity as compared to extra NO 3 − as evident from [fig_ref] Table 2: Parameters for the interaction of HSA with Ag [/fig_ref] (K values). is exponential increase of the magnitude of 10 4 says a lot about the role of anions in the stronger binding affinity of the silver compounds with HSA. e order of binding of Ag(I) compounds with HSA upon extra anion addition was found to be 1 > 2 > 4 > 3. Thus, it is evident that the number of the binding sites (n) increases upon addition of extra anions.
## Binding and conformational changes in circular
Dichroism. CD spectroscopy is an ideal technique for monitoring the conformational changes of proteins. As shown in [fig_ref] Figure 8: Binding of compounds [/fig_ref] , the CD spectrum of free HSA (line 1) exhibits two negative bands at 208 nm and 222 nm in the ultraviolet region, attributed to n-π * transfer for the peptide bond, with a positive ellipticity at 193 nm, which is the characteristic of an α-helix structure of HSA [bib_ref] Determination of the secondary structures of proteins by circular dichroism and optical..., Chen [/bib_ref]. CD often allows obtaining information for almost all secondary structural variants, such as α-helices, β-sheets, β-turns, and random coil structures. All of these structures provide origins to bands of distinct forms and degrees in the far-UV region.
Modifications of the ellipticity at 222 nm (−MRE222) are convenient methods for observing and quantifying changes in the α-helical content [bib_ref] Insight into the binding mechanism of imipenem to human serum albumin by..., Rehman [/bib_ref]. CD spectra recorded in the presence and absence of various concentrations of Ag(I) compounds are presented in [fig_ref] Figure 8: Binding of compounds [/fig_ref]. Similar CD curves for the other three compounds are shown in [fig_ref] Figure 8: Binding of compounds [/fig_ref] , as they show comparable behavior. e CD signal, expressed in millidegree, obtained over the wavelength range of 190-250 nm, was converted to a mean residue ellipticity (MRE, θ), using the following conversion:
[formula] MRE � θ obs (mdeg) 10 × n × C p × l ,(5) [/formula]
where θ obs is the CD in millidegree, the number of amino acid residues (585) is given by n, "l" is the path length (cm), and C p is the molarity. e unit of MRE is deg·cm 2 ·dmol −1 . e α-helix contents of free and combined HSA were calculated from the MRE value at 222 nm, using the following equation [bib_ref] Determination of the secondary structures of proteins by circular dichroism and optical..., Chen [/bib_ref] [bib_ref] Interaction of meropenem with "N" and "B" isoforms of human serum albumin:..., Rehman [/bib_ref] :
[formula] % α − helix � MRE 222 nm − 2, 340 30, 300 × 100.(6) [/formula]
With increasing concentrations of the Ag(I) compounds, the CD signal exhibits significant changes in ellipticity, and this change corroborates with the binding of the compounds with the HSA backbone (spectra 2-6, [fig_ref] Figure 8: Binding of compounds [/fig_ref]. In fact with increasing concentration of the compounds the ellipticity decreases. However, at high concentration of silver compounds, a transition of the secondary structure (i.e., α-helix to β-sheet) was observed. e results confirm that the compound binds to the amino acid residues of the primary polypeptide chain in HSA, which has distorted its hydrogen-bonding networks. Interestingly, the decrease in the α-helical content is indicative of substantial microenvironmental changes of the polypeptide chains of HSA, which increased the exposure of some hydrophobic regions that were previously submerged. e microenvironmental changes of the protein were found more pronounced in the case of 1 (ClO 4 − ) and 2 (NO 3 − ), as compared to other two compounds [fig_ref] Table 3: Effect of the Ag [/fig_ref]. e results of all studies corroborate well with the findings of fluorescence and UV studies.
## Molecular docking.
To provide further and deeper insight into the interactions of HSA with compounds 1 and 2, a molecular docking technique was employed to learn more about the exact binding sites inside the molecular target HSA. From the 3D structure of crystalline albumin, it is known that HSA comprises three homologous domains that assemble to form a heart-shaped molecule, denoted I, II, and III: I (residues , , and III (384-585). e principal region of compound 1 binding sites of HSA is located in hydrophobic cavities in the subdomains IIA and IIIA, corresponding to sites I and II, respectively, and the tryptophan residue (Trp-214) of HSA in the subdomain IIA. A large hydrophobic cavity in the subdomain IIA (a binding site at I) to accommodate compound 1 is present, while compound 2 preferentially appears to the bind at site III. The minimum energy docked pattern ) indicates that compound 1 is primarily located within the subdomain IIA of HSA, forming numerous hydrophobic contacts (π-σ, π-π stacked, and π-alkyl) with GLN196, HIS242, LYS199, LYS195, CYS200, CYS245, and CYS245 residues of the hydrophobic binding site IIA [fig_ref] 3. 1: General Observations and Synthesis [/fig_ref]. Furthermore, also a number of hydrogen bonds and specific electrostatic interaction formed by the compound 1 are observed [fig_ref] Table 4: Noncovalent interactions of compound 1 with the HSA binding site IIA [/fig_ref]. e resulting docked pattern ) indicates that compound 2 is located in the subdomains IA and IB, forming various noncovalent interactions like hydrogen bond, electrostatic, and hydrophobic within the binding cavity residues. A detailed description is given in Table 5 [fig_ref] 3. 1: General Observations and Synthesis [/fig_ref]. ese noncovalent interactions formed by both the compounds 1 and 2 are dominated by hydrophobic contacts, with additional stabilization also assisted by the hydrogen bonding and electrostatic interaction with the polar residues of the binding site cavity. It has been previously observed [bib_ref] Synthesis, characterization and interaction studies of copper based drug with Human Serum..., Tabassum [/bib_ref] [bib_ref] Interaction of erucic acid with bovine serum albumin using a multi-spectroscopic method..., Shua [/bib_ref] that hydrogen bonding and electrostatic interaction decreased the hydrophilicity and increased the hydrophobicity to keep the compounds-HSA system stable. e relative binding energy of the docked structures was found to be −344.74 and −326.31 kJ/mol for 1 and 2, respectively. The results obtained from the molecular docking studies revealed that the hydrophobic forces dominated the interaction of Ag compounds with the HSA. Notably, the binding of the compounds 1 and 2 to different sites can be largely associated with the difference of ionic sphere.
To find out the effect of counterion in the HSA binding, we also perform the docking of the individual counterion (nitrate ion and perchlorate ion) with the HSA . In the minimum energy docked pose, the nitrate and perchlorate ion are found in the outer environment of the binding site's IIA and IIIA domains, respectively. ese anions form electrostatic and other noncovalent interaction at these domains, which could be responsible for the microenvironmental changes or conformational changes of proteins. So that presence of the nitrate and perchlorate ions in the complex may have facilitated or enhanced the binding propensity (see Tables S1 and S2 in Supplementary Materials), which is also evident from the binding constant values in the presence of the anions. us, it plays an influential role in binding to the biomolecule. is observation confirms the significant role of the anions in the mode of interaction with the biomolecule.
## Concluding remarks
β-Carbolines motif crosses blood-brain membrane (BBM) and are known to inhibit the phosphorylated form of the tau protein [bib_ref] Noble metals in medicine: latest advance, Medici [/bib_ref]. ese inhibitions are the essential features to study for any new compound to treat diseases such as Alzheimer's disease. Spectrophotometric methods have been presence of Hnor, despite being a labile ligand, appears to be important for the binding to HSA. e effect of anions was further validated by the molecular modeling studies which corroborate well with our findings of binding studies and ascertain that anions play an importance role in binding to HSA. [fig_ref] 3. 1: General Observations and Synthesis [/fig_ref] : UV absorption spectra of HSA in the absence and presence of compounds 1-4; concentration [HSA] � 5 μM. [fig_ref] Figure 2: displays the UV absorption spectra of HSA in the absence and presence... [/fig_ref] : UV absorption difference of HSA (the difference UV absorption spectra were obtained by HSA-Ag compound spectra minus Ag compound spectra). : fluorescence emission spectra of HSA (5 μM) in the presence of various concentrations of compounds 2, 3, and 4; curves from 1 to 10 correspond to compound concentrations of 0, 0.5, 1, 1.5, 2, 3, 4, 5, 7.5, and 10 µM, respectively, when excited at 295 nm. : the Stern-Volmer plot for the quenching of the HSA fluorescence by compounds 2, 3, and 4 at 295 nm. Inset: plot of log (F 0 -F)/F as a function of log (complex). [fig_ref] Figure 5 1: [/fig_ref] : fluorescence emission spectra of HSA (5 μM) in the presence of various concentrations of compounds 1-4, corresponding to compound concentrations of 0, 1, 2, 4, 7.5, and 10 µM with the addition of extra KClO 4 in a 1 : 4 ratio, respectively, when excited at 295 nm. : fluorescence emission spectra of HSA (5 μM) in the presence of various concentrations of compounds 1-4, corresponding to compound concentrations of 0, 1, 2, 4, 7.5, and 10 µM with the addition of extra KNO 3 in 1 : 4 ratio, respectively, when excited at 295 nm. : CD spectra of the HSA-compound system (HSA � 20 μM) in the presence of various concentrations of compounds 2, 3, and 4; curves from 1 to 6 corresponding to compound concentrations of 0, 20, 40, 60, 80, and 100 mM, respectively. [fig_ref] Figure 8: Binding of compounds [/fig_ref] : absorption spectra of Ag complexes only, 1: ClO 4 (red line) and 2: NO 3 (black line) of 1.0 mM solution. : molecular docked model of HSA in presence of anions. [fig_ref] Table 1: Stern-Volmer quenching constants and bimolecular quenching rate constant for the interaction of... [/fig_ref] : noncovalent interactions of nitrate ion with the HSA.
# Supplementary materials
[fig] 3. 1: General Observations and Synthesis. Synthesis of the four Ag(I) compounds, namely, [Ag(Hnor) 2 ](ClO 4 ) (1), [Ag(Hnor) 2 ](NO 3 ) (2), [Ag(Hnor) 2 (MeCN)](PF 6 ) (3), and [Ag(Hnor) 2 ](BF 4 ) (4) [/fig]
[fig] Figure 2: displays the UV absorption spectra of HSA in the absence and presence of [Ag(I)(Hnor) 2 ]ClO 4 . e behavior of the other 3 compounds(compounds 2-4)is quite similar, and these data are presented inFigure S1(see Supplementary Materials). [/fig]
[fig] Figure 3, Figure 4: Fluorescence emission spectra of HSA (5 µM) in the presence of various concentrations of [Ag(I)(Hnor) : e Stern-Volmer plot of HSA fluorescence quenching by [Ag(I)(Hnor) 2 ]ClO 4 at 295 nm. Inset: plot of log (F 0 −F)/F as a function of log [complex]. [/fig]
[fig] − > 4 BF 4 −: All estimated values of n are approximately 1, indicating the existence of just one major binding site in HSA for the present Ag(I) compounds. e extent of interaction of Ag(I) compounds with HSA was found in the order 1 (ClO 4 − ) > 2 (NO 3 > 3 (PF6 − ), which can be associated with the differences in the effect of counteranions. us, compounds 1 (ClO 4 − ) and 2 (NO 3 − ) exhibit significantly higher binding affinities towards HSA, compared to other two compounds. [/fig]
[fig] Figure 5 1: (1.0 μM) + extra ClO 4 1 (2.0 μM) + extra ClO 4 1 (4.0 μM) + extra ClO 4 1 (7.5 μM) + extra ClO 4 1 (10 μM) 2 (1.0 μM) + extra ClO 4 2 (2.0 μM) + extra ClO 4 2 (4.0 μM) + extra ClO 4 2 (7.5 μM) + extra ClO 4 2 (10 μM) Fluorescence emission spectra of HSA (5 µM) in the presence of various concentrations of (a) [Ag(I)(Hnor) 2 ]ClO 4 compound 1 + extra KClO 4 , and (b) [Ag(I)(Hnor) 2 ]NO 3 compound 2 + extra KClO 4 corresponding to compound concentrations of 1.0, 2.0, 4.0, 7.5, and 10 µM, respectively, when excited at 295 nm. [/fig]
[fig] Figure 6, Figure 7: Stern-Volmer plot of HSA fluorescence quenching by (Far-UV CD spectra of HSA-compound system (HSA � 20 µM) in the presence of various concentrations of [Ag(I) (Hnor) 2 ]ClO 4 complex. Curves from 1 to 6 correspond to compound concentrations of 0, 20, 40, 60, 80, and 100 mM, respectively. [/fig]
[fig] Figure 8: Binding of compounds (a) 1 and (b) 2 at different binding sites of HSA. [/fig]
[fig] Figure 9, Figure 10: (a) Molecular docked model of compound 1 (stick representation) located within the hydrophobic pocket in the subdomain IIA of HSA; (b) molecular docked model of compound 2 (stick representation) located within the hydrophobic pocket in the subdomain IB of HSA. used to investigate the interaction between HSA and the present Ag(I) compounds. All Ag(I) compounds showed a strong affinity towards HSA. An extra effect of the perchlorate and nitrate anions was found in the binding studies, which ascertained the higher binding propensity of the Ag(I) compounds with counteranions ClO 4 − and NO 3 − . e moderate HSA binding in the case of BF 4 − and PF 6 − salts could be enhanced by the addition of extra anions, namely, ClO 4 − and NO 3 − . e results have shown a significant role of the anion in Ag-HSA binding and even showed an exponential increase in the binding propensity of the Ag(I) compounds. is affinity has been attributed to the increased exposure of the Ag-binding subdomains of HSA, as a result of the electrostatic and hydrophobic interactions. Noncovalent interactions of compounds (a) 1 and (b) 2 at the subdomains IIA and IB of HSA, respectively. [/fig]
[table] Table 1: Stern-Volmer quenching constants and bimolecular quenching rate constant for the interaction of HSA with four Ag(I) Hnor compounds. [/table]
[table] Table 2: Parameters for the interaction of HSA with Ag(I) compounds with the addition of extra anion, namely, ClO 4 [/table]
[table] Table 3: Effect of the Ag(I) compounds on the α-helical structure of HSA (model protein) expressed as percentage α-helix character. [/table]
[table] Table 4: Noncovalent interactions of compound 1 with the HSA binding site IIA. [/table]
[table] Table 5: Noncovalent interactions of compound 2 with the HSA binding site III. [/table]
[table] Table S2: noncovalent interactions of perchlorate ion with the HSA. (Supplementary Materials) [/table]
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10.7759/cureus.6769
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7039359
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32140336
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Massive Gouty Tophi Presenting as Pseudotumor of the Elbow: A Rare Presentation
# Introduction
Gout is a systemic metabolic disorder with elevated serum urate levels and deposition of monosodium urate crystals in synovial and non-articular tissues resulting in repeated attacks of arthritis. The cutaneous manifestations of gout are represented by intradermal lesions or subcutaneous nodules called tophi, commonly seen in avascular tissue over the ears, olecranon and prepatellar bursae or in acral sites, often associated with tendons. Although tophi are seen in 10% of the patients with chronic gout, literature is sporadic on massive gouty tophi [bib_ref] Tophaceous gout: a clinical and radiographic assessment, Nakayama [/bib_ref]. We are presenting a case of massive elbow tophi simulating a tumor in a known patient of chronic gouty arthritis.
## Case presentation
A 39-year-old male patient, a known case of chronic gouty arthritis, presented to the orthopaedic clinic with massive swelling over the extensor aspect of left elbow, measuring 18x10 cm. which was progressively increasing over the past 10 years.The swelling was insidious in onset, slowly progressive with a waxing and waning course with occasional pain. There was no associated fever or any other constitutional symptoms. Past and family history were not significant.
On physical examination, the skin over the swelling was tense, shiny with venous prominence and superficial ulceration [fig_ref] FIGURE 1: Clinical photograph of the left elbow showing massive swelling with central ulceration [/fig_ref]. There was chalky white discharge from the ulceration. The patient had full, pain free range of motion of the elbow joint without any neurovascular deficit. The patient had multiple small swellings over the right pinna, bilateral hands, ankle and both feet. The systemic examination was unremarkable.
Hematological investigations revealed raised serum uric acid level (11 mg/dl), erythrocyte sedimentation rate of 38 mm/hour for the first hour and highly sensitive C-reactive protein level of 44.92 mg/l. Radiographs of the left elbow showed a huge soft tissue shadow with calcification [fig_ref] FIGURE 2: AP and lateral radiograph of the left elbow showing a huge soft... [/fig_ref]. Direct fine needle aspiration cytology was done from the left elbow swelling, which yielded blood mixed brownish material, and from the right index finger swelling, which yielded whitish chalky material. On microscopic examination, smears from both the sites showed similar cytological material comprising of numerous scattered and aggregates of non-branching needle-shaped urate crystals in a fluffy to amorphous dirty background. Few scattered histiocytes and occasional lymphocytes were also visualized along with red blood cells.
The patient was initially put on dietary restrictions, plenty of fluids and drug therapy in the form of anti-inflammatory medications and oral allopurinol 100 mg three times daily for three months. At the end of three months, the serum uric acid levels reduced to 6.6 mg/dl, tophi over the pinna disappeared, the swellings over the hand and feet decreased in size, but the swelling and ulceration over the left elbow tophi continued to increase in size.
After informed consent, the patient was planned for surgical excision of the massive left elbow tophi. The patient was positioned in a right lateral position under general anesthesia and en bloc excision of the swelling was performed through a standard posterior approach. The excised mass weighing around 1.5 kg was sent for histopathological examination and the wound was closed after excising the redundant skin margins [fig_ref] FIGURE 4: Excised mass weighing around 1,500 g [/fig_ref]. Gross examination of soft tissue specimen revealed a skin-covered globular mass measuring 16x16x8 cm with an area of ulceration. On sectioning, a thick paste-like brownish material with whitish chalky deposits was observed. Microscopic examination showed skin with hyperkeratosis, parakeratosis and an ulcer covered by acute inflammatory exudate, crystalline deposits and fibrin and dense perivascular lymphocytic infiltrate in the dermis. Extensive crystal deposition (mainly needle-shaped crystals present in sheaves and bunches) and associated calcification within the dermis and fibrocollagenous areas were associated with multinucleated giant cell reaction and chronic inflammatory cells. These features were consistent with urate arthropathy .
# Discussion
Gout (also known as Podagra) is a systemic disorder of purine metabolism resulting in increased uric acid levels with recurrent attacks of arthritis [bib_ref] Gout: an update, Eggebeen [/bib_ref]. The male-to-female ratio is 3.6:1 [bib_ref] The last defence? Surgical aspects of gouty arthritis of hand and wrist, Tang [/bib_ref]. Chronic gouty arthritis is often associated with tophi, which are deposits of monosodium urate crystals in and around the joints and tendons. Distal extremities are most commonly involved with predilection for extensor surfaces. These tophi are considered pathognomonic for gout [bib_ref] Gout: an old disease in new perspective-a review, Ragab [/bib_ref]. Conservative management with uricosuric drugs and xanthine oxidase inhibitors is effective in stabilizing and reducing the size of the tophi. However, 5%-10% of the patients do not respond completely to the conservative treatment.
The underlying pathology is invasion and destruction of skin, ligament, tendon, cartilage, and bone by deposition of urates. The process is accompanied by an acute or chronic inflammatory response at the site of involvement. The stages of gout include asymptomatic hyperuricemia, acute gouty arthritis, intercritical gout and chronic tophaceous gout which develops after 10 years of the intercritical gout phase [bib_ref] Management of acute and chronic gouty arthritis: present state-of-the-art, Schlesinger [/bib_ref].Tophi vary from semiliquid to inspissated, chalk-like deposits. These chalky materials reveal negatively birefringent needle-shaped crystals.
Early diagnosis and treatment prevent severe crippling from the disease. Although majority of the cases respond to conservative management, relative surgical indications in chronic tophaceous gout are unsightly painful tophi, infection, impairment of tendon function, nerve compression due to tophi, impending skin necrosis, ulceration and discharging sinus, painful destruction of joint, a decrease in uric acid level in the body by excision of massive tophi and cosmesis [bib_ref] A survey of indications, results and complications of surgery for tophaceous, Kumar [/bib_ref] [bib_ref] The surgical management of chronic tophaceous gout, Larmon [/bib_ref] [bib_ref] Surgical treatment of the chronic tophaceous deformity in upper extremities: the shaving..., Lee [/bib_ref]. Curettage and debridement can be done to remove tophi; however, it is associated with high rates of delayed wound healing and skin necrosis [bib_ref] Surgical treatment of subcutaneous tophaceous gout, Lee [/bib_ref]. Other surgical methods include shaving, hydrotherapy and en bloc excision. We chose surgical treatment in our case as the mass was progressively increasing despite treatment, has developed ulceration and was cosmetically disfiguring.
Postoperative wound healing is generally poor after excision of large tophi because of decreased circulation to the overlying skin which may require skin grafting [bib_ref] A survey of indications, results and complications of surgery for tophaceous, Kumar [/bib_ref]. In our case, the wound healed uneventfully as entire avascular skin was excised along with the mass.
# Conclusions
This case highlights one of the rare presentations of chronic tophaceous gout with a massive elbow tophus weighing around 1.5 kg and locally mimicking a soft tissue tumor. A high index of suspicion is required in these cases, and a simple test like aspiration of the material for crystal analysis and cytology can help to distinguish this from soft tissue sarcoma.
# Additional information disclosures
Human subjects: Consent was obtained by all participants in this study.
## Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.
[fig] FIGURE 1: Clinical photograph of the left elbow showing massive swelling with central ulceration. [/fig]
[fig] FIGURE 2: AP and lateral radiograph of the left elbow showing a huge soft tissue shadow with calcifications. [/fig]
[fig] FIGURE 3: AP radiograph of bilateral hands showing soft tissue shadows in phalanges and periarticular punched-out erosions. [/fig]
[fig] FIGURE 4: Excised mass weighing around 1,500 g. Imprint smear of the exudative material from the specimen confirmed gouty tophi with negatively birefringent needle-shaped sodium urate crystals (Figure 5). [/fig]
[fig] FIGURE 5: Imprint smear of the exudative material showing negatively birefringent needle-shaped sodium urate crystals. [/fig]
[fig] FIGURE 7: Clinical image showing healed scar and full function at the elbow. [/fig]
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10.1371/journal.pone.0041723
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22848579
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Photoperiodic Influences on Ultradian Rhythms of Male Siberian Hamsters
Seasonal changes in mammalian physiology and behavior are proximately controlled by the annual variation in day length. Long summer and short winter day lengths markedly alter the amplitude of endogenous circadian rhythms and may affect ultradian oscillations, but the threshold photoperiods for inducing these changes are not known. We assessed the effects of short and intermediate day lengths and changes in reproductive physiology on circadian and ultradian rhythms of locomotor activity in Siberian hamsters. Males were maintained in a long photoperiod from birth (15 h light/day; 15 L) and transferred in adulthood to 1 of 7 experimental photoperiods ranging from 14 L to 9 L. Decreases in circadian rhythm (CR) robustness, mesor and amplitude were evident in photoperiods #14 L, as were delays in the timing of CR acrophase and expansion of nocturnal activity duration. Nocturnal ultradian rhythms (URs) were comparably prevalent in all day lengths, but 15 L markedly inhibited the expression of light-phase URs. The period (t'), amplitude and complexity of URs increased in day lengths #13 L. Among hamsters that failed to undergo gonadal regression in short day lengths (nonresponders), t' of the dark-phase UR was longer than in photoresponsive hamsters; in 13 L the incidence and amplitude of light-phase URs were greater in hamsters that did not undergo testicular regression. Day lengths as long as 14 L were sufficient to trigger changes in the waveform of CRs without affecting UR waveform. The transition from a long-to a short-day ultradian phenotype occurred for most UR components at day lengths of 12 L-13 L, thereby establishing different thresholds for CR and UR responses to day length. At the UR-threshold photoperiod of 13 L, differences in gonadal status were largely without effect on most UR parameters.
# Introduction
Ultradian rhythms (URs) have been described for many taxa and persist at multiple levels of biological organization [bib_ref] Ultradian and Circadian Rhythms: Experiments and Models, Fuentes-Pardo [/bib_ref] [bib_ref] Ultradian rhythms as the dynamic signature of life, Yates [/bib_ref]. Prominent functionally significant URs of hormone secretion are well-described in the gonadal, pituitary and adrenal axes of mammals [bib_ref] The wisdom of the body revisited, Knobil [/bib_ref] [bib_ref] The crucial role of pulsatile activity of the HPA axis for continuous..., Lightman [/bib_ref].
In contrast to the abundant research on circadian rhythms, little is known about ultradian rhythms of behavior, with the exception of feeding and locomotor activity of voles and shrews [bib_ref] Differential elimination of circadian and ultradian rhythmicity by hypothalamic lesions in the..., Gerkema [/bib_ref] [bib_ref] Phase control of ultradian feeding rhythms in the common vole (Microtus arvalis):..., Gerkema [/bib_ref] [bib_ref] Polyphasic activity patterns in small mammals, Halle [/bib_ref]. and torpor in Siberian hamsters [bib_ref] Photoperiod and thermoregulation in vertebrates: body temperature rhythms and thermogenic acclimation, Heldmaier [/bib_ref]. Seasonal (photoperiod-driven and circannual) changes in the mammalian circadian system have been well-elaborated at formal [bib_ref] Photoperiod and thermoregulation in vertebrates: body temperature rhythms and thermogenic acclimation, Heldmaier [/bib_ref] [bib_ref] Circannual variations in circadian rhythms of ground squirrels, Lee [/bib_ref] [bib_ref] Complex circadian regulation of pineal melatonin and wheel-running in Syrian hamsters, Elliott [/bib_ref] [bib_ref] Photoperiod differentially modulates photic and nonphotic phase response curves of hamsters, Evans [/bib_ref]. and molecular levels of analysis [bib_ref] The rat suprachiasmatic nucleus is a clock for all seasons, Sumová [/bib_ref] [bib_ref] Neuroendocrine regulation of seasonal gonadotrophin and prolactin rhythms: lessons from the Soay..., Lincoln [/bib_ref] [bib_ref] Photoperiod differentially regulates circadian oscillators in central and peripheral tissues of the..., Carr [/bib_ref] [bib_ref] Photoperiod regulates multiple gene expression in the suprachiasmatic nuclei and pars tuberalis..., Johnston [/bib_ref] [bib_ref] Photoperiod differentially regulates gene expression rhythms in the rostral and caudal SCN, Hazlerigg [/bib_ref] [bib_ref] Influence of photoperiod duration and light-dark transitions on entrainment of Per1 and..., Sosniyenko [/bib_ref] [bib_ref] Distinct patterns of Period gene expression in the suprachiasmatic nucleus underlie circadian..., Schwartz [/bib_ref] , but only a few studies have addressed seasonal variations in mammalian URs. In the common vole, a 2 h rhythm in daytime trap catches was detected in winter but not in summer [bib_ref] Seasonal change in the daily timing of behavior of the common vole, Hoogenboom [/bib_ref]. In reindeer, URs of locomotor activity were significantly shorter in summer than in winter [bib_ref] Where clocks are redundant: weak circadian mechanisms in reindeer living under polar..., Van Oort [/bib_ref]. In Siberian hamsters, the dominant period of the body temperature rhythm also was shorter in long than in short day lengths [bib_ref] Photoperiod and thermoregulation in vertebrates: body temperature rhythms and thermogenic acclimation, Heldmaier [/bib_ref]. The precise day length at which the UR period change occurs, and whether it differs across decreasing short day lengths, has not been investigated. In female Siberian hamsters entrained to long day lengths, multiple quantitative aspects of URs (robustness, mesor, amplitude) differed between the light and dark phases of the photocycle, and the circadian system mediated most of these effects [bib_ref] a) Dissociation of Ultradian and Circadian Phenotypes in Female and Male Siberian..., Prendergast [/bib_ref]. In addition, an earlier study of Syrian hamsters documented increases in the robustness and amplitude of URs paralleling decreases in the robustness and amplitude of CRs over the course of gestation and lactation [bib_ref] b) Enhancement and suppression of ultradian and circadian rhythms across the female..., Prendergast [/bib_ref] along with apparent influences of ovarian hormones on the period and amplitude of URs. Whether changes in entrainment of the circadian system that occur as photoperiods decrease impact the ultradian system is unknown, as is the extent to which testicular hormones affect URs. To address these issues we monitored home cage locomotor activity of adult male Siberian hamsters transferred from a long photoperiod (15 h light/day; 15 L) to one of several day lengths ranging between 14 L to 9 L. This permitted titration of the critical day lengths for induction of photoperiodic responses in the ultradian, circadian and reproductive systems.
# Methods
## Animals and housing
Siberian hamsters (Phodopus sungorus) from a local breeding colony maintained on a light:dark cycle of 15 L (lights off at 18:00 CST) were housed in polypropylene cages (28617612 cm) on wood shaving bedding (Harlan Sani-Chips, Harlan Inc., India-napolis, IN) with cotton nesting material continuously available. Ambient temperature was 2060.5uC, and relative humidity 5362%. Food (Teklad Rodent Diet 8604, Harlan Inc.) and filtered tap water were provided ad libitum. In all photoperiods, illuminance was 400-700 lux at cage levls. All procedures conformed to the USDA Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Chicago.
## Activity measurements
Many studies of URs measure foraging or feeding behavior [bib_ref] Differential elimination of circadian and ultradian rhythmicity by hypothalamic lesions in the..., Gerkema [/bib_ref] [bib_ref] Phase control of ultradian feeding rhythms in the common vole (Microtus arvalis):..., Gerkema [/bib_ref] [bib_ref] Short-term rhythms in foraging behavior in the common vole, Microtus arvalis, Daan [/bib_ref] [bib_ref] Impact of behavior on central and peripheral circadian clocks in the common..., Van Der Veen [/bib_ref]. We measured URs and CRs of spontaneous general locomotor activity-a non-food-specific behavior that correlates highly with daily rhythms of sleep-wakefulness, body temperature, and drinking behavior [bib_ref] Neural regulation of circadian rhythms, Rusak [/bib_ref] [bib_ref] Circadian rhythms in the endocrine system, Kriegsfeld [/bib_ref] ; in the ultradian domain, locomotor activity correlates positively with feeding rhythms [bib_ref] Phase control of ultradian feeding rhythms in the common vole (Microtus arvalis):..., Gerkema [/bib_ref]. Locomotor activity studies address qualitative and quantitative aspects of underlying circadian and ultradian timing systems. Hereafter, when referring to ''URs'' and ''CRs'' we are referencing locomotor behavior rhythms.
Locomotor activity data were collected in the home cage for a minimum of 10 consecutive days with passive infrared motion detectors (Coral Plus, Visonic, Bloomfield, CT) positioned 22 cm above the cage floor. Motion detectors registered activity when 3 of 27 zones were crossed. Activity triggered closure of an electronic relay recorded by a computer running ClockLab software (Actimetrics, Evanston, IL). Cumulative activity counts were collected at 6 min intervals.
## Photoperiod manipulations
Male hamsters (n = 95), 60-90 days of age maintained from birth in a 15 L photoperiod were transferred on week 0 to one of seven day lengths: 9 L (n = 11), 10 L (n = 15), 11 L (n = 15), 12 L (n = 15), 13 L (n = 16), 14 L (n = 14), or 15 L (n = 9). The onset of darkness remained constant (18:00 CST) in all photoperiods to facilitate entrainment [bib_ref] Photoperiodism in hamsters: abrupt versus gradual changes in day length differentially entrain..., Gorman [/bib_ref]. Home cage activity data were collected between weeks 6 and 12. Reproductive and somatic responses in testis sizes, body mass, and pelage color were monitored at regular intervals. Estimated testis volume (ETV, the product of testis length and the square of testis width) was determined for each hamster on weeks 0, 3, 6, and 12, by measuring the length and width of the left testis under light isoflurane anesthesia through the scrotal skin with analog calipers. ETV is positively correlated with testis mass, circulating testosterone concentrations and spermatogenesis [bib_ref] Seasonal adaptations of Siberian hamsters. II. Pattern of change in day length..., Gorman [/bib_ref] [bib_ref] Spontaneous recrudescence of spermatogenesis in the photoinhibited male Djungarian hamster, Phodopus sungorus, Schlatt [/bib_ref]. On weeks 0, 6, and 12, hamsters were weighed (60.1 g), pelage color was assessed using a scale of 1 to 4 (1 = agouti, summer fur, 4 = white, winter fur, as described in [bib_ref] Hormonal regulation of the annual pelage color cycle in the Djungarian hamster,..., Duncan [/bib_ref] , without knowledge of the hamster's treatment group. For analyses of UR and CR waveforms (see below), sample sizes were increased by incorporating home cage locomotor data from 61 additional hamsters that were subjected to photoperiod manipulations identical to those described above in a study of photoperiod history effects on immune function [bib_ref] Photoperiod history differentially impacts reproduction and immune function in adult Siberian hamsters, Prendergast [/bib_ref] which was conducted concurrently with the present study.
Hamsters that failed to exhibit gonadal regression (week 12 ETV$300) and also did not exhibit a winter pelage (fur score = 1) in photoperiods #12 L were designated nonresponders (NR). Hamsters in 13 L and 14 L with large testes were not categorized as responders or nonresponders, but rather as having developed (ETV$300) or undeveloped (ETV,300) testes. Unlike hamsters that fail to exhibit gonadal regression in categorically short days (#12 L), because of aberrant entrainment of the circadian system and failure to expand nocturnal melatonin secretion [bib_ref] Photoperiodic polyphenisms in rodents: neuroendocrine mechanisms, costs, and functions, Prendergast [/bib_ref] , heterogeneous responses in 13 L and 14 L may be unrelated to circadian anomalies and instead reflect photoperiod history and non-photic effects [bib_ref] Establishment and persistence of photoperiodic memory in hamsters, Prendergast [/bib_ref] [bib_ref] Intermediate-duration day lengths unmask reproductive responses to nonphotic environmental cues, Paul [/bib_ref]. Data from hamsters with equivalent circadian entrainment in 13 L and 14 L, but exhibiting divergent reproductive responses, are instructive in determining the impact of reproductive status on URs and CRs.
## Activity analyses -data reduction
Ultradian rhythms (URs). Activity data collected at 6 min intervals were parsed into light-phase only (90-150 data points/ 24 h) and dark-phase only (90-150 data points/24 h) files. For hamsters in each day length, the number of days and nights sampled was adjusted to approximately equalize the number of data points to 900. Thus, for 15 L hamsters, 10 consecutive nights and 6 consecutive days generated dark-phase and light-phase activity files, each with 900 points; the same arrangement was achieved for 12 L hamsters by sampling 7.5 nights and 7.5 days. Successive days of photophase activity data were concatenated into a single file, as were successive nights of scotophase activity, and separately subjected to Lomb-Scargle periodogram (LSP) and cosinor periodogram analyses, as described in detail elsewhere [bib_ref] a) Dissociation of Ultradian and Circadian Phenotypes in Female and Male Siberian..., Prendergast [/bib_ref].
Circadian rhythms (CRs). Unparsed files (240 data points/ 24 h) 10 days in length, were subjected to LSP and cosinor periodogram analyses to extract quantitative CR parameters.
## Activity analyses -statistical analyses
Lomb-Scargle periodogram analyses [bib_ref] Least-squares frequency analysis of unequally spaced data, Lomb [/bib_ref] identified the statistical presence/absence of URs and CRs, and UR complexitythe number of significant peaks (distinct periods) in the UR spectrum (range: 0.1-7.9 h; [bib_ref] b) Enhancement and suppression of ultradian and circadian rhythms across the female..., Prendergast [/bib_ref]. The level of statistical significance (a) was set to 0.01. Cosinor analyses determined several quantitative measures of behavioral URs (range: 0.1-7.9 h) and CRs (range: 22-26 h): robustness (or 'prominence', the percent of variance accounted for by the best-fit cosine model, which corresponds to the coefficient of determination R 2 in regression analyses; [bib_ref] Procedures for numerical analysis of circadian rhythms, Refinetti [/bib_ref] ; mesor (rhythm-adjusted mean value around which the waveform oscillates); amplitude (the difference between the peak or trough value and the mesor), expressed as absolute values (activity counts) and relative values referenced to the photophase-specific mesor values); the latter measure incorporates baseline activity levels during each photophase in determining rhythm amplitude. Lastly acrophase was computed as the mean time (relative to the onset or offset of light) at which the waveform peaks. The level of statistical significance was set to 0.05.
The LSP detects ultradian periodicities from incomplete evenlysampled time series, is well-suited for measurement of data binned into separate scotophase/photophase files and optimizes detection of URs by not displaying peaks at multiples of all rhythms detected [bib_ref] The Lomb-Scargle periodogram in biological rhythm research: analysis of incomplete and unequally..., Ruf [/bib_ref] [bib_ref] A procedure of multiple period searching in unequally spaced time series with..., Van Dongen [/bib_ref]. Supplemental analyses after completion of LSP analysis [bib_ref] Analysis of problematic time series with the Lomb-Scargle method, a reply to..., Van Dongen [/bib_ref] were adopted as recommended by Refinetti et al. [bib_ref] Procedures for numerical analysis of circadian rhythms, Refinetti [/bib_ref]. The cosinor periodogram [bib_ref] Inferential statistical methods for estimating and comparing cosinor parameters, Bingham [/bib_ref] is a reliable, preferred curve-fitting tool to quantify rhythm parameters [bib_ref] Procedures for numerical analysis of circadian rhythms, Refinetti [/bib_ref].
## General statistical analyses
ANOVAs and pairwise comparisons were performed on a computer with Statview 5.0 (SAS Institute, Cary, NC, USA) and LSP and cosinor analyses with software written by R. Refinetti (available at http://www.circadian.org/softwar.html). The proportion of hamsters displaying URs and CRs was evaluated with chi-square tests. The hypothesis being tested was that transfer from 15 L to one of several shorter photoperiods caused a change in UR waveform. To this end, effects of day length on quantitative aspects of URs and CRs, were first examined with ANOVA, and a priori planned comparisons were pairwise contrasts between 15 L and each of the 6 shorter day lengths. Planned comparisons were calculated using Fisher's PLSD tests or unpaired, two-tailed t tests. Effects of day length on reproductive and somatic measures were evaluated similarly. Omnibus analyses of pelage scores were performed using the Kruskal-Wallis H test, followed by Mann-Whitney U tests for pairwise comparisons. Differences were considered significant if P#0.05.
## Multiple regression and correlation analyses
Pearson correlations were calculated to examine the relation between several potential predictor variables (photoperiod, testis size, body mass, and circadian waveform) and two features of the dark-phase UR waveform that respond robustly to decreases in photoperiod: 1) dark-phase UR t' and 2) dark-phase UR amplitude. In addition, a multiple regression assessed the relative contributions of photoperiod, reproductive status (ETV) and body mass to dark-phase UR t' and amplitude.
To further characterize the manner in which photoperiod affected URs and CRs, non-linear regressions were performed on quantitative parameters of both URs and CRs. Orthogonal polynomial contrast codes were assigned for each of the 7 experimental photoperiods to represent linear, quadratic, and cubic effects. Significant correlations following contrast coding assess whether or not the effect of incrementally-decreasing experimental photoperiods can be characterized by linear, quadratic, or cubic functions, and permit insight into whether photoperiod affects URs and CRs in a similar manner.
# Results
## Reproductive and somatic responses to photoperiod
All 11 hamsters in 9 L exhibited gonadal regression. Reproductively nonresponsive hamsters identified in 10 L (n = 4), 11 L (n = 6), and 12 L (n = 5) were removed from the main analysis. In 13 L, 14 L and 15 L, 50%, 21% and 0% of hamsters exhibited gonadal regression (13 L vs. 14 L:
## Ultradian rhythms
For the analysis of locomotor activity data, sample sizes were increased by incorporating data from 61 additional hamsters (9 L: n = 9, 10 L: n = 9, 11 L: n = 8, 12 L: n = 8, 13 L: n = 8, 14 L: n = 9, 15 L: n = 10), treated concurrently and in an identical fashion in a study of immune function [bib_ref] Photoperiod history differentially impacts reproduction and immune function in adult Siberian hamsters, Prendergast [/bib_ref]. None of the data in the present study were included in the prior report, which did not investigate URs, their relation to CRs, or the several circadian components affected by day length considered herein.
Dark-phase URs. Most hamsters exhibited dark-phase URs [fig_ref] Figure 2: Ultradian and circadian rhythms in locomotor activity [/fig_ref]. UR incidence ranged from 60-100%, but was not influenced by day length (P.0.10, all comparisons). UR complexity [fig_ref] Figure 3: Prevalence, complexity and period of URs in decreasing photoperiods [/fig_ref] and period [fig_ref] Figure 3: Prevalence, complexity and period of URs in decreasing photoperiods [/fig_ref] , P,0.005) increased in day lengths #13 L; UR robustness increased in photoperiods #12 L [fig_ref] Figure 4: Robustness, amplitude, and mesor of URs in decreasing photoperiods [/fig_ref] , P,0.001). UR amplitude increased in day lengths #13 L [fig_ref] Figure 4: Robustness, amplitude, and mesor of URs in decreasing photoperiods [/fig_ref] , and mesor activity levels were significantly lower in all day lengths shorter than 15 L [fig_ref] Figure 4: Robustness, amplitude, and mesor of URs in decreasing photoperiods [/fig_ref] , P,0.001). Short day lengths shifted the acrophase of dark-phase URs to later times in
## Circadian rhythms
Circadian organization was markedly altered by decreases in day length [fig_ref] Figure 6: Effects of decreasing photoperiods on robustness, mesor, and amplitude of circadian rhythms [/fig_ref]. CR robustness was greater in 15 L than in all other photoperiods [fig_ref] Figure 6: Effects of decreasing photoperiods on robustness, mesor, and amplitude of circadian rhythms [/fig_ref] , P,0.005, all comparisons), and did not differ among hamsters in day lengths from 9 L through 13 L. Mesor values were greater in 15 L than in all in other day lengths [fig_ref] Figure 6: Effects of decreasing photoperiods on robustness, mesor, and amplitude of circadian rhythms [/fig_ref] , P,0.05, all comparisons), except 11 L (P.0.40). CR amplitude was lower in 9 L than in all other day lengths [fig_ref] Figure 6: Effects of decreasing photoperiods on robustness, mesor, and amplitude of circadian rhythms [/fig_ref] , P,0.001, all comparisons), and higher in 15 L than in all other photoperiods (P,0.01, all comparisons). Circadian acrophases occurred progressively later in shorter photoperiods (P,0.001) and the duration of the nocturnal active phase (a) increased incrementally from 8.960.06 h in 15 L to 11.460.19 h in 9 L (mean 6 SEM; P,0.001).
## Urs and crs in nonresponder (nr) hamsters
URs were compared between short-day (9 L through 12 L, inclusive) hamsters that underwent gonadal regression (responders, SD-R) and those that maintained large testes (nonresponders, SD-NR). Because there were no quantitative differences in URs of SD- NR hamsters in 10 L, 11 L and 12 L (n = 4-6 per group), a single SD-NR group was constituted for purposes of analysis (n = 16). UR waveforms of SD-R and SD-NR hamsters were compared to those of 15 L hamsters (n = 19).
Dark-phase URs. UR prevalence was high (79-90%) and did not differ significantly between SD-R and SD-NR hamsters (P.0.80; [fig_ref] Figure 7: Ultradian rhythms of reproductively non-responsive hamsters [/fig_ref] , but UR complexity [fig_ref] Figure 7: Ultradian rhythms of reproductively non-responsive hamsters [/fig_ref].001) and UR robustness [fig_ref] Figure 7: Ultradian rhythms of reproductively non-responsive hamsters [/fig_ref] , P = 0.05) were significantly lower in SD-NR relative to SD-R hamsters; on these measures, SD-NR hamsters were comparable to 15 L hamsters. Dark-phase UR period was substantially longer in SD-NR hamsters than in SD-R hamsters [fig_ref] Figure 1: Reproductive, somatic, and pelage responses to decreasing photoperiods [/fig_ref]. Mesor and amplitude [fig_ref] Figure 7: Ultradian rhythms of reproductively non-responsive hamsters [/fig_ref] values were comparable in SD-NR and SD-R hamsters (P.0.05; all comparisons), but acrophases of SD-NR hamsters occurred significantly later than those of SD-R hamsters [fig_ref] Figure 7: Ultradian rhythms of reproductively non-responsive hamsters [/fig_ref] , P,0.001).
Light-phase URs. URs were less prevalent in SD-NR than in SD-R hamsters [fig_ref] Figure 7: Ultradian rhythms of reproductively non-responsive hamsters [/fig_ref] , P = 0.01). UR complexity (P,0.05) and robustness (P = 0.01) were lower in SD-NR than SD-R hamsters [fig_ref] Figure 7: Ultradian rhythms of reproductively non-responsive hamsters [/fig_ref] , but t' did not differ between these groups [fig_ref] Figure 7: Ultradian rhythms of reproductively non-responsive hamsters [/fig_ref] , P.0.30). Mesor values were low and comparable in SD-NR and SD-R hamsters (P.0.70), but UR amplitude was lower in SD-NR than SD-R hamsters [fig_ref] Figure 7: Ultradian rhythms of reproductively non-responsive hamsters [/fig_ref] , P,0.001). Acrophases did not differ significantly between SD-R and SD-NR hamsters [fig_ref] Figure 7: Ultradian rhythms of reproductively non-responsive hamsters [/fig_ref].
Circadian rhythms. CR robustness [fig_ref] Figure 8: Circadian rhythms of reproductively non-responsive hamsters [/fig_ref] , P,0.05) and amplitude [fig_ref] Figure 8: Circadian rhythms of reproductively non-responsive hamsters [/fig_ref] , P,0.005) were significantly greater in SD-NR than SD-R hamsters; CR acrophases occurred .3 h later in SD-NR hamsters [fig_ref] Figure 1: Reproductive, somatic, and pelage responses to decreasing photoperiods [/fig_ref]. Duration of the active phase was substantially shorter in SD-NR hamsters [fig_ref] Figure 8: Circadian rhythms of reproductively non-responsive hamsters [/fig_ref] , P,0.001; cf. [bib_ref] Characterization of circadian function in Djungarian hamsters insensitive to short day photoperiod, Puchalski [/bib_ref] [bib_ref] Evidence that the circadian system mediates photoperiodic nonresponsiveness in Siberian hamsters: the..., Freeman [/bib_ref]. Ultradian Rhythms. Dark-phase URs were evident in 94% of 13 L2 and 88% of 13 L+ hamsters , P.0.60). In contrast, light phase URs were present in 100% of 13 L2 but in only 63% of 13 L+ hamsters , P,0.05).
## Crs and urs in the intermediate-duration photoperiod
Quantitative aspects of dark-phase URs (complexity, t', robustness, mesor, amplitude, acrophase) did not differ between 13 L+ and 13 L2 hamsters. Light-phase UR complexity, t', robustness, mesor and acrophase also were similar in 13 L + and 13 L2 hamsters (P.0.10, all comparisons), but amplitude of the light-phase UR was greater in 13 L2 than in 13 L+ hamsters [fig_ref] Figure 5: Ultradian rhythm acrophase in decreasing photoperiods [/fig_ref].
Circadian Rhythms. CR acrophase and circadian a were comparable in 13 L+ and 13 L2 hamsters ; P.0.40, both comparisons). CR robustness, mesor and amplitude were also indistinguishable between 13 L+ and 13 L2 groups (P.0.10, all comparisons).
## Simple and multiple regression analyses
Day length (R = 20.36; P,0.001) and testis size (R = 20.21; P,0.01) were negatively correlated with dark-phase UR t', whereas body mass did not predict t' (P.0.50; [fig_ref] Table 1: Least squares regression models of single [/fig_ref]. Robustness of the circadian waveform (R = 0.17; P,0.05), mesor activity levels (R = 0.27, P,0.01) and CR acrophase (R = 0.29, [fig_ref] Table 1: Least squares regression models of single [/fig_ref].
To examine if effects of photoperiod on URs are mediated by concurrent changes in reproductive condition or body mass, a multiple regression model constructed of 3 components (photoperiod, week 12 body mass and week 12 ETV) was constructed. This model significantly predicted dark-phase UR t' (R 2 = 0.22, F3,130 = 7.94, P,0.001; standard error of the estimate = 1.42 h) and dark-phase UR amplitude (R 2 = 0.47, F3,129 = 36.6, P,0.001; standard error = 0.129; [fig_ref] Table 1: Least squares regression models of single [/fig_ref]. The effect of photoperiod on both t' and UR amplitude was significant (P,0.001) in this model [fig_ref] Table 1: Least squares regression models of single [/fig_ref]. The effects of testis size on t' and on UR amplitude, which were present as zero-order effects in the simple linear regression, were not significant when photoperiod was included in the model. However, a significant negative effect of body mass on UR amplitude was obtained in the multiple regression model (partial correlation coefficient = 20.009, P,0.001; [fig_ref] Table 1: Least squares regression models of single [/fig_ref].
## Linear, quadratic and cubic contrasts
Linear regression analyses on quantitative parameters of darkphase URs revealed significant simple (linear) effects of photoperiod on all measures of the waveform [fig_ref] Table 2: UR and CR responses to photoperiod [/fig_ref] ; higher-order (quadratic, cubic) contrasts were not significant with the exception of dark-phase UR mesor. Linear effects were significant for most measures of the light-phase UR waveform, except UR mesor and UR acrophase. Higher-order quadratic contrasts were significant for all measures of the light-phase UR waveform except UR mesor, but cubic contrasts were non-significant. Lastly, analyses of the CR waveform revealed significant simple linear contrasts for robustness, amplitude and acrophase. Quadratic effects of photoperiod change were largely absent, but higher-order cubic contrasts were significant for all measures of the CR waveform [fig_ref] Table 2: UR and CR responses to photoperiod [/fig_ref].
# Discussion
Earlier reports suggested that ultradian body temperature rhythms of Siberian hamsters are substantially longer in a short (8 L) than a long (16 L) day length [bib_ref] Photoperiod and thermoregulation in vertebrates: body temperature rhythms and thermogenic acclimation, Heldmaier [/bib_ref]. The period estimates of 1.6 h in long days and 3.7 h in short days were based on 24 h analyses that encompassed both the light and dark phases [bib_ref] Photoperiod and thermoregulation in vertebrates: body temperature rhythms and thermogenic acclimation, Heldmaier [/bib_ref]. The present investigation revealed substantial effects of photoperiod on multiple components of the ultradian waveform, which in many cases differed in the active (dark) versus the inactive (light) phases, suggesting that behavioral analyses are most informative if restricted to a given photophase. This approach established period lengthening of the ultradian locomotor rhythm in both photophases as day lengths decreased from 15 h to #13 h. The expression of dark phase locomotor URs was not affected by variations in day length, but hamsters were significantly more likely to express light phase URs in short than in long days, establishing photophase-specific seasonal variation in ultradian organization.
The amplitude of URs was enhanced in both the light and dark phases after the transition from a long to one of several shorter day lengths. The critical day length for these transitions ranges from 12 to 14 h for the several ultradian components. The latency for instatement of the short-day ultradian phenotype is presently unspecified.
Day length induced parallel changes in circadian organization. Circadian amplitude, and robustness were greater in 15 L than in all short day lengths #13 L. The acrophase of the circadian locomotor rhythm occurred later in short days, and nocturnal a expanded as day length decreased, as previously noted [bib_ref] Photoperiodism in hamsters: abrupt versus gradual changes in day length differentially entrain..., Gorman [/bib_ref]. A functional circadian system is not required for the generation of URs. URs persist in Siberian hamsters rendered arrhythmic after a regimen of disruptive phase shifts [bib_ref] a) Dissociation of Ultradian and Circadian Phenotypes in Female and Male Siberian..., Prendergast [/bib_ref] [bib_ref] Siberian hamsters free run or become arrhythmic after a phase delay of..., Ruby [/bib_ref] ; in rats, Syrian hamsters and common voles, URs in behavior and physiology also persist after surgical ablation of the suprachiasmatic nucleus [bib_ref] Suprachiasmatic nuclei lesions eliminate circadian temperature and sleep rhythms in the rat, Eastman [/bib_ref] [bib_ref] The role of the suprachiasmatic nuclei in the generation of circadian rhythms..., Rusak [/bib_ref] [bib_ref] Circadian modulation of ultradian oscillation in the body temperature of the golden..., Refinetti [/bib_ref] [bib_ref] Daily torpor in the absence of the suprachiasmatic nucleus in Siberian hamsters, Ruby [/bib_ref] , but see [bib_ref] SCN lesions abolish ultradian and circadian components of activity rhythms in LEW/Ztm..., Wollnik [/bib_ref]. Although URs are not dependent on a functional circadian system, circadian activity exerts modest influences on URs. An increase in the number of significant URs is positively correlated with the power of Syrian hamster free-running circadian rhythms [bib_ref] Circadian modulation of ultradian oscillation in the body temperature of the golden..., Refinetti [/bib_ref] and hamsters bearing the tau mutation have shorter UR periods in feeding [bib_ref] Temporal organization of feeding in Syrian hamsters with a genetically altered circadian..., Oklejewicz [/bib_ref] and locomotor activity [bib_ref] Ultradian rhythms of body temperature and locomotor activity in wild-type and tau-mutant..., Refinetti [/bib_ref] relative to wild-type hamsters. And, absent circadian organization, day-night rhythms in quantitative features of the UR waveform (robustness, mesor activity levels, amplitude) are abolished [bib_ref] a) Dissociation of Ultradian and Circadian Phenotypes in Female and Male Siberian..., Prendergast [/bib_ref]. In the present study, across all day lengths, decreases in the amplitude of CRs were significantly correlated with increases in the amplitude of URs. A similar relation was recently observed in Syrian hamster dams-beginning early in gestation and persisting through weaning, CR amplitude and robustness were significantly diminished, whereas UR complexity, robustness and amplitude were markedly increased [bib_ref] b) Enhancement and suppression of ultradian and circadian rhythms across the female..., Prendergast [/bib_ref]. Decrements in the amplitude of the circadian system, whether a consequence of short photoperiods or gestation, may be a prerequisite for emergence of ultradian power. Some Siberian hamsters fail to undergo testicular regression in short day lengths (nonresponders; reviewed 33, 53). The dark phase t'was substantially longer (5.5 h) in males that sustained large testes in short day lengths (10 L-12 L) than in those whose testes were regressed (3.8 h); such differences were absent in the light phase, emphasizing the importance of photophase-specific analyses. At present the increase in t' in nonresponder hamsters appears paradoxical. In Siberian hamsters, blood testosterone concentrations of SD responders are reduced to about 10% of LD values (e.g., [bib_ref] Short-day increases in aggression are inversely related to circulating testosterone concentrations in..., Jasnow [/bib_ref] , but this decrease is unlikely to account for the above t' differences; nonresponder t' s are much longer than those of hamsters housed in long day lengths (15 L), yet both groups have equally large testes and presumably generate comparable blood androgen concentrations. The duration of nightly melatonin secretion is substantially shorter in nonresponder than responder hamsters [bib_ref] Evidence for differences in the circadian organization of hamsters exposed to short..., Puchalski [/bib_ref] ; if the nocturnal melatonin signal influences t', as suggested by Heldmaier et al. [bib_ref] Photoperiod and thermoregulation in vertebrates: body temperature rhythms and thermogenic acclimation, Heldmaier [/bib_ref] , then one would anticipate that t' would be shorter in nonresponders than responders and comparable to that of long day (15 L) hamsters, with comparably short duration nocturnal melatonin secretion, but this outcome was not observed. Modified circadian organization in nonresponders, including the delayed acrophase [fig_ref] Figure 7: Ultradian rhythms of reproductively non-responsive hamsters [/fig_ref] , and altered phase response curves to light pulses [bib_ref] Characterization of circadian function in Djungarian hamsters insensitive to short day photoperiod, Puchalski [/bib_ref] , rather than changes in hormone secretion, may be causally related to changes in the ultradian period (cf. [bib_ref] Phase control of ultradian feeding rhythms in the common vole (Microtus arvalis):..., Gerkema [/bib_ref] [bib_ref] b) Enhancement and suppression of ultradian and circadian rhythms across the female..., Prendergast [/bib_ref]. On the other hand, the robustness and amplitude of CRs, which are greater in nonresponder than responder hamsters may reflect decreased androgen secretion. In Syrian hamsters [bib_ref] Photoperiodic regulation of the hamster testis: dependence on circadian rhythms, Eskes [/bib_ref] [bib_ref] Effect of surgical or photoperiodic castration, testosterone replacement or pinealectomy on male..., Morin [/bib_ref] the integrity of wheel running circadian rhythms decreases in short day lengths.
Gonadal steroid modulation of seasonal changes in URs remains to be elaborated. In Syrian hamsters ovarian hormones profoundly influence ultradian locomotor organization [bib_ref] b) Enhancement and suppression of ultradian and circadian rhythms across the female..., Prendergast [/bib_ref] hamsters with elevated estradiol and progesterone during pregnancy exhibit increases in complexity, robustness, and amplitude of dark-phase URs. In Siberian hamsters, SD-induced changes in circadian and gonadal function are usually tightly linked [bib_ref] Characterization of circadian function in Djungarian hamsters insensitive to short day photoperiod, Puchalski [/bib_ref] [bib_ref] Evidence that the circadian system mediates photoperiodic nonresponsiveness in Siberian hamsters: the..., Freeman [/bib_ref]. The increase in dark-phase ultradian t', complexity, robustness and amplitude in shorter day lengths is correlated with decreased gonadal androgen and gonadotrophin secretion in shorter days [bib_ref] Short-day increases in aggression are inversely related to circulating testosterone concentrations in..., Jasnow [/bib_ref] [bib_ref] Blockade of singular follicle-stimulating hormone secretion and testicular development in photostimulated Djungarian..., Wolfe [/bib_ref] , but it is also correlated with changes in the entrainment state of the circadian system. Manipulations of gonadal steroids in hamsters maintained in a fixed LD photoperiod are required to assess the relative contributions of circadian and gonadal responses to seasonal changes in ultradian structure.
Divergent responses of hamsters to the 13 L photoperiod provide additional insight into seasonal modulation of ultradian structure by the circadian system and gonadal steroids. Circadian entrainment (acrophase, a) and power (robustness, mesor, amplitude) were comparable in 13 L+ and 13 L2 hamsters, but these groups, by definition, exhibited profound differences in gonadal function. Nevertheless, gonadal phenotype did not affect dark-phase URs in 13 L. Only a modest increase in light-phase UR amplitude was evident in 13 L hamsters. The absence of any systematic effect of reproductive phenotype in 13 L hamsters suggests that photoperiodic changes in quantitative aspects of URs occur via mechanisms largely independent of concurrent changes in gonadal hormone secretion.
Threshold photoperiods for initiating the transition to the shortday phenotype differed for the circadian and ultradian systems. Photoperiods as long as 14 L were sufficient to trigger decreases in CR robustness, mesor, amplitude, acrophase and a. In contrast, increases in dark-phase UR complexity, period, and amplitude required photoperiods #13 L; increases in robustness occurred at 12 L, and delays in acrophase occurred at 10 L. This suggests that significant decreases in the amplitude or robustness of circadian pacemaker output are not sufficient to induce SD-like enhancements in ultradian rhythm amplitude. Photoperiod-driven changes in CR amplitude may interact with putative gonadal hormone effects to influence the UR waveform.
Quantitative comparison of circadian and ultradian responses to day length [fig_ref] Table 2: UR and CR responses to photoperiod [/fig_ref] with regression analyses revealed significant linear effects of decreasing photoperiod on all quantitative aspects of dark-phase URs except mesor activity. Higher-order effects of photoperiod were absent on dark-phase URs, indicating that incremental decreases in photoperiod induce proportional effects on dark-phase UR complexity, t', robustness, amplitude, and acrophase (cf. [fig_ref] Figure 3: Prevalence, complexity and period of URs in decreasing photoperiods [/fig_ref]. In contrast, higher-order effects of photoperiod were evident on light-phase URs. These were primarily quadratic effects, indicating that as day lengths decrease, there is a non-linear acceleration of the impact of photoperiod change on light-phase URs (cf. [fig_ref] Figure 3: Prevalence, complexity and period of URs in decreasing photoperiods [/fig_ref]. Higher-order responses to day length were also evident in all measures of CRs (robustness, mesor, amplitude, acrophase), but these were uniformly cubic in nature, indicating that circadian responses to decreasing day lengths are best characterized by a step function. For example, decreases in CR amplitude occur abruptly upon transfer from 15 L to 14 L, followed by a plateau from 13 L through 10 L, followed by further decreases in 9 L (cf. [fig_ref] Figure 6: Effects of decreasing photoperiods on robustness, mesor, and amplitude of circadian rhythms [/fig_ref]. The mechanisms responsible for these asymmetries between circadian and ultradian responses to photoperiod remain unspecified.
Correlation and multiple regression analyses examined the relation between aspects of the dark-phase UR waveform that exhibit robust responses to decreasing day length (t' and amplitude) and various potential predictors. Photoperiod and testis size were each negatively and significantly correlated with increases in t'; similar effects were also evident on dark-phase UR amplitude; in addition, week 12 body mass was a significant negative predictor of UR amplitude [fig_ref] Table 1: Least squares regression models of single [/fig_ref]. When photoperiod is included in the model, effects of testis size on t' and UR amplitude disappear, suggesting that any effects of reproductive condition on UR t' and amplitude may be mediated via photoperiod effects on the reproductive system. However, the partial correlation between body mass and UR amplitude persists in the multiple regression model, indicating a contribution of body mass to UR amplitude even when strong effects of photoperiod are taken into account. The magnitude of such photoperiod-independent effects of body mass are modest: according to this model, a 1 g decrease in body mass would be expected to yield an increase in UR amplitude of ,1%. The prediction based on these data is that heavier hamsters would be predisposed to lower-amplitude URs, independent of photoperiod. [fig_ref] Table 1: Least squares regression models of single [/fig_ref] also summarizes results of a simple regression analyses of the relative contributions of several components of the circadian waveform to dark-phase t' and amplitude. Small positive correlations were obtained between CR mesor and CR acrophase, and t'. More robust simple effects were evident on UR amplitude. Nocturnal a was a strong positive predictor of UR amplitude; shorter as were linked to higher-amplitude dark-phase URs. The close temporal relation between nocturnal melatonin secretion and the duration of a [bib_ref] Complex circadian regulation of pineal melatonin and wheel-running in Syrian hamsters, Elliott [/bib_ref] [bib_ref] Characterization of circadian function in Djungarian hamsters insensitive to short day photoperiod, Puchalski [/bib_ref] [bib_ref] Adjustment of pineal melatonin and N-acetyltransferase rhythms to change from long to..., Illnerová [/bib_ref] suggests that photoperiod-mediated changes in melatonin secretion, independent of changes in the circadian waveform, influence this aspect of dark-phase URs. CR robustness, mesor and amplitude were each large negative predictors of UR amplitude, indicating that hamsters with robust, high-amplitude CR waveforms and high activity levels tended to have low-amplitude URs.
The functional significance of photoperiodic changes in behavioral URs remains a matter of conjecture. The short-day ultradian phenotype can be induced by maintaining long-day hamsters in low ambient temperatures [bib_ref] Photoperiod and thermoregulation in vertebrates: body temperature rhythms and thermogenic acclimation, Heldmaier [/bib_ref] , suggesting that the longer period of the UR in short days, which in nature coincides with lower environmental temperatures, imposes longer rest periods that conserve energy [bib_ref] Photoperiod and thermoregulation in vertebrates: body temperature rhythms and thermogenic acclimation, Heldmaier [/bib_ref].
In summary the present work titrated effects of photoperiod on quantitative aspects of CRs and URs. A significant negative relation exists between amplitude of the circadian and ultradian systems in this species, but SD-like changes in several quantitative aspects of the CR waveform were evident at photoperiods inadequate to trigger changes in the UR waveform. Future studies will be necessary to disentangle the respective contributions of gonadal hormones from direct or indirect contributions of the circadian system to the genesis of short-day induced changes in the UR waveform. However, at the threshold photoperiod of 13 L, categorical differences in reproductive condition were without effect on most UR measures, suggesting that changes in gonadal hormone secretion are not sufficient mediators of seasonal changes in URs.
[fig] Figure 1: Reproductive, somatic, and pelage responses to decreasing photoperiods. Mean 6SEM (A) estimated testis volume, (B) body mass, and (C) fur score of male Siberian hamsters raised in 15 L and transferred to one of seven experimental photoperiods ranging from 9 L to 15 L *P#0.05 and **P,0.001 vs. 15 L value. doi:10.1371/journal.pone.0041723.g001 [/fig]
[fig] Figure 2: Ultradian and circadian rhythms in locomotor activity. Representative double-plotted home-cage locomotor activity records of two hamsters housed in each photoperiod (15 L,left column), (13 L, center column), and (9 L, right column). Clock time is indicated on the horizontal axis at the top of each actogram, along with light (white) and dark (black) phases of the photocycle. The shaded area overlapping the activity record denotes the daily dark phase. doi:10.1371/journal.pone.0041723.g002 [/fig]
[fig] Figure 3: Prevalence, complexity and period of URs in decreasing photoperiods. (A) Percent hamsters exhibiting significant URs during the dark phase (filled/black bars) and during the light phase (open/white bars). (B) Mean 6 SEM complexity, and (C) period (t') of male Siberian hamsters raised in 15 L and transferred to one of seven experimental photoperiods ranging from 9 L to 15 L *P#0.05 and **P#0.005 vs. 15 L value, within photophase. doi:10.1371/journal.pone.0041723.g003 9 L and 10 L, as compared to15 L (Fig. 5; P,0.05 both comparisons).Light-phase URs. The proportion of hamsters displaying light-phase URs was greater in photoperiods #13 L compared to 15 L (Fig. 2; Fig. 3A, 13 L through 10 L: P,0.05, all comparisons; 9 L: P = 0.08). URs were more prevalent in the dark-than the light-phase in 15 L (P,0.05), but not in other day lengths (P.0.05, all comparisons). Most day lengths #13 L increased UR complexity(Fig. 3B, P,0.001). A main effect of day length on light-phase UR period (t') fell short of statistical significance(Fig. 3C, P = 0.07), but t' was significantly longer in 12 L, 11 L and 9 L than in 15 L (P,0.05, all comparisons). UR robustness was greater in all day lengths #13 L (Fig. 4A, P,0.005). UR amplitude(Fig. 4B, P,0.005) was significantly greater in 12 L, 11 L, and 10 L than in 15 L (P,0.005, all comparisons). Mesor values were not affected by changes in day length(Fig. 4C) and no main effect of photoperiod was evident on the timing of acrophases(Fig. 5, P.0.15). [/fig]
[fig] Figure 4: Robustness, amplitude, and mesor of URs in decreasing photoperiods. Mean 6 SEM (A) robustness, (B) amplitude, and (C) mesor of the dark phase (filled/black bars) and light phase (open/white bars) ultradian waveforms in 15 L and after transfer to one of seven experimental photoperiods ranging from 9 L to 15 L (indicated along the abscissae). *P#0.05 and **P#0.005 vs. 15 L value, within photophase. doi:10.1371/journal.pone.0041723.g004 [/fig]
[fig] Figure 5: Ultradian rhythm acrophase in decreasing photoperiods. Mean 6 SEM acrophase of the ultradian rhythm in 15 L and one of seven experimental photoperiods.*P#0.05 and **P#0.005 vs. 15 L value, within photophase. doi:10.1371/journal.pone.0041723.g005 [/fig]
[fig] Figure 6: Effects of decreasing photoperiods on robustness, mesor, and amplitude of circadian rhythms. Mean 6 SEM (A) robustness, (B) mesor, and (C) amplitude of the circadian waveforms of male Siberian hamsters raised in 15 L and transferred to one of seven experimental photoperiods ranging from 9 L to 15 L (indicated along the abscissae). *P#0.05 and **P#0.005 vs. 15 L value. doi:10.1371/journal.pone.0041723.g006 P,0.001) were each positively correlated with dark-phase UR t'. Significant negative correlations were observed between darkphase UR amplitude and photoperiod (R = 20.62), testis size (R = 20.57) and body mass (R = 20.57; P,0.001 all correlations). In the circadian waveform, CR robustness (R = 20.64), mesor (R = 20.47) and amplitude (R = 20.44) were negatively correlated with UR amplitude (P,0.001, all correlations), and CR acrophase (R = 0.19, P,0.05) and nocturnal a (R = 0.47; P,0.001) were positive predictors of dark-phase UR amplitude [/fig]
[fig] Figure 7: Ultradian rhythms of reproductively non-responsive hamsters. Mean 6 SEM (A) testis volumesof 15 L hamsters classified as reproductively responsive (ETV#300; SD-R) or non-responsive (ETV.300 and fur score = 1; SD-NR) to short photoperiods #12 L. (B) Percent hamsters exhibiting significant URs during the dark phase (left) and light phase (right). Mean 6 SEM (C) complexity, (D) robustness, (E) period (t'), (F) amplitude, and (G) acrophase of the dark phase and light phase ultradian waveforms of 15 L (white bars/symbols), SD-NR (crosshatched bars/symbols) and SD-R (black bars/symbols) hamsters. *P#0.05 and **P#0.005 vs. SD-R value. doi:10.1371/journal.pone.0041723.g007 [/fig]
[fig] Figure 8: Circadian rhythms of reproductively non-responsive hamsters. Mean 6 SEM (A) robustness, (B) amplitude, and (C) acrophase of the circadian waveforms of 15 L (white bars/symbols), SD-NR (crosshatched bars/symbols) and SD-R (black bars/symbols) hamsters. (D) Mean 6 SEM duration of nocturnal locomotor activity. *P#0.05 and **P#0.005 vs. SD-R value. doi:10.1371/journal.pone.0041723.g008 [/fig]
[table] Table 2: UR and CR responses to photoperiod: linear, quadratic, and cubic contrasts. Symbols indicate significance at *P,0.05, **P,0.01, ***P,0.001. doi:10.1371/journal.pone.0041723.t002Figure 9. Ultradian rhythms of hamsters exhibiting divergent reproductive responses to the intermediate-duration (13 L) photoperiod. Mean 6 SEM (A) testis volumes of hamsters that exhibited gonadal regression (ETV#300; 13 L2; black bars/symbols) or maintained developed gonads (ETV.300; 13 L+; grey bars/symbols) in a 13 L photoperiod. (B) Percent of 13 L+ and 13 L2 hamsters exhibiting significant URs during the dark phase (left) and light phase (right). (C) Mean 6 SEM amplitude of the ultradian waveform of 13 L+ and 13 L2 hamsters. (D) Acrophase of the circadian waveform and (E) duration of nocturnal locomotor activity (a) of 13 L+ and 13 L2 hamsters. *P#0.05 and **P#0.005 vs. 13 L+ value, within photophase. doi:10.1371/journal.pone.0041723.g009 [/table]
[table] Table 1: Least squares regression models of single (top) and multiple (bottom) predictor variables on dark-phase UR t' and darkphase UR amplitude after photoperiod manipulations.Pearson correlations (R) [/table]
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10.3390/cancers13174338
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CCBY
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8430947
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34503148
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s2orc_pubmed_articles
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Long-Term Prognostic Value of the Response to Therapy Assessed by Laboratory and Imaging Findings in Patients with Differentiated Thyroid Cancer
Citation: Klain, M.; Zampella, E.; Piscopo, L.; Volpe, F.; Manganelli, M.; Masone, S.; Pace, L.; Salvatore, D.; Schlumberger, M.; Cuocolo, A.
# Introduction
Although the overall long-term survival of patients with differentiated thyroid cancer (DTC) is excellent, disease recurrence is relatively common in some subsets of DTC patients who can be identified by an accurate risk stratification system [bib_ref] Long-Term Outcome of 444 Patients with Distant Metastases from Papillary and Follicular..., Durante [/bib_ref] [bib_ref] Survival and Death Causes in Differentiated Thyroid Carcinoma, Eustatia-Rutten [/bib_ref] [bib_ref] Papillary and follicular thyroid carcinoma, Schlumberger [/bib_ref]. The American Joint Committee on Cancer/Union for International Cancer Control Tumor Node Metastasis is an initial staging system that predicts mortality. The American Thyroid Association (ATA) initial risk stratification system has been proposed to assess the risk of recurrent or persistent disease in DTC patients [bib_ref] American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and..., Haugen [/bib_ref]. The patients are classified into low, intermediate, and high-risk categories [bib_ref] Estimating Risk of Recurrence in Differentiated Thyroid Cancer After Total Thyroidectomy and..., Tuttle [/bib_ref]. In addition, a dynamic risk stratification system has been proposed by the ATA based on clinical, biochemical, and imaging data obtained during follow-up [bib_ref] American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and..., Haugen [/bib_ref] [bib_ref] Estimating Risk of Recurrence in Differentiated Thyroid Cancer After Total Thyroidectomy and..., Tuttle [/bib_ref]. Radioiodine diagnostic whole-body scanning (WBS) has been largely used in the past for assessment of disease status [bib_ref] Is diagnostic iodine-131 scanning with recombinant human TSH useful in the follow-up..., Mazzaferri [/bib_ref] [bib_ref] Modified dynamic risk stratification for predicting recurrence using the response to initial..., Jeon [/bib_ref]. Currently, WBS scanning is performed less frequently and has been replaced by a combination of serum thyroglobulin (Tg) determination and neck ultrasonography (US). It has been recently reported that the distribution of response to therapy could differ according to the follow-up protocols when diagnostic WBS scanning is considered [bib_ref] Clinicopathologic risk factors of radioactive iodine therapy based on response assessment in..., Kwon [/bib_ref]. However, the prognostic implications of these findings has not yet been fully elucidated. Hence, we assessed the long-term predictive value of the response to therapy at 12 months, evaluated by serum thyroglobulin determination and neck ultrasound, and estimated the potential additional impact of diagnostic WBS in patients with DTC treated with surgery and radioactive iodine (RAI) therapy.
# Materials and methods
## Patients
A total of 712 patients with DTC who were referred to our center between 1992 and 2002 were included. All patients underwent total thyroidectomy, with central and/or lateral neck dissection when appropriate, followed by 131 I therapy. In particular, 142 patients had total thyroidectomy, 446 had total thyroidectomy and central neck dissection, and 124 patients had total thyroidectomy and lateral neck dissection. Histopathological data were collected, and patients were classified as low, intermediate, or high risk in terms of structural recurrence of disease according to ATA guidelines [bib_ref] American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and..., Haugen [/bib_ref]. Before 131 I therapy, L-thyroxine treatment was discontinued for 20-30 days until serum TSH levels increased above an arbitrary level of 30 mIU/L. At that time, serum thyroglobulin (Tg) concentration was measured, and 131 I was administered (range 1110-6808 MBq). Serum Tg levels were determined by immunoradiometric assay (Dynotest Tg, Henning, Berlin, Germany), with a sensitivity of 1 ng/mL up to 1996 and then by chemiluminescence system (Immulite, Diagnostic Products Corp, Los Angeles, CA, USA) with a detection limit of 0.2 ng/mL. According to post-operative Tg values obtained following thyroid hormone withdrawal at the time of 131 I administration, patients were categorized into two groups: ≥10 ng/mL and <10 ng/mL [bib_ref] Preablative Stimulated Thyroglobulin Correlates to New Therapy Response System in Differentiated Thyroid..., Yang [/bib_ref] [bib_ref] Prognostic Role of 18F-FDG PET/CT in the Postoperative Evaluation of Differentiated Thyroid..., Pace [/bib_ref]. Five to seven days later, a post-therapy WBS was performed using a dual-head gamma camera (E.CAM, Siemens Medical Systems, Hoffman Estates, IL, USA) equipped with thick crystals and high energy collimators [bib_ref] Outcome of Patients with Differentiated Thyroid Cancer Treated With 131-Iodine on the..., Klain [/bib_ref]. The patients with evidence of distant metastasis at post-therapy WBS were excluded from the study.
## Therapy response evaluation
The response to 131 I therapy at 12 months was assessed with serum Tg measurement obtained on LT4 treatment or following thyroid hormone withdrawal, neck US, and diagnostic WBS. WBS was performed after LT4-withdrawal until the TSH increased above an arbitrary level of 30 mIU/L, and two days after the administration of 185 MBq of 131 I, using a dual-head gamma camera (E.CAM, Siemens Medical Systems, Hoffman Estates, IL, USA). According to the 2015 ATA guidelines [bib_ref] Estimating Risk of Recurrence in Differentiated Thyroid Cancer After Total Thyroidectomy and..., Tuttle [/bib_ref] [bib_ref] Preablative Stimulated Thyroglobulin Correlates to New Therapy Response System in Differentiated Thyroid..., Yang [/bib_ref] , definitions of responses to therapy were: (1) excellent response (ER), negative imaging and either Tg < 0.2 ng/mL on LT4 treatment or TSH-stimulated Tg < 1 ng/mL; (2) indeterminate response (IR), non-specific findings on imaging studies or Tg levels on LT4 treatment that are detectable but <1 ng/mL or stimulated Tg levels between 1 and 10 ng/mL or stable or declining titer of Anti-Tg antibodies in the absence of structural disease; (3) biochemical incomplete response (BIR), negative imaging and Tg ≥ 1 ng/mL on LT4 treatment or stimulated Tg ≥ 10 ng/mL or rising titer of anti-Tg antibody; and (4) structural incomplete response (SIR), structural evidence of disease with any level of serum Tg or of anti-Tg antibodies. Response to therapy was assessed considering Tg and US findings and then the results of the diagnostic WBS were analyzed according to the response to therapy. The patients without neck US or diagnostic radioiodine scan at the 12-month evaluation were excluded from the study.
## Follow-up
After the evaluation at 12-months, all patients were followed up with serum Tg level determinations (on L-thyroxine and in some patients off L-thyroxine therapy when withdrawal was performed for a 131 I WBS) every 6-12 months, and imaging procedures as appropriate. Disease status was recorded at each evaluation. Recurrence of disease was defined by histology or imaging procedures; suspicious nodal abnormalities at neck ultrasonography were confirmed by fine needle aspiration cytology, histology, and the presence of RAI uptake when it corresponded to abnormal findings at neck ultrasonography. All patients with recurrent disease were submitted to further treatments. Patients last known to be alive and progression free were censored at the date of last contact.
# Statistical analysis
Continuous data are expressed as mean ± standard deviation and categorical data as percentage. A student two-sample t test and χ 2 test were used to compare the differences in continuous and categorical variables, respectively. Hazard ratios with 95% confidence intervals (CI) were first calculated by univariate regression analysis. Variables showing a p value < 0.05 at univariate analysis were considered statistically significant and were included in the multivariate Cox regression analysis. Statistical analysis was performed with Stata Statistical Software (StataCorp. 2015.: Release 14. StataCorp, College Station, TX, USA).
# Results
Of the total of 712 patients with DTC, 39 with initial distant metastases and 32 without neck US or diagnostic WBS at 12 months were excluded. Of the remaining 641 patients, follow-up was not available in 35 (5%), leaving 606 subjects for the analysis. With a median follow-up of 105 months (range 10-384), 42 (7%) events occurred and required additional treatments. Twenty-five patients had additional 131 I therapy, 11 with structural disease in the thyroid bed, eight in both thyroid bed and lymph nodes, four for lung metastases and two for bone metastases. The other 17 patients with nodal disease had additional surgery and 131 I therapy.
Characteristics of the study population at the time of RAI therapy according to events are reported in [fig_ref] Table 1: Characteristics of the study population at the time of RAI therapy according... [/fig_ref]. The prevalence of high ATA risk and Tg ≥ 10 ng/mL was higher in patients with events as compared to those without (both p < 0.001).
Characteristics of the study population at 12 months evaluation according to events are reported in [fig_ref] Table 2: Characteristics of the study population at 12 months evaluation according to events [/fig_ref]. The prevalence of detectable Tg, abnormal US and positive diagnostic WBS was higher in patients with events as compared to those without. According to Tg level and US, 219 patients were classified as ER and 387 as no-ER. Among no-ER patients, 182 were classified as IR, 203 as BIR and 2 as SIR for lymph node metastases confirmed by FNA. Individual characteristics of 42 patients with events at the time of recurrence are depicted in [fig_ref] Table 3: Characteristics of 42 patients with events at the time of recurrence [/fig_ref].
## Predictors of events
The rate of events was higher in the ATA high risk patients compared to intermediate and low risk patients (p for trend < 0.01) [fig_ref] Figure 1: Event rate according to ATA risk categories [/fig_ref]. However, the 198 patients with pretherapy Tg ≥ 10 ng/mL had a higher rate of recurrence as compared to the 408 patients with lower Tg values (14% vs. 3%, p < 0.001). Similarly, the rate of structural recurrence was higher in no-ER patients as compared to ER (9% vs. 2%, p < 0.01).
Univariable and multivariable predictors of recurrence are reported in [fig_ref] Table 4: Univariable and multivariable predictors of recurrence [/fig_ref]. ATA risk categories, pre therapy Tg ≥ 10 ng/mL, response to therapy evaluated with serum Tg and neck US at 12 months, were predictors of events at both univariable and multivariable analyses. At Kaplan Meier analysis, disease free survival was lower in patients with no-ER at 12 months after RAI therapy, as compared with those with ER (p < 0.001) [fig_ref] Figure 2: Disease-free survival by Kaplan-Meier in patients with ER and with no-ER at... [/fig_ref]. To assess the role of diagnostic WBS, the outcome of patients according to 12-months response to therapy and WBS findings was evaluated [fig_ref] Figure 3: Outcome of patients according to 12-months response to therapy and diagnostic WBS... [/fig_ref]. Among the 219 patients with ER according to serum Tg level and neck US findings, 20 (9%) had detectable foci of uptake at diagnostic WBS. During the subsequent follow-up, only five of these 20 (25%) patients had a structural recurrence with high serum Tg. One (1%) patient without detectable uptake at diagnostic WBS had recurrence in neck lymph nodes during follow-up. Among the 387 with no-ER patients, diagnostic WBS demonstrated uptake in 57 (15%) patients and 24 (42%) had recurrence during follow-up. On the other hand, among the 330 patients with no-ER and without detectable uptake at 12 months, diagnostic WBS recurrence occurred during follow-up in 12 (2%). Finally, in 156 patients with intermediate/high ATA risk and pre-therapy Tg ≥10 ng/mL, the rate of events was higher in patients with abnormal diagnostic WBS in both ER (37% vs. 2%) and no-ER (68% vs. 5%) patients (both p < 0.001) [fig_ref] Figure 4: Event rate in patients at intermediate/high ATA risk and pre-therapy Tg ≥... [/fig_ref].
# Discussion
In this retrospective study we tested the incremental prognostic value of diagnostic WBS in the evaluation of response to 131 I therapy in DTC patients treated with surgery and RAI. In our series, ATA risk classification and Tg level before RAI administration, as well as Tg level and US evaluation at 12 months, are the main predictors of outcome. The addition of diagnostic WBS at 12 months seems to be useless in low-risk patients considered in ER according to serum Tg level and neck US findings and it may still have a role in patients with intermediate/high ATA risk and pre-therapy Tg ≥ 10 ng/mL.
In patients with DTC an accurate risk stratification is essential in order to guide RAI treatment and follow-up. The AJCC/UICC TNM staging system has been developed to predict mortality according to histopathological findings and the presence of distant metastases [bib_ref] Differentiated and anaplastic thyroid carcinoma: Major changes in the American Joint Committee..., Perrier [/bib_ref]. However, TNM system is able to predict mortality but it revealed not ideal in assessing the risk of recurrence. The ATA initial risk stratification system is designed to predict the risk of recurrent or persistent disease in DTC patients and has been validated in different cohorts of DTC patients [bib_ref] Estimating Risk of Recurrence in Differentiated Thyroid Cancer After Total Thyroidectomy and..., Tuttle [/bib_ref] [bib_ref] Clinicopathologic risk factors of radioactive iodine therapy based on response assessment in..., Kwon [/bib_ref] [bib_ref] Delayed risk stratification, to include the response to initial treatment (surgery and..., Castagna [/bib_ref]. However, many DTC patients initially categorized as intermediate risk have varied clinical outcomes [bib_ref] Dynamic risk assessment in patients with differentiated thyroid cancer, Pitoia [/bib_ref]. The addition of laboratories and imaging findings obtained during the first 12-24 months after treatment, including serum Tg and TgAb determinations or imaging procedures, can improve this initial risk assessment, and for this reason a dynamic approach has been proposed which considers different responses to treatment, such as ER, IR, BIR and SIR [bib_ref] Estimating Risk of Recurrence in Differentiated Thyroid Cancer After Total Thyroidectomy and..., Tuttle [/bib_ref] [bib_ref] Delayed risk stratification, to include the response to initial treatment (surgery and..., Castagna [/bib_ref]. Integrative prognostic stratification systems, including molecular markers closely associated with non-RAI avidity, have also been proposed [bib_ref] Refining Dynamic Risk Stratification and Prognostic Groups for Differentiated Thyroid Cancer with..., Kim [/bib_ref]. During followup, diagnostic WBS was not routinely performed to detect recurrent disease, due to its low sensitivity [bib_ref] Diagnostic 131-iodine whole-body scan may be avoided in thyroid cancer patients who..., Pacini [/bib_ref] [bib_ref] Is diagnostic iodine-131 scanning useful after total thyroid ablation for differentiated thyroid..., Cailleux [/bib_ref] [bib_ref] Diagnostic Whole-Body Scan May Not Be Necessary for Intermediate-Risk Patients with Differentiated..., Jeon [/bib_ref]. Undetectable levels of serum Tg is highly predictive of complete remission and the present data confirm that a diagnostic WBS may be avoided in these patients [bib_ref] Diagnostic 131-iodine whole-body scan may be avoided in thyroid cancer patients who..., Pacini [/bib_ref]. Furthermore, serum Tg may remain detectable during some months after initial treatment and subsequently disappear without any further treatment [bib_ref] American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and..., Haugen [/bib_ref] [bib_ref] Positive Predictive Value of Serum Thyroglobulin Levels, Measured during the First Year..., Baudin [/bib_ref].
Recently, Known et al. [bib_ref] Clinicopathologic risk factors of radioactive iodine therapy based on response assessment in..., Kwon [/bib_ref] found that the proportion of patients without ER significantly increased when a radioiodine scan was added to the follow-up protocol. In our retrospective cohort, only eight patients with undetectable Tg and negative US were reclassified according to WBS results. The other 19 patients had persistent uptake in the thyroid bed, but this may only indicate the persistence of normal-non tumoral thyroid cells that have not been totally eradicated with initial treatment. These cells have been irradiated and will probably disappear with time and in fact none of these 19 patients experienced a structural recurrence and serum Tg remained undetectable.
Only one of the 219 patients classified as ER according to serum Tg level and neck US showed pathological uptake in the thoracic region and was re-classified as SIR. The presence of positive WBS despite negative Tg or TgAb values has been previously observed in some patients with cervical and mediastinal lymph nodes or small lung metastases [bib_ref] Recurrent/metastatic thyroid carcinomas false negative for serum thyroglobulin but positive by posttherapy..., Park [/bib_ref] [bib_ref] Possible explanations for patients with discordant findings of serum thyroglobulin and 131I..., Ma [/bib_ref]. Also, in the absence of SPECT-CT, the location of foci of uptake might be unreliable, as observed in one patient with uptake in the lateral neck that was not subsequently confirmed and that was probably thyroid bed uptake due to thyroid remnants.
Moreover, seven patients with detectable Tg levels, first classified as either IR for 2 or BIR for 5, were reclassified as SIR according to the discovery of abnormal uptake in the chest. These findings agree with current ATA guidelines [bib_ref] American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and..., Haugen [/bib_ref] which recommend the use of diagnostic WBS only in patients with high or intermediate risk of persistent disease, in those patients with some evidence of disease such as detectable serum Tg level or suspicious findings at neck US [bib_ref] American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and..., Haugen [/bib_ref]. In patients with detectable serum Tg during the first months after initial therapy, the slope of serum Tg levels over time might help to select patients for imaging (those with an increasing trend) and to follow-up those with a decreasing trend for whom the risk of recurrence is low.
# Conclusions
Diagnostic WBS at 12 months has no significant incremental value in the identification of patients at higher risk of recurrence beyond Tg and US findings in low-risk patients with an excellent response to therapy. Diagnostic WBS may still have a role only in patients at intermediate-high ATA risk and in those with high pre-therapy Tg values.
[fig] Figure 1: Event rate according to ATA risk categories. [/fig]
[fig] Figure 2: Disease-free survival by Kaplan-Meier in patients with ER and with no-ER at 12 months after RAI therapy. ER (excellent response). [/fig]
[fig] Figure 3: Outcome of patients according to 12-months response to therapy and diagnostic WBS findings. ER (excellent response), WBS (whole body scan), TB (thyroid bed), LN (lymph nodes). [/fig]
[fig] Figure 4: Event rate in patients at intermediate/high ATA risk and pre-therapy Tg ≥ 10 ng/mL, according to 12-months response to therapy by Tg and US and diagnostic WBS findings. ER (excellent response), WBS (whole body scan). [/fig]
[table] Table 1: Characteristics of the study population at the time of RAI therapy according to events. [/table]
[table] Table 2: Characteristics of the study population at 12 months evaluation according to events. [/table]
[table] Table 3: Characteristics of 42 patients with events at the time of recurrence. [/table]
[table] Table 4: Univariable and multivariable predictors of recurrence. [/table]
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10.3389/fpls.2017.01350
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CCBY
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5551076
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28848568
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s2orc_pubmed_articles
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Effects of Low Temperature Stress on Spikelet-Related Parameters during Anthesis in Indica–Japonica Hybrid Rice
Poor spikelet fertility under low temperature (LT) stress during anthesis limits the possibility of high yield potential in indica-japonica hybrid rice, leading to reduced stability of grain yield. However, the cause for it is still unclear. In this study, three indica-japonica hybrid rice cultivars, Yongyou9, Yongyou17 (both cold sensitive), and Yongyou538, and one japonica inbred rice cultivar, Zhejing88 (cold tolerant), were grown under LT (17 - C) and ambient temperature (AT) (25 - C) during anthesis to test for their response with respect to spikelet fertility, pollen germination, and spikelet flowering. The results indicated that LT resulted in lower spikelet fertility in cold-sensitive cultivars than in cold-tolerant cultivars. Spikelet fertility was highly correlated with pollen germination on the stigma. The number of pollen grains and germinated pollen were higher in cold-tolerant cultivars than in cold-sensitive cultivars. Pollen fertility and pollen diameter were also higher in cold-tolerant cultivars, although the latter could achieve a high number of spikelets at anthesis in flowering patterns throughout the duration of LT stress. There were significant differences in anther width and volume between genotypes and treatments according to microscopic analyses, but no differences were observed in anther dehiscence. Moreover, variation in the number of pollen grains on stigmas and in spikelet fertility was not related to either the number of spikelets reaching anthesis or anther dehiscence. Overall, improved anther size, better pollen function, and higher spikelet fertility under LT stress were observed in cold-tolerant cultivars than in cold-sensitive cultivars. The results suggest that the increase in spikelet fertility is due to enhanced pollen germination rather than the number of spikelets reaching anthesis.
# Introduction
Rice (Oryza sativa L.) is a staple grain crop. Six hundred million tons of rice are produced annually, and rice is widely cultivated throughout the world. Low temperature (LT) is a major environmental factor causing reductions in yield [bib_ref] Plant productivity and environment, Boyer [/bib_ref]. In general, the mean critical temperature for rice is +4.7 - C, and once the ambient temperature (AT) is below 5-10 - C, irreversible LT-induced damage to seedling growth and development can occur [bib_ref] Temperatures and the growth and development of maize and rice: a review, Sanchez [/bib_ref]. During different developmental stages, rice shows growth responses to LT stress. The reproductive stage in rice is a critical period. [bib_ref] The cost of low temperature to the NSW rice industry, Farrell [/bib_ref] [bib_ref] Genotypic variation for cold tolerance during reproductive development in rice: screening with..., Farrell [/bib_ref] reported that rice was susceptible to abundant LT-induced damage during the young microspore stage, and 12 h of exposure to LT can cause spikelet sterility. LT-induced spikelet sterility during the microsporogenesis stage is considered genotype dependent, which for cold-tolerant genotypes was a night-time air temperature of 15 - C for 4 consecutive days and for cold-sensitive genotypes was a night-time air temperature between 17 and 19 - C [bib_ref] Research on cold injury of paddy rice plants in Japan, Satake [/bib_ref]. Hence, developing and verifying cold-tolerant cultivars will be beneficial for overcoming the influence of LT stress on rice growth and development.
Low temperature-induced spikelet sterility, in particular during the reproductive period, could be related to the physiological response mechanisms in the anther, pollen, and stigma. Previous studies have indicated that short anther dehiscence, poor pollen grains, and low pollen germination on stigmas were all highly correlated with spikelet sterility under LT-induced stress [bib_ref] The difference in sterility due to high temperatures during the flowering period..., Matsui [/bib_ref] [bib_ref] Species, ecotype and cultivar differences in spikelet fertility and harvest index of..., Prasad [/bib_ref]. Moreover, [bib_ref] Determination of the most sensitive stage to sterile-type cool injury in rice..., Satake [/bib_ref] considered that the main reason for this could be attributed to LT-induced damage of male organs, such as pollen, not of female organs, such as the stigma. Hence, if pollen grains were deficient at anthesis, they could impede pollination, leading to spikelet sterility. These results might be similar to high temperature stress (HTs) in rice. A larger anther and longer stigma [bib_ref] Cold tolerance in rice plants with special reference to the floral characters...., Suzuki [/bib_ref] [bib_ref] Cold tolerance with rice plants with special reference to the floral characters...., Suzuki [/bib_ref] were found to contribute to the increase in the basal pore length and found to improve LT tolerance during the reproductive stage, and larger anthers and longer stigmas also ultimately benefit successful pollination [bib_ref] Characteristics of floral organs related to reliable self pollination in rice, Matsui [/bib_ref]. In addition, panicle exsertion (PE) [bib_ref] Effect of high temperature and water stress on pollen germination and spikelet..., Rang [/bib_ref] and peduncle length (PeL) [bib_ref] Changes in OsXTH gene expression, ABA content, and peduncle elongation in rice..., He [/bib_ref] have been reported to be responsible for enhanced spikelet sterility under water stress, as has HTs [bib_ref] Effect of high temperature and water stress on pollen germination and spikelet..., Rang [/bib_ref] , but similar results have not been documented under LT stress.
In most rice genotypes, flowering and anthesis undergo a 5-day period with most spikelets at anthesis between 1000 and 1200 h; the similar patterns of flowering have been reported in indica and japonica subspecies [bib_ref] Avoidance of high temperature sterility by flower opening in the early morning, Nishiyama [/bib_ref] [bib_ref] Species, ecotype and cultivar differences in spikelet fertility and harvest index of..., Prasad [/bib_ref]. Studies have shown that flowering (containing anthesis and fertilization), as a developmental stage in rice, in particular at the booting stage (microsporogenesis), is considered most susceptible to LT [bib_ref] Genotypic variation for cold tolerance during reproductive development in rice: screening with..., Farrell [/bib_ref] [bib_ref] Temporal sensitivities of rice seed development from spikelet fertility to viable mature..., Maite [/bib_ref]. The influences on spikelet flowering in rice have been verified under HTs [bib_ref] Effect of high temperature and water stress on pollen germination and spikelet..., Rang [/bib_ref]. The climatic effects on the timing of day and the duration of flowering in rice also differ [bib_ref] Variation in time of day of anthesis in rice in different climatic..., Julia [/bib_ref]. [bib_ref] High temperature stress and spikelet fertility in rice (Oryza sativa L.), Jagadish [/bib_ref] analyzed the flowering patterns and the time of day of spikelets reaching anthesis under HTs in indica and japonica rice. Furthermore, spikelet fertility under stress conditions could be generally measured by marking individual spikelets during anthesis, which was adopted as an effective method. Indica-japonica hybrid cultivars (Yongyou series) have been successfully bred and have been widely used for rice production due to their exceptionally high grain yield potential. However, they are very susceptible to LT, and can succumb to spikelet sterility. Previous studies have not systematically investigated the effects of LT on spikelet flowering and spikelet fertility in indica-japonica hybrid cultivars [bib_ref] Determination of the most sensitive stage to sterile-type cool injury in rice..., Satake [/bib_ref] [bib_ref] Male sterility caused by cooling treatment at the young microspore stage in..., Nishiyama [/bib_ref] [bib_ref] Genotypic variation for cold tolerance during reproductive development in rice: screening with..., Farrell [/bib_ref] [bib_ref] Variation in time of day of anthesis in rice in different climatic..., Julia [/bib_ref] [bib_ref] Temporal sensitivities of rice seed development from spikelet fertility to viable mature..., Maite [/bib_ref] [bib_ref] Evaluation of chilling resistance in Yongyou series of hybrid rice at flowering..., Zeng [/bib_ref].
The overall objectives in the present study were therefore to evaluate spikelet fertility of indica-japonica hybrid rice under LT stress and study the main traits of anthesis and pollen related to spikelet sterility. The experiments were conducted under AT (25 - C) and LT (17 - C) and involved the following:
(1) recording the effect of LT on peduncle length (PeL) and PE in rice; (2) studying the influences on spikelet fertility, anther dehiscence, pollen grains, and pollen germination on stigmas under LT during anthesis; and (3) examining the relationship between spikelet flowering and spikelet fertility across cultivars.
# Materials and methods
## Experimental design and crop husbandry
Pot experiments were conducted during the period from June to November in 2013 and in 2014 at the China National Rice Research Institute (CNRRI), Fuyang district (longitude: 119 - 55 48 E; latitude: 30 - 2 24 N), Zhejiang Province, China. A split-plot design was used for the experiments. Temperature treatments (i.e., LT and AT) were used for the main plots, and cultivars were the subplots with three replications. Half of the plants were established under the AT treatment, and the other half were established under the LT treatment. Each section comprised 40 plants of each cultivar in 20 pots (2 hills per pot, 2 seedlings per hill) under each temperature regime, and each pot was considered a sample. The height and diameter of each rice pot was 33 and 23 cm, respectively. Eight kilograms of paddy soil common to rice planting in the area was placed into each pot. Four rice cultivars were used with different LT sensitivities during anthesis. The growth characteristics of cultivars are described in [fig_ref] TABLE 1 |: Information on four rice cultivars selected for the study [/fig_ref] , including sowing date, growth duration, and flowering dates. The cold-tolerant cultivar Yongyou538, which has a shorter growth duration, was sown 1 week later than the other cultivars to maintain the synchronous time of anthesis.
Pre-germinated seeds were sown in seed trays covered with a matrix containing vermiculite, charcoal, soil, and slow-release fertilizer. After 20 days, the seedlings were transplanted into the pots. The pots were established in an open space under natural environmental conditions except for temperature treatments. Alternate wetting and drying irrigation was conducted throughout the cropping season. Two grams (g) of calcium superphosphate (P 2 O 5 ) and 4.0 g of nitrogen, phosphorus, and potassium (NPK) compound fertilizer were applied to each pot for 1 day before transplanting. One gram of potassium chloride was applied identically at tillering and panicle initiation, and 4.0 g of urea was supplied at planting (50%), at tillering (20%), [bib_ref] Evaluation of chilling resistance in Yongyou series of hybrid rice at flowering..., Zeng [/bib_ref]. c Yongyou538, which has a shorter growth duration, was sown 1 week later than other cultivars to maintain a synchronous time of flowering. d The booting stage, anthesis, and grain filling were recorded in both 2013 and 2014.
and at panicle initiation (30%). Pest-, disease-, and weedrelated problems were intensively controlled. Other management practices for high grain yield cultivation were in accordance with local recommendations. At the end of productive tiller stage, redundant tillers were removed, leaving approximately 12 large tillers.
## Stress treatments
On the day of spikelet flowering (i.e., anthers appearing or spikelets expanding), the plants of each cultivar were transferred to growth chambers (PGV-36, Conviron Corporation, Winnipeg, MB, Canada) with automatically controlled temperatures using sensing elements. The temperature threshold of the rice plants during the reproductive growth stage (booting stage and heading) is often described as 17 - C [bib_ref] Research on cold injury of paddy rice plants in Japan, Satake [/bib_ref] [bib_ref] Genotypic variation for cold tolerance during reproductive development in rice: screening with..., Farrell [/bib_ref]. Therefore, in this study, LT was set at 17 - C, and the growth chamber was held at an average LT of 17 - C (20/14 - C max/min; actual: 16.7 - C [SD = 1.16]). The plants of the AT treatment were maintained at an elevated average temperature of 25 - C (28/22 - C max/min; actual: 23.7 - C [SD = 0.64]) for five consecutive flowering days, such that the AT was nearly equal to the natural environmental temperature during the past 10 years during flowering days, as described by [bib_ref] Evaluation of chilling resistance in Yongyou series of hybrid rice at flowering..., Zeng [/bib_ref]. The time window of the temperature treatment day was divided into four time frames on five consecutive flowering days , and the relative humidity (RH) was 75% (actual: 81.7% [SD = 1.10]). Plants were spaced at a distance of approximately 15-20 cm (0.15-0.20 m) to reduce crowding and shading effects. Five days after the temperature treatment, the plants were placed under natural environmental conditions. Temperature and RH were both monitored independently through recordings by a micrometeorological station (HOBO, H08-002-21, Onset Computer Corporation, Barnstable, MA, United States) with different sensors/loggers every 10 s and averaged for each 30 min. Temperatures surrounding the plants were controlled and maintained consistently within the set points of 0.5 - C for temperature and 5% for RH in order to reduce any chamber effects on the observations recorded of plants. In addition, the level of photosynthetic photon flux density for each temperature treatment was maintained at 640 µmol m −2 s −1 . Natural environmental conditions were FIGURE 1 | Temperature treatments, AT, ambient temperature; LT, low temperature.
adopted for plant growth and development, and no unusual HT or LT was observed.
## Measurements and data collection
In the study, five pots (10 plants) were used for testing PeL, PE, and wrap-around panicle length (WPL) under both LT and AT treatments; another five pots were used to determine spikelet fertility and spikelet flowering, while the remaining 10 pots in the set were used for the collection of spikelets for microscopic analyses.
## Pel, pe, and wpl
PeL and PE were determined using a ruler according to the methods of [bib_ref] Effect of high temperature and water stress on pollen germination and spikelet..., Rang [/bib_ref]. The elongation of panicle exsertion (EPE) was calculated as the ratio of PeL to the duration of exposure to LT. EPE was to express the degree of LT stress on peduncle exsertion. PeL, panicle length (PaL), and WPL were measured in 2013.
## Microscopic analysis of spikelets
Eight to 10 uniform panicles of each pot were marked for exposure to temperature treatment during anthesis. Twenty to 25 spikelets, positioned on the three primary branches on the top rachis in each panicle [bib_ref] Characteristics of floral organs related to reliable self pollination in rice, Matsui [/bib_ref] , were collected Data indicate the means ± standard errors, n = 60. Means within a column followed by a different lowercase letter differ significantly at a level of 0.05. G: genotypes; T: treatments. * P < 0.05, * * P < 0.01, ns, not significant.
FIGURE 2 | Effects of LT and AT on anther dehiscence of rice plants (n > 60 anthers). The three parameters were analyzed separately for each anther dehiscence. Bars with different lowercase letters denote a particular anther dehiscence across cultivars, which differ at a significance level of 0.05. Bars indicate standard error. FAD, full anther dehiscence; PAD, partial anther dehiscence; AID, anther indehiscence; YY9, Yongyou9; YY17, Yongyou17; YY538, Yongyou538; ZJ88, Zhejing88; AT, ambient temperature; LT, low temperature.
simultaneously between 1030 and 1200 h and between 1300 and 1400 h for different treatments and cultivars every day. Therefore, spikelets that approximately opened (i.e., with an observable anther) on the main tiller and on some primary tillers were both marked using black acrylic paint. Then, marked spikelets were collected in brown glass vials filled with formalin-acetic acid-alcohol (FAA) fixative (including 90 mL of 70% ethanol, 5 mL of acetic acid, and 38% formaldehyde) after flowering and pollination, following the methods of [bib_ref] Physiological and Proteomic approaches to address heat tolerance during anthesis in rice..., Jagadish [/bib_ref]. The spikelets collected from the tillers were used for microscopic analyses (i.e., anther dehiscence, total pollen grain number on stigmas, and pollen germination), excluding spikelet fertility estimations. The spikelets were dissected under a stereoscopic microscope (Olympus SZX7, Olympus Corporation, Tokyo, Japan). Then, using a digital camera (DP70) affixed to an Axioplane 2 microscope (Carl Zeiss, Oberkochen, Germany) at 100× [fig_ref] FIGURE 5 |: Pictorial illustration of LT and AT affecting the number of pollen grains... [/fig_ref] , images were taken following the methods of [bib_ref] Physiological and Proteomic approaches to address heat tolerance during anthesis in rice..., Jagadish [/bib_ref]. The anthers were isolated from the fixed spikelets to score the numbers of dehisced anthers, which were described as full anther dehiscence (FAD) if both apical and basal pores were present, partial anther dehiscence (PAD) if only one pore was present, and anther indehiscence (AID) if no pore was present. The ratio of the number of dehiscent anthers to the total number of anthers (containing both dehiscent and indehiscent anthers) was calculated and described as anther dehiscence [bib_ref] Effect of high temperature and water stress on pollen germination and spikelet..., Rang [/bib_ref]. Anther length and width were also determined, and anther volume was calculated using the following equation: V = 0.34 LW 2 [bib_ref] Male sterility caused by cooling treatment at the young microspore stage in..., Nishiyama [/bib_ref]. Anthers were separated on a glass slide and colored with 1% I 2 -KI with the aid of a stereoscopic microscope (Olympus SZX7, Olympus Corporation, Tokyo, Japan), and the number of pollen grains and pollen diameter were recorded. Stigmas separated from spikelets with minimum disturbance were cleared using 8 N NaOH for 3-5 h at room temperature, after which they were stained with 0.05% aniline blue dissolved in a solution of 0.1 M dipotassium phosphate for 5-10 min. The pollen grains and germination on the stigma were recorded using a fluorescence microscope (Olympus BX43, Olympus Corporation, Tokyo, Japan). The amount of germinated pollen on the stigma was scored according to the procedures of [bib_ref] Physiological and Proteomic approaches to address heat tolerance during anthesis in rice..., Jagadish [/bib_ref] , and the per cent pollen germination (PPG) was calculated as the percentage of germinated pollen of the total amount of pollen (germinated + non-germinated). All the above measurements were performed using calibrated Image Pro-Plus 6.0 software (Media Cybernetics, Inc., Bethesda, MD, United States).
## Spikelet fertility and spikelet flowering
The main-tiller panicles on the day before the heading stage were selected and tagged for each cultivar in the treatments, and then the panicles were harvested at physiological maturity. PaL and the number of filled (i.e., completely or partially filled with grain) and unfilled grains per panicle were both determined, and spikelet fertility (seed set) was recorded for 2 years following the methods of [bib_ref] High nighttime temperatures affect rice productivity through altered pollen germination and spikelet..., Mohammed [/bib_ref]. Each grain was evaluated for whether it was filled or not by pressing the grain between the forefinger and the thumb. Since the grain filling of the spikelets on different positions of the panicle was not Data indicate the means ± standard errors. Means within a column followed by a different lowercase letter differ significantly at a level of 0.05 for a particular parameter. G: genotypes; T: treatments. * P < 0.05, * * P < 0.01, ns, not significant. Data indicate the means ± standard errors, n = 25. Means within a column followed by a different lowercase letter differ significantly at a level of 0.05. G: genotypes; T: treatments. * P < 0.05, * * P < 0.01, ns, not significant. synchronous, spikelet fertility ultimately differed between the upper part and the lower part. Panicles were separated into the upper (i.e., superior grains), middle, and lower (i.e., inferior grains) parts described as spikelet positions in accordance with the protocol of [bib_ref] Abscisic acid and ethylene interact in wheat grains in response to soil..., Yang [/bib_ref] to test the effects on spikelet fertility in each part. Spikelet fertility was evaluated by dividing the number of filled grains by the total number of grains (i.e., florets) and was described as the per cent. In addition, in 2014, the effect of temperature on spikelet opening, marked by different tinctorial acrylic paints, was also examined on five consecutive flowering days in accordance with the methods of [bib_ref] High temperature stress and spikelet fertility in rice (Oryza sativa L.), Jagadish [/bib_ref] [bib_ref] Phenotyping parents of mapping populations of rice for heat tolerance during anthesis, Jagadish [/bib_ref]. The effect of flowering patterns was also identified using blue paint to distinguish spikelets undergoing anthesis at different time periods between 0830 and 1630 h Beijing Summer time (BST), and spikelets were counted every 2 h on the third flowering day. Spikelet flowering in this study was described as the per cent of the numbers of spikelets at anthesis of the total number of spikelets in the panicle (anthesis + non-anthesis) per 2 h period on the third day of anthesis in the four cultivars among the treatments. Spikelet fertility of the marked panicles was also ultimately recorded.
# Statistical analysis
All data of several traits were analyzed as a spilt-plot design, in which the main plot consisted of temperature treatments (low temperature and ambient temperature) with three replications and the subplots consisted of cultivars, using PROC Generalized Linear Mixed Models in the SAS 9.1 (Cary, NC, United States). The data regarding morphology at anthesis, anther dehiscence, pollen germination traits, spikelet flowering and spikelet fertility were used to test the analysis of variance (ANOVA) of temperature treatments, cultivars and their interaction. Linear regressions among pollen traits, anther dehiscence, spikelet fertility, and spikelet flowering were also conducted using GLM procedures. The values in this study were expressed as the means and errors. The means were examined by Tukey's least significant difference (LSD) test to compare the differences at the probability level of 0.05.
# Results
## Pel, pe, and wpl
Elongation of panicle exsertion significantly decreased (P < 0.05) among cultivars subjected to LT stress compared with AT [fig_ref] TABLE 2 |: Effects of LT and AT on morphological traits of the panicles in... [/fig_ref]. The interaction between cultivars and treatments was also significantly different. Across treatments, the EPE of cold-sensitive cultivars, Yongyou9 and Yongyou17, was higher than that of cold-tolerant cultivars, Yongyou538 and Zhejing88. Similarly, the PE of all cultivars also significantly (P < 0.05) decreased by 44.3% under LT [fig_ref] TABLE 2 |: Effects of LT and AT on morphological traits of the panicles in... [/fig_ref] ; Yongyou9 had the highest PE, ranging from 32.0 to 58.0%. PeL among the cultivars was consistently maintained in response to LT stress and was 68.1% longer in cold-sensitive cultivars than in cold-tolerant cultivars. Across all the cultivars, PeL was reduced by 6.81 cm under LT, compared with AT. Furthermore, the WPL of Yongyou17 was significantly longer by 114.1% under LT than under AT, but there was no difference (P > 0.05) in Yongyou9. Compared with AT, the percent of spikelets trapped inside panicle was enhanced by 3.65% in all the cultivars under LT.
## Anther dehiscence and anther characteristics
Low temperature have an influence on anther dehiscence (including FAD, PAD, and AID), and there were obvious differences among cultivars . All cultivars showed less FAD under LT than under AT, and a significant difference (P < 0.05) was observed in the majority of the cultivars. However, the cultivars had higher PAD under LT than under AT, and Yongyou17 was significantly affected (P < 0.05).
Overall, LT increased anther indehiscence in cold-sensitive cultivars.
Anther size (anther length, width, and volume) varied among temperature treatments and cultivars [fig_ref] TABLE 3 |: Effects of LT on the anther size of rice plants [/fig_ref] , and the effects on the cultivars were significantly different (P < 0.05). Anther width and volume indicated the same decreasing trends under LT compared with those under AT; however, a significant difference was observed in cold-sensitive cultivars regarding anther volume. There were no significant differences among cold-tolerant cultivars.
## Spikelet fertility and pollen grain on the stigma
There were significant (P < 0.01) differences among cultivars and temperature treatments on spikelet positions regarding spikelet fertility [fig_ref] TABLE 4 |: Effects of LT and spikelet position on the spikelet fertility of rice... [/fig_ref]. Spikelet fertility significantly (P < 0.05) decreased in Yongyou9 (9.5%) and Yongyou17 (12.5%) under LT [fig_ref] TABLE 4 |: Effects of LT and spikelet position on the spikelet fertility of rice... [/fig_ref] , whereas no significant differences were observed in Yongyou538 and Zhejing88. Based on spikelet position, LT also significantly reduced spikelet fertility in the upper, middle, and lower parts of the panicle, with a significant impact on cold-sensitive cultivars, particularly in the upper part of the panicle.
Spikelet fertility on five consecutive flowering days decreased significantly due to being subjected to LT for both Yongyou9 and Yongyou17. Zhejing88 and Yongyou538, however, exhibited much higher spikelet fertility under LT than did Yongyou9 and Yongyou17 [fig_ref] FIGURE 3 |: Spikelet fertility during five consecutive days of anthesis of four rice varieties... [/fig_ref]. Spikelet fertility under AT was not significantly different among cultivars. There was less spikelet fertility reduction in Yongyou9 (−29.4%) and Yongyou17 (−25.5%) under LT than in Yongyou538 (−9.8%) and Zhejing88 (2.1%).
Variational differences in the numbers of pollen grains, germinated pollen grains, and PPG between treatments followed the same trends in all cultivars [fig_ref] FIGURE 4 |: Effects of LT and AT on the number of pollen grains on... [/fig_ref]. These traits significantly decreased under LT in Yongyou9 and Yongyou17 compared with those under AT but not in Yongyou538 and Zhejing88. The results are shown as pictorial illustrations of pollen count and pollen germination [fig_ref] FIGURE 5 |: Pictorial illustration of LT and AT affecting the number of pollen grains... [/fig_ref]. There were significant differences between the cultivars and treatments and their interaction for pollen fertility and diameter [fig_ref] TABLE 5 |: Effects of LT on pollen fertility and pollen diameter [/fig_ref]. Moreover, under LT stress, pollen fertility and diameter significantly decreased in cold-sensitive cultivars compared with those under AT, but no differences in cold-tolerant cultivars were observed (P > 0.05). Pollen fertility and diameter were both positively related to the number of germinated pollen grains (r = 0.77 and r = 0.79, respectively; n = 15).
## Spikelet flowering at anthesis
The time of day of spikelets undergoing anthesis for 5 days under AT showed similar trends among cultivars, and flowering peaked on the third (or fourth) day [fig_ref] FIGURE 6 |: Spikelet flowering per day during five consecutive days of anthesis in indica-japonica... [/fig_ref]. On average, LT significantly reduced the total number of spikelets at anthesis in all cultivars. However, spikelet flowering under LT was higher in Yongyou9 and Yongyou17 than in Yongyou538 and almost decreased to 0% during the 5-day flowering period for Zhejing88.
The pattern of flowering under AT showed similar trends among cultivars of increasing but then decreasing on the third flowering day, peaking at 1230 or 1030 h (Yongyou9) [fig_ref] FIGURE 7 |: Spikelet flowering every 2 h on the third day of anthesis in... [/fig_ref]. However, LT significantly reduced spikelet flowering between 1030 and 1230 h. Under LT, spikelet flowering decreased on the third flowering day and was higher on average in Yongyou9 (2.14%) and Yongyou17 (1.82%) compared to that of Yongyou538 (0.63%) and Zhejing88 (0.01%).
Spikelet fertility was positively correlated with the number of pollen grains, germinated pollen grains, and PPG but not with anther dehiscence or spikelet flowering [fig_ref] TABLE 6 |: Relationship among pollen count on the stigma, pollen germination, anther dehiscence, spikelet... [/fig_ref]. Moreover, Data indicate the means ± standard errors. Means within a column followed by a different lowercase letter differ significantly at a level of 0.05. G: genotypes; T: treatments. * P < 0.05, * * P < 0.01, ns, not significant.
Frontiers in Plant Science | www.frontiersin.org anther dehiscence was not correlated with spikelet flowering (P > 0.05).
# Discussion
With changing global climate, extreme cold conditions will be more frequent in the future, which make rice subject to adverse abiotic stresses. Improving the temperature stress tolerance in rice at anthesis during the susceptible reproductive stage is beneficial for adapting to highly dynamic climatic conditions in the future [bib_ref] Effect of elevated CO2 and global climate change on rice yield in..., Horie [/bib_ref]. In addition, increasing the absolute stress tolerance of rice could facilitate important physiological processes (e.g., anther dehiscence, pollen germination, pollination, and fertilization) being carried out with high spikelet fertility under stress [bib_ref] Phenotyping parents of mapping populations of rice for heat tolerance during anthesis, Jagadish [/bib_ref] [bib_ref] Physiological and Proteomic approaches to address heat tolerance during anthesis in rice..., Jagadish [/bib_ref] [bib_ref] Effect of high temperature and water stress on pollen germination and spikelet..., Rang [/bib_ref]. [bib_ref] Effect of high temperature and water stress on pollen germination and spikelet..., Rang [/bib_ref] considered that spikelet fertility might be largely determined by morphological conditions at anthesis under different abiotic stresses. PE, which was primarily affected by PeL, could be described as affecting rice spikelet fertility when exposed to LT. However, in the present study, the panicles were partially exserted under LT; some spikelets within the panicles were still stuck in the leaf sheath of the four cultivars regardless of the degree of cold tolerance and EPE [fig_ref] TABLE 2 |: Effects of LT and AT on morphological traits of the panicles in... [/fig_ref]. Interestingly, we found that PeL decreased significantly (P < 0.05) under LT in all cultivars apart from Yongyou9. These results were similar to the previous findings under HTs [bib_ref] Effect of high temperature and water stress on pollen germination and spikelet..., Rang [/bib_ref]. Although spikelets stuck in the leaf sheath could be reflective of spikelet fertility through altered anthesis and fertilization [bib_ref] Role of panicle exertion in waterstress induced sterility, O'toole [/bib_ref] [bib_ref] Dryland rice response to an irrigation gradient at flowering stage, Cruz [/bib_ref] , the physiological process of spikelet fertility under LT did not invariably coincide with phenology. In general, anther dehiscence can affect the number of pollen grains on the stigma [bib_ref] Effect of high temperature and water stress on pollen germination and spikelet..., Rang [/bib_ref]. In the present study, anther indehiscence under LT was higher in cold-sensitive cultivars than in cold-sensitive cultivars . It was mainly due to a sharply reduced FAD, resulting in decreased pollen grains on the stigma. [bib_ref] Genetic variation in the sensitivity of anther dehiscence to drought stress in..., Liu [/bib_ref] reported that the rupturing of the septa, the expansion of locule walls, the swelling of pollen grains, and then the rupturing of the stomium were all highly related to anther dehiscence. However, in the present study, this phenomenon was not observed [fig_ref] TABLE 6 |: Relationship among pollen count on the stigma, pollen germination, anther dehiscence, spikelet... [/fig_ref]. The reason was that anthers still dehisced under stress due to spikelet flowering; pollen grains could not swell, and they lost viability and did not shed, resulting in unfertilized pollen [bib_ref] Genotypic variation for cold tolerance during reproductive development in rice: screening with..., Farrell [/bib_ref] [bib_ref] Genetic variation in the sensitivity of anther dehiscence to drought stress in..., Liu [/bib_ref] [bib_ref] Physiological and Proteomic approaches to address heat tolerance during anthesis in rice..., Jagadish [/bib_ref]. There was no relationship between greater anther dehiscence and more pollen grains on the stigma, as was described by [bib_ref] Physiological and Proteomic approaches to address heat tolerance during anthesis in rice..., Jagadish [/bib_ref]. However, spikelet fertility under cool temperature conditions was strongly correlated with large anthers [bib_ref] Cold tolerance in rice plants with special reference to the floral characters...., Suzuki [/bib_ref] , and anther length, width and area benefit increasingly large pollen grains with respect to fertilization [bib_ref] Genotypic variation for cold tolerance during reproductive development in rice: screening with..., Farrell [/bib_ref]. Our findings also showed that anther volume, which was significantly affected in cold-sensitive cultivars, was more closely related to anther width (r = 0.96, P < 0.01; n = 17, data not shown) than anther length (r = 0.69, P < 0.01) under LT. In addition, spikelets scored for high fertility had at least 10 or more than 20 germinated pollen grains, which could be identified as the threshold number [bib_ref] Physiological and Proteomic approaches to address heat tolerance during anthesis in rice..., Jagadish [/bib_ref]. It was therefore thought that cold-tolerant cultivars obtained more germinated pollen on the stigma [fig_ref] FIGURE 4 |: Effects of LT and AT on the number of pollen grains on... [/fig_ref] , and increased seed set compared with that of cold-sensitive cultivars under LT was ultimately confirmed [fig_ref] TABLE 4 |: Effects of LT and spikelet position on the spikelet fertility of rice... [/fig_ref]. It has been reported that pollen germination alters spikelet fertility, ultimately affecting rice productivity under high night-time temperatures [bib_ref] High nighttime temperatures affect rice productivity through altered pollen germination and spikelet..., Mohammed [/bib_ref] as well as under LT. [bib_ref] Genetic variation in the sensitivity of anther dehiscence to drought stress in..., Liu [/bib_ref] reported that improved pollen stickiness, which could restrict pollen grains from shedding whether the anther pores dehisced or not, might affect pollen count on the stigma. Our results suggest that a small pollen count and germinated pollen grains under LT were observed in cold-sensitive cultivars [fig_ref] FIGURE 4 |: Effects of LT and AT on the number of pollen grains on... [/fig_ref] , mainly due to poor pollen fertility and shortened pollen diameters [fig_ref] TABLE 5 |: Effects of LT on pollen fertility and pollen diameter [/fig_ref]. Moreover, there was strong positive relationship between spikelet fertility and pollen germination [fig_ref] TABLE 6 |: Relationship among pollen count on the stigma, pollen germination, anther dehiscence, spikelet... [/fig_ref]. However, the mechanism controlling high pollen germination with low anther dehiscence in spikelets under LT stress is not clear. Although a uniform level of PE, PeL, and WPL between treatments was observed among the cultivars, rice plants exposed to LT did have lower spikelet fertility across cold-sensitive cultivars [fig_ref] TABLE 4 |: Effects of LT and spikelet position on the spikelet fertility of rice... [/fig_ref]. This was caused by different spikelet positions within the panicle (P < 0.05); the upper parts of the panicle under LT stress and LT alone could especially aggravate poor spikelet fertility. However, under stress conditions, the grains at the upper parts of the panicle are usually filled primarily [bib_ref] The rice plant -its development and yield, Stansel [/bib_ref]. The upper parts are also exposed to LT first, which can enhance the extent of cold-induced damage and lead to spikelet sterility, even when the panicles are exserted. However, the lower and middle parts of the panicle were correspondingly subjected to less damage due to the site of PE. Similar results on the grain characteristics of rice under high night-time temperatures and spikelet position can be obtained from the report of [bib_ref] Effects of high night temperature and spikelet position on yield-related parameters of..., Mohammed [/bib_ref]. Furthermore, the effect of spikelet position within the panicle on spikelet fertility across cultivars was also related to the exsertion of the panicle.
The cumulative negative influence of extended LT stress on consecutive flowering days was examined in terms of spikelet fertility (seed set) using the individual spikelets marked. In the study, we observed a significant reduction in spikelet fertility with the duration of flowering in cold-sensitive cultivars but not in cold-tolerant cultivars [fig_ref] FIGURE 3 |: Spikelet fertility during five consecutive days of anthesis of four rice varieties... [/fig_ref]. This result was in accordance with that in the studies of [bib_ref] High temperature stress and spikelet fertility in rice (Oryza sativa L.), Jagadish [/bib_ref] and [bib_ref] Effect of high temperature and water stress on pollen germination and spikelet..., Rang [/bib_ref] on the effects of HTs. Flowering under AT peaked within a 5-day period earlier in indica-japonica hybrid cultivars than in japonica rice [fig_ref] FIGURE 6 |: Spikelet flowering per day during five consecutive days of anthesis in indica-japonica... [/fig_ref]. The former cultivars contain the indica gene besides the japonica gene [bib_ref] Species, ecotype and cultivar differences in spikelet fertility and harvest index of..., Prasad [/bib_ref] , resulting in early flowering, even though the pattern of flowering, which reached peaked between 1030 and 1230 h, on the third day was similar among cultivars [fig_ref] FIGURE 7 |: Spikelet flowering every 2 h on the third day of anthesis in... [/fig_ref]. Compared with cold-sensitive cultivars, cold-tolerant cultivars still had fewer numbers of flowering spikelets under LT [fig_ref] FIGURE 6 |: Spikelet flowering per day during five consecutive days of anthesis in indica-japonica... [/fig_ref] , allowing floral organs to avoid damage from LT. [bib_ref] High temperature stress and spikelet fertility in rice (Oryza sativa L.), Jagadish [/bib_ref] proposed that more spikelet anthesis is correlated with a higher spikelet fertility under HTs. However, this phenomenon was not verified in our study, because spikelet flowering did not significantly affect spikelet fertility [fig_ref] TABLE 6 |: Relationship among pollen count on the stigma, pollen germination, anther dehiscence, spikelet... [/fig_ref]. Hence, our results stated that spikelet fertility was not the consequence of spikelet flowering. It had been understood that the normal action of dehisced pollen sacs in anthers and pollen vitality might be affected by LT stress as well as by HTs a day ahead flowering [bib_ref] Rice (Oryza sativa L.) cultivars tolerant to high temperature at flowering: anther..., Matsui [/bib_ref]. It is possible that in cold-susceptible cultivars such as Yongyou9 and Yongyou17, other processes that occur before fertilization were affected, such as pollen shrinkage and anther volume reduction [bib_ref] Genotypic variation for cold tolerance during reproductive development in rice: screening with..., Farrell [/bib_ref] as well as effects on pollen tube growth rate and pollen germination [bib_ref] Response of in vitro pollen germination and pollen tube growth of groundnut..., Kakani [/bib_ref] [bib_ref] Resilience of rice (Oryza spp.) pollen germination and tube growth to temperature..., Coast [/bib_ref]. This could confirm that high pollen germination was observed in Yongyou538 and Zhejing88. Nonetheless, it is unlikely that ovary development was unaffected after fertilization, although spikelets subjected to LT after opening were fertile in both cold-sensitive cultivars and cold-tolerant cultivars. This phenomenon could also result in spikelet sterility.
In general, indica species are more tolerant to HTs; conversely, japonica species are more tolerant to LT stress [bib_ref] Genotypic variation for cold tolerance during reproductive development in rice: screening with..., Farrell [/bib_ref]. Moreover, indica-japonica hybrid rice, which simultaneously contains indica and japonica genes, was sensitive to temperature fluctuations, resulting in spikelet sterility under LT, despite the neutral alleles at the two loci being used to solve pollen sterility in indica-japonica hybrid rice [bib_ref] Identification of segregationdistortion-neutral alleles to improve pollen fertility of indica-japonica hybrids in..., Lu [/bib_ref]. In this study, indica-japonica hybrid rice cultivars, which were tested for the gene frequencies of indica and japonica using SSR molecular markers (data not shown), indeed had different responses to LT. Our study may provide evidence of indica-japonica hybrid rice adapting to extreme climatic changes for breeding purposes.
# Conclusion
Our study has shown that LT induces spikelet sterility by shrinking anther size and decreasing pollen function (the number of pollen grains, pollen fertility, and pollen germination on stigmas) in cold-sensitive cultivars during anthesis. We found that spikelet fertility was strongly correlated with pollen germination rather than spikelet flowering across cultivars and treatments. Spikelet fertility was not attributed to the patterns of flowering and the number of spikelets reaching anthesis. Therefore, this aspect should be considered for breeding indica-japonica hybrid rice varieties to tolerance during flowering.
# Author contributions
DZ conceived and designed the experiments. YPZ, JX, NU, and XP provided experimental opinions and assistance. YPZ and YHZ performed the experiments. YHZ analyzed the data and wrote the paper.
Natural Science Foundation of China (Y13C130013), the Special Fund for Chinese Academy of Agricultural Sciences (2012RG004-2), the Earmarked Fund for Modern Agro-Industry Technology Research System of China (CARS-01-09B) and the Jiangxi Province Natural Science Foundation of China (20161BAB214171).
[fig] FIGURE 3 |: Spikelet fertility during five consecutive days of anthesis of four rice varieties under LT and AT. On different days, individual spikelet fertility was marked with different colored acrylic paints (n = 10), and the fertility of spikelets undergoing anthesis was scored separately. Bars with different lowercase letters denote spikelet fertility, which differ at a significance level of 0.05. Smaller bars indicate standard error. AT, ambient temperature; LT, low temperature. [/fig]
[fig] FIGURE 4 |: Effects of LT and AT on the number of pollen grains on stigmas (A, n > 60 spikelets) and per cent pollen germination (B) of four rice cultivars. Smaller bars with different lowercase letters indicate significant differences at the level of 0.05. Smaller bars indicate standard error. PRS, pollen reception on the stigma; GPS, number of germinated pollen grains on stigmas; AT, ambient temperature; LT, low temperature. [/fig]
[fig] FIGURE 5 |: Pictorial illustration of LT and AT affecting the number of pollen grains and pollen germination of four rice genotypes. The luminous circle symbol indicates pollen, and the threadlike light line represents the pollen tube. Pollen was recorded as germinated when the length of pollen tube was at least equivalent to the pollen diameter. AT, ambient temperature; LT, low temperature. [/fig]
[fig] FIGURE 6 |: Spikelet flowering per day during five consecutive days of anthesis in indica-japonica hybrid rice Yongyou9 (A), Yongyou17 (B) and Yongyou538 (C) and in japonica rice Zhejing88 (D) under LT and AT. Individual spikelet flowering per panicle was marked with different colored acrylic paints on different days (n = 10), and spikelet fertility was scored separately. Smaller bars indicate standard error. AT, ambient temperature; LT, low temperature. [/fig]
[fig] FIGURE 7 |: Spikelet flowering every 2 h on the third day of anthesis in four rice genotypes under LT and AT. Individual spikelets flowering per panicle at different times of day during anthesis were marked with different colored acrylic paints (n = 10), and spikelet fertility was scored separately. Smaller bars indicate standard error. BST, Beijing Summer time; AT, ambient temperature; LT, low temperature. [/fig]
[table] TABLE 1 |: Information on four rice cultivars selected for the study. [/table]
[table] TABLE 2 |: Effects of LT and AT on morphological traits of the panicles in four cultivars. [/table]
[table] TABLE 3 |: Effects of LT on the anther size of rice plants (n = 60) during the flowering period. [/table]
[table] TABLE 4 |: Effects of LT and spikelet position on the spikelet fertility of rice plants (%). [/table]
[table] TABLE 5 |: Effects of LT on pollen fertility and pollen diameter (n = 60) during the flowering period. [/table]
[table] TABLE 6 |: Relationship among pollen count on the stigma, pollen germination, anther dehiscence, spikelet fertility, and spikelet flowering (n = 15). * P < 0.05, * * P < 0.01, ns, not significant. [/table]
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10.18632/oncotarget.12997
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s2orc_pubmed_articles
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Defective ciliogenesis in thyroid hürthle cell tumors is associated with increased autophagy
Primary cilia are found in the apical membrane of thyrocytes, where they may play a role in the maintenance of follicular homeostasis. In this study, we examined the distribution of primary cilia in the human thyroid cancer to address the involvement of abnormal ciliogenesis in different thyroid cancers. We examined 92 human thyroid tissues, including nodular hyperplasia, Hashimoto's thyroiditis, follicular tumor, Hürthle cell tumor, and papillary carcinoma to observe the distribution of primary cilia. The distribution and length of primary cilia facing the follicular lumen were uniform across variable-sized follicles in the normal thyroid gland. However, most Hürthle cells found in benign and malignant thyroid diseases were devoid of primary cilia. Conventional variant of papillary carcinoma (PTC) displayed longer primary cilia than those of healthy tissue, whereas both the frequency and length of primary cilia were decreased in oncocytic variant of PTC. In addition, ciliogenesis was markedly defective in primary Hürthle cell tumors, including Hürthle cell adenomas and carcinomas, which showed higher level of autophagosome biogenesis. Remarkably, inhibition of autophagosome formation by Atg5 silencing or treatment with pharmacological inhibitors of autophagosome formation restored ciliogenesis in the Hürthle cell carcinoma cell line XTC.UC1 which exhibits a high basal autophagic flux. Moreover, the inhibition of autophagy promoted the accumulation of two factors critical for ciliogenesis, IFT88 and ARL13B. These results suggest that abnormal ciliogenesis, a common feature of Hürthle cells in diseased thyroid glands, is associated with increased basal autophagy.
# Introduction
Primary cilia in mammalian cells are crucial organelles for sensory reception and signal transduction, and their roles are closely linked with cell type-specific functions [bib_ref] The primary cilium as a novel extracellular sensor in bone, Hoey [/bib_ref]. In thyroid epithelial cells, primary cilia protrude from the apical surface into the follicular luminal space, which contains colloid. It is generally accepted that, in thyroid epithelial cells, primary cilia sense the environment of the follicular lumen and contribute to follicular function, including the production of hormones [bib_ref] Molecular mechanisms of protein and lipid targeting to ciliary membranes, Emmer [/bib_ref] [bib_ref] Structural and ultrastructural differentiation of the thyroid gland during embryogenesis in the..., Rupik [/bib_ref]. The maintenance of primary ciliary function in Research Paper specific cell types requires highly regulated mechanisms of ciliogenesis, which, if altered, can lead to primary ciliopathy, a disease that may show clinical phenotypes of congenital hypothyroidism [bib_ref] Mutations in GLIS3 are responsible for a rare syndrome with neonatal diabetes..., Senee [/bib_ref]. Ciliogenesis is tightly regulated by molecular programs that control the steps required for both the assembly of the axoneme and the biogenesis of the ciliary membrane. The first step of ciliogenesis is the migration of the centriole-derived basal body to the cell surface where distal appendages of the basal body establish the link between the plasma membrane and the basal body. Thereafter, axonemal microtubules extend from the basal body through the process of intraflagellar transport (IFT), and docking of transport vesicles at the base of the growing cilia promotes ciliary membrane biogenesis [bib_ref] Ciliogenesis: building the cell's antenna, Ishikawa [/bib_ref].
Recently, it has been shown that ciliogenesis and autophagy are bidirectionally regulated [bib_ref] Autophagy and regulation of cilia function and assembly, Orhon [/bib_ref] [bib_ref] Functional interaction between autophagy and ciliogenesis, Pampliega [/bib_ref] [bib_ref] Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites, Tang [/bib_ref] [bib_ref] Dysregulation of Parkin-mediated mitophagy in thyroid Hurthle cell tumors, Lee [/bib_ref]. Autophagy is a lysosome-dependent degradation process that removes cell constituents, cellular organelles, and protein aggregates [bib_ref] The biology and the genetics of Hurthle cell tumors of the thyroid, Maximo [/bib_ref]. Basal autophagy appears to prevent ciliary growth through the degradation of ciliary proteins such as IFT20, a key component in IFT [bib_ref] Functional interaction between autophagy and ciliogenesis, Pampliega [/bib_ref]. By contrast, enhanced autophagy triggered by starvation stimulates the degradation of oral-facial-digital syndrome 1 (OFD1), the endogenous inhibitor of ciliogenesis, thereby promoting ciliogenesis [bib_ref] Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites, Tang [/bib_ref]. However, abnormal activation of autophagy results in IFT20 degradation, which impedes the unlimited growth of the cilia [bib_ref] Reciprocal regulation of cilia and autophagy via the MTOR and proteasome pathways, Wang [/bib_ref]. On the other hand, the loss of primary cilia has been shown to partially inhibit autophagy [bib_ref] Functional interaction between autophagy and ciliogenesis, Pampliega [/bib_ref].
Hürthle cells, which are found in chronic inflammatory and tumorigenic thyroid glands, are oncocytic cells with higher amounts of abnormal mitochondria, which results in an abundantly acidophilic, granular cytoplasm [bib_ref] The biology and the genetics of Hurthle cell tumors of the thyroid, Maximo [/bib_ref]. Increases in mitochondrial content are caused by the accumulation of damaged mitochondria possessing mitochondrial DNA (mtDNA) mutations in respiratory complex genes that cause severe bioenergetic crisis [bib_ref] The genetic and metabolic signature of oncocytic transformation implicates HIF1alpha destabilization, Porcelli [/bib_ref] [bib_ref] A subset of the follicular variant of papillary thyroid carcinoma harbors the..., Castro [/bib_ref]. Recently, we found that XTC. UC1 cells derived from Hürthle cell tumors have higher levels of autophagosome formation [bib_ref] Dysregulation of Parkin-mediated mitophagy in thyroid Hurthle cell tumors, Lee [/bib_ref]. However, it remains unclear whether this feature is linked to the cellular functions of Hürthle cells, which are biologically less active than those of normal epithelial cells [bib_ref] Hurthle cell neoplasms of the thyroid, Mcleod [/bib_ref].
In this study, we examined the appearance of primary cilia in the human thyroid gland and the alternations in ciliogenesis in thyroid diseases, namely, Hashimoto's thyroiditis, follicular tumors, Hürthle cell tumors, and papillary thyroid carcinomas. Interestingly, primary cilia were abnormal in Hürthle cells in Hashimoto's thyroiditis, papillary thyroid cancer and primary Hürthle cell tumors. In addition, ciliogenesis was suppressed in the Hürthle cell line XTC.UC1, which shows a relatively high level of autophagic activity. We found that genetic and pharmacologic inhibition of autophagy turnover in XTC.UC1 cells promoted ciliogenesis as well as ciliary elongation. We propose that activated autophagy flux impedes ciliogenesis in Hürthle cells with compromised mitochondrial oxidative phosphorylation (OxPhos). The identification of the molecular mechanism underlying defective ciliogenesis in Hürthle cell tumors could help understand the clinical features of thyroid diseases.
# Results
## Identification of primary cilia in normal human thyroid gland
Thyroid follicles lined with a single layer of epithelial cells are structural and functional units that produce thyroid hormone. We identified primary cilia using antibodies against acetylated α-tubulin and ARL13B, a cilia-enriched small GTPase. The staining of acetylated α -tubulin and Arl13B has been widely used for the determination of ciliated cell frequency and cilia length [bib_ref] Primary cilia are lost in preinvasive and invasive prostate cancer, Hassounah [/bib_ref] [bib_ref] Loss of primary cilia occurs early in breast cancer development, Menzl [/bib_ref]. The detection of basal bodies, the root of primary cilia, was performed by immunofluorescent staining using an antibody against γ-tubulin, which localized in the basal bodies. We assessed ciliated cell frequency in the normal follicular epithelium in vivo by immunofluorescent staining of five specimens taken from the contralateral lobe of thyroid cancer. The tissue cross-sections were stained with haematoxylin and eosin (H&E) to identify normal follicles [fig_ref] Figure 1: Distribution of primary cilia in thyroid tissue with normal and nodular hyperplasia [/fig_ref]. As shown in [fig_ref] Figure 1: Distribution of primary cilia in thyroid tissue with normal and nodular hyperplasia [/fig_ref] , primary cilia were detected in both follicular epithelial cells and parafollicular cells. It has been reported that primary cilia usually extend from the apical surface of secretory cells [bib_ref] The primary cilia of secretory cells in the human oviduct mucosa, Hagiwara [/bib_ref]. As expected, primary cilia in follicular cells extended from the apical membrane toward the colloid-rich follicular lumen. More than 50% of the epithelial cells showed uniformly ciliated patterns in normal follicles [fig_ref] Figure 1: Distribution of primary cilia in thyroid tissue with normal and nodular hyperplasia [/fig_ref].
## Expression patterns of primary cilia in benign thyroid diseases
One of the representative thyroid diseases exhibiting follicular heterogeneity is benign nodular hyperplasia (NH), which shows structural variability in follicles [fig_ref] Figure 1: Distribution of primary cilia in thyroid tissue with normal and nodular hyperplasia [/fig_ref]. No remarkable changes in either the frequency of ciliated cells or the lengths of cilia were found in benign nodular hyperplasia when they were compared with those in normal thyroid glands (normal 67.8 ± 3.6% vs NH 64.8 ± 18.3%, p = 0.363) [fig_ref] Figure 1: Distribution of primary cilia in thyroid tissue with normal and nodular hyperplasia [/fig_ref] and 1E). This finding indicates that benign structural variability found in nodular hyperplasia does not associate with abnormalities in ciliogenesis.
Hashimoto's thyroiditis (HT) is a representative chronic thyroiditis accompanied by variable degrees of follicular damage with heavy infiltration of immune cells into the stroma surrounding the thyroid follicles [bib_ref] Autoantibodies in Hashimoto's disease (lymphadenoid goitre), Roitt [/bib_ref]. Follicles found in areas close to lymphocyte infiltrations were smaller and filled with scanty colloid [fig_ref] Figure 2: Distribution of primary cilia in Hashimoto's thyroiditis [/fig_ref].
These follicular epithelial cells showed normal features of the primary cilia, and the percentage of ciliated epithelial cells was similar to that of the normal thyroid gland (normal 67.8 ± 3.6% vs HT 67.5 ± 13.4%, p = 0.472) [fig_ref] Figure 2: Distribution of primary cilia in Hashimoto's thyroiditis [/fig_ref] and 2F). The follicles infiltrated with lymphocytes also showed primary cilia [fig_ref] Figure 2: Distribution of primary cilia in Hashimoto's thyroiditis [/fig_ref]. The atrophic follicles with abundant Hürthle cells were observed as isolated follicular structures [fig_ref] Figure 2: Distribution of primary cilia in Hashimoto's thyroiditis [/fig_ref]. Interestingly, Hürthle cells of Hashimoto's thyroiditis rarely displayed primary cilia (normal 67.8 ± 3.6% vs Hürthle cell of HT 3.6 ± 1.9%, p = 0.0007) [fig_ref] Figure 2: Distribution of primary cilia in Hashimoto's thyroiditis [/fig_ref] and 2F). The pathogenesis of Hürthle cells in Hashimoto's thyroiditis may be secondary to a mutation in mtDNA that causes mitochondrial dysfunction. The staining of Hürthle cells with an antibody against acetylated α-tubulin showed a diffuse distribution pattern in the cytoplasm, unlike normal thyroid cells [fig_ref] Figure 2: Distribution of primary cilia in Hashimoto's thyroiditis [/fig_ref]. a-Tubulin is an intrinsic mitochondrial structural protein required for molecular transport, and a significant portion of α-tubulin is acetylated in mitochondria [bib_ref] Tubulin is an inherent component of mitochondrial membranes that interacts with the..., Carre [/bib_ref]. Thus, excessive accumulation of mitochondria may cause increased immunoreactivity of acetylated α-tubulin in the cytoplasm. Translocase of outer mitochondrial membrane 40 (TOM40) was used as a marker of mitochondria density. Hürthle cells in Hashimoto's thyroiditis showed strong expression of TOM40 but showed decreased distribution of primary cilia [fig_ref] Figure 2: Distribution of primary cilia in Hashimoto's thyroiditis [/fig_ref]. Together, these results suggest that Hürthle cells have altered primary ciliogenesis that may be linked to defects in mitochondrial oxidative phosphorylation.
## Expression patterns of primary cilia in malignant thyroid tumors
To further substantiate the finding of abnormal ciliogenesis in Hürthle cells in malignant thyroid diseases, we examined the distribution of primary cilia in papillary thyroid cancer (PTC). PTC is the most common type of thyroid cancer and has multiple histopathological variants, including conventional, follicular, oncocytic (Hürthle cell), solid, and tall cell variants . We examined the expression of primary cilia in histopathological variants of PTC. The conventional PTC was characterized by complex papillae with thin fibrovascular cores, and the cancer cells in this PTC variant showed well-expressed primary cilia [fig_ref] Figure 3: Distribution of primary cilia in papillary thyroid carcinoma [/fig_ref]. The other relatively common variant of PTC, follicular variant of PTC characterized by follicular architecture with PTC nuclear features, showed a similar expression pattern of primary cilia compared to that of the conventional type [fig_ref] Figure 3: Distribution of primary cilia in papillary thyroid carcinoma [/fig_ref]. The frequency of ciliated cells was comparable in four different variant types, namely, conventional (conv), follicular variant (FV), solid variant (SV), tall cell variant [fig_ref] Figure 3: Distribution of primary cilia in papillary thyroid carcinoma [/fig_ref]. However, the length of primary cilia showed characteristic alterations according to histopathological variants [fig_ref] Figure 3: Distribution of primary cilia in papillary thyroid carcinoma [/fig_ref]. The conventional variant showed significantly longer primary cilia, whereas the solid variant showed shorter primary cilia compared to that of normal thyroid cells. Similar to Hürthle cells found in Hashimoto's thyroiditis, the oncocytic variant of PTC had remarkably fewer ciliated cells compared to normal follicular cells (normal 67.8 ± 3.6% vs PTC-OV 17.6 ± 11.7%, p = 0.0002) or the conventional type of PTC (PTCconv 68.7 ± 18.0% vs PTC-OV 17.6 ± 11.7%, p = 0.0027). In addition, we observed increased diffuse cytoplasmic staining of anti-acetylated α-tubulin in the oncocytic variant, suggesting excessive accumulation of mitochondria [fig_ref] Figure 3: Distribution of primary cilia in papillary thyroid carcinoma [/fig_ref]. Taken together, these findings suggest that ciliogenesis is influenced by the variant-specific pathogenesis of PTC, particularly the oncocytic variant of PTC.
## Defective ciliogenesis in hürthle cell tumors
A primary Hürthle cell tumor of the thyroid gland is a relatively rare type of differentiated thyroid cancer [bib_ref] Oncocytic neoplasms of the thyroid gland, Tallini [/bib_ref] [bib_ref] Genomic dissection of Hurthle cell carcinoma reveals a unique class of thyroid..., Ganly [/bib_ref]. It has a poorer clinical course than that of other differentiated thyroid cancers, and is considered a variant of the follicular tumor of the thyroid known as follicular carcinoma, oxyphilic type [bib_ref] Oncocytic neoplasms of the thyroid gland, Tallini [/bib_ref] [bib_ref] Genomic dissection of Hurthle cell carcinoma reveals a unique class of thyroid..., Ganly [/bib_ref]. However, several investigators contend that a Hürthle cell tumor is a distinct form of thyroid neoplasm differentiated from follicular neoplasms [bib_ref] Oncocytic neoplasms of the thyroid gland, Tallini [/bib_ref] [bib_ref] Genomic dissection of Hurthle cell carcinoma reveals a unique class of thyroid..., Ganly [/bib_ref]. We observed the formation of primary cilia in follicular adenomas (n = 10), follicular carcinomas (n = 10), Hürthle cell adenomas (n = 10) and Hürthle cell carcinomas (n = 10). As shown in [fig_ref] Figure 4: Distribution of primary cilia in follicular tumors and Hürthle cell tumors [/fig_ref] , tumor cells of the follicular adenoma showed primary cilia in the apical membrane oriented toward the follicular lumen similar to normal thyroid follicles. By contrast, ciliogenesis was markedly decreased in Hürthle cell carcinomas (normal 67.8 ± 3.6% vs FC 60.5 ± 11.5% vs HC 4.4 ± 2.2%) [fig_ref] Figure 4: Distribution of primary cilia in follicular tumors and Hürthle cell tumors [/fig_ref] and 4C). Microtuble-associated protein light chain 3 (LC3) has been used as a specific marker for the monitoring of autophagy. As we previously reported, normal follicular cells and non-oncocytic cells of follicular tumors show rare expression of LC3, whereas oncocytes in Hürthle cell tumors consistently express high levels of LC3 [bib_ref] Dysregulation of Parkin-mediated mitophagy in thyroid Hurthle cell tumors, Lee [/bib_ref]. Therefore, higher expression of LC3 has been used as a specific marker of Hürthle cells. As shown in [fig_ref] Figure 4: Distribution of primary cilia in follicular tumors and Hürthle cell tumors [/fig_ref] , Hürthle cells that were positive for LC3 did not show primary cilia. It is likely that the suppression of ciliogenesis is a common feature of Hürthle cells, and the loss of primary cilia may contribute to the dysregulation of biological activities in Hürthle cells.
## Defective ciliogenesis in the hürthle cell carcinoma cell line xtc.uc1
We examined primary cilia in Nthy-ori 3-1 cells, an untransformed human thyroid follicular cell line. Primary cilia were analyzed not only in the presence of serum but also in the absence of serum, because serum starvation is commonly used to induce ciliogenesis in cell culture [bib_ref] The resorption of primary cilia during mitosis in a vertebrate (PtK1) cell..., Rieder [/bib_ref] [bib_ref] Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells, Tucker [/bib_ref]. Serum starvation induced a moderate increase in ciliated cell numbers in Nthy-ori 3-1 cells [fig_ref] Figure 5: Primary cilia in human normal thyroid follicular cell lines and thyroid cancer... [/fig_ref]. Approximately 26.2% of Nthy-ori 3-1 cells cultured in serum-supplemented conditions exhibited primary cilia, and approximately 32.6% of cells were ciliated after 36 hr of serum starvation (p = 0.037). Serum starvation clearly facilitated the elongation of primary cilia. The average length of primary cilia in Nthy-ori 3-1 cells was 3.62 ± 0.21 μm in serum-supplemented conditions and 9.06 ± 4.09 μm in serum-starved conditions for 36 hr (p = 0.014) [fig_ref] Figure 5: Primary cilia in human normal thyroid follicular cell lines and thyroid cancer... [/fig_ref]. To test whether ciliogenesis is affected by tumor transformation, we observed primary cilia in the thyroid cancer cell line TPC-1 cultured in the presence or absence of serum. The TPC-1 cell line was originally derived from a human papillary thyroid carcinoma containing rearrangements of the RET gene (RET/PTC). Although the difference in ciliated cell number was not statistically significant (serum-supplemented conditions 27.5 ± 1.6% vs serum-starved conditions 30.5 ± 10.2%, p = 0.43), serum starvation clearly facilitated the elongation of primary cilia [fig_ref] Figure 5: Primary cilia in human normal thyroid follicular cell lines and thyroid cancer... [/fig_ref] and 5D). The average length of the primary cilia was 4.97 ± 2.48 μm in serumsupplemented and 13.60 ± 4.23 μm in serum-starved conditions (p = 0.003) [fig_ref] Figure 5: Primary cilia in human normal thyroid follicular cell lines and thyroid cancer... [/fig_ref].
We then assessed ciliogenesis in XTC.UC1 cells derived from Hürthle cell carcinoma. XTC.UC1 cells showed markedly decreased numbers of primary cilia compared with those of Nthy-ori 3-1 and TPC-1 cells [fig_ref] Figure 5: Primary cilia in human normal thyroid follicular cell lines and thyroid cancer... [/fig_ref]. The frequency of primary cilia in XTC.UC1 cells was 7.75 ± 3.42% in serum-supplemented conditions and 8.19 ± 4.53% in serum-starved conditions (p = 0.74) [fig_ref] Figure 5: Primary cilia in human normal thyroid follicular cell lines and thyroid cancer... [/fig_ref] and 5F). Serum starvation-induced ciliary elongation was not observed in XTC.UC1 cells. The average length of the primary cilia was 4.77 ± 1.82 μm in serum-supplemented and 5.60 ± 1.57 μm in serum-starved conditions (p = 0.27) [fig_ref] Figure 5: Primary cilia in human normal thyroid follicular cell lines and thyroid cancer... [/fig_ref] and 5F). These results further support the idea that loss of cilia is a characteristic feature of Hürthle cells. It is likely that certain cellular factors that allow a thyrocyte to differentiate into a Hürthle cell may affect ciliogenesis.
## Pharmacological and genetic inhibition of autophagosome formation restores ciliogeneis in xtc.uc1 cells
Our previous study demonstrated that XTC.UC1 cells have higher basal autophagic flux, which can be further augmented by CCCP and bafilomycin A1 (BAF) treatment [bib_ref] Dysregulation of Parkin-mediated mitophagy in thyroid Hurthle cell tumors, Lee [/bib_ref]. Consistently, we found a higher level of LC3-II under basal and BAF-treated conditions in XTC.UC1 cells than in Nthy-ori 3-1 cells [fig_ref] Figure 6: Relationship between primary ciliogenesis and autophagy in XTC [/fig_ref]. To determine whether increased autophagic flux is associated with defective ciliogenesis in XTC.UC1 cells, we examined the frequency of ciliated cells as well as the length of cilia following treatment with inhibitors of phagopore and autophagolysosome formation. 3-MA inhibits autophagy by blocking autophagosome formation via the inhibition of class III phosphatidylinositol 3-kinases (PI3K) [bib_ref] Synthesis and screening of 3-MA derivatives for autophagy inhibitors, Wu [/bib_ref]. To test if increased autophagy in XTC.UC1 cells interferes with ciliogenesis by degrading proteins critical for ciliogenesis, we examined cilia expression in 3-MA-treated XTC.UC1 cells. As shown in [fig_ref] Figure 6: Relationship between primary ciliogenesis and autophagy in XTC [/fig_ref] , the levels of IFT88 and ARL13B, which regulate ciliogenesis, increased in response to 3-MA treatment. Interestingly, the frequency of ciliated cells was markedly higher with 3-MA treatment (10 mM, 89.2% ± 5.6%) than without treatment (8.1% ± 0.4%, p < 0.001) [fig_ref] Figure 5: Primary cilia in human normal thyroid follicular cell lines and thyroid cancer... [/fig_ref]. The lengths of the primary cilia were also significantly increased (13.6 μm for 3-MA versus 4.8 μm for the control, p = 0.0003) [fig_ref] Figure 6: Relationship between primary ciliogenesis and autophagy in XTC [/fig_ref]. On the other hand, BAF prevents the maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes. BAF treatment also increased ciliated cell number, although its effect on ciliogenesis was milder than that of 3-MA. BAF treatment did not promote ciliary H&E. Immunofluorescent staining of primary cilia in tumorigenic follicular cells using anti-acetylated α-tubulin (green) and anti-γ-tubulin (red). Scale bar, 10 µm. (B) Hürthle cell adenoma stained with H&E. Immunofluorescent staining of primary cilia in tumorigenic Hürthle cells using anti-acetylated α-tubulin (green). Scale bar, 10 µm (first panels). Immunofluorescent staining of Hürthle cell tumors using anti-LC3A/B-I & II (green) and primary cilia using anti-acetylated α-tubulin (red). Scale bar, 10 µm (second, third, and fourth panels). (C) The average frequency of primary cilia in Hürthle cell carcinoma compared with that in normal thyroid (p = 0.0002) or follicular carcinoma (p = 0.008). Normal follicular cells were used as controls. **p < 0.01, N.S.; not significant. elongation (p = 0.27 with 10 nM BAF; p = 0.47 with 20 nM BAF) [fig_ref] Figure 6: Relationship between primary ciliogenesis and autophagy in XTC [/fig_ref]. Although deprivation of serum increases LC3-II processing in XTC.UC1 cells, it did not further decrease ciliogenesis [fig_ref] Figure 6: Relationship between primary ciliogenesis and autophagy in XTC [/fig_ref]. These observations indicate that ciliogenesis in the XTC.UC1 cell line is maintained at a maximally suppressed level that may be unresponsive to further activation of autophagic processes.
To further substantiate the role of autophagosome formation in the regulation of ciliogenesis, we used Atg5 siRNA to specifically inactivate the autophagosome in XTC.UC1 cells. Atg5 siRNA achieved efficient knockdown of ATG5 protein expression and inhibited autophagic activity, as indicated by the decreased level of LC3-II [fig_ref] Figure 6: Relationship between primary ciliogenesis and autophagy in XTC [/fig_ref]. The knockdown of ATG5 is normally able to prevent mitochondrial protein degradation. The levels of IFT88 and ARL13B increased in response to Atg5 siRNA. Inhibition of autophagy or prevention of mitochondrial protein degradation induced ciliogenesis, as shown by the increases in the frequency of ciliated cells (13.8% ± 8.3%, p < 0.01) and in the lengths of primary cilia (18.5 ± 4.0 μm, p < 0.01) [fig_ref] Figure 6: Relationship between primary ciliogenesis and autophagy in XTC [/fig_ref] and 6I) in XTC. UC1 cells.
Taken together, these results suggest that ciliogenesis in the Hürthle cell cancer cell line, XTC.UC1, is negatively influenced by higher autophagic activity, a bona fide feature of this cell line. Defective ciliogenesis in Hürthle cells in benign and malignant diseases may be Immunofluorescent staining of primary cilia using anti-acetylated α-tubulin (green) and anti-γ-tubulin (red). Scale bar, 10 µm. The average frequencies and the average lengths of primary cilia in Nthy-ori 3-1, TPC-1, and XTC.UC1 cells. serum +; serum-supplemented conditions, serum -; serum-starved conditions. caused by persistent sequestration of ciliogeneic proteins, such as IFT88 and ARL13B [fig_ref] Figure 7: Schematic model for the regulation of ciliogenesis in XTC [/fig_ref].
# Discussion
Primary cilia are thought to function as sensors of the follicular lumen environment, which plays a crucial role in maintaining follicular homeostasis. Although there is no clear evidence on the role of primary cilia in follicular homeostasis, patients with primary ciliopathy and animal models of defective ciliogenesis show profound hypothyroidism [bib_ref] Mechanistic insights into Bardet-Biedl syndrome, a model ciliopathy, Zaghloul [/bib_ref]. These observations indicate that abnormal ciliogenesis may be important for the development of thyroid diseases. In this study, we observed ciliogenesis by analyzing the frequency of ciliated cells and the lengths of cilia in the normal thyroid gland and in benign and malignant human thyroid diseases. We found that a common benign thyroid disease, nodular hyperplasia, had no marked abnormalities in ciliogeneis compared with ciliogeneis in the normal thyroid. Hashimoto's thyroiditis is a representative chronic autoimmune thyroid disease characterized by the infiltration of immune cells and the presence of autoantibodies against thyroid autoantigens. We found the follicles in Hashimoto's thyroiditis to have a normal pattern of primary cilia. Patients with Hashimoto's thyroiditis examined in this study were euthyroid. Therefore, it is unclear how TSH regulates the ciliogenesis of thyroid epithelial cells in vivo. Immune cells and their production of multiple proinflammatory cytokines results in functional and structural abnormalities in thyroid epithelial cells. Hürthle cells found in Hashimoto's thyroiditis are thought to arise due to chronic inflammatory stress on epithelial cells. Therefore, markedly defective ciliogenesis in Hürthle cells of Hashimoto's thyroiditis may have been caused by inflammatory stress or defective oxidative metabolism in Hürthle cells.
Differentiated thyroid cancers such as PTC lose follicular structures because of the loss of epithelial cell polarity. However, we found that primary cilia were well preserved in PTC cells. Therefore, follicular structures are not the prerequisite structure for the expression of primary cilia in thyroid cancer. The clinical prognostic outcome of PTC is determined by several histological subclasses [bib_ref] Differential Clinicopathological Risk and Prognosis of Major Papillary Thyroid Cancer Variants, Shi [/bib_ref]. It was reported that the subtypes of the oncocytic, solid, and tall cell variants showed a more unfavorable clinical course than that of the conventional type of PTC [bib_ref] Differential Clinicopathological Risk and Prognosis of Major Papillary Thyroid Cancer Variants, Shi [/bib_ref]. The frequency of ciliated cells was not significantly different between histological subtypes, except for the oncocytic variant. Furthermore, the average length of cilia was also reduced in the oncocytic variant of PTC. These findings indicated that abnormal ciliogenesis was an inherent feature of Hürthle cells in inflammatory and tumorous thyroid diseases. The presence of Hürthle cells in the thyroid was attributed to impaired mitochondrial oxidative function caused by oxidative stress or a mutation in mtDNA [bib_ref] The biology and the genetics of Hurthle cell tumors of the thyroid, Maximo [/bib_ref]. Hürthle cell tumors are defined as being composed of at least 75% Hürthle cells [bib_ref] The WHO histological classification of thyroid tumors: a commentary on the second..., Hedinger [/bib_ref]. Although several investigators propose that they are distinct from other follicular cell neoplasms [bib_ref] The WHO histological classification of thyroid tumors: a commentary on the second..., Hedinger [/bib_ref] , others consider them to be subtypes of follicular adenoma or carcinoma. Our results showed that Hürthle cells found in both primary and secondary thyroid lesions exhibited abnormal ciliogenesis.
It is interesting that abnormal ciliogenesis in Hürthle cell lesions was observed in both benign and malignant thyroid diseases. Because Hürthle cells display defective oxidative phosphorylation and increased basal autophagy that includes mitophagy, abnormal ciliogenesis may be associated with mitophagy or autophagy in Hürthle cells. The tumorigenic Hürthle cell line XTC.UC1 shows higher autophagosome formation; however, actual mitophagy turnover is lower because of dysfunctions in the regulation of PINK1 and Parkin-mediated mitophagy [bib_ref] Dysregulation of Parkin-mediated mitophagy in thyroid Hurthle cell tumors, Lee [/bib_ref]. These molecular features might explain the inefficient clearance of abnormal mitochondria in Hürthle cell tumors. Recent studies show that autophagy and ciliogenesis are intricately linked. Tang et al. demonstrate that autophagy promotes ciliogenesis by degrading OFD1 at centriolar satellites [bib_ref] Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites, Tang [/bib_ref]. By contrast, Pampliega et al. reveal that autophagy negatively regulates ciliogenesis by degrading the essential ciliary protein IFT20 [bib_ref] Functional interaction between autophagy and ciliogenesis, Pampliega [/bib_ref]. In addition, Cuervo et al. reported that autophagy dysfunction can be attributed to impaired ciliary signaling [bib_ref] Autophagy and regulation of cilia function and assembly, Orhon [/bib_ref]. The relationship between autophagy and primary cilia is bidirectionally regulated and specific to different cell types, environments, growth conditions, and stimuli. These observations indicate that the relationship between ciliogenesis and autophagy needs to be interpreted in the context of the specific disease. In this study, Hürthle cell tumors and XTC.UC1 cells showed high basal levels of autophagosome formation, and exhibited defects in primary ciliogenesis in vitro and in vivo. Upon formation, the autophagosome undergoes a stepwise maturation process that includes fusion events with the lysosome. Bafilomycin A1, a vacuolar H(+)-ATPase inhibitor, inhibits the fusion of the autophagosome with the lysosome. The ciliogenesis phenotype of XTC.UC1 cells was not affected by bafilomycin A1. Collectively, the data suggest that autophagosome formation, not lysosome fusion, is the critical step leading to defective ciliogenesis in XTC.UC1 cells. The findings that Atg5 siRNA and 3-MA increased the levels of IFT88 and ARL13B in XTC. UC1 cells indicates sequestration of structural proteins for ciliogenesis during enhanced autophagosome formation [fig_ref] Figure 7: Schematic model for the regulation of ciliogenesis in XTC [/fig_ref]. It is interesting that improved ciliogenesis was not observed in XTC.UC1 cells treated with BAF, which inhibits the fusion of the autophagosome, suggesting that the impairment of ciliogenesis in Hürthle cell tumors may affect an early stage of autophagy in the thyroid gland.
The population study based on the Surveillance, Epidemiology, and End Results (SEER) database has confirmed that Hürthle cell carcinoma is more aggressive and that patients with Hürthle cell carcinoma have a lower survival rate than those with other differentiated thyroid cancer subtypes [bib_ref] Hurthle cell carcinoma: a population-level analysis of 3311 patients, Goffredo [/bib_ref]. Improved survival associated with small tumors confined to the thyroid without local and distant metastasis and with those treated with radioiodine therapy [bib_ref] Hurthle cell carcinoma: a population-level analysis of 3311 patients, Goffredo [/bib_ref]. Ciliogenesis and autophagy are the determining factors in the prognosis of human cancers [bib_ref] Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites, Tang [/bib_ref]. Abnormal ciliogenesis and increased autophagy correlate with poor prognosis in these specific forms of cancer [bib_ref] Cilia in autophagy and cancer, Cao [/bib_ref]. Therefore, the impairment of ciliogenesis in Hürthle cell tumors may explain the relatively poor prognosis of differentiated thyroid cancers. In this study, we demonstrated that defects in the expression of genes involved in ciliogenesis is a hallmark of Hürthle cell tumors in the thyroid gland.
# Materials and methods
## Human thyroid specimens
Patients that underwent a thyroidectomy between January 2002 and December 2005 at St. Mary's Hospital, Daejeon, South Korea, were retrospectively enrolled. The baseline characteristics of each patient are summarized in [fig_ref] Table 1: Summary of patients characteristics and clinical data [/fig_ref]. All patients presented with nodular hyperplasia of the thyroid, Hashimoto's thyroiditis, follicular adenoma and carcinoma, Hürthle cell adenoma and carcinoma, or PTC (conventional, follicular variant, oncocytic variant, and solid variant, tall cell variant). Contralateral normal tissues were obtained and used as controls. Two pathologists independently reviewed H&Estained tissue cross-sections, and a diagnosis was made according to the World Health Organization classification of endocrine organ tumors [bib_ref] Poorly differentiated thyroid carcinoma: 5 years after the 2004 WHO classification of..., Volante [/bib_ref]. The study protocol was reviewed and approved by the Institutional Review Board and the methods were carried out in accordance with the approved guidelines of the Daejeon St. Mary's Hospital, College of Medicine, the Catholic University of Korea. All participants provided signed, written informed consent.
## Cell lines, culture conditions, and chemicals
The Hürthle cell carcinoma cell line XTC.UC1 and TPC-1 cells were cultured in Dulbecco's Modified Eagle medium (Gibco ® ) supplemented with 5% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO 2 [bib_ref] Defective oxidative phosphorylation in thyroid oncocytic carcinoma is associated with pathogenic mitochondrial..., Bonora [/bib_ref]. The untransformed human thyroid cell line Nthy-ori 3-1 was provided by the European Collection of Authenticated Cell Cultures and maintained in RPMI 1640 medium (Gibco ® ) supplemented with 5% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO 2 .
## Immunofluorescence staining
Paraffin-embedded 7 μm-thick tissue cross-sections were placed in an oven and incubated at 56°C for 3 hr. Thereafter, cross-sections were deparaffinized in xylene and rehydrated through a graded-series of ethanol baths. Antigens were retrieved in antigen retrieval buffer (0.01 M citric acid-sodium citrate, pH 6.0) by heating the cross-sections in an autoclave at 121°C for 25 min. After washing, the cross-sections were air-dried for 30 min and then re-washed with 1 × phosphate-buffered saline (PBS, 10 mM Na 2 HPO 4 , pH 7.4 and 150 mM NaCl). Cells were cultured on round coverslips in 12-well plates for 48 hr after seeding. After incubation under the indicated conditions, the cells were washed with 1 × PBS. The tissue cross-sections and cultured cells were fixed with 4% paraformaldehyde in PBS for 15 min, and then permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. Permeabilized cells were blocked with 5% bovine serum albumin in PBS for 30 min at room temperature. Thereafter, tissue cross-sections and cultured cells were incubated with primary antibodies for 24 hr at 4°C. On the following day, the slides and coverslips were washed three times with 1 × PBS and incubated at 4°C for 12 hr with secondary antibodies. Primary antibodies against LC3 (Sigma-Aldrich), acetylated α-tubulin (Sigma-Aldrich), Arl13B (ProteinTech Group), and γ-tubulin (Sigma-Aldrich) were used. Goat anti-mouse and goat anti-rabbit secondary antibodies conjugated to Alexa Fluor dyes (Invitrogen/Life Technologies) were used for indirect fluorescent detection. The stained slides were observed under an Olympus FluoView FV1000 microscope equipped with a charge-coupled device camera (Olympus Corp.).
## Analysis of cilia frequency in cell lines and thyroid tissue
The frequency of ciliated cells in cultures and thyroid tissue was determined by counting acetylated α-tubulin-or Arl13B-positive cilia in 1000 cell nuclei. We determined the frequency of ciliated cells in human thyroid tissues by the following method. We prepared five paraffin blocks with control and diseased tissues, and prepared two slides from each paraffin block. Cross-sections were immunostained with the indicated antibodies and 1000 follicles were inspected. Primary cilia were manually counted. Primary cilia length was measured using the length measurement tool within the software package (Olympus Corp.).
## Detection of primary cilia after inhibition of autophagosome formation
To silence the function of ATG5, XTC.UC1 cells were transfected with Atg5 siRNA (100 nM) using Lipofectamine ® RNAiMAX (Invitrogenm Carlsbad, CA, USA) according to the manufacturer's protocol. The medium was replaced after 6 hr and cells were incubated for a further 48 hr.
Knockdown of ATG5 was confirmed for each experiment by performing western blot analysis with anti-ATG5 antibody (Cell signaling). XTC.UC1 cells were cultured on coverslips in 12-well plates for 48 hr after seeding. Thereafter, cells were treated with 5 mM and 10 mM 3-methyladenine (3-MA; Sigma-Aldrich) or 10 and 20 nM bafilomycin A1 (BAF; Sigma-Aldrich) for 0, 12, 24, and 36 hr. After immunofluorescent staining, the stained slides were observed under a confocal microscope (Olympus Corp.).
# Western blot analysis
Cells were washed twice with PBS and lysed in RIPA lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1% Nonidet P-40) supplemented with a broadspectrum protease inhibitor cocktail (Roche). Protein concentrations were measured using the Bradford assay. Proteins were denatured by boiling for 5 min. Samples were resolved by 10% SDS-PAGE and transferred to Hybond ECL membranes (Amersham Pharmacia Biotech). The membranes were blocked for 30 min in Tris-buffered saline containing 0.1% Tween 20 (TBS/T) and 5% non-fat milk, and then incubated overnight at 4°C with primary antibodies against LC3 (Sigma-Aldrich), IFT88 (ProteinTech Group), Arl13B (ProteinTech Group), Polyglutamylation Modification (GT335, AdipoGen), and GAPDH (Abcam). The membranes were washed three times with TBS/T and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Phototope-HRP Western blot detection Kit; New England Biolabs) for 2 hr at room temperature. After three washes for 10 min each, the blots were developed using the LumiGLO chemiluminescent substrate (Cell Signaling Technology).
[fig] Figure 1: Distribution of primary cilia in thyroid tissue with normal and nodular hyperplasia. (A) Normal thyroid stained with H&E. F, follicles; P, parafollicular cells. Scale bar, 30 µm. (B) Immunofluorescent staining of primary cilia in follicular and parafollicular cells of the normal thyroid using anti-acetylated α-tubulin (green) and anti-γ-tubulin (red). F, follicles; P, parafollicular cells. Scale bar, 5 µm. (C) The average frequency of primary cilia in the follicles of five cases with normal thyroid glands. (D) Nodular hyperplasia of thyroid stained with H&E. Scale bar, 20 µm. Immunofluorescent staining of primary cilia in the nodular hyperplasia using anti-acetylated α-tubulin (green) and anti-γ-tubulin (red). Scale bar, 5 µm. (E) The average frequency of primary cilia in follicles of five patients with nodular hyperplasia. Normal follicular cells were used as controls. N.S.; not significant. [/fig]
[fig] Figure 2: Distribution of primary cilia in Hashimoto's thyroiditis. (A) Small and atrophic follicles with infiltrating lymphoplasma cells stained with H&E. Scale bar, 30 µm. Arrows indicate follicles. Immunofluorescent staining of primary cilia using anti-acetylated α-tubulin (green). Scale bar, 10 µm. (B) Follicles damaged by infiltrating lymphoplasma cells and fibrosis stained with H&E. Scale bar, 30 µm. Immunofluorescent staining of primary cilia using anti-acetylated α-tubulin (green). Scale bar, 10 µm. (C) Hürthle cells with abundant eosinophilic cytoplasm stained with H&E. Scale bar, 30 µm. The red dotted line indicates Hürthle cells. Immunofluorescent staining of primary cilia using anti-acetylated α-tubulin (green). Scale bar, 10 µm. (D) Immunostaining of rare primary cilia in Hürthle cells using antiacetylated α-tubulin (green). Arrows indicate non-Hürthle cells with primary cilia. The square box indicates Hürthle cells. Scale bar, 10 µm. (E) Hürthle cells showed intense expression of mitochondrial proteins, TOM40 (green). Immunofluorescent staining of primary cilia using anti-GT335 (red). Scale bar, 10 µm. (F) The average frequency of primary cilia in non-Hürthle and Hürthle cells in the thyroid glands of patients with Hashimoto's thyroiditis. Normal follicular cells were used as controls. **p < 0.01, N.S.; not significant. www.impactjournals.com/oncotarget [/fig]
[fig] Figure 3: Distribution of primary cilia in papillary thyroid carcinoma. (A) Conventional PTC (PTC-conv) composed of complex papillae with thin fibrovascular cores stained with H&E. Scale bar, 30 µm. Immunofluorescent staining of primary cilia using antiacetylated α-tubulin (green). Scale bar, 10 µm. (B) Follicular variant of PTC (PTC-FV) showing follicular morphology with PTC nuclear features stained with H&E. Scale bar, 30 µm. Immunofluorescent staining of primary cilia using anti-acetylated α-tubulin (green). Scale bar, 10 µm. (C) Solid variant of PTC (PTC-SV) composed of round solid nests stained with H&E. Scale bar, 30 µm. Immunofluorescent staining of primary cilia using anti-acetylated α-tubulin (red). Scale bar, 10 µm. (D) Oncocytic variant of PTC (PTC-OV) composed of Hürthle cells with PTC nuclear features stained with H&E. Scale bar, 30 µm. Immunofluorescent staining of primary cilia using anti-acetylated α-tubulin (green). Scale bar, 10 µm. (E) The average frequency of primary cilia in different PTC variants compared with that of the normal thyroid. (F) The average length of primary cilia in different PTC variants compared with that of the normal thyroid. PTC-conv, conventional; PTC-FV, follicular variant; PTC-SV, solid variant; PTC-TV, tall cell variant; PTC-OV, oncocytic variant of papillary carcinoma. Normal follicular cells were used as controls. *p < 0.05, **p < 0.01, N.S.; not significant. www.impactjournals.com/oncotarget (TC), and oncocytic variant (OV) ( [/fig]
[fig] Figure 4: Distribution of primary cilia in follicular tumors and Hürthle cell tumors. (A) Follicular adenoma stained with [/fig]
[fig] Figure 5: Primary cilia in human normal thyroid follicular cell lines and thyroid cancer cell lines. (A, C, E) [/fig]
[fig] Figure 6: Relationship between primary ciliogenesis and autophagy in XTC.UC1 cells. (A) Western blot analyses of LC3 levels in XTC.UC1 and Nthy-ori 3-1 cells after serum deprivation and BAF treatment. β-actin served as the loading control. (B) Western blot analyses of LC3, IFT88, ARL13B, and GT335 levels in XTC.UC1 after treatment with 10 mM 3-MA for 6, 12, or 24 hr. GAPDH served as the loading control. (C) Immunofluorescent staining of primary cilia (arrows) using anti-acetylated α-tubulin (green) and anti-γtubulin (red). Scale bar, 10 µm. (D) Immunofluorescent staining of primary cilia (arrows) using anti-acetylated α-tubulin (red) and anti-γtubulin (green). Scale bar, 10 µm. (E) The average frequency of primary cilia after the induction of autophagy by serum deprivation or its inhibition by 3-MA or BAF treatment. (F) The average length of primary cilia after the induction of autophagy by serum starvation or its inhibition by 3-MA or BAF treatment. *p < 0.05, **p < 0.01, N.S.; not significant. (G) Western blot analyses of ATG5, LC3, IFT88, GT335 and ARL13B expression in XTC.UC1 after transfection with Atg5 siRNA. (H) Immunofluorescent staining of primary cilia of XTC.UC1 after transfection with Atg5 siRNA using anti-Arl13B (green). Scale bar, 10 µm. (I) The average frequency and length of primary cilia after the inhibition of autophagy by Atg5 siRNA. **p < 0.01. [/fig]
[fig] Figure 7: Schematic model for the regulation of ciliogenesis in XTC.UC1. Cells of the Hürthle cell carcinoma cell line, XTC.UC1, show high basal levels of autophagosome formation. 3-MA inhibits the cleavage of LC3-I and increases the levels of IFT88 and ARL13B by sequestrating structural proteins for ciliogenesis. Inhibition of autophagosome formation restores ciliogenesis via the accumulation of IFT88 and ARL13. Improved ciliogenesis is not observed in cells treated with BAF, an inhibitor of autophagosome fusion. [/fig]
[table] Table 1: Summary of patients characteristics and clinical data [/table]
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10.3390/ijms21144837
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CCBY
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7402304
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32650589
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s2orc_pubmed_articles
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Bone Regeneration Capability of 3D Printed Ceramic Scaffolds
In this study, we evaluated the bone regenerative capability of a customizable hydroxyapatite (HA) and tricalcium phosphate (TCP) scaffold using a digital light processing (DLP)-type 3D printing system. Twelve healthy adult male beagle dogs were the study subjects. A total of 48 defects were created, with two defects on each side of the mandible in all the dogs. The defect sites in the negative control group (sixteen defects) were left untreated (the NS group), whereas those in the positive control group (sixteen defects) were filled with a particle-type substitute (the PS group). The defect sites in the experimental groups (sixteen defects) were filled with a 3D printed substitute (the 3DS group). Six dogs each were exterminated after healing periods of 4 and 8 weeks. Radiological and histomorphometrical evaluations were then performed. None of the groups showed any specific problems. In radiological evaluation, there was a significant difference in the amount of new bone formation after 4 weeks (p < 0.05) between the PS and 3DS groups. For both of the evaluations, the difference in the total amount of bone after 8 weeks was statistically significant (p < 0.05). There was no statistically significant difference in new bone between the PS and 3DS groups in both evaluations after 8 weeks (p > 0.05). The proposed HA/TCP scaffold without polymers, obtained using the DLP-type 3D printing system, can be applied for bone regeneration. The 3D printing of a HA/TCP scaffold without polymers can be used for fabricating customized bone grafting substitutes.Int. J. Mol. Sci. 2020, 21, 4837 2 of 13 and osteoinductivity. However, some studies have reported disadvantages, such as donor site morbidity and limited availability[3,4]. Allografts and xenografts also have infection risks[5]. Among the alloplastic grafts, calcium phosphate bioceramic is mainly used because its composition is similar to that of bone mineral[6]. Biphasic calcium phosphate (BCP) ceramic is made up of hydroxyapatite (HA) and tricalcium phosphate (TCP) in different proportions[7]. HA can be fabricated into solid scaffolds with porosities for cell attachment, migration, and bone formation[8]. HA has attracted attention in the fields of bone tissue engineering, implant coatings, and the hybridization of materials[9]. The resorption rate of HA is slower than the rate of bone regeneration because of chemical stability[10]. To improve the resorption rate of HA, TCP, which has a fast resorption rate, has been introduced and suggested for use in combination with HA [10]. The composition and degradation of ceramic scaffolds are easily controlled, making them attractive scaffolds for use in bone regeneration[11].Past studies showed how HA/TCP macropores were interconnected and induced growth into soft tissues and blood vessels. Fu et al. reported a ceramic scaffold with a proper compressive strength (166 MPa) comparable to that of cortical bone[12]. The scaffold had a large average pore size (1.2 mm) and high interconnectivity (100%) between the pores, with 60% porosity. Habibovic et al. showed a compressive strength value of 6.3 MPa for HA scaffolds, with 80% porosity and an average pore size of 450 µm; however, the interconnectivity between the pores was drastically compromised (~50%)[11,13]. Porosity allowed bone-forming proteins (BMP) to adhere on the surface of the material and induce bone formation[14][15][16].This study compared the bone forming ability of 3D printed scaffolds versus particle-type bone, both of which are made up of this HA/TCP. Generally, particle-type or block bone substitutes are used for GBR. Block bone substitutes can provide better mechanical support than particle-type bone substitutes. Therefore, GBR sites with block bone substitutes show less collapse in the grafted area and better maintenance of their volume and contours than GBR sites with particle-type bone substitutes[17]. However, block bone substitutes usually need to be fixed and require additional manipulation, such as trimming to fit the defect area. When grafted block-bone substitutes are exposed, they are more likely than the particle-type bone substitutes to cause problems such as infection. To overcome the disadvantages of the two types of bone substitutes, demand for the manufacturing of customizable block bones has increased.Personalized medicine is challenging for adapting the bone substitute to the patient's specific bone geometry[18]. Customized block bones are made using computer-aided design and computer-aided manufacturing (CAD/CAM) or 3D printing[19]. The advantages of 3D printing include reduced material waste, optimizable surface and porous structures, and a reduced operation time[20]. For this reason, there is great demand for 3D printing technology, and many studies on 3D printing technology have been published[19,21,22]. 3D printing technology for block grafts is currently applicable to alloplastic materials. In general, BCP is widely used in 3D printing with synthetic polymers. Synthetic polymers are moldable to the required shapes and have excellent mechanical properties[23]. As the synthetic polymers degrade, they release acidic by-products that often lead to tissue necrosis and subsequent scaffold failure[24]. Therefore, it is desirable to remove these polymers before applying the grafts to the defect area. Using 3D printing technology, the block bone substitutes could be produced with the desired shape and structure. The 3D printed block bone substitutes would not need to be trimmed to fit the defects, which could shorten the operation time and result in good suitability for the defect area.In this study, the HA/TCP scaffold was manufactured without polymers using 3D printing. To our knowledge, animal experiments for evaluating the bone regenerative ability of 3D printed HA/TCP scaffolds without polymer are lacking. Our hypothesis is that 3D printed HA/TCP scaffolds without polymer will have the required mechanical strength to endure the pressure from the surrounding structure and sufficient biocompatibility for enhancing bone regeneration. This study was conducted to evaluate the bone regeneration of the customizable HA/TCP scaffold without polymers using digital light processing (DLP)-type 3D printing. Int. J. Mol. Sci. 2020, 21, 4837 10 of 13 A 3D printed HA/TCP scaffold without polymers had sufficient bone-forming ability and provided superior stability for the defect area. The 3D printed HA/TCP scaffold is easy to use for large or complex bone defects. The 3D printing of a HA/TCP scaffold without polymers could be used for fabricating customized bone-grafting substitutes.
# Introduction
Guided bone regeneration (GBR) is used to increase the volume of the alveolar ridge or preserve the extraction socket. Currently, the bone graft materials used in GBR are classified as autogenous, allogenic, xenogeneic, and alloplastic bone [bib_ref] A comparison of demineralized freeze-dried bone and autologous bone to induce bone..., Becker [/bib_ref] [bib_ref] Xenograft versus extraction alone for ridge preservation after tooth removal: A clinical..., Barone [/bib_ref]. Autogenous bone is regarded as the ideal material for restoring bone defects. It has advantages for new bone formation, such as osteogenicity, osteoconductivity,
# Materials and methods
Twelve healthy adult male beagle dogs that were approximately 22-26 weeks old and 10-12 kg in weight were enrolled for this study. The study protocol was approved by the Animal Ethical Committee for Animal Research at Genoss ® (Suwon, Korea), (GEN-IACUC-1908-01). Before the experiment, the dogs had free access to food and water and were given one week to become accustomed to their new environment prior to the operation.
## Manufacture of 3d printed ha/tcp scaffold
The 3D printed scaffold substitute was a synthetic bone graft material produced by a DLP-type 3D printer (Cubicon Lux, Cubicon ® , Sungnam, Korea). The 3D printer had a resolution of 100 µm and could print with a thickness of 20-100 µm [fig_ref] Figure 1: Structure of 3D printed hydroxyapatite [/fig_ref]. After designing the bone graft material for the defect size (7 × 3 × 5 mm), the design data were converted into a stereolithography (STL) file. The file was printed with the 3D printer using a photoreactive ceramic-resin composite. The slurry was made up of HA/TCP (6:4 ratio), a dispersant, acrylic monomers, and a photo initiator. The resin component was removed completely through heat treatment after 3D printing [fig_ref] Figure 2: Digital light processing [/fig_ref]. The detailed method of the manufacture of 3D printed HA/TCP scaffolds without polymer is a trade secret of Genoss ® , so the procedure and technique cannot be described in this article.
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 13 without polymer will have the required mechanical strength to endure the pressure from the surrounding structure and sufficient biocompatibility for enhancing bone regeneration. This study was conducted to evaluate the bone regeneration of the customizable HA/TCP scaffold without polymers using digital light processing (DLP)-type 3D printing.
# Materials and methods
Twelve healthy adult male beagle dogs that were approximately 22-26 weeks old and 10-12 kg in weight were enrolled for this study. The study protocol was approved by the Animal Ethical Committee for Animal Research at Genoss ® (Suwon, Korea), (GEN-IACUC-1908-01). Before the experiment, the dogs had free access to food and water and were given one week to become accustomed to their new environment prior to the operation.
## Manufacture of 3d printed ha/tcp scaffold
The 3D printed scaffold substitute was a synthetic bone graft material produced by a DLP-type 3D printer (Cubicon Lux, Cubicon ® , Sungnam, Korea). The 3D printer had a resolution of 100 μm and could print with a thickness of 20-100 μm [fig_ref] Figure 1: Structure of 3D printed hydroxyapatite [/fig_ref]. After designing the bone graft material for the defect size (7 × 3 × 5 mm), the design data were converted into a stereolithography (STL) file. The file was printed with the 3D printer using a photoreactive ceramic-resin composite. The slurry was made up of HA/TCP (6:4 ratio), a dispersant, acrylic monomers, and a photo initiator. The resin component was removed completely through heat treatment after 3D printing [fig_ref] Figure 2: Digital light processing [/fig_ref]. The detailed method of the manufacture of 3D printed HA/TCP scaffolds without polymer is a trade secret of Genoss ® , so the procedure and technique cannot be described in this article.
## Compression test of 3d printed ha/tcp scaffold block
The compressive strength of the specimens was evaluated (ASTM D695). After specimen placement, the rod of the universal testing machine (H50K-T UTM ® , Tinius Olsen corp., Surrey, UK) was lowered at a rate of 0.5 ± 0.1 mm/min, and the load applied to the sample was measured. The extracted load data were calculated per the distance divided by the cross-sectional area of the specimen. The values were plotted as distance (μm, linear scale) versus stress (N/cm 2 , log scale). The compression stress values were derived from the intersection of rapid and gradual stress increases. without polymer will have the required mechanical strength to endure the pressure from the surrounding structure and sufficient biocompatibility for enhancing bone regeneration. This study was conducted to evaluate the bone regeneration of the customizable HA/TCP scaffold without polymers using digital light processing (DLP)-type 3D printing.
# Materials and methods
Twelve healthy adult male beagle dogs that were approximately 22-26 weeks old and 10-12 kg in weight were enrolled for this study. The study protocol was approved by the Animal Ethical Committee for Animal Research at Genoss ® (Suwon, Korea), (GEN-IACUC-1908-01). Before the experiment, the dogs had free access to food and water and were given one week to become accustomed to their new environment prior to the operation.
## Manufacture of 3d printed ha/tcp scaffold
The 3D printed scaffold substitute was a synthetic bone graft material produced by a DLP-type 3D printer (Cubicon Lux, Cubicon ® , Sungnam, Korea). The 3D printer had a resolution of 100 μm and could print with a thickness of 20-100 μm [fig_ref] Figure 1: Structure of 3D printed hydroxyapatite [/fig_ref]. After designing the bone graft material for the defect size (7 × 3 × 5 mm), the design data were converted into a stereolithography (STL) file. The file was printed with the 3D printer using a photoreactive ceramic-resin composite. The slurry was made up of HA/TCP (6:4 ratio), a dispersant, acrylic monomers, and a photo initiator. The resin component was removed completely through heat treatment after 3D printing [fig_ref] Figure 2: Digital light processing [/fig_ref]. The detailed method of the manufacture of 3D printed HA/TCP scaffolds without polymer is a trade secret of Genoss ® , so the procedure and technique cannot be described in this article.
## Compression test of 3d printed ha/tcp scaffold block
The compressive strength of the specimens was evaluated (ASTM D695). After specimen placement, the rod of the universal testing machine (H50K-T UTM ® , Tinius Olsen corp., Surrey, UK) was lowered at a rate of 0.5 ± 0.1 mm/min, and the load applied to the sample was measured. The extracted load data were calculated per the distance divided by the cross-sectional area of the specimen. The values were plotted as distance (μm, linear scale) versus stress (N/cm 2 , log scale). The compression stress values were derived from the intersection of rapid and gradual stress increases.
## Compression test of 3d printed ha/tcp scaffold block
The compressive strength of the specimens was evaluated (ASTM D695). After specimen placement, the rod of the universal testing machine (H50K-T UTM ® , Tinius Olsen corp., Surrey, UK) was lowered at a rate of 0.5 ± 0.1 mm/min, and the load applied to the sample was measured. The extracted load data were calculated per the distance divided by the cross-sectional area of the specimen. The values were plotted as distance (µm, linear scale) versus stress (N/cm 2 , log scale). The compression stress values were derived from the intersection of rapid and gradual stress increases.
## Cytotoxic test of 3d printed ha/tcp scaffold block
The in vitro cytotoxicity test standard (ISO 10993-12) was used to determine the cytotoxicity of the specimen (three specimens each). The 4g 3D printed HA/TCP specimen was eluted for 24 h at 37 - C in 20 mL of elution solvent. Meanwhile, 500 mL of minimal essential medium was prepared by mixing 4 of 13 50 mL of fetal bovine serum (Gibco ® , Thermo Fisher Scientific, Green Island, NY, USA) and 10 mL of penicillin-streptomycin solution (Welgene ® , Gyeongsan-si, Korea). High density polyethylene and natural rubber were used as negative and positive control materials, respectively. The control solutions were eluted under the same conditions. Mouse fibroblasts (ATCC CCL 1, clone 929 of strain L, Korean Research Institute of Bioscience and Biotechnology, Daejeon, Korea) were injected into a flask containing the antibiotics and fetal bovine serum, and the flask was maintained in an incubator at 37 - C. The culture solution in the flask was changed three times a week.
One day after injecting the cell culture medium into a 6-well plate, the cells had attained 80% confluence. After the complete removal of the cell culture medium, 2 mL each of the elution solvent of the negative control, positive control, and experimental specimen was applied to and cultured in the 6-well plate. After culturing for 24 h and 48 h, cell growth and lysis levels were measured using a microscope. The condition of the cells was evaluated by the grade of the cells. When the grade of the negative control eluate was zero and that of the positive control eluate was higher than 4, the test was considered reliable. If the grade of the experimental specimen was lower than 2, no cytotoxicity occurred [fig_ref] Table 1: Reactivity grades for elution test [/fig_ref]. Severe Nearly completely destruction of the cell layers.
## Surgical procedure
General anesthesia was performed intravenously with 0.1-0.14 mL/kg of xylazine and 0.01 mg/kg of tiletamine-zolazepam. Orotracheal intubation was performed using size 6 tubes without ballooning for securing the airway. The tube was fixed to the anterior part of the mandible using paper tape, and the fixed part was marked so that the position of the tube remained constant. Upon intubation, anesthesia was maintained using isoflurane (Piramal Critical Care, Inc., Bethlehem, PA, USA) and oxygen by inhalation. After the operation, each animal received analgesic medication (meloxicam, 0.2 mg/kg) and antibiotic medication (enrofloxacin, 0.2 mL/kg) through intramuscular injection for seven days. Each animal was fed a liquid diet for one week after the operation.
## First surgery for tooth extraction
After the crestal incision, the mucoperiosteal flaps were elevated bilaterally from the mandibular first premolar (P1) to the first molar (M1), and the teeth from P1 to M1 were carefully extracted on both sides. X-rays were taken to identify the existence of retained roots in the extraction sockets. Finally, the flap was repositioned and sutured with nylon.
## Second surgery for preparation of defect (gbr)
After 10 weeks, incision and flap elevation were done similarly to prepare the defects for GBR. The bone defects were made using a prepared defect guide (7 × 3 × 5 mm 3 ) and high-speed dental burs. A total of 48 defects were created, with two defects on each side of the mandible in all the dogs. The defect sites for the positive control groups (PS group, sixteen defects) were filled with a particle-type substitute (OSTEON 3, Genoss ® , Suwon, Korea), whereas defect sites for the experimental groups (3D printed substitute (3DS) group, sixteen defects) were filled with a 3D printed substitute grafted bone. The defect sites for the negative control group (NS group, sixteen defects) were left untreated for spontaneous healing. The grafted bone and defect sites were covered with a collagen membrane (Collagen Membrane, Genoss ® , Suwon, Korea). Fixation pins (Membrane Pin, Dentium ® , Suwon, Korea) were used for stabilizing the grafted bones and membranes. Finally, the flap was repositioned and sutured using 4-0 Vicryl (Vicryl ® , Ethicon, Somerville, NJ, USA). The sutures were removed two weeks after the second surgery.
The dogs were sacrificed after healing periods of four and eight weeks. Six dogs were sacrificed after each healing period. The mandibles were dissected, and the experimental specimens were harvested. All the specimens obtained were fixed in a 10% neutral buffered formalin solution.
## Radiological examination
X-ray microcomputed tomography was conducted using a Skyscan 1173 (Bruker-micro CT, Belgium) four and eight weeks after sacrifice. After processing in a CT Analyzer 1.13 (Bruker-micro CT, Belgium), the X-ray transmittance intensity of the remaining grafted materials and new bone was analyzed. The percent bone volume was calculated using the following formula:
Percent bone volume (%) = bone volume/tissue volume × 100.
## Histomorphometric examination
The fixation of the specimens was performed in neutral buffered formalin at a temperature of 4 - C for one week. A tissue slide was made by making a longitudinal section through the bone tissue.
A total of 48 slides were stained with H&E/Goldner's trichrome staining.
The slide scanning was performed with a digital slide scanner (Pannoramic 250 Flash III, 3DHISTECH Ltd. Budapest, Hungary). The digital images were evaluated and quantified using histomorphometrical measurements. The examination was performed with a 200× magnified microscope and CaseViewer (3DHISTECH Ltd., Budapest, Hungary). The digital image was evaluated using Image-Pro Plus (Jenoptik, Seoul, Korea), and the data were summarized in Excel. The percentage of new bone was calculated using the following formula:
New bone (%) = [Area of new bone/Total area of defect] × 100.
# Statistical analysis
The statistical analysis was conducted using the Statistical Package for the Social Sciences (Version 12.0K; SPSS, Inc., Chicago, IL, USA). The differences between three groups were evaluated with the Kruskal-Wallis-test. Comparisons between groups were performed with the Mann-Whitney U test, and results with p-values < 0.05 were considered as statistically significant.
# Results
## Compression test of 3d-printed ha/tcp scaffold block
A total of 16 specimens were evaluated. The compressive strength of the HA/TCP scaffold block was 2.80 ± 0.8246 MPa.
## Cytotoxic test of 3d printed ha/tcp scaffold block
At 24 h and 48 h, the grades of cells using the negative and positive control eluate were zero and four, respectively. The eluate of the control showed no change. The grades of the cells in the HA/TCP specimens were zero at both 24 and 48 h, thereby confirming no cytotoxicity [fig_ref] Table 2: Results of cytotoxicity tests [/fig_ref].
## Clinical evaluation
No dog in any group was lost during the experimental period. None of the groups showed specific problems such as inflammation, wound dehiscence, swelling, or infection. The collagen membrane overlying the bone graft material was not absorbed completely at 4 or 8 weeks.
## Radiological evaluation
At 4 weeks, the percentage formation of new bone was 45.31 ± 3.74% in the PS group, and 27.44 ± 5.86% in the 3DS group. The remaining bone in the grafted material was 31.02 ± 1.35% in the 3DS group [fig_ref] Figure 3: Radiological evaluation at 4 and 8 weeks [/fig_ref]. [fig_ref] Table 1: Reactivity grades for elution test [/fig_ref] shows that the new bone formation in the 3DS group was 41.08 ± 3.96% at 8 weeks. At 4 and 8 weeks, the total amounts of remaining and new bone in the 3DS group after GBR were 58.47 ± 6.36% and 67.72 ± 11.25%, respectively [fig_ref] Figure 3: Radiological evaluation at 4 and 8 weeks [/fig_ref].
## Cytotoxicity grades
## Clinical evaluation
No dog in any group was lost during the experimental period. None of the groups showed specific problems such as inflammation, wound dehiscence, swelling, or infection. The collagen membrane overlying the bone graft material was not absorbed completely at 4 or 8 weeks.
## Radiological evaluation
At 4 weeks, the percentage formation of new bone was 45.31 ± 3.74% in the PS group, and 27.44 ± 5.86% in the 3DS group. The remaining bone in the grafted material was 31.02 ± 1.35% in the 3DS group [fig_ref] Figure 3: Radiological evaluation at 4 and 8 weeks [/fig_ref]. [fig_ref] Table 1: Reactivity grades for elution test [/fig_ref] shows that the new bone formation in the 3DS group was 41.08 ± 3.96% at 8 weeks. At 4 and 8 weeks, the total amounts of remaining and new bone in the 3DS group after GBR were 58.47 ± 6.36% and 67.72 ± 11.25%, respectively [fig_ref] Figure 3: Radiological evaluation at 4 and 8 weeks [/fig_ref]. The NS and PS groups had a significant difference in the amount of new bone formation at 4 weeks. A statistically significant difference between the PS and 3DS groups at 4 weeks (p < 0.05) was observed [fig_ref] Figure 3: Radiological evaluation at 4 and 8 weeks [/fig_ref]. The PS group showed a larger amount of new bone formation at 4 weeks than that of the NS and 3DS groups. Moreover, the total amount of bone showed a statistically significant difference between the NS and PS groups and between the NS and 3DS groups at 4 weeks (p < 0.05) [fig_ref] Figure 3: Radiological evaluation at 4 and 8 weeks [/fig_ref]. The 3DS and PS groups showed higher values of the total amount of bone than the NS group at 4 weeks. However, no statistically significant difference was shown between the PS and 3DS groups in the total amounts of bone at 4 weeks (p > 0.05).
There were no significant differences in the amounts of new bone at 8 weeks among all the groups. At this time, the total amount of bone showed a statistically significant difference between the NS and PS groups and between the NS and 3DS groups (p < 0.05) [fig_ref] Figure 3: Radiological evaluation at 4 and 8 weeks [/fig_ref]. A statistically significant difference in the total amount of bone was observed between the PS and 3DS groups at 8 weeks as well (p < 0.05). The three groups from the greatest to least relative total amounts of bone were 3DS, PS, and NS.
## Histomorphometric evaluation
There were no specific inflammatory reactions associated with foreign body reactions, including necrosis and granuloma, in any of the groups. There were no other specific reactions in any group. The NS and PS groups had a significant difference in the amount of new bone formation at 4 weeks. A statistically significant difference between the PS and 3DS groups at 4 weeks (p < 0.05) was observed [fig_ref] Figure 3: Radiological evaluation at 4 and 8 weeks [/fig_ref]. The PS group showed a larger amount of new bone formation at 4 weeks than that of the NS and 3DS groups. Moreover, the total amount of bone showed a statistically significant difference between the NS and PS groups and between the NS and 3DS groups at 4 weeks (p < 0.05) [fig_ref] Figure 3: Radiological evaluation at 4 and 8 weeks [/fig_ref]. The 3DS and PS groups showed higher values of the total amount of bone than the NS group at 4 weeks. However, no statistically significant difference was shown between the PS and 3DS groups in the total amounts of bone at 4 weeks (p > 0.05).
There were no significant differences in the amounts of new bone at 8 weeks among all the groups. At this time, the total amount of bone showed a statistically significant difference between the NS and PS groups and between the NS and 3DS groups (p < 0.05) [fig_ref] Figure 3: Radiological evaluation at 4 and 8 weeks [/fig_ref]. A statistically significant difference in the total amount of bone was observed between the PS and 3DS groups at 8 weeks as well (p < 0.05). The three groups from the greatest to least relative total amounts of bone were 3DS, PS, and NS.
## Histomorphometric evaluation
There were no specific inflammatory reactions associated with foreign body reactions, including necrosis and granuloma, in any of the groups. There were no other specific reactions in any group.
In the PS and 3DS groups, granulation tissue was observed in the defect area at 4 weeks, but little or no granulation tissue was observed at 8 weeks; however, hematopoietic cells were observed. Vascularization was observed at 4 and 8 weeks in the PS and 3DS groups. New bone formation was hardly observed at 4 weeks; however, it was easily observed at 8 weeks [fig_ref] Figure 4: Histomorphometric evaluation at 4 and 8 weeks in the 3D printed substitute [/fig_ref].
## Int. j. mol. sci. 2020, 21, x for peer review 7 of 13
In the PS and 3DS groups, granulation tissue was observed in the defect area at 4 weeks, but little or no granulation tissue was observed at 8 weeks; however, hematopoietic cells were observed. Vascularization was observed at 4 and 8 weeks in the PS and 3DS groups. New bone formation was hardly observed at 4 weeks; however, it was easily observed at 8 weeks [fig_ref] Figure 4: Histomorphometric evaluation at 4 and 8 weeks in the 3D printed substitute [/fig_ref]. The 3DS group clearly showed the ingrowth of new bone from the lower side to the center at 8 weeks. In the PS group and 3DS groups, more ingrowth of new bone was observed at 8 weeks than at 4 weeks [fig_ref] Figure 5: Histomorphometric evaluation at 4 and 8 weeks [/fig_ref]. [fig_ref] Figure 5: Histomorphometric evaluation at 4 and 8 weeks [/fig_ref] shows that at 4 weeks, the amount of new bone formation was 9.73% and 7.61% in the PS and 3DS groups, respectively. In addition, at 8 weeks, the amount of new bone formation was 19.24% and 20.70% in the PS and 3DS groups, respectively. At 4 weeks, the new bone formation of the PS group showed a statistically significant difference compared to that of the NS group, and the 3DS group showed a statistically significant difference compared to the NS group (p < 0.05). The PS group and 3DS group showed lower values of new bone than that in the NS group at 4 weeks. There was no statistically significant difference in new bone between the PS group and the 3DS group (p > 0.05). Statistically significant differences were shown among the groups regarding the total amount of bone (p < 0.05). The 3DS group showed a higher value of the total amount of bone than the NS group and PS group, and the PS group showed a higher value of total amount of bone than the NS group at 4 weeks. At 8 weeks, the new bone formation of the PS group and 3DS group was statistically different from that of the NS group (p < 0.05). The new bone formation of the PS group and 3DS group was lower than that of the NS group. There was no statistically significant difference in new bone between the PS and 3DS groups (p > 0.05). Statistically significant differences in the total amount of bone (p < 0.05) were observed between the NS and PS groups and between the PS and 3DS groups. At 8 weeks, the total amount of bone was the highest in the 3DS group, followed by that in the PS group and then that in the NS group. The 3DS group clearly showed the ingrowth of new bone from the lower side to the center at 8 weeks. In the PS group and 3DS groups, more ingrowth of new bone was observed at 8 weeks than at 4 weeks [fig_ref] Figure 5: Histomorphometric evaluation at 4 and 8 weeks [/fig_ref]. [fig_ref] Figure 5: Histomorphometric evaluation at 4 and 8 weeks [/fig_ref] shows that at 4 weeks, the amount of new bone formation was 9.73% and 7.61% in the PS and 3DS groups, respectively. In addition, at 8 weeks, the amount of new bone formation was 19.24% and 20.70% in the PS and 3DS groups, respectively. At 4 weeks, the new bone formation of the PS group showed a statistically significant difference compared to that of the NS group, and the 3DS group showed a statistically significant difference compared to the NS group (p < 0.05). The PS group and 3DS group showed lower values of new bone than that in the NS group at 4 weeks. There was no statistically significant difference in new bone between the PS group and the 3DS group (p > 0.05). Statistically significant differences were shown among the groups regarding the total amount of bone (p < 0.05). The 3DS group showed a higher value of the total amount of bone than the NS group and PS group, and the PS group showed a higher value of total amount of bone than the NS group at 4 weeks. In the PS and 3DS groups, granulation tissue was observed in the defect area at 4 weeks, but little or no granulation tissue was observed at 8 weeks; however, hematopoietic cells were observed. Vascularization was observed at 4 and 8 weeks in the PS and 3DS groups. New bone formation was hardly observed at 4 weeks; however, it was easily observed at 8 weeks [fig_ref] Figure 4: Histomorphometric evaluation at 4 and 8 weeks in the 3D printed substitute [/fig_ref]. The 3DS group clearly showed the ingrowth of new bone from the lower side to the center at 8 weeks. In the PS group and 3DS groups, more ingrowth of new bone was observed at 8 weeks than at 4 weeks [fig_ref] Figure 5: Histomorphometric evaluation at 4 and 8 weeks [/fig_ref]. [fig_ref] Figure 5: Histomorphometric evaluation at 4 and 8 weeks [/fig_ref] shows that at 4 weeks, the amount of new bone formation was 9.73% and 7.61% in the PS and 3DS groups, respectively. In addition, at 8 weeks, the amount of new bone formation was 19.24% and 20.70% in the PS and 3DS groups, respectively. At 4 weeks, the new bone formation of the PS group showed a statistically significant difference compared to that of the NS group, and the 3DS group showed a statistically significant difference compared to the NS group (p < 0.05). The PS group and 3DS group showed lower values of new bone than that in the NS group at 4 weeks. There was no statistically significant difference in new bone between the PS group and the 3DS group (p > 0.05). Statistically significant differences were shown among the groups regarding the total amount of bone (p < 0.05). The 3DS group showed a higher value of the total amount of bone than the NS group and PS group, and the PS group showed a higher value of total amount of bone than the NS group at 4 weeks. At 8 weeks, the new bone formation of the PS group and 3DS group was statistically different from that of the NS group (p < 0.05). The new bone formation of the PS group and 3DS group was lower than that of the NS group. There was no statistically significant difference in new bone between the PS and 3DS groups (p > 0.05). Statistically significant differences in the total amount of bone (p < 0.05) were observed between the NS and PS groups and between the PS and 3DS groups. At 8 weeks, the total amount of bone was the highest in the 3DS group, followed by that in the PS group and then that in the NS group. At 8 weeks, the new bone formation of the PS group and 3DS group was statistically different from that of the NS group (p < 0.05). The new bone formation of the PS group and 3DS group was lower than that of the NS group. There was no statistically significant difference in new bone between the PS and 3DS groups (p > 0.05). Statistically significant differences in the total amount of bone (p < 0.05) were observed between the NS and PS groups and between the PS and 3DS groups. At 8 weeks, the total amount of bone was the highest in the 3DS group, followed by that in the PS group and then that in the NS group.
# Discussion
In non-segmental defects, there were five different types of defect: rectangular, box, arc, saddle, and cylindrical [bib_ref] Critical Size Defects for Bone Regeneration Experiments in the Dog Mandible: A..., Marei [/bib_ref]. Calculating the critical size defect in order to compare different sizes is difficult because all the experimental animals were exterminated at different points in time [bib_ref] Critical Size Defects for Bone Regeneration Experiments in the Dog Mandible: A..., Marei [/bib_ref]. Previous studies set the defect size as 3 × 5 mm for the rectangular defects and 8 × 12 mm for the box-type defects in a mandible model of a beagle dog [bib_ref] Critical Size Defects for Bone Regeneration Experiments in the Dog Mandible: A..., Marei [/bib_ref] [bib_ref] Healing of alveolar bone in resorbable and non-resorbable membrane-protected defects. A histologic..., Imbronito [/bib_ref]. If the defect size is large, the possibility of complications-such as mandible fracture, bleeding, and infection-increases. Based on previous studies, this study set the defect size as 7 × 3 × 5 mm 3 , but there was new bone formation at 4 weeks and 8 weeks in the negative control group. Huh et al. reported that, when the periosteum was preserved, the defect length in the beagle dog needed to be greater than 50 mm in order to fail to heal. During the surgical procedure of this study, the periosteum was preserved; thus, new bone formation could occur.
It was assumed that the bone formation percentage for the 3D printed HA/TCP scaffold would be lower than that for the particle-type bone substitutes; however, this was not the case. This study calculated the new bone formation as follows: New bone (%) = [Area of new bone/Total area of defect] × 100. The total area of the defect was the same in the three groups (7 × 3 × 5 mm 3 ). However, the new bone could only be formed in the remaining area of the grafted material area in the defect, since the grafted ceramic scaffold would not be absorbed within 8 weeks. Thus, the PS and 3DS groups had smaller areas for generating the new bone than the NS group. Although the rest area in the PS and 3DS groups was smaller than that in the NS group, the new bone formation of the PS and 3D groups was greater than that of the NS group as observed in radiological evaluation at 8 weeks. The amount of remaining grafted material at 8 weeks was found to be the highest in the 3DS group. Moreover, the 3DS group maintained the largest volume of grafted material of all the groups at 4 and 8 weeks. The PS and 3DS groups showed vascularization after 4 weeks. Kuboki et al. observed higher bone formation in porous hydroxyapatite scaffolds, with pore sizes in the range of 300-400 µm after 4 weeks of implantation in rats [bib_ref] Geometry of carriers controlling phenotypic expression in BMP-induced osteogenesis and chondrogenesis, Kuboki [/bib_ref]. This result was explained by the rapid vascularization within the implanted scaffolds, which provided a proper microenvironment for osteogenesis. These results indicate that the average pore size of scaffolds can be manipulated to potentially improve the formation of bone and vascular networks in ceramic scaffolds [bib_ref] Geometry of carriers controlling phenotypic expression in BMP-induced osteogenesis and chondrogenesis, Kuboki [/bib_ref].
Guided bone regeneration is conducted with bone substitutes to prevent volume loss or to increase the bone width and height [bib_ref] Ridge preservation techniques for implant therapy, Darby [/bib_ref] [bib_ref] Does ridge preservation following tooth extraction improve implant treatment outcomes: A systematic..., Mardas [/bib_ref] [bib_ref] The Effects of Alveolar Ridge Preservation: A Meta-Analysis, Willenbacher [/bib_ref]. There are many studies comparing block-type autogenous bone and particle-type allogenic bone, and most studies have demonstrated that the absorption rate of block-type bone substitutes is low [bib_ref] Guided bone regeneration of peri-implant defects with particulated and block xenogenic bone..., Benic [/bib_ref] [bib_ref] Bone augmentation procedures in localized defects in the alveolar ridge: Clinical results..., Jensen [/bib_ref] [bib_ref] Influence of wound closure on volume stability with the application of different..., Naenni [/bib_ref] [bib_ref] Influence of wound closure on the volume stability of particulate and non-particulate..., Mir-Mari [/bib_ref]. It is known that the use of block bone substitutes is more effective for large defects than particulate substitutes because the block bone can properly support the surrounding structure [bib_ref] Ridge augmentation by applying bioresorbable membranes and deproteinized bovine bone mineral: A..., Hammerle [/bib_ref]. However, when the block bone substitutes cannot fit the defect site well [bib_ref] Biodegradation property of beta-tricalcium phosphate-collagen composite in accordance with bone formation: A..., Kato [/bib_ref] [bib_ref] Ridge preservation of extraction sockets with buccal bone deficiency using poly lactide-co-glycolide..., Okada [/bib_ref] [bib_ref] Effects of additional collagen in biphasic calcium phosphates: A study in a..., Schaller [/bib_ref] , 3D printing technology has made it possible to preoperatively create customized shapes as desired [bib_ref] 3D printed porous ceramic scaffolds for bone tissue engineering: A review, Wen [/bib_ref] [bib_ref] Roohani, I. 3D Printing of Bioceramic Scaffolds-Barriers to the Clinical Translation: From..., Lin [/bib_ref]. Using this technology, the block-type bone can be custom-manufactured with the appropriate size and shape. Polymers are mainly used as the material in 3D printed bone substitutes. However, the molecular weight of the polymer-which causes incompleteness in pore size, porosity, and interconnectivity-negatively affects cell adhesion to the scaffold [bib_ref] Correlation between porous texture and cell seeding efficiency of gas foaming and..., Costantini [/bib_ref] [bib_ref] Formation of porous HPCL/LPCL/HA scaffolds with supercritical CO2 gas foaming method, Moghadam [/bib_ref]. In general, it is known that polymers have less bone conduction ability than calcium phosphate-based artificial bone [bib_ref] Biodegradable Polymers as Drug Delivery Systems for Bone Regeneration, Aoki [/bib_ref]. HA/TCP is a suitable ceramic material for manufacturing bone-grafting materials using 3D printing. It is known to exhibit excellent biocompatibility, biodegradation, osteoconductivity, and osteoinductivity [bib_ref] Comparative Efficacies of Collagen-Based 3D Printed PCL/PLGA/beta-TCP Composite Block Bone Grafts and..., Hwang [/bib_ref] [bib_ref] Calcium phosphate cements: Study of the beta-tricalcium phosphate-monocalcium phosphate system, Mirtchi [/bib_ref]. In addition, the osteoinduction property depends on the surface chemistry and charge of calcium phosphate ceramics, which can influence protein adsorption and, in turn, promote cell differentiation through cell-extracellular matrix interactions [bib_ref] Calcium phosphate ceramics in bone tissue engineering: A review of properties and..., Samavedi [/bib_ref]. The resolution, porosity, and strength of 3D printed TCP scaffolds strongly depend on the particle size, depowering efficiency, binder droplet size, and scaffold geometry [bib_ref] SrO-and MgO-doped microwave sintered 3D printed tricalcium phosphate scaffolds: Mechanical properties and..., Tarafder [/bib_ref]. Beta-TCP is the most favorable form of TCP scaffold, owing to its mechanical strength and chemical stability.
Most scaffolds with HA/TCP are fabricated by conventional methods such as gas foaming, fiber bonding, freeze drying, phase separation/inversion, and particulate leaching [bib_ref] Additive manufacturing techniques for the production of tissue engineering constructs, Mota [/bib_ref]. However, the pore shape, geometry, porosity, and interconnectivity of the scaffolds cannot be controlled by these methods, and the fabricated scaffolds cannot be specifically adapted to promote cell growth and tissue regeneration [bib_ref] Tailor-made biopolymers porous scaffold fabrication for tissue engineering: Application of radiant energy..., Jaya [/bib_ref]. To overcome the limitations of conventional methods for the fabrication of ceramic scaffolds, 3D printing technology has been widely used [bib_ref] 3D-printed bioceramic scaffolds: From bone tissue engineering to tumor therapy, Ma [/bib_ref]. There are various types of 3D printing systems in the biomedical field. Most previous studies used fuse deposition modeling (FDM)-type 3D printing to manufacture bone material [bib_ref] In vivo biocompatibility and degradation of novel Polycaprolactone-Biphasic Calcium phosphate scaffolds used..., Thuaksuban [/bib_ref] [bib_ref] 3D-printed PLA/HA composite structures as synthetic trabecular bone: A feasibility study using..., Wu [/bib_ref]. FDM is usually performed by mixing HA or TCP powders with polymers and stacking them through a nozzle. It is necessary to use polymers-such as polycaprolactone, polylactic acid, and poly-DL-lactic acid-that melt at high temperatures as a solvent for TCP to maintain the shape and structure of the 3D printed ceramic materials. HA/TCP material with polymer is inferior in terms of its biocompatibility and new bone-forming ability due to the polymer [bib_ref] Development of a PCL/gelatin/chitosan/beta-TCP electrospun composite for guided bone regeneration, Ezati [/bib_ref] [bib_ref] Effects of 3D-Printed Polycaprolactone/beta-Tricalcium Phosphate Membranes on Guided Bone Regeneration, Shim [/bib_ref]. In addition, the nozzle diameter of an FDM-type 3D printer is approximately 0.4 to 0.5 mm; therefore, it cannot print the bone material precisely and in detail.
Very few studies have reported the fabrication of HA/TCP scaffolds using a DLP-type 3D printing system. Unlike FDM-based 3D printers, the DLP-type 3D printing system used herein reduces the processing time by conducting the simultaneous irradiation of the entire desired cross-section. It can produce biomaterial with a high resolution compared to other systems [bib_ref] Application of high resolution DLP stereolithography for fabrication of tricalcium phosphate scaffolds..., Schmidleithner [/bib_ref]. DLP technology uses a digital projector as a light source, which significantly reduces the printing time and enables high-accuracy printing [bib_ref] Precision and trueness of dental models manufactured with different 3-dimensional printing techniques, Kim [/bib_ref]. A DLP system uses a ceramic slurry stacked layer by layer for the production of ceramic scaffolds [bib_ref] Hydrophilic excipients in digital light processing (DLP) printing of sustained release tablets:..., Krkobabic [/bib_ref] [bib_ref] Fabrication of an interim complete removable dental prosthesis with an in-office digital..., Lin [/bib_ref]. Light-unresponsive HA and TCP should be mixed with other polymers for DLP printing [bib_ref] Current state of fabrication technologies and materials for bone tissue engineering, Wubneh [/bib_ref] because each layer is solidified by exposure to light. Subsequent heat treatment is used to remove the polymers completely and produce biocompatible substitutes. In DLP-type 3D printing systems, developing a technology that can completely remove the photopolymer by heat treatment after printing is key [bib_ref] A Hybrid Dental Model Concept Utilizing Fused Deposition Modeling and Digital Light..., Lee [/bib_ref]. There are several essential conditions that need to be satisfied for complete removal, such as an appropriate heating rate, temperature, and time. The methods for removing polymers, methods for making the HA/TCP slurry, and 3D printing sequences are trade secrets in the field of bioceramic 3D printing. In this study, the safety and appropriateness of 3D printed bone were verified through biological safety assessments.
This study showed the possibility to manufacture 3D printed HA/TCP bone with complete polymer removal after 3D printing. This study also proved the biocompatibility and bone regenerative abilities of the 3D printed bone substitutes with the same composition as that of conventional HA/TCP alloplastic bone but with additional structural stability.
The bone regeneration and biocompatibility of the 3D printed HA/TCP scaffold were confirmed through this study. Further studies on scaffold structure and porosity in 3D printed bone are needed.
# Conclusions
In this study, a 3D printed HA/TCP scaffold was fabricated using a DLP-type 3D printer. The 3D printing of HA/TCP scaffolds using a DLP system is rare and innovative, and it will enable customized bone grafting for biomedical applications. However, this study has a few limitations. The mechanical strength and bone regenerative ability of the 3D printed ceramic scaffold could be affected by the pore structure. Further study is required for accurately determining the pore size, scaffold geometry, and interconnectivity. In addition, growth factor and bioprinting techniques could enhance the bone regeneration facilitated by the ceramic scaffold. This study did not include those techniques; therefore, additional research will be helpful for increasing the utility of 3D printed HA/TCP scaffolds without polymer.
[fig] Figure 1: Structure of 3D printed hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffold (SEM, 20.0kV). (A) 100×, (B) 500×, (C) 1000×. [/fig]
[fig] Figure 2: Digital light processing (DLP)-type 3D-printing process for pure HA/TCP scaffold. [/fig]
[fig] Figure 3: Radiological evaluation at 4 and 8 weeks. [/fig]
[fig] Figure 4: Histomorphometric evaluation at 4 and 8 weeks in the 3D printed substitute (3DS) group. (A) After 4 weeks. (B) After 8 weeks (white asterisk: grafted bone; black arrow: osteoblast). [/fig]
[fig] Figure 5: Histomorphometric evaluation at 4 and 8 weeks. [/fig]
[table] Table 1: Reactivity grades for elution test. [/table]
[table] Table 2: Results of cytotoxicity tests. [/table]
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10.3389/fpls.2017.00337
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CCBY
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5344897
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28344588
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s2orc_pubmed_articles
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AtLSG1-2 Regulates Leaf Growth by Affecting Cell Proliferation and the Onset of Endoreduplication and Synergistically Interacts with AtNMD3 during Cell Proliferation Process
AtLSG1-2 is a circularly permuted GTPase required for ribosome biogenesis and recently shown to be involved in early leaf development, although it was unclear how AtLSG1-2 affects leaf growth. Here, we found that atlsg1-2 mutants had reduced leaf size as a result of decreased cell size and cell number. Leaf kinematic analysis and CYCB1;1::GUS expression pattern in atlsg1-2 mutant indicated that loss of function of AtLSG1-2 delays the transition from cell division to cell expansion. Decreases in ploidy levels and trichome branch number suggest that AtLSG1-2 deficiency suppresses endoreduplication. Real-time PCR analysis showed that genes specifically expressed in the proliferation stage were highly expressed and those involved in endoreduplication were differentially regulated. LSG1 is known to mediate the recruitment of nucleocytoplasmic shuttling protein NMD3 back to the nucleus in yeast, yet their relationship was unclear in plants. Our genetic analysis revealed that the atlsg1 atnmd3 double mutant displayed enhanced phenotypes as compared with the respective single mutant and that AtLSG1-2 and AtNMD3 synergistically affect the cell proliferation process.
# Introduction
The leaves of higher plants are important structures where photosynthesis takes place that provides carbon and energy for plant growth. Leaf development is a complicated process that is coordinately regulated by internal factors and environmental conditions. The final size of a leaf is determined by two factors: cell size and cell number. Cell size is influenced by vacuolar volume, cell wall expansion, macromolecular synthesis in the cytoplasm and nuclei size [bib_ref] What determines cell size?, Marshall [/bib_ref]. Meanwhile, cell division controls cell number. Many genes that control cell size, cell number or both have been identified [bib_ref] Shaping up: the genetic control of leaf shape, Kessler [/bib_ref] [bib_ref] Leaf size control: complex coordination of cell division and expansion, Gonzalez [/bib_ref] [bib_ref] Plant-specific features of ribosome biogenesis, Weis [/bib_ref]. Genes controlling cell division include, for example, transcription factor genes, microRNAs, genes involved in hormone biosynthesis or signaling, cell-cycle-related genes, ribosome biogenesis, etc. On the other hand, genes involved in cell expansion are functionally related with cell wall formation, transcription, and DNA replication [bib_ref] Leaf size control: complex coordination of cell division and expansion, Gonzalez [/bib_ref] [bib_ref] Plant-specific features of ribosome biogenesis, Weis [/bib_ref].
Ribosome is the basic machine for protein production. Ribosome biogenesis and function are tightly linked to development in various species. Mutations in ribosome proteins (RP) genes cause either lethal effects or pleiotropic phenotypes [bib_ref] Plant-specific features of ribosome biogenesis, Weis [/bib_ref]. Certain RP genes involved in leaf development have been identified. Mutations in these genes affect either cell number or size or both. For instance, Arabidopsis OLIGOCELLULA2(OLI2), OLI5, and OLI7 encode a yeast Nop2 homolog, RPL5A or RPL5B, respectively. Whereas these oli mutants display decreased cell numbers [bib_ref] Coordination of cell proliferation and cell expansion mediated by ribosomerelated processes in..., Fujikura [/bib_ref] , leaves of the rpl18c-1, rps21b-1, and rps28b-1 mutants are smaller than those of the wild-type because of their reduced cell areas. On the other hand, rps6a-1 and rps6a-3 mutants showed strong reductions both in the cell size and in cell number in leaves [bib_ref] Differential contributions of ribosomal protein genes to Arabidopsis thaliana leaf development, Horiguchi [/bib_ref]. The deficiency of three ribosome biogenesis factors PESCADILLO, BLOCK OF PROLIFERATION1, and WD REPEAT DOMAIN12 inhibits cell-cycle progression, which results in the defective cell growth and proliferation [bib_ref] Functional characterization of the ribosome biogenesis factors PES, BOP1, and WDR12 (PeBoW),..., Ahn [/bib_ref]. These studies point to an important role of ribosomal proteins in leaf development, although the mechanisms are still under investigation.
LSG1 is a circularly permuted GTPase whose function has been well studied in yeast. The nucleocytoplasmic shuttling protein NMD3 is an adaptor for the export of the large ribosomal subunit (60S) from the nucleus. LSG1 appears to recycle NMD3 from the cytosol to the nucleus and its deficiency causes an accumulation of NMD3 in the cytoplasm and indirectly affects the export of the large ribosomal subunit (60S) from the nucleus [bib_ref] Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires..., Hedges [/bib_ref]. In humans, its ortholog HLsg1 is essential for cell growth and it shuttles between the nucleus and the cytoplasm [bib_ref] Human Lsg1 defines a family of essential GTPases that correlates with the..., Reynaud [/bib_ref]. In Drosophila, the ortholog of Lsg1 Nucleostemin 3 (NS3) is essential for ribosome production and autonomous cell growth. Overexpression of NS3 in yeast lsg1 mutants partially rescues this lethal mutant, suggesting that it conserves functions in ribosome biogenesis [bib_ref] Regulation of ribosome biogenesis by nucleostemin 3 promotes local and systemic growth..., Hartl [/bib_ref].
Whereas yeast, human, and Drosophila only have one copy of the LSG1 gene, Arabidopsis has two copies, AtLSG1-1 and AtLSG1-2. The protein sequences of the two LSG genes share high identity, suggesting their functional redundancy. Expression analysis with fluorescent fusion proteins showed that the two proteins are cytosolic [bib_ref] The Arabidopsis DIG6 gene encodes the large 60S subunit GTPase 1 that..., Zhao [/bib_ref] , similar to their yeast orthologs. Results from our work [bib_ref] The Arabidopsis DIG6 gene encodes the large 60S subunit GTPase 1 that..., Zhao [/bib_ref] and those of a recent study showed that the expression of AtLSG1-1 or AtLSG1-2 can partially rescue the yeast lsg1 mutant [bib_ref] The Arabidopsis DIG6 gene encodes the large 60S subunit GTPase 1 that..., Zhao [/bib_ref] , suggesting that the two proteins share similar functions as their yeast orthologs. In plants, AtLSG1-2 appears to play a dominant role since the atlsg1-2 null mutant showed pleiotropic phenotypes, including small size, short roots, delayed lateral root emergence, and distorted auxin homeostasis [bib_ref] The Arabidopsis DIG6 gene encodes the large 60S subunit GTPase 1 that..., Zhao [/bib_ref] , whereas AtLSG1-1 deficiency only subtly effected plant development. Furthermore, the expression level of AtLSG1-2 is higher than that of AtLSG1-1 [bib_ref] The Arabidopsis DIG6 gene encodes the large 60S subunit GTPase 1 that..., Zhao [/bib_ref].
In this study, we focus on the roles of AtLSG1-2 on leaf development and investigated how loss of function of AtLSG1-2 may affect leaf growth. Compared to wild-type plant, atlsg1-2 mutant had reduced leaf size. Leaf kinematic analysis and flow cytometry analysis revealed that cell division, differentiation and endoreduplication processes were obviously affected in atlsg1-2 mutant. We also investigate the relationship between LSG1 and NMD3 in plants by exploring their genetic interactions between AtLSG1-2 and AtNMD3.
# Materials and methods
## Plant material and growth conditions
T-DNA insertion lines (atlsg1-2: Salk_114083 and atnmd3: WiscDsLox257G09) were obtained from Arabidopsis Biological Research Center. Wild-type (Accession Columbia-0) and mutant seeds were surface-sterilized in 50% bleach solution for 5 min and rinsed with water five times. The sterilized seeds were sown on agar-solidified half-strength Murashige and Skoog medium and incubated at 4 - C for 3 days before being transferred to a growth chamber at 22 - C with a 16-h-light/8-h-dark photoperiod. For soil-grown plants, 12-day-old seedlings growing on a petri dish were transferred to soil and grown in a growth room at 22 - C with a 16-h-light/8-h-dark photoperiod.
For T-DNA line WiscDsLox257G09, the mutant allele was verified by PCR genotyping and by sequencing the PCR products. T-DNA insertion was detected using the left border primer (LB) combined with gene-specific primers LP and RP. Primer sequences are given in [fig_ref] TABLE S2 |: List of primers used in this study [/fig_ref].
# Phenotype analysis
Leaf size, cell number, and size of abaxial epidermis were measured in the fifth leaf of 4-week-old plants growing in soil. A small droplet of superglue was applied on the glass slide. The leaf abaxial side was tightly imprinted on the slide coated with superglue for 30 s and the leaf was then quickly removed. The imprinted slides were observed and photographed with a microscope (Axio Imager Z2). Data from 5 to 8 leaves were used for statistical analysis.
## Plasmid construction and plant transformation
To generate promoter deletion fusion constructs, different promoter deletions were cloned into the pENTRTM/D-TOPO vector using pENTRTM directional TOPO R cloning kit (Invitrogen) and then subcloned into the binary vector pMDC162 by the LR recombination reaction. The plasmids were transferred into Agrobacterium tumefaciens and Arabidopsis plants were transformed by the floral dipping method [bib_ref] Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Clough [/bib_ref].
# Leaf kinematic analysis
Leaf kinematic analysis was performed essentially as described by [bib_ref] Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis, De Veylder [/bib_ref] with some modifications. Leaves were submerged in 10 µM FM4-64 for 10 min and destained in water. The epidermal cells of stained leaf abaxial side were observed and photographed with a microscope (Axio Imager Z2). The experiment was repeated three times with similar results obtained. Results from only one set of the experiment were presented in this study.
## Gus staining
Whole seedlings were incubated in ice-cold 90% acetone for 2 h, washed in 100 mM Na 3 PO 4 (pH 7.0) and subsequently immersed in 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) buffer (100 mM Na 3 PO 4 buffer, pH 7.0, 10 mM Tris, pH 8.0, 1 mM EDTA, 0.05% Triton X-100, 1 mg/ml X-Gluc) at 37 - C overnight. Chlorophyll was cleared in 70% ethanol. The cleared samples were photographed with a Nikon SMZ25 stereomicroscope.
# Flow cytometry analysis
Preparation of plant materials for flow cytometric assays was performed as described in [bib_ref] Estimation of nuclear DNA content in plants using flow cytometry, Dolezel [/bib_ref]. In brief, leaves were quickly chopped with a razor blade in ice-cold Galbraith's buffer [45 mM MgCl 2 , 20 mM MOPS, 30 mM sodium citrate, and 0.1% (vol/vol) Triton X-100, pH 7.0]. The homogenate was filtered through 70 µM nylon mesh. Fifty microgram per milliliter propidium iodide (PI) combined with 50 µg/ml of RNase A was added into the filtered homogenate and mixed for flow cytometry analysis. The sample was analyzed in a BD LSRFortessa flow cytometer equipped with a 50 mW 561 nm laser.
## Rna sequencing and quantitative real-time pcr
Total RNA was extracted from the first pair of 11-day-old wild-type plant and 13-day-old mutant leaves using an RNasy Plant Mini Kit (Qiagen). Residual DNA was removed with DNase I (NEB) and 2 µg of cleaned RNA were used for reverse transcription. RNA sequencing was performed according to. For real-time PCR, reverse transcription was conducted using the SuperScript III first-strand synthesis SuperMix (Invitrogen) and PCR was conducted using Power SYBRgreen PCR Master Mix (Applied Biosystems) in a 7900 HT Fast Real-Time PCR System (Applied Biosystems). ACTIN2 was used as the internal control. Three biological replicates were performed for real-time PCR analysis. For checking the transcript level of AtNMD3 in the T-DNA insertion line WiscDsLox257G09, RNA was extracted from the wild-type plant and the mutant seedlings and real-time PCR was performed as described above.
# Results
## Small-sized atlsg1-2 leaves are caused by reduced cell size and cell number
In a genetic screen for mutants defective in lateral root response to drought and abscisic acid, we isolated a mutant, dig6 (drought inhibited growth of lateral roots), which showed Frontiers in Plant Science | www.frontiersin.org reduced lateral root numbers. Map-based cloning identified that the mutation occurred in the AtLSG1-2 gene [bib_ref] The Arabidopsis DIG6 gene encodes the large 60S subunit GTPase 1 that..., Zhao [/bib_ref]. The dig6 mutant and a T-DNA insertion mutant allele atlsg1-2 (Salk_114083) had nearly identical phenotypes [bib_ref] The Arabidopsis DIG6 gene encodes the large 60S subunit GTPase 1 that..., Zhao [/bib_ref]. Here, we focused on the T-DNA insertion mutant atlsg1-2. Young leaves of the mutant exhibited significantly retarded growth and mature leaves remained small [fig_ref] FIGURE 1 |: The atlsg1-2 mutant has smaller leaf size, cell number and cell size [/fig_ref]. By checking the fifth leaves of the wild-type and atlsg1-2 mutants in detail, we found that the leaf area of the mutant was half that of the wild-type, as shown in [fig_ref] FIGURE 1 |: The atlsg1-2 mutant has smaller leaf size, cell number and cell size [/fig_ref]. To determine which factor contributed to leaf size reduction, we evaluated cell size and cell number and found that in the mutant, cell size and cell number decreased to 73.5 and 70.9% that of the wild-type plant, respectively.
# Leaf kinematic analysis
Cell size and cell number are closely associated with cell expansion and cell proliferation activity, respectively. We conducted a leaf kinematic analysis to investigate how loss of function of AtLSG1-2 affects cell division and expansion. Leaf development is divided into three stages: cell division, expansion, and maturation [bib_ref] Genome-wide analysis of gene expression profiles associated with cell cycle transitions in..., Beemster [/bib_ref]. We evaluated leaf development in the first pair of atlsg1-2 leaves. As shown in , compared to the wild-type, leaf emergence was delayed by 2 days in the atlsg1-2 mutant. Furthermore, leaf growth was relatively slower in the mutant and cell size and cell number also increased more slowly compared with the wild-type, suggesting that cell proliferation and expansion activity were simultaneously suppressed in the atlsg1-2 mutant . Furthermore, the cell expansion phase was noticeably delayed in the atlsg1-2 mutant and Supplementary [fig_ref] FIGURE 1 |: The atlsg1-2 mutant has smaller leaf size, cell number and cell size [/fig_ref]. In wild-type plants, cell size was approximately 100 µm 2 5-8 days after stratification (DAS), but after 9 DAS, cell size rapidly increased, suggesting that cell differentiation has begun. In the atlsg1-2 mutant, cell size from 7 to 11 DAS was similar to that of the wild-type plant from 5 to 8 DAS, and although cell size increased after 11 DAS, the rate was apparently slower . As shown in Supplementary [fig_ref] FIGURE 1 |: The atlsg1-2 mutant has smaller leaf size, cell number and cell size [/fig_ref] , whereas most of the cells in 9-DAS-old wild-type leaves had already underwent expansion, those in the mutant leaves began to expand only at 13-DAS. This suggests that cell expansion activity was apparently impaired in the atlsg1-2 mutant.
To substantiate the inhibitory effects of AtLSG1-2 deficiency on the normal progression of cell division to differentiation, we examined the CYCB1;1::GUS activity in the wild-type and atlsg1-2 mutants; CYCB1;1 is specifically expressed in dividing cells [bib_ref] Developmental expression of the arabidopsis cyclin gene cyc1At, Ferreira [/bib_ref]. We found that GUS signals greatly diminished in 12-day-old wild-type leaves but were still strongly expressed in the 14-day-old mutant leaves [fig_ref] FIGURE 3 |: Expression pattern of CYCB1 [/fig_ref]. As shown in Supplementary Figure S1, many cells in 13-DAS-old mutants still underwent division whereas the cells in wild-type plant were expanded. This was consistent with CYCB1;1 staining results. Therefore, loss of function of AtLSG1-2 hindered the transition from cell division to expansion. FIGURE 2 | Kinematic analysis of the first pair of leaves. The first pair of leaves was collected daily from the wild-type and mutants. At least five samples were used for statistical analyses. The experiment was repeated three times with similar results obtained.
## Atlsg1-2 deficiency inhibits endoreduplication
The leaf kinematic analysis showed that cell expansion processes were suppressed in the atlsg1-2 mutant. Because cell expansion is closely related with the endoreduplication process, we examined the ploidy distribution by flow cytometry in the first pair of wild-type and atlsg1-2 mutant leaves. Leaf samples were collected during cell division, cell expansion, and maturation phases. As shown in , the 2C population decreased more slowly in atlsg1-2 mutants than in wild-type leaves, while the 4C population slowly increased in the mutant. This suggests that cell expansion phase was delayed in the mutant. Although both 8C and 16C populations represent the onset of endoreduplication, the 16C population contributes minimally and thus will not be discussed here. The atlsg1-2 mutant plants also had a more slowly increasing 8C population such that the final 8C frequency was around 30% in wild-type leaves but only about 20% in the atlsg1-2 mutant , suggesting that the endoreduplication process was affected in the mutants. The ploidy distribution measured by flow cytometry can also be expressed as an endoreduplication index (EI), which represents the average number of endocycles undergone by a given nucleus. The EI, developmentally regulated during leaf growth, was lower in the atlsg1-2 mutant throughout development, indicating that the average number of endocycles is reduced in the atlsg1-2 mutant.
To verify these results, we also checked the ratio of trichomes with different numbers of branches because trichome branch number is positively correlated with ploidy level [bib_ref] Endoreplication controls cell fate maintenance, Bramsiepe [/bib_ref]. In the first leaf, the ratio of two-branched trichome was significantly higher in atlsg1-2 mutant leaves than in wild-type plants [fig_ref] FIGURE 5 |: The ratio of different types of trichomes in the wild-type and atlsg1-2... [/fig_ref]. In atlsg1-2 mutants, most trichomes are two or three-branched, while wild-type plant leaves contain a relatively high ratio of four-branched trichomes and small number of five-branched trichomes [fig_ref] FIGURE 5 |: The ratio of different types of trichomes in the wild-type and atlsg1-2... [/fig_ref]. These results combined with those from flow cytometry suggest that an AtLSG1-2 deficiency suppresses endoreduplication.
## Genes specifically expressed in proliferation stage are upregulated in atlsg1-2 mutants and genes associated with endoreduplication were differentially regulated
Both leaf kinematic analysis and pCYCB1;1::GUS expression showed that the progression from cell division to differentiation was delayed in the atlsg1-2 mutant, suggesting that the expression of those genes associated with this process might be differentially regulated. Because leaf emergence in the mutant was delayed 2 days compared with wild-type plants, to eliminate the time difference in leaf emergence, we chose the first pair of 11-day-old wild-type and 13-day-old atlsg1-2 mutant leaves for RNAseq and qRT-PCR analysis. In 11-day-old wild-type plant leaves, small amount of cells in the tip of wild plant leaves expand and undergo differentiation. RNAseq results showed that cell-cycle process was obviously perturbed in the atlsg1-2 mutant (Supplementary [fig_ref] TABLE S1 |: Differentially regulated genes [/fig_ref]. We further checked the expression of those genes specifically expressed in proliferation stage [bib_ref] Genome-wide analysis of gene expression profiles associated with cell cycle transitions in..., Beemster [/bib_ref] , which includes CYCA2;3, CYCA3;2, CDKB2;1, CYCB1;5, CYCB2;1, and CYCB2;4, etc. Most of these genes were highly expressed in atlsg1-2 mutant (Supplementary [fig_ref] TABLE S1 |: Differentially regulated genes [/fig_ref]. While the cause and effect relation of these events is unclear, one possibility is that AtLSG1 deficiency probably affects ribosome biogenesis and compromises the capacity of protein synthesis. The insufficient ability for protein translation may cause slower proliferation and delayed cell-cycle exit, which resulted in higher expression of cell-cycle genes later in proliferation stage in the mutant. Since RNA-seq analysis was only performed once, the expression patterns of some genes specifically expressed in proliferation stage were further confirmed by using real-time PCR . It could be concluded that the prolonged cell division phase in the atlsg1 mutant correlated with high levels of these genes specifically expressed in proliferation stage.
Leaf kinematic analysis showed that the onset of endoreduplication was affected in the atlsg1-2 mutant, suggesting that the expression of some genes associated with this process might be altered. Our RNA-seq analysis (Supplementary Table S1) showed that several key regulators of endoreduplication including KRP1, CYCA2;3, CDKB1;1, and LGO were differentially regulated in atlsg1-2 mutants. Among these genes, KRP1 and LGO are positive regulators of endoreduplication. KRP1 encodes a cyclin-dependent kinase FIGURE 4 | Cytometry analysis of ploidy levels in the wild-type and atlsg1-2 mutant. The first pair of leaves was collected for flow cytometry analysis every 2 days starting from 11 days after stratification. The percentage of each ploidy type among total types was calculated. Endoreduplication index indicates the average number of endocycles undergone by a given nucleus. Data represent means and standard derivations from three biological replicates.
inhibitor protein that negatively regulates cell division and promotes endoreduplication [bib_ref] Novel functions of plant cyclin-dependent kinase inhibitors, ICK1/KRP1, can act non-cell-autonomously and..., Weinl [/bib_ref].
LGO is a member of a plant-specific cell-cycle-inhibitor-family SIAMESE and is essential for endoreduplication of sepal giant cells. Its loss has caused the absence of giant cells in leaves and sepals [bib_ref] Variability in the control of cell division underlies sepal epidermal patterning in..., Roeder [/bib_ref]. RNA-seq data showed that the transcript levels of KRP1 and LGO were reduced considerably in atlsg1-2 mutants. On the other hand, the expression of CDKB1;1 and CYCA2;3, which encode negative regulators of endoreduplication, significantly increased in the mutant (Supplementary [fig_ref] TABLE S1 |: Differentially regulated genes [/fig_ref]. CYCA2;3, encoding A-type cyclin, could form a functional complex with CDKB1;1 and suppress the onset of endoreduplication [bib_ref] CDKB1;1 forms a functional complex with CYCA2;3 to suppress endocycle onset, Boudolf [/bib_ref]. Other genes associated with endoreduplication were also differentially expressed in the mutant (Supplementary Table S1); these genes were associated with signaling pathways or transcription factors. For example, CALMODULIN LIKE 42 (CML42), a calmodulin-related calcium sensor: a cml42 knockout mutant had abnormal trichomes with increased branching [bib_ref] The calmodulin-related calcium sensor CML42 plays a role in trichome branching, Dobney [/bib_ref] , suggesting that it is a negative regulator of trichome branching. Its expression was significantly upregulated in the atlsg1-2 mutant. HOMEODOMAIN GLABROUS 11 and 12, belonging to the HD-ZIP IV family, also negative regulators of trichome branching [bib_ref] Characterization of the class IV homeodomain-Leucine Zipper gene family in Arabidopsis, Nakamura [/bib_ref] , had upregulated expression levels in the atlsg1-2 mutant. These results from RNA-seq analysis suggest that the expression of these genes might be affected in the mutant. We thus conducted real-time PCR to validate and quantify the expression level of these genes. Our real-time PCR results are consistent with those of the RNAseq analysis . Therefore, the differential expression of these endoreduplication regulatory genes may lead to disturbed endoreduplication as observed in the mutant.
Simultaneous Knockout of AtLSG1-2 and AtNMD3 Exhibits Synergistic Effects LSG1 is known to mediate the export of the ribosome export factor NMD3 from the nucleus in yeast, but how plant NMD3 homologs interplay with LSG1 remains unknown. To understand the genetic interaction between AtLSG1-2 and AtNMD3 in Arabidopsis, we obtained an atnmd3 knockdown mutant (WiscDsLox257G09) because a atnmd3 knockout mutant is lethal. In the atnmd3 knockdown mutant, a T-DNA fragment was inserted in its 3 -untranslated region. The expression level of the AtNMD3 transcripts in the homozygous line was examined by RT-PCR using genespecific primers (Supplementary [fig_ref] FIGURE S2 |: Molecular characterization of the atnmd3 mutant [/fig_ref] and [fig_ref] TABLE S2 |: List of primers used in this study [/fig_ref] and was found significantly reduced (Supplementary [fig_ref] FIGURE S2 |: Molecular characterization of the atnmd3 mutant [/fig_ref]. Nonetheless, this mutant showed only subtle phenotype changes at a very early stage such as retarded growth, delayed leaf emergence, and stunted roots [fig_ref] FIGURE 7 |: Phenotypes of atlsg1, atnmd3, and atlsg1 atnmd3 mutants [/fig_ref]. We generated an atlsg1 atnmd3 double mutant by genetic crossing of the two single mutants and examined the phenotype of the resulting double mutant. The growth of the primary root of the double mutant was very slow and leaf emergence was also significantly delayed in the early seedling stage [fig_ref] FIGURE 7 |: Phenotypes of atlsg1, atnmd3, and atlsg1 atnmd3 mutants [/fig_ref]. The stature of the adult double mutant was much shorter than either single mutant [fig_ref] FIGURE 7 |: Phenotypes of atlsg1, atnmd3, and atlsg1 atnmd3 mutants [/fig_ref]. The dwarf and bushy phenotypes along with short siliques and low fertility [fig_ref] FIGURE 7 |: Phenotypes of atlsg1, atnmd3, and atlsg1 atnmd3 mutants [/fig_ref] were not seen in either single mutant. We thus conclude that the attenuation of AtNMD3 function enhances the phenotypes caused by AtLSG1-2 loss. We examined the phenotypes of the first pair of leaves in more details. As shown in [fig_ref] FIGURE 7 |: Phenotypes of atlsg1, atnmd3, and atlsg1 atnmd3 mutants [/fig_ref] and Supplementary [fig_ref] FIGURE 3 |: Expression pattern of CYCB1 [/fig_ref] , atlsg1-2 leaf area was reduced, whereas that of the atnmd3 mutant was similar to that of the wild-type. FIGURE 6 | Real-time PCR analysis of differentially expressed genes in the atlsg1-2 mutant. RNA was extracted from the first pair of leaves of 11-day-old wild-type and 13-day-old atlsg1-2 mutant. ACTIN2 was used as an internal control. Data are means and standard derivations (n = 3, * P < 0.01 by Student's t-test). Error bars in the graph indicate standard derivations of three biological replicates for each gene.
The leaf area of the double mutant was considerably less than either single mutant. Although cell size of the double mutant was similar to that of the atlsg1-2 mutant, cell number was significantly reduced compared with either single mutant [fig_ref] FIGURE 7 |: Phenotypes of atlsg1, atnmd3, and atlsg1 atnmd3 mutants [/fig_ref]. These results suggest that AtLSG1-2 and AtNMD3 synergistically control cell proliferation activity during leaf development.
# Discussion
The functions of LSG1 proteins have been studied in yeasts, humans, Drosophila, and now in plants. Despite their different subcellular localization patterns, their roles in ribosome biogenesis have consistently been confirmed. Moreover, the complete loss of these genes is usually lethal, suggesting that the ribosome biogenesis processes they are involved in are essential for cell viability. The identification of weak alleles makes it feasible to investigate the roles of these genes in growth and development. ns3 is a knockdown fly mutant with a P-element insertion in the NS3 gene [bib_ref] A nucleostemin family GTPase, NS3, acts in serotonergic neurons to regulate insulin..., Kaplan [/bib_ref] that results in small body size because fewer and smaller cells are produced. However, further study showed that these phenotypic defects could be rescued by the expression of AKT1, a central effector of the insulin-signaling pathway, whose activation affects a number of downstream effectors to stimulate ribosome biogenesis [bib_ref] A nucleostemin family GTPase, NS3, acts in serotonergic neurons to regulate insulin..., Kaplan [/bib_ref]. Therefore, NS3-mediated body size acts through the insulin-signaling pathway. In Arabidopsis, there are two LSG1 paralogs. Because of their conserved functions in ribosome biogenesis and functional redundancy, loss of either gene only mildly affects plant growth [bib_ref] The Arabidopsis DIG6 gene encodes the large 60S subunit GTPase 1 that..., Zhao [/bib_ref]. Nonetheless, whereas the loss of AtLSG1-1 had little effect on plant growth, atlsg1-2 mutants showed pleiotropic phenotypes, suggesting that AtLSG1-2 has more important roles. A recent study identified that the AtLSG1-2 gene is involved in the 40S ribosome maturing process. Our study showed that AtLSG1-2 loss of function caused the decreased levels of 40S, 60S, and 80S ribosomes [bib_ref] The Arabidopsis DIG6 gene encodes the large 60S subunit GTPase 1 that..., Zhao [/bib_ref] , demonstrating its importance in ribosome biogenesis. Furthermore, defective ribosome biogenesis seems to be closely related with the phenotypes observed in atlsg1-2 mutants; some similar phenotypes were shown in other mutants with defective ribosome biogenesis [bib_ref] Disruption of an Arabidopsis cytoplasmic ribosomal protein S13-homologous gene by transposon-mediated mutagenesis..., Ito [/bib_ref] [bib_ref] An Arabidopsis Minute-like phenotype caused by a semidominant mutation in a RIBOSOMAL..., Weijers [/bib_ref]. Because there is no insulin pathway in plants, AtLSG1-2 must act through different pathways to control leaf growth. Despite the possibility of different regulatory pathways of NS3 and AtLSG1, NS3 and AtLSG1-2 loss of function mutants share some common phenotypes: defective ribosome biogenesis, retarded growth and small size as a result of decreased cell size and reduced cell number, suggesting that NS3 and AtLSG1 share some conserved functions and that ribosome biogenesis which both genes are involved in is necessary for maintaining normal cell sizes. The various phenotypes caused by AtLSG1-2 loss of function also overlapped with those reported for several RP gene mutants [bib_ref] Coordination of cell proliferation and cell expansion mediated by ribosomerelated processes in..., Fujikura [/bib_ref] [bib_ref] Differential contributions of ribosomal protein genes to Arabidopsis thaliana leaf development, Horiguchi [/bib_ref] [bib_ref] Plant-specific features of ribosome biogenesis, Weis [/bib_ref]. The reason for these phenotypes might be inefficient global protein synthesis, which impairs the cell-cycle progression and thus affects normal cell division and expansion activity. We also noticed that the atlsg1-2 leaves displayed abnormal leaf polarity and auxin-defective phenotypes [bib_ref] The Arabidopsis DIG6 gene encodes the large 60S subunit GTPase 1 that..., Zhao [/bib_ref]. Thus it is also likely that dysfunctional ribosomes due to the lack specific ribosomal proteins may affect the translation of some specific mRNA involved in the leaf development process.
The interaction of LSG1 and NMD3 was well studied in yeast, but is still unknown in plants. In yeast, LSG1 participates in the nuclear export of NMD3 during ribosome biogenesis. The NMD3 ortholog in Arabidopsis was also demonstrated to be required for the nuclear export of 60S ribosomal subunit. Our current genetic analysis showed that atlsg1 atnmd3 double mutant displayed smaller leaves compared to parental single mutants. Decreased leaf size is mainly associated with the further reduction of cell number in the double mutant [fig_ref] FIGURE 7 |: Phenotypes of atlsg1, atnmd3, and atlsg1 atnmd3 mutants [/fig_ref] , suggesting that AtNMD3 may mainly affect cell division, which is different from AtLSG1-2 that controls both cell number and cell size. Specific roles by NMD3 in cell proliferation have also been demonstrated in rice, where overexpression of a dominant-negative form of truncated OsNMD3 led to a dwarf phenotype as a result of decreased cell number [bib_ref] Retention of OsNMD3 in the cytoplasm disturbs protein synthesis efficiency and affects..., Shi [/bib_ref]. These data suggest conserved functions of NMD3 in both monocotyledonous and dicotyledonous plants. The synergistic effects seen in the double mutant also implies that AtLSG1-2 and AtNMD3 act through common or shared pathways to regulate cell division, which is consistent with findings in other systems where the two proteins work together in ribosome biogenesis. Meanwhile, we found that AtLSG1-2 and AtNMD3 also have their own specific functions. atlsg1-2 mutants displayed incurvate leaves and auxin-defective phenotypes [bib_ref] The Arabidopsis DIG6 gene encodes the large 60S subunit GTPase 1 that..., Zhao [/bib_ref] , which were not present in atnmd3 mutants, suggesting unique roles by AtLSG1-2 in leaf polarity and auxin homeostasis. NMD3 proteins were found to be involved in secondary wall thickening and to control some agronomic traits including internode growth and panicle and seed development [bib_ref] Retention of OsNMD3 in the cytoplasm disturbs protein synthesis efficiency and affects..., Shi [/bib_ref]. These studies suggested that AtLSG1-2 might participate in the 60S subunit nuclear export mediated by AtNMD3, but that NMD3 may be also involved in other unknown pathways.
Primer sequence in this study was listed in [fig_ref] TABLE S2 |: List of primers used in this study [/fig_ref] AUTHOR CONTRIBUTIONS HZ designed, conducted the experiments, and wrote the manuscript. SL performed leaf kinematic analysis. LX supervised this work and revised the manuscript.
# Funding
This study was supported by King Abdullah University of Science and Technology (KAUST) and Texas A&M Agrilife Research.
# Supplementary material
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.00337/ full#supplementary-material FIGURE S1 | Morphology of abaxial epidermal cells in the wild-type and atlsg1 in different days after stratification (DAS). Scale bars indicate 20 µm. The detection of T-DNA insertion by PCR analysis. Genomic DNA was extracted from wild-type and atnmd3. PCR was performed using the gene -specific primer LP and RP [fig_ref] FIGURE 1 |: The atlsg1-2 mutant has smaller leaf size, cell number and cell size [/fig_ref] or left border -specific primer (LB) and RP (lanes 2,4). (B) Transcript levels of AtNMD3 in the wild-type and atnmd3 mutant. RNA was extracted from the rosette leaves of the wild-type and the atnmd3 mutant. Real-time PCR was preformed with primers OXH329 and OXH330. ACTIN2 was used as internal control. Data are means and standard deviations (n = 3, * P < 0.01 by Student's t-test).
[fig] FIGURE 1 |: The atlsg1-2 mutant has smaller leaf size, cell number and cell size. (A) Morphology of 4-week-old wild-type (upper panel) and atlsg1-2 mutants (lower panel) growing in soil. Scale bars indicate 1 cm. (B) Morphology of detached leaves of the wild-type (upper panel) and atlsg1-2 mutants (lower panel). Cotyledon and leaves 1-12 are arranged from left to right. Scale bars indicate 1 cm. (C) Statistical analysis of leaf area, cell size and cell number of the fifth leaf. Data are means and standard derivations (n = 5, * P < 0.01 by Student's t-test). [/fig]
[fig] FIGURE 3 |: Expression pattern of CYCB1;1::GUS in the wild-type and atlsg1-2 mutant leaves. The first pair of leaves was collected from 8 (A,F), 10 (B,G), 11 (C,H), 12 (D,I), and 14 (E,J) days after stratification. Six to ten plants were used for staining with similar results and the representative images are shown. Scale bars indicate 0.5 mm. [/fig]
[fig] FIGURE 5 |: The ratio of different types of trichomes in the wild-type and atlsg1-2 mutant leaves. Data are means and standard derivations from three biological replicates each with at least 5-8 leaves examined. [/fig]
[fig] FIGURE 7 |: Phenotypes of atlsg1, atnmd3, and atlsg1 atnmd3 mutants. (A,B) Morphology of 11-day-old seedlings growing on horizontally (A) and vertically (B) placed MS agar plates. The atlsg1 atnmd3 double mutant has two (D1) or three cotyledons (D2). (C) Morphology of 44-day-old plants growing in soil. Bars in (A,B) represent 0.5 and 1 mm, respectively. (D) (insert) A close up of siliques. From left to right the plants are wild-type, single-mutant atlsg1, single-mutant atnmd3, and double-mutant atlsg1 atnmd3. The scale bar represents 0.5 cm. (E) Leaf phenotype of single and double mutants. Leaf area, cell size and cell number were examined in fifth leaves of 4-week-old plants. At least five plants each genotype was used for statistical analysis. [/fig]
[fig] FIGURE S2 |: Molecular characterization of the atnmd3 mutant. (A) [/fig]
[fig] FIGURE S3 |: Leaf phenotype of 4-week-old plants. Scale bars indicate 1 cm. [/fig]
[table] TABLE S1 |: Differentially regulated genes. [/table]
[table] TABLE S2 |: List of primers used in this study. [/table]
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10.18295/squmj.2019.19.02.007
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Significance of Simple Steatosis: An Update on the Clinical and Molecular Evidence
Non-alcoholic fatty liver disease (NAFLD) is defined clinicopathologically by the accumulation of lipids in >5% of hepatocytes and the exclusion of secondary causes of fat accumulation. NAFLD encompasses a wide spectrum of liver damage, extending from simple steatosis or non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH)-the latter is characterized by inflammation and hepatocyte ballooning degeneration, in addition to the steatosis, with or without fibrosis. NAFLD is now the most common cause of chronic liver disease in Western countries and affects around one quarter of the general population. It is a multisystem disorder, which is associated with an increased risk of type 2 diabetes mellitus as well as liver-and cardiovascular-related mortality. Although earlier studies had suggested that NAFL is benign (i.e., non-progressive), cumulative evidence challenges this dogma, and recent data suggest that nearly 25% of those with NAFL may develop fibrosis. Importantly, NAFLD patients are more susceptible to the toxic effects of alcohol, drugs, and other insults to the liver. This is likely due to the functional impairment of steatotic hepatocytes, which is virtually undetectable by current clinical tests. This review provides an overview of the current evidence on the clinical significance of NAFL and discusses the molecular basis for NAFL development and progression.
# Introduction
Nonalcoholic fatty liver disease (NAFLD) is now the leading cause of chronic liver disease worldwide [bib_ref] Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and..., Younossi [/bib_ref] , and represents a major cause of severe liver complications, including cirrhosis and hepatocellular carcinoma (HCC) [bib_ref] Nonalcoholic Fatty Liver Disease: Pathogenesis and Disease Spectrum, Hardy [/bib_ref]. The clinicopathological definition of NAFLD is the accumulation
## Can patients with nafl progress to a more advanced disease stage?
Without doubt, NASH and NASH fibrosis are associated with an increased risk of progression to end-stage hepatic disease and development or worsening of a variety of extra-hepatic metabolic complications [bib_ref] NAFLD: A multisystem disease, Byrne [/bib_ref]. In contrast major uncertainties remain if simple steatosis is truly a benign liver condition. As mentioned above, more data on prospective disease monitoring by (multiple) repeat biopsies are urgently required for a better understanding of the natural course of NAFLD. Reasons for currently open questions and discrepancies between published reports include the mis-diagnosis of liver disease, where patients with cryptogenic cirrhosis actually had NASH-related cirrhosis [bib_ref] Cryptogenic cirrhosis: Clinical characterization and risk factors for underlying disease, Caldwell [/bib_ref] [bib_ref] Prevalence of obesity and diabetes in patients with cryptogenic cirrhosis: A case-control..., Poonawala [/bib_ref] , variable study duration and follow-up [bib_ref] Nonalcoholic Steatohepatitis and Endpoints in Clinical Trials, Hannah [/bib_ref] [bib_ref] Natural history of nonalcoholic fatty liver disease: A prospective follow-up study with..., Nasr [/bib_ref] , as well as sampling variability from liver biopsy [bib_ref] Sampling variability of liver biopsy in nonalcoholic fatty liver disease, Ratziu [/bib_ref]. In the following section, we try to compile data on the one hand suggesting that NAFL or simple steatosis is a benign condition [fig_ref] Table 1: Evidence in favor of the good prognosis of NAFL patients [/fig_ref] , and on the other hand, data suggesting that NAFL confers a substantial risk of disease progression and the development of comorbidities [fig_ref] Table 2: Evidence in favor of the poor prognosis of NAFL patients [/fig_ref]. natural history retrospective no progression to cirrhosis or liver-related complications; low number of patients [bib_ref] Natural history of NAFLD: Remarkably benign in the absence of cirrhosis, Day [/bib_ref] no 132 cirrhosis outcome, overall mortality, liver-related mortality retrospective poor outcome only in patients with ballooning, Mallory hyaline or fibrosis [bib_ref] Risk of cardiovascular disease in patients with nonalcoholic fatty liver disease, Targher [/bib_ref] no 209 liver-related mortality retrospective only fibrosis as independent factor of liver-related mortality [bib_ref] Fibrosis stage but not NASH predicts mortality and time to development of..., Hagström [/bib_ref] no 646 liver-related mortality, overall survival retrospective only fibrosis associated with liver-related mortality and overall survival [bib_ref] Systematic review of risk factors for fibrosis progression in non-alcoholic steatohepatitis, Argo [/bib_ref] yes 221 natural history of fibrosis progression; predictors of progression to F3 fibrosis retrospective systematic review age and inflammation are predictors of progression to advanced fibrosis [bib_ref] Long-term follow-up of patients with NAFLD and elevated liver enzymes, Ekstedt [/bib_ref] Yes (n = 60) 129 survival and cause of death retrospective survival is lower in NASH but not in simple steatosis; low number of patients; heterogeneity of patient population; [bib_ref] Final results of a long-term, clinical follow-up in fatty liver patients, Dam-Larsen [/bib_ref] no 170 NAFLD 246 ALD risk of cirrhosis development; risk of death retrospective patients with simple steatosis have similar survival to Danish population [bib_ref] Disease progression of non-alcoholic fatty liver disease: A prospective study with paired..., Wong [/bib_ref] no 547 potential risk factors (index biopsy) for survival and cirrhosis development retrospective long-term follow-up study of ref. #31 [bib_ref] Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: Implications..., Mcpherson [/bib_ref] No 619 long-term prognostic relevance of histologic features retrospective only fibrosis showed decreased overall survival, low number of patients with NASH and without fibrosis [bib_ref] Liver Fibrosis, but No Other Histologic Features, Is Associated With Long-term Outcomes..., Angulo [/bib_ref] Abbreviations: NAFLD, non-alcoholic fatty liver disease; ALD, alcoholic liver disease. Abbreviations: NAFLD, non-alcoholic fatty liver disease; NAFL, non-alcoholic fatty liver; NASH, non-alcoholic steatohepatitis.
## Evidence in favor of the good prognosis of nafl patients
Several studies have argued against the possibility that simple steatosis can progress to fibrosis progression and is associated with poor clinical outcome [bib_ref] Natural history of NAFLD: Remarkably benign in the absence of cirrhosis, Day [/bib_ref]. In 12 patients with bland steatosis (no inflammation at all), a repeat liver biopsy was performed after 7.6-16 years, which did not show progression to cirrhosis and patients had no serious liver complications [bib_ref] The natural history of nonalcoholic fatty liver: A follow-up study, Teli [/bib_ref]. Another study from Younossi et al. demonstrated that only the presence of fibrosis of any degree remained an independent predictor of liver-related mortality in the multivariate analysis [bib_ref] Fibrosis stage but not NASH predicts mortality and time to development of..., Hagström [/bib_ref]. In a large study (n = 646 patients) with a mean follow up of 20 years (0-40 years range), Hagstrom et al. showed that fibrosis stage is directly associated with liver-related mortality and overall survival [bib_ref] Systematic review of risk factors for fibrosis progression in non-alcoholic steatohepatitis, Argo [/bib_ref]. A systematic review including 10 longitudinal NASH biopsy studies comprising a total of 221 patients described that age and portal inflammation on initial biopsy are independent predictors of progression to advanced fibrosis in patients with NASH [bib_ref] Long-term follow-up of patients with NAFLD and elevated liver enzymes, Ekstedt [/bib_ref]. In a study comprising 728 adults and 205 children from the Nonalcoholic Steatohepatitis Clinical Research Network increased portal chronic inflammation was associated with progressive NAFLD in both adults and children [bib_ref] Long term prognosis of fatty liver: Risk of chronic liver disease and..., Dam-Larsen [/bib_ref]. One hallmark study of Ekstedt et al. analyzed survival and causes of death in a cohort study of 129 patients diagnosed with biopsy-proven NAFLD in comparison with a matched reference population. From the initial cohort, 88 patients were reevaluated, and in 68, a repeat liver biopsy could be performed. The authors observed that the overall survival of NAFLD patients was significantly lower than that of the reference population. When separating this population in histologically confirmed NASH or steatosis with no or mild inflammation survival was significantly reduced only in NASH patients [bib_ref] Final results of a long-term, clinical follow-up in fatty liver patients, Dam-Larsen [/bib_ref]. It is important to recognize that this study was limited by heterogeneity of the patient population and the low number of patients included. A follow-up study on this important work by the same group focused on different causes of mortality [bib_ref] Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after..., Ekstedt [/bib_ref]. The core finding of this work was that a fibrosis stage of 3 or 4, independent of NAS/presence of NASH, was associated with an increased risk for overall, cardiovascular and liver related mortality. NASH without significant fibrosis (stages 0-2) was associated only with an increased mortality risk from HCC. Patients with a NAS < 5 (NAFL or borderline NASH) with fibrosis stages 0-2 exhibit no significantly increased mortality risk compared to the control population. Of note, the proportion of patients with stage 3-4 fibrosis was similar in patients with NAS < 5 (5%) and in patients with NAS ≥ 5 (5%). A limitation of this study was that the reference cohort was selected from population data, without any information on BMI, factors of the metabolic syndrome or hepatic steatosis.
Two studies by Dam-Larsen et al. on individuals with histologically confirmed steatosis/fatty liver without inflammation or fibrosis compared those with NAFLD (170) to patients with alcoholic liver disease (AFLD; 246). In this comparison it was observed that the prognosis in patients with NAFLD was good as only 1.2% progressed to cirrhosis compared to 22% of AFLD [bib_ref] Disease progression of non-alcoholic fatty liver disease: A prospective study with paired..., Wong [/bib_ref] [bib_ref] Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: Implications..., Mcpherson [/bib_ref] ; 48 (28%) of NAFLD patients and 188 (76%) of AFLD patients died during the follow up period (median 12.8 years). Cardiovascular events (37%) and extrahepatic cancer (17%) were the leading causes in NAFLD, and cardiovascular events (20%) followed by cirrhosis (17%) were the main causes of death in AFLD. Central limitations of this study was that histological assessment was limited to steatosis and fibrosis and that BMI was only available for 35% of AFLD patients.
Angulo et al. performed a retrospective longitudinal analysis of 619 patients with NAFLD at centers in the United States, Europe, and Thailand over a period between 1975 and 2005 [bib_ref] Liver Fibrosis, but No Other Histologic Features, Is Associated With Long-term Outcomes..., Angulo [/bib_ref]. The central finding was that a total of 193 patients (33.2%) died or underwent liver transplantation. In multivariable analysis, only patients with fibrosis had shorter overall survival in comparison with patients without fibrosis. Limitations of the study included the low number of patients with NASH but without fibrosis, and that the mortality in the control group without NASH and fibrosis was significantly higher than previously reported.
The studies summarized herein [fig_ref] Table 1: Evidence in favor of the good prognosis of NAFL patients [/fig_ref] show unmistakably that the main liver-related risk in NAFLD arises from advanced fibrosis and cirrhosis and that at least liver-related mortality is driven by NASH. However, this does not in itself imply that NAFL or steatosis without significant inflammation is without risk.
## Evidence of progression from steatosis to fibrosing-steatohepatitis and mortality in nafl
Although the above described early studies demonstrated a different outcome in NAFLD patients based on the presence or absence of NASH, there is growing evidence challenging the dogma that NAFL is a non-progressive disease. Contrary to the claim that NAFL is a benign condition, several studies provide data objecting this statement [fig_ref] Table 2: Evidence in favor of the poor prognosis of NAFL patients [/fig_ref]. In a prospective study of 52 patients with paired liver biopsies, it was observed that 28% of patients with a NAS below 5 had fibrosis progression. Moreover, 17 of 29 (58%) NAFL patients (NAS < 3) progressed to borderline NASH or NASH [bib_ref] Causal relationship of hepatic fat with liver damage and insulin resistance in..., Dongiovanni [/bib_ref]. The conclusions of this study should be taken with caution because all patients in this study received lifestyle advice and metabolic monitoring, which might have altered the natural course of the disease and because the sample size in each subgroup is small.
In another study data of 108 patients with NAFLD were analyzed, who had serial liver biopsies after a median of 6.6 years (1.3-22.6 years) [bib_ref] Fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: A systematic review..., Singh [/bib_ref]. The authors observed progression to NASH in 44% of patients with baseline NAFL. Of 27 NAFL patients 10 (37%) progressed to fibrosis, all 10 exhibited NASH in the follow-up biopsy. Of those NAFL patients with fibrosis progression 6 reached stage 3 fibrosis at follow-up biopsy. T2DM was present in 80% of NAFL patients with fibrosis progression but only in 25% of NAFL patients without progression. These findings are in line with a study on seventy patients with untreated NAFLD (25 with simple steatosis and 45 with NASH) with follow up biopsy after a mean duration of 3.7 years between 1998 and 2009 [bib_ref] LIDO Study Group. A systematic review of follow-up biopsies reveals disease progression..., Pais [/bib_ref]. A substantial proportion (64%) of the 25 patients with simple steatosis on initial biopsy progressed to NASH. Severe ballooning was observed in eight and bridging fibrosis in six NAFL patients progressing to NASH. Of note ballooning progression and fibrosis was paralleled with a reduction in ALT. This finding supports the notion that classic liver enzymes (ALT and AST) are not useful for diagnosis, follow up, or disease monitoring in NAFLD [bib_ref] The histological course of nonalcoholic fatty liver disease: A longitudinal study of..., Adams [/bib_ref]. Previous studies found high proportions of patients with liver enzymes in normal range with NASH or even advanced fibrosis [bib_ref] Pathologic criteria for nonalcoholic steatohepatitis: Interprotocol agreement and ability to predict liver-related..., Younossi [/bib_ref] [bib_ref] A list of members of the Nonalcoholic Steatohepatitis Clinical Research Network can..., Brunt [/bib_ref]. Our own unpublished data of over 250 obese individuals with biopsy proven NAFLD resulted in significant differences of AST and ALT between patients with steatosis and patients with NASH. However, to allow discrimination of steatosis and NASH cut off concentrations around 19 U/l would have to be chosen with insufficient sensitivities and specificities (unpublished data). In a sample of outpatients in a gastroenterology unit, who were diagnosed with liver steatosis by ultrasound only, we found that cardiovascular and metabolic risk factors were significantly more common than in patients without liver steatosis [bib_ref] Evaluation of Biomarkers of NAFLD in a Cohort of Morbidly Obese Patients, Kälsch [/bib_ref]. This effect was independent of the underlying cause of the outpatient visit. This finding suggests that liver steatosis detectable by ultrasound may already be sufficient to indicate elevated cardiovascular risk, independent of definite NAFL or NASH diagnosis.
Mendelian randomization is an epidemiological method that avoids confounding factors (such as diet or physical activity) and is aimed to avoid reverse causation in observational studies [bib_ref] Clinical and histologic spectrum of nonalcoholic fatty liver disease associated with normal..., Mofrad [/bib_ref]. Applying mendelian randomization Dongiovanni et al. analyzed data of more than 9000 individuals from the liver biopsy cohort (LBC), the Swedish Obese Subjects Study (SOS), and the population-based Dallas Heart Study (DHS) [bib_ref] Nonalcoholic fatty liver disease in severely obese subjects, Gholam [/bib_ref]. The risk alleles of PNPLA3, TM6SF2, GCKR and MBOAT7 and their effect on hepatic steatosis were evaluated in a polygenic risk score. The extent of hepatic steatosis was associated with liver damage, insulin resistance, dyslipidemia and hypertension. Strikingly the impact of genetic variants on liver damage was proportional to their effect on lipid accumulation in the liver. Within the LBC, hepatic fat and fibrosis were associated independently of inflammation, suggesting a causal effect of steatosis on fibrogenesis. Overall, the data of this meticulous analysis of a very large number of patients suggests that at least the genetically determined proportion of hepatic steatosis is likely to be a causal risk factor for the development of liver fibrosis and extent of liver damage in NAFLD.
A systematic review and meta-analysis was performed by Singh et al. on 11 studies, including 411 patients with biopsy-proven NAFL and NASH who underwent paired liver biopsies at least one year apart. From six studies, the fibrosis progression rate (FBR) in 133 NAFL patients could be calculated, with 52 (39.1%) patients developing progressive fibrosis. From seven studies, FBR for 116 patients with NASH was derived, with 40 (34.5%) developing progressive fibrosis. The annual FBR was 0.07 stages in NAFL and 0.14 stages in NASH, resulting in a progression of one stage in 14.3 years for NAFL and in 7.1 years for NASH. It should be noted that while the rate of progression was slower in NAFL, the proportion of patients who actually exhibited fibrosis progression did not differ significantly between NAFL and NASH [bib_ref] Risk of severe liver disease in nonalcoholic fatty liver disease with normal..., Fracanzani [/bib_ref]. Limitations of this meta-analysis include the heterogeneity of the studies analyzed, and that seven of them enrolled less than 25 patients.
Adams et al. analyzed the natural history of 103 patients, who underwent serial liver biopsies with a mean interval between biopsies of 3.2 ± 3.0 years [bib_ref] Patients with ultrasound diagnosis of hepatic steatosis are at high metabolic risk, Kälsch [/bib_ref]. When excluding cirrhotic patients, who cannot further progress, the annual FBR in these patients was 0.09 ± 0.67, which corresponds to the overall FBR of NAFLD found by Singh et al. [bib_ref] Risk of severe liver disease in nonalcoholic fatty liver disease with normal..., Fracanzani [/bib_ref]. NASH was present in 96 (93%) of the 103 patients. Of the three patients with bland steatosis on initial biopsy, two developed NASH. All four patients with steatosis and nonspecific inflammation on initial biopsy developed NASH. The proportion of patients who progressed in fibrosis stage (overall 37%) did not differ significantly between NAFL (34.4%) and NASH (53.8%). The main determinant of fibrosis progression over time was the fibrosis stage on initial biopsy, with lower stages (0-2) at higher risk. Progressive inflammation, steatosis, and ballooning were not associated with fibrosis progression. Of note, progression of fibrosis stage was associated with a reduction in ALT and AST as well as severity of steatosis. The findings of this study are limited by the very low number of NAFL patients (9).
In the above described study by Eksted et al. that demonstrated increased mortality only in NASH patients [bib_ref] Final results of a long-term, clinical follow-up in fatty liver patients, Dam-Larsen [/bib_ref] , a histological course was also analyzed in available repeat biopsies. Progressive fibrosis was observed in 29 of 70 (41%) of NAFLD patients. In patients with steatosis without fibrosis, 17 of 36 (47%) developed fibrosis. It should be noted that this study was performed in patients with elevated serum AST or ALT, which preselects patients with more severe disease course. Moreover, patients with NASH in this cohort were significantly older (54.5 ± 12.4 years) than those with steatosis, with or without unspecific inflammation (46.7 ± 12.3 years). The most recent analysis from a matched cohort study of all Swedish patients with biopsy confirmed NAFLD (10568 patients) found an increased risk of mortality for all histological stages [bib_ref] Mendelian randomization: Using genes as instruments for making causal inferences in epidemiology, Lawlor [/bib_ref]. Compared to age, sex, and location matched controls of the general population, the mortality risk was 1.71-times higher in simple steatosis, 2.14-times higher in non-fibrotic NASH, 2.44-times higher in non-cirrhotic fibrosis, and 3.79-times higher in cirrhosis. The NAFLD-associated excess mortality derived from extrahepatic cancer, cirrhosis, cardiovascular disease and hepatocellular carcinoma.
The results summarized above collectively support the concept that NAFL can progress to NASH with fibrosis, mainly in patients who have poor metabolic control. Some studies suggest that fibrosis can even develop or progress without a transition from NAFL to NASH. Overall roughly 30-40% of NAFL patients seem to exhibit progression of fibrosis in studies with sequential biopsies. In some cases, this progression occurred within a relatively short time frame. These observations should be taken with some caution. On the one hand, sampling variability between two liver biopsies due to the inhomogeneous distribution of inflammation and ballooning might lead to incorrect classification of a region with lower disease activity, and subsequently 'disease progression' upon second biopsy in a region of representative disease activity. On the other hand, we need to consider the possibility of selection bias in studies which examined only those with follow-up biopsies, as these are usually individuals most at risk and/or likely to progress [bib_ref] Non-alcoholic fatty liver-perhaps not so benign, Adams [/bib_ref]. However, a proportion of 30-40% with disease progression would imply a substantial variability or observer error in histological assessment.
Regarding mortality, there is no doubt that histologically proven NASH and in particular advanced fibrosis and cirrhosis are associated with a higher risk of overall and especially liver-related mortality. Though, NAFLD as a whole exhibits increased mortality rates compared to the general population. A recent analysis suggested that up to 29% of healthy controls may actually have NAFLD [bib_ref] How healthy are the "healthy volunteers, Takyar [/bib_ref] , masking a part of the impact of NAFLD on health and clinical outcomes. Some studies referred to above used population based controls without correction for BMI or metabolic morbidities to compare mortality to NAFLD or NAFL patients. This would probably underestimate the true effect of NAFLD on mortality, especially as non-cirrhotic stages and even compensated cirrhosis in NAFLD are still widely underdiagnosed in daily practice [bib_ref] Patterns and predictors of mortality and disease progression among patients with non-alcoholic..., Canbay [/bib_ref].
EASL Clinical Practice Guidelines indicate that NAFL patients without worsening metabolic risk factors should be monitored at 2-3-year intervals considering the low risk of progression for this group of patients, although this may be a too long an interval for patients with an underestimated risk of progression. Thus, it is imperative to identify reliable risk factors for progression and subsequently improve the surveillance of patients with NAFL at risk of progression. Currently the disease progression of any NAFLD patient cannot be excluded with certainty, and thus it is mandatory to improve the efficacy of surveillance to prevent as many liver-related and other metabolic complications as possible.
## Pathophysiological and molecular mechanisms of non-alcoholic fatty liver disease
NAFLD is the consequence of excess calorie intake (from overnutrition and inactivity) and subsequent adipose tissue hypertrophy, with different combinations of genetic, epigenetic, environmental and metabolic factors interacting in development and influencing the severity of the disease. This complex genesis makes understanding of this disease a true challenge; though, it is now widely accepted that NAFLD is a multifactorial disease . The previous "two sequential hits pathogenesis" theory was based on insulin resistance (IR) and its stimulation of triglyceride (TG) deposits within the liver as initial hit, which is followed by a "second hit" induced by oxidative stress or ATP depletion, which further induces apoptosis, inflammation and fibrosis [bib_ref] Steatohepatitis: A tale of two "hits, Day [/bib_ref]. This theory has been replaced by the "multiple parallel hits theory", in which several factors act in parallel and different combinations, sometimes synergistically, generating NAFLD [bib_ref] The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD), Buzzetti [/bib_ref]. This situation limits the development of pharmacological interventions, which could help the majority of patients, in contrast to HBV or HCV, where a single factor is causative for the disease. Patients with NAFLD seem to have individually different disease courses with varying contribution of IR and genetics [bib_ref] Nonalcoholic fatty liver disease in severely obese subjects, Gholam [/bib_ref] and some of them directly develop NASH while others (seem to) progress sequentially from NAFL to NASH [bib_ref] Mechanisms of NAFLD development and therapeutic strategies, Friedman [/bib_ref]. Approximately 25-30% of patients with simple steatosis will develop NASH, and thus it is important to understand the molecular mechanisms involved and how to treat or prevent this complex disease. In this chapter of the review, we will discuss known key pathogenic factors involved in the genesis of NAFLD on a molecular level.
## Genetic factors involved in nafld
By now it is clear that genetic factors contribute to the pathogenesis of NAFLD. Genome-wide association and gene expression profiling have identified genes with a number of polymorphisms associated with NAFLD development such as Patatin-like phospholipase domain containing 3 (PNPLA3), Transmembrane protein 6 superfamily member 2 (TM6SF2), GCKR, CETO, MBOAT7, MTTP, SOD2, GST, APOC3, IL-6, TNF-a, PPAR-a, just to mention a few. In general, these genes or their products are involved in one or more of the following processes: lipid and glucose metabolism, insulin signaling pathways, oxidative stress (OS), detoxification, inflammatory pathways, and fibrogenesis. The two most studied and best characterized are PNPLA3 and TM6SF2. PNPLA3 is highly expressed in the liver and adipose tissue [bib_ref] Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic..., Huang [/bib_ref].
Although the function of PNPLA3 is not completely defined, it is considered to have lipogenic transacetylase activity. Presence of the PNPLA3 SNP rs738409 (I148M) promotes lipid storage in the liver, mainly because of a deficient glycerolipid hydrolase activity [bib_ref] Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic..., Huang [/bib_ref]. This effect seems to be more pronounced in normal-weight subjects than in overweight subjects [bib_ref] Influence of the PNPLA3 rs738409 Polymorphism on Non-Alcoholic Fatty Liver Disease and..., Oniki [/bib_ref] [bib_ref] PNPLA3 gene polymorphism and response to lifestyle modification in patients with nonalcoholic..., Shen [/bib_ref]. The PNPLA3 variant allele rs738409 C>G has been associated with the risk and severity of NAFLD (inflammation, and progression to fibrosis) and even with HCC development [bib_ref] PNPLA3 as a Genetic Determinant of Risk for and Severity of Non-alcoholic..., Salameh [/bib_ref]. PNPLA3 is a strong modifier of the natural history of NAFLD and can be considered as a potential target for therapy. . Factors contributing to development of NASH or progression of NAFL to NASH. NAFLD develops due to excess calorie intake, which leads to adipose tissue hypertrophy. Adipose tissue confronted with nutrient overload will increase lipolysis and change secretion of adipokines. This altered systemic situation of nutrients, lipids and adipokines, ultimately resulting in NAFLD, is modulated by genetic, epigenetic, environmental and metabolic factors (outer ring). Various molecular and cellular mechanisms (cogs) interact in development of NAFLD and NASH. Abbrevations: ATP: adenosine triphosphate; β-oxidation: beta oxidation of lipid components within mitochondria; DNL: de novo lipogenesis; ER stress: endoplasmatic reticulum stress; FFA: free fatty acids; UPR: unfolded protein response.
## Genetic factors involved in nafld
By now it is clear that genetic factors contribute to the pathogenesis of NAFLD. Genome-wide association and gene expression profiling have identified genes with a number of polymorphisms associated with NAFLD development such as Patatin-like phospholipase domain containing 3 (PNPLA3), Transmembrane protein 6 superfamily member 2 (TM6SF2), GCKR, CETO, MBOAT7, MTTP, SOD2, GST, APOC3, IL-6, TNF-a, PPAR-a, just to mention a few. In general, these genes or their products are involved in one or more of the following processes: lipid and glucose metabolism, insulin signaling pathways, oxidative stress (OS), detoxification, inflammatory pathways, and fibrogenesis. The two most studied and best characterized are PNPLA3 and TM6SF2. PNPLA3 is highly expressed in the liver and adipose tissue [bib_ref] Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic..., Huang [/bib_ref].
Although the function of PNPLA3 is not completely defined, it is considered to have lipogenic transacetylase activity. Presence of the PNPLA3 SNP rs738409 (I148M) promotes lipid storage in the liver, mainly because of a deficient glycerolipid hydrolase activity [bib_ref] Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic..., Huang [/bib_ref]. This effect seems to be more pronounced in normal-weight subjects than in overweight subjects [bib_ref] Influence of the PNPLA3 rs738409 Polymorphism on Non-Alcoholic Fatty Liver Disease and..., Oniki [/bib_ref] [bib_ref] PNPLA3 gene polymorphism and response to lifestyle modification in patients with nonalcoholic..., Shen [/bib_ref]. The PNPLA3 variant allele rs738409 C>G has been associated with the risk and severity of NAFLD (inflammation, and progression to fibrosis) and even with HCC development [bib_ref] PNPLA3 as a Genetic Determinant of Risk for and Severity of Non-alcoholic..., Salameh [/bib_ref]. PNPLA3 is a strong modifier of the natural history of NAFLD and can be considered as a potential target for therapy. . Factors contributing to development of NASH or progression of NAFL to NASH. NAFLD develops due to excess calorie intake, which leads to adipose tissue hypertrophy. Adipose tissue confronted with nutrient overload will increase lipolysis and change secretion of adipokines. This altered systemic situation of nutrients, lipids and adipokines, ultimately resulting in NAFLD, is modulated by genetic, epigenetic, environmental and metabolic factors (outer ring). Various molecular and cellular mechanisms (cogs) interact in development of NAFLD and NASH. Abbrevations: ATP: adenosine triphosphate; β-oxidation: beta oxidation of lipid components within mitochondria; DNL: de novo lipogenesis; ER stress: endoplasmatic reticulum stress; FFA: free fatty acids; UPR: unfolded protein response.
The TM6SF2 SNP rs58542926 C>T confers a significant genetic susceptibility for NAFLD and disease severity [bib_ref] Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic..., Kozlitina [/bib_ref] [bib_ref] The PNPLA3 variant associated with fatty liver disease (I148M) accumulates on lipid..., Basuray [/bib_ref] ; surprisingly, this mutation might confer cardiovascular protection [bib_ref] The dual and opposite role of the TM6SF2-rs58542926 variant in protecting against..., Pirola [/bib_ref]. The TM6SF2 variant has been associated with higher serum ALT and AST levels, and reduced plasma levels of TG and LDL-cholesterol [bib_ref] Circulating triacylglycerol signatures and insulin sensitivity in NAFLD associated with the E167K..., Zhou [/bib_ref]. In a mouse model, TM6SF2 silencing using an adeno-associated virus (AAV) vector increases hepatic TG levels and decreases plasma levels of TG, LDL-and HDL-cholesterols, demonstrating a key role of the TM6SF2 gene in hepatic TG secretion regulation [bib_ref] Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic..., Kozlitina [/bib_ref].
Although genetic testing in NAFLD currently remains in the realm of research, it is likely that this could soon be in routine clinical practice, either as a prognostic indicator, or to risk stratify individuals for treatment. For example, individuals who are carriers of one or both alleles may be more at risk of progressive liver disease and therefore, could be targeted for more intensive surveillance, and/or receive therapy. However, it must be clear that the presence of the above described SNPs increases the risk to develop NAFLD or for a more severe course, but do not inevitably result in NASH.
## Insulin resistance
IR is one of the first metabolic changes occurring in overweight and obese individuals, associated with the systemic inflammatory state induced by obesity, often leading to more severe metabolic consequences. Adipose tissue inflammation and an altered adipokine profile promote peripheral IR. Hepatic IR with continued nutrient excess leads to lipotoxicity, ER stress, mitochondrial dysfunction, impaired autophagy, altered gut microbiota, which all promote progression from NAFL to NASH [bib_ref] The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD), Buzzetti [/bib_ref].
The detailed molecular mechanisms of IR that promote NAFLD development remain unclear. What is known is that under a chronic over-nutrition state and adipose tissue inflammation, an altered adipokine profile promotes a systemic inflammatory state, which further induces peripheral IR [bib_ref] Inflammation is necessary for long-term but not short-term high-fat diet-induced insulin resistance, Lee [/bib_ref] [bib_ref] Adipokines in inflammation and metabolic disease, Ouchi [/bib_ref]. Adipose tissue IR promotes lipolysis, thus leading to an increase in circulating free fatty acids (FFA), which are then taken up by the liver for lipogenesis. In addition, gluconeogenesis and de novo lipogenesis (DNL) are stimulated [bib_ref] The Subtle Balance between Lipolysis and Lipogenesis: A Critical Point in Metabolic..., Saponaro [/bib_ref]. In general, the transition from NAFL to NASH occurs in the presence of IR, however, the triggering events and molecular mechanisms regulating disease progression remain poorly understood.
## Lipid metabolism and lipotoxicity
Excessive hepatic accumulation of fatty acids constitutes the initial step of steatosis. In NAFLD patients, 59% of FFA are derived from deregulated lipolysis in adipose tissue, while the remaining 26% come from hepatic DNL, and only 15% originate from the diet [bib_ref] Sources of fatty acids stored in liver and secreted via lipoproteins in..., Donnelly [/bib_ref]. Patients with high liver fat content exhibit at least threefold higher levels of DNL when compared with individuals with low liver fat content despite similar levels of FFA flux from the adipose tissue; therefore, DNL is a major contributor to the development of steatosis, apart from lipolysis [bib_ref] Increased de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic..., Lambert [/bib_ref]. Activation of the transcription factors sterol regulatory binding protein-1c (SREBP-1c) and the carbohydrate response element-binding protein (ChREBP) are crucial steps for DNL and FFA uptake [bib_ref] SREBP-regulated lipid metabolism: Convergent physiology-divergent pathophysiology, Shimano [/bib_ref]. Dietary cholesterol, dietary saturated fatty acid or hyperinsulinemia activate SREBP1c; chREBP is activated by hyperglycemia [bib_ref] The Scap/SREBP pathway is essential for developing diabetic fatty liver and carbohydrate-induced..., Moon [/bib_ref]. SREBP1c and ChREBP transactivate genes involved in FFA metabolism, including those required for FFA uptake (e.g., FABP3, FABP4, CD36), DNL (e.g., FASN and ACC1) and triglycerides (TG) synthesis (e.g., GPAT). Nevertheless, lipid accumulation is not necessarily harmful. The type of lipid and not the amount of lipids seems to determine lipotoxicity in hepatocytes [bib_ref] Good fat/bad fat, Mcclain [/bib_ref]. Both TG storage in lipid droplets or TG export via VLDL secretion appear to have a protective role for the liver. In fact, transgenic mice overexpressing diacylglycerol acyltransferase 2 (DGAT2), which catalyzes the final step of TG synthesis, developed hepatic steatosis with reduced necro-inflammation [bib_ref] Hepatic insulin resistance in mice with hepatic overexpression of diacylglycerol acyltransferase 2, Jornayvaz [/bib_ref]. Not lipid accumulation per se, but lipid composition seems to be crucial for disease progression from NAFL to NASH. While some lipid species are lipotoxic, others may protect cells from lipotoxicity. In contrast to unsaturated FFA, saturated FFA are detrimental to cell viability [bib_ref] Hepatic lipid partitioning and liver damage in nonalcoholic fatty liver disease: Role..., Li [/bib_ref]. Unsaturated FFA (e.g., oleic acid) induce TG formation, which results in a mechanism of defense against the pro-apoptotic stimuli of large loads of saturated FFA [bib_ref] Triglyceride accumulation protects against fatty acid-induced lipotoxicity, Listenberger [/bib_ref] [bib_ref] Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis..., Ricchi [/bib_ref]. Several other lipid metabolites, such as diacylglycerol, lysophosphatidyl choline, free cholesterol, cholesterol ester, ceramide, and bile acids, also accumulate in hepatic cells, and they promote ER stress, which leads to the execution of pro-apoptotic pathways and stimulates pro-inflammatory signals that activate Kupffer cells and HSCs, and induce mitochondrial dysfunction and ROS [bib_ref] The interaction of hepatic lipid and glucose metabolism in liver diseases, Bechmann [/bib_ref] [bib_ref] Free fatty acids repress small heterodimer partner (SHP) activation and adiponectin counteracts..., Bechmann [/bib_ref] [bib_ref] Cell death mechanisms in human chronic liver diseases: A far cry from..., Mazzolini [/bib_ref] [bib_ref] Molecular mechanisms of lipotoxicity and glucotoxicity in nonalcoholic fatty liver disease, Mota [/bib_ref]. Alterations in bile acid composition and excretion contribute additionally to liver injury in NAFLD [bib_ref] Free fatty acids repress small heterodimer partner (SHP) activation and adiponectin counteracts..., Bechmann [/bib_ref] [bib_ref] Molecular mechanisms of lipotoxicity and glucotoxicity in nonalcoholic fatty liver disease, Mota [/bib_ref] [bib_ref] The role of bile acids in nonalcoholic fatty liver disease and nonalcoholic..., Chow [/bib_ref].
## Er stress
Endoplasmic reticulum (ER) stress is the result of the accumulation of unfolded/misfolded proteins within the ER, which is enhanced by oxidative stress (OS) [bib_ref] Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease, Lebeaupin [/bib_ref]. Under healthy physiological conditions, reactive oxygen species (ROS) are produced in hepatocytes (mainly within the mitochondria), i.e., by FFA oxidation. However, when ROS are generated in excess, are not eliminated, or converted (e.g., to a less toxic compound) they can initiate cell death [bib_ref] Mitochondrial dysfunction in NASH: Causes, consequences and possible means to prevent it, Begriche [/bib_ref]. Excessive ROS production leads to lipid peroxidation and protein misfolding due to oxidation of polyunsaturated fatty acids and sulfhydryl group proteins, respectively, which are common events in NAFLD [bib_ref] Mutual interaction between oxidative stress and endoplasmic reticulum stress in the pathogenesis..., Fujii [/bib_ref]. ER stress stimulates lipid droplets accumulation in the liver in a SREBP-dependent process [bib_ref] SREBP transcription factors: Master regulators of lipid homeostasis, Eberlé [/bib_ref]. It has been demonstrated that SREBPs, transcriptional master-regulators for fatty acid and cholesterol synthesis, are upregulated by OS and ER stress leading to lipid accumulation generated by DNL, TG, and cholesterol deposits in the liver [bib_ref] Transcriptional mediators of lipid homeostasis, Horton [/bib_ref]. Conversely, increased hepatocyte lipid content can also initiate and perpetuate chronic ER stress [bib_ref] Lipid-induced endoplasmic reticulum stress in liver cells results in two distinct outcomes:..., Achard [/bib_ref]. In addition, OS and ER stress interfere with lipoprotein secretion from the liver [bib_ref] The unfolded protein response transducer IRE1α prevents ER stress-induced hepatic steatosis, Zhang [/bib_ref]. In addition, the unfolded protein response (UPR) is linked to the activation of DNL pathways, further increasing steatosis [bib_ref] Role of mitochondria in nonalcoholic fatty liver disease-from origin to propagation, Grattagliano [/bib_ref]. Chronic activation of UPR also induces hepatocyte death and inflammation. The interaction of increased lipid uptake, DNL, and reduced lipoprotein secretion result in a fatty liver. The continued generation of ROS, i.e., by sustained FFA oxidation, can promote progression of NAFL to NASH.
Thus, the presence of OS and, consequently, ER stress are key players in the pathogenesis of NAFLD and progression to NASH. Importantly, ER stress can be pharmacologically targeted, e.g., by the use of antioxidants (vitamin E) aiming at reducing excessive ROS production.
## Mitochondrial dysfunction
Increased mitochondrial activity is crucial to protect hepatocytes from the deleterious effect of FFA deposition since FFAs are oxidized by mitochondrial β-oxidation [bib_ref] Mice in the early stage of liver steatosis caused by a high..., Lee [/bib_ref] [bib_ref] CHOP links endoplasmic reticulum stress to NF-κB activation in the pathogenesis of..., Willy [/bib_ref]. NAFL induces PPAR-α, which promotes FFA delivery to the mitochondria via CPT-1. However, during NAFLD development enhanced mitochondrial FFA β-oxidation leads to OS due to exacerbated leakage of electrons from the electron transport chain. This disrupts the balance between FFA removal and ROS generation [bib_ref] Mitochondrial abnormalities in non-alcoholic steatohepatitis, Caldwell [/bib_ref]. Elevated ROS concentrations in the mitochondrial environment due to excess FFA may further damage the organelle function establishing a vicious circle. Hepatic mitochondria are structurally and molecularly altered in NAFLD [bib_ref] Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease, Begriche [/bib_ref] , including electron transport chain alterations, and reduced ATP synthesis, which further induces ER stress. Increased mitochondrial cholesterol deposits induce changes in membrane permeability, and mitochondrial dysfunction; both processes have been linked to the progression of NAFL to NASH [bib_ref] Mitochondrial dysfunction in NASH: Causes, consequences and possible means to prevent it, Begriche [/bib_ref]. When ROS are produced in excess, both mitochondrial and nuclear DNA are damaged, impairing the transcription of mitochondrial proteins involved in metabolism and organelle biogenesis [bib_ref] Fatty liver is associated with impaired activity of PPARγ-coactivator 1α (PGC1α) and..., Aharoni-Simon [/bib_ref] [bib_ref] Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is..., Koliaki [/bib_ref] , which further aggravates the situation. Taken together impaired mitochondrial lipid peroxidation under a continuous supply of FFA leads to a vicious circle of excess ROS generation, ER stress, lipid accumulation, and mitochondrial impairment or damage. Currently, there are no known agents to inhibit this vicious circle apart from drastically reducing the influx of additional FFA, i.e., radical lifestyle change.
## Nafld patients are more susceptible to liver injury generated by drugs and alcohol
As described above simple steatosis is still considered generally a benign disorder, unlike NASH. Apart from the above described direct liver related and cardiovascular consequences of NAFL and NASH, several studies have shown that NAFLD worsens most conditions that injure the liver, suggesting increased sensitivity to viral hepatitides, alcohol, and drugs [bib_ref] Overweight patients are more susceptible for acute liver failure, Canbay [/bib_ref]. It is generally accepted that NAFLD patients will be more likely to be injured than healthy individuals by the same drug and dose [bib_ref] Fat accretion in a subpopulation of hepatocytes as a strategy to protect..., Fromenty [/bib_ref] [bib_ref] Acetaminophen-induced liver injury in obesity and nonalcoholic fatty liver disease, Michaut [/bib_ref]. In addition, drug-induced liver injury (DILI) remains a major concern for investigators in NASH clinical trials, and there are no evidence-based recommendations for best practices related to DILI in NAFLD patients [bib_ref] Consensus: Guidelines: Best practices for detection, assessment and management of suspected acute..., Regev [/bib_ref]. In fact, the US Drug Induced Liver Injury Network (DILIN) reported recently that DILI in patients with chronic liver disease (including hepatitis C and NAFLD) was associated with more frequent adverse events, including mortality [bib_ref] Features and Outcomes of 899 Patients with Drug-Induced Liver Injury: The DILIN..., Chalasani [/bib_ref]. Different predisposing factors inherent to patients are known to enhance the risk of DILI including NAFLD and obesity [bib_ref] A prospective study of acute drug-induced liver injury in patients suffering from..., Tarantino [/bib_ref] [bib_ref] Drug-induced toxicity on mitochondria and lipid metabolism: Mechanistic diversity and deleterious consequences..., Begriche [/bib_ref] [bib_ref] Patients with Chronic Liver Disease Suggestive of Nonalcoholic Fatty Liver Disease May..., Lammert [/bib_ref]. In a prospective study on 248 patients with either HCV or NAFLD, NAFLD patients had a 3.95-fold risk of DILI compared to HCV [bib_ref] A prospective study of acute drug-induced liver injury in patients suffering from..., Tarantino [/bib_ref]. NAFLD is associated with obesity, diabetes, hyperlipidemia, and other chronic diseases that require long-term and multiple drug administration. Therefore, increasing the risk of hepatic adverse effects or toxicity. As such, clinicians should be aware of the added risk of liver injury in patients with chronic liver diseases, especially in NAFLD [bib_ref] Drug-induced liver disease, Lewis [/bib_ref] [bib_ref] Risk factors for idiosyncratic drug-induced liver injury, Chalasani [/bib_ref]. Apparently patients with NAFLD and/or obesity are more likely to suffer from drug-induced acute liver injury. NAFLD patients are also at risk of progression from NAFL to NASH as a result of DILI [bib_ref] Patients with Chronic Liver Disease Suggestive of Nonalcoholic Fatty Liver Disease May..., Lammert [/bib_ref] , at least for some types of drugs (e.g., acetaminophen, halothane, methotrexaye, pentoxufylline, rosiglitazone, tamoxifen) [bib_ref] Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity, Massart [/bib_ref].
The increased susceptibility for drug-induced acute liver damage could be caused by various mechanisms, separately or in combination, such as increased expression of CYP2E1, inflammation, and reduced mitochondria respiratory chain activity; these might lead to increased ROS production, decreased ATP synthesis and production of pro-inflammatory cytokines (e.g., TNF-α) [bib_ref] Role of Oxidative Stress in Pathophysiology of Nonalcoholic Fatty Liver Disease, Masarone [/bib_ref]. In addition, drug-induced liver damage (more frequently by isoflurane, acetaminophen, losartan, ticlopidine, and omeprazole) could be aggravated by the generation of higher levels of toxic metabolites produced by increased activity of CYP isoforms including CYP2E1 in obese individuals with NAFLD [bib_ref] Fat accretion in a subpopulation of hepatocytes as a strategy to protect..., Fromenty [/bib_ref] [bib_ref] A prospective study of acute drug-induced liver injury in patients suffering from..., Tarantino [/bib_ref] [bib_ref] Drug-induced toxicity on mitochondria and lipid metabolism: Mechanistic diversity and deleterious consequences..., Begriche [/bib_ref] [bib_ref] Hepatic cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis, Weltman [/bib_ref] [bib_ref] Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: Mechanisms..., Aubert [/bib_ref]. It has been shown that, in human liver samples, steatosis was associated with decreased hepatic cytochrome P450 3A (CYP3A) activity, which seems to be related to the severity of hepatic steatosis [bib_ref] Association between nonalcoholic hepatic steatosis and hepatic cytochrome P-450 3A activity, Kolwankar [/bib_ref]. However, the link between decreased CYP3A activity and DILI in NAFLD patients is not robust.
Caregivers should consider that chronic administration of some drugs can worsen pre-existing NAFLD. Chronic alcohol intake in particular is detrimental to the steatotic liver as it may worsen the clinical course of NAFLD [bib_ref] Excess weight risk factor for alcoholic liver disease, Naveau [/bib_ref] [bib_ref] Effect of body mass index and alcohol consumption on liver disease: Analysis..., Hart [/bib_ref] [bib_ref] Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin..., Xu [/bib_ref] [bib_ref] Ethanol administration exacerbates the abnormalities in hepatic lipid oxidation in genetically obese..., Everitt [/bib_ref]. One study demonstrated a 5.3-fold risk of mortality for obese males drinking 1-14 drinks per week and a 18.9-fold risk of mortality for obese males drinking 15 or more drinks per week compared to normal weight non-drinkers [bib_ref] Effect of body mass index and alcohol consumption on liver disease: Analysis..., Hart [/bib_ref]. Indeed, both drugs and alcohol can accelerate the transition from NAFL to NASH by increasing DNL, limiting the excretion of VLDL, and reducing fatty acid oxidation. In parallel the expression of pro-inflammatory cytokines, ROS production, and ER stress are further increased and mitochondrial respiratory chain activity is reduced [bib_ref] Fat accretion in a subpopulation of hepatocytes as a strategy to protect..., Fromenty [/bib_ref] [bib_ref] Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity, Massart [/bib_ref] [bib_ref] Ethanol administration exacerbates the abnormalities in hepatic lipid oxidation in genetically obese..., Everitt [/bib_ref] [bib_ref] Excess iron modulates endoplasmic reticulum stress-associated pathways in a mouse model of..., Tan [/bib_ref].
Taken together, it is likely that NAFLD predisposes an individual to more severe liver injury by factors causing acute injury of hepatocytes as drugs, viral hepatitides, and alcohol. It should be noted that many of the above-referenced studies do not histologically discern NAFL from NASH. Therefore, it cannot be excluded that NAFL or liver steatosis alone could already enhance the risk of liver injury and/or aggravate the severity of liver injury, albeit probably to a lower extent than NASH.
## Covid-19 and nafld
Coronavirus disease 2019 (COVID-19) is caused by a pathogen named Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) [bib_ref] A Novel Coronavirus from Patients with Pneumonia in China, Zhu [/bib_ref]. In general, liver injury in COVID-19 patients was frequent but mild, and a clear association between any underlying hepatic disease with the course of the SARS-CoV-2 infection was not established. Underlying pathophysiological mechanisms of liver injury in COVID-19 remain largely unknown. However, in a series of 202 consecutive patients admitted (from January through February) to two COVID-19-designated hospitals in China up to March 2020, 76 (37.2%) had NAFLD [bib_ref] Implication of non-alcoholic fatty liver diseases (NAFLD) in patients with COVID-19: A..., Ji [/bib_ref]. The authors observed that patients with NAFLD showed a higher risk of disease progression to a more severe disease, and more persistent viral shedding in comparison with non-NAFLD patients [bib_ref] Implication of non-alcoholic fatty liver diseases (NAFLD) in patients with COVID-19: A..., Ji [/bib_ref]. In line with this, the EASL and ESCMID recommend to patients with NAFLD or NASH, which in addition may suffer from hypertension, diabetes and obesity, to adhere to physical distancing because they are at risk of a severe course of COVID-19 [bib_ref] Centre for Mathematical Modelling of Infectious Diseases COVID-19 working group. Early dynamics..., Kucharski [/bib_ref] [bib_ref] Care of patients with liver disease during the COVID-19 pandemic: EASL-ESCMID position..., Boettler [/bib_ref]. Another important issue in the scenario of COVID-19 in patients with chronic liver diseases, which includes NAFLD or NASH, is related to the use of experimental drugs that have been tested (such as chloroquine, hydroxychloroquine, lopinavir/ritonavir, remdesivir, camostat, tocilizumab, emapalumab, anakinra, among many others) to mitigate the most severe complications. Although clinicians have to keep in mind potential side effects of these drugs, it is also recommended to consider these patients for early treatment as they are at risk of progression [bib_ref] Care of patients with liver disease during the COVID-19 pandemic: EASL-ESCMID position..., Boettler [/bib_ref]. Altogether, efforts should be made to better understand the role of NAFLD in COVID-19, and to be alert to the consequences of COVID-19 in patients with NALFD.
## Concluding remarks
In summary, cumulative data show that NAFLD (which includes NAFL and NASH) may progress to severe chronic liver disease. NAFLD is also a risk factor for the development of other metabolic diseases (diabetes, cardiovascular disease) and may aggravate any other underlying liver disease. As such, clinical providers will need to recognize that NAFL/liver steatosis is not necessarily benign. As there is currently no non-invasive marker that can accurately and reliably distinguish NAFL from NASH, providers will have to use their clinical judgement in association with available tools to identify individuals likely to progress. While it seems obvious that NASH confers a greater risk of liver-related and overall morbidity and mortality than NAFL, NAFL is not without risk. In particular, progressive fibrosis and the development of cardiovascular morbidity occur in a similar proportion of NAFL and NASH patients. In conclusion, any patient with NAFLD must be considered an at-risk patient for the progression of liver disease and for the development of components of metabolic syndrome.
## Conflicts of interest:
The authors declare no conflict of interest.
[fig] Author: Contributions: Drafting of the manuscript, G.M. and Ö.K.; critical revision of the manuscript, G.M., J.-P.S., C.A., W.-K.S. and A.C.; preparation of figures, J.-P.S.; supervision, A.C. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This research was funded by the German Research Foundation (DFG CA267/13-3; CA267/14-1 to A.C.) and by the Wilhelm Laupitz Foundation (A.C. and O.K.). [/fig]
[table] Table 1: Evidence in favor of the good prognosis of NAFL patients. [/table]
[table] Table 2: Evidence in favor of the poor prognosis of NAFL patients (5 out of 6 with paired liver biopsies). [/table]
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10.3390/v9060149
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CCBY
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5490825
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28608818
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s2orc_pubmed_articles
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Impact of Parvovirus B19 Viremia in Liver Transplanted Children on Anemia: A Retrospective Study
Acute parvovirus B19 (B19V) infection in immunocompromised patients may lead to severe anemia. However, in adult transplant recipients, B19V reactivations without anemia and low-level viremia are common. The impact of B19V in pediatric transplant patients, with high risk of primary infection, is investigated here. In a six-month period, 159 blood samples of 54 pediatric liver transplant recipients were tested for B19V DNA by quantitative real-time PCR. Viremia was correlated with anemia and immunosuppression and compared with rates in adult transplant recipients. B19V DNA was detected in 5/54 patients. Primary B19V infections were observed in four patients prior to and in one patient after transplantation. Rates of viremia were significantly higher in pediatric recipients than in adults. Prolonged virus shedding after primary infection prior to transplantation accounts for most viremic cases. Anemia was significantly more frequent in samples from viremic patients, but remained mild. In 15% of anemic samples, B19V DNA was detected. Therefore, in anemic pediatric transplant recipients, diagnostics for B19V seem reasonable.
# Introduction
In immunocompetent individuals, acute parvovirus B19 (B19V) infection is a mild disease associated with transient anemia, rash and arthritis. In contrast, in immunocompromised patients, cases of persisting B19 viremia with severe life-threatening chronic anemia have been reported [bib_ref] Long-term parvovirus B19 viremia associated with pure red cell aplasia after allogeneic..., Plentz [/bib_ref] [bib_ref] Recurrent high level parvovirus B19/genotype 2 viremia in a renal transplant recipient..., Liefeldt [/bib_ref] [bib_ref] Human parvovirus B19 genotype 3 associated with chronic anemia after stem cell..., Knoester [/bib_ref]. Those clinical courses occur in acute infections with high B19V DNA levels after solid organ transplantation, whereas B19V reactivations are more likely in adult transplant recipients and lead to low-level DNAemia, which is not associated with notable anemia [bib_ref] Low-level DNAemia of parvovirus B19 (genotypes 1-3) in adult transplant recipients is..., Plentz [/bib_ref] [bib_ref] Frequent occurrence of parvovirus B19 DNAemia in the first year after kidney..., Porignaux [/bib_ref]. Therefore, B19V does not seem to be a frequently harmful pathogen in adult transplant recipients. However, children are at higher risk of a primary B19V infection due to the low level of immunoglobulin G (IgG) seroprevalence [bib_ref] Seroprevalence of parvovirus B19 in the German population, Röhrer [/bib_ref]. It is unclear if there is a higher rate of acute B19V infections and consequently severe anemia in pediatric transplant recipients since larger studies are lacking.
The aims of this retrospective anonymous study were: [bib_ref] Long-term parvovirus B19 viremia associated with pure red cell aplasia after allogeneic..., Plentz [/bib_ref] to assess the prevalence and quantity of B19V DNA, to determine the respective B19V genotypes and the serostatus at liver transplantation among pediatric patients, and [bib_ref] Recurrent high level parvovirus B19/genotype 2 viremia in a renal transplant recipient..., Liefeldt [/bib_ref] to correlate the occurrence of B19 viremia with hemoglobin levels and reticulocyte counts as surrogate markers of pure red cell aplasia and to compare these findings with previously published data on B19V prevalence in adult transplant recipients [bib_ref] Low-level DNAemia of parvovirus B19 (genotypes 1-3) in adult transplant recipients is..., Plentz [/bib_ref].
# Materials and methods
For retrospective analysis, pediatric patients between 0 and 18 years who had undergone liver transplantation in the pediatric university hospital of Regensburg between 2008 and 2012 were eligible for the study. Remaining samples from routine blood analyses derived from a period of six months after transplantation were tested for B19V DNA. Follow-up samples were selected at intervals of 30 days or as close as possible to these dates. In case of re-transplantation follow-up was extended or extra cases were defined. All samples were analyzed for B19V DNA (genotypes 1-3) by quantitative real-time PCR. Hemoglobin levels and reticulocyte counts of each analyzed sample were recorded. For assessment of the patients' serostatus, B19V IgG was tested in pre-transplantation samples or, if not available, shortly after transplantation. Because of intravenous immunoglobulins (IVIG) routinely transfused in a large number of patients for cytomegalo virus (CMV) prophylaxis, which contributes to the serostatus for several months, B19V IgG negative patients were tested again at one month to assess the postoperative serostatus. Clinical data on underlying disease, immunosuppression, IVIG transfusions and symptoms characteristic of B19V infection were obtained from the patient records. All data were pseudonymized. The study was approved by the Ethics Committee of the University Hospital of Regensburg on 31 March 2011 (reference number 11-101-0036).
B19V DNA was amplified using an in-house real-time quantitative TaqMan PCR assay including all three genotypes according to the previously published protocol [bib_ref] Low-level DNAemia of parvovirus B19 (genotypes 1-3) in adult transplant recipients is..., Plentz [/bib_ref]. Purified B19V plasmids were included as positive controls and for calibration and B19V-seronegative samples as negative controls. The detection limit of the assay, defined as the DNA concentration with at least 95% of positive samples detectable, was 600 genome equivalents (geq)/mL (215 IU/mL) for each genotype. Reproducible positive results below the detection limit (<600 geq/mL) were described as 'weakly positive'.
B19V specific IgG, immunoglobulin M (IgM), and IgG avidity were analyzed by enzyme immune assay (EIA) and immunoblot according to the manufacturer's instructions (recomWell parvovirus B19 IgG and IgM assay, Mikrogen, Neuried, Germany).
Reference levels of the Department of Clinical Chemistry and Laboratory Medicine of the University Hospital of Regensburg were used for definition of anemia [fig_ref] Table 1: Characteristics of parvovirus B19 [/fig_ref].
# Results
## Patients
A total of 51 pediatric liver transplant recipients met the inclusion criteria. Due to re-transplantations, a total of 54 cases were analyzed. The median age was two years, 56% were female, 44% male. Thirty-six liver transplantations were performed in infants, 15 patients where six years or older. In patients in infancy almost 2/3 suffered from biliary atresia, whereas patients aged six years or older mostly suffered from cystic fibrosis and autoimmune hepatitis. A total of 159 samples were available for analysis.
## B19v dna detection
Within the first six months post-transplantation (post-Tx), in 12 samples (7.6%) from five patients (9.3%) B19V DNA was detected after transplantation, in all cases B19V genotype 1. In 7/12 samples, the B19V DNA load was <600 geq/mL, >10 3 geq/mL B19V-DNA/genotype 1 were detectable in four and >10 5 geq/mL in one sample. In 3/5 (60%) of these patients, initial B19V DNA detection post-Tx was at month 1, in one patient at month 3. In these four patients B19V DNA was already found in pre-transplantation (pre-Tx) specimens, as well. In one pre-Tx DNA-negative patient initial B19V DNA detection was at month 5, three days after re-transplantation (month 0). Thus, in all cases viremia was observed within the first three months after transplantation.
## B19v igg serostatus
B19V IgG was detected in 31.5% (17/54 patients) pre-Tx. In patients younger than one year, 5/20 were seropositive (0-4 months old). The four patients with viremia pre-Tx were also B19V IgG positive. That means that 4 (23.5%) of 17 children with an earlier infection were still viremic. As five patients had probably maternal antibodies, this number is raised to 4/12 (33.3%). In month 1, 70.4% (38/54) were seropositive due to IVIG.
## Dependence of viremia on immunosuppressive regimen
In all cases an induction therapy with basiliximab was implemented. Mostly, immunosuppressive regimens were used combining prednisolone with cyclosporine (56%) or tacrolimus (20.8%). Changes of medication from cyclosporine to other immunosuppressive drugs were performed in the further course of the observation period. Thus, we compared the immunosuppressive regimens with B19V viremia in each sample. B19V viremia was more frequent in tacrolimus-based than in cyclosporine-based regimens (p = 0.0003): B19V viremia was observed in 10/50 tacrolimus-based regimens (20%), whereas viral genomes were found only in 2/106 cyclosporine-based regimens (1.9%).
Furthermore, the correlation of B19V viremia with high dose prednisolone therapy (≥60 mg/m 2 /day) used for treatment of acute rejection (13 patients, one patient twice) or for initiation of immunosuppression (two patients) was evaluated. In 3/15 samples, B19V viremia was detectable simultaneously to high-dose prednisolone administration (20%), whereas B19V DNA could be found only in 9/144 of the remaining samples (6.3%). This difference was significant in Fisher's exact test (p = 0.036).
## Association of viremia and hemoglobin levels
Anemia was found in 17/27 samples (63.0%) from patients with B19V viremia, in contrast only in 43/132 samples (32.6%) of all 49 B19V negative patients. Also, anemia was more frequent in all B19V DNA positive (9/12, 75%) samples than in all B19V DNA negative samples (50/145, 34.5%). Both comparisons were highly significant (p = 0.003 and 0.006). There were also higher rates of reticulocytopenia in viremic patients (20.0% vs. 14.3%) and samples (33.3% vs. 13.4%), respectively. Due to low number of samples with reticulocyte counts, the significance of these findings remains unclear.
## Case report of a primary b19v infection after pediatric liver transplantation
Patient PLTX_19, a three-year old boy suffering from progressive familial intrahepatic cholestasis, primary B19V infection occurred after liver transplantation. He underwent re-transplantation after 143 days due to acute refractory rejection. B19V IgG was lacking despite IVIG transfusion after first transplantation. He received high doses of prednisolone (≥60 mg/m 2 /day) and a tacrolimus-based maintenance therapy in temporal relation to re-transplantation.
Parvovirus B19 DNA (<600 geq/mL) was detected for the first time three days after re-transplantation. Unfortunately, neither the donor organ nor donor blood was available for testing, so it remains unknown if the organ was the source of infection. The patient developed an itching exanthema with target lesions and slapped cheeks two days after transplantation, consistent with the clinical manifestation of the fifth disease. The course of viremia and other markers are depicted in [fig_ref] Figure 1: Figure 1 [/fig_ref]. immunosuppression (two patients) was evaluated. In 3/15 samples, B19V viremia was detectable simultaneously to high-dose prednisolone administration (20%), whereas B19V DNA could be found only in 9/144 of the remaining samples (6.3%). This difference was significant in Fisher's exact test (p = 0.036).
## Association of viremia and hemoglobin levels
Anemia was found in 17/27 samples (63.0%) from patients with B19V viremia, in contrast only in 43/132 samples (32.6%) of all 49 B19V negative patients. Also, anemia was more frequent in all B19V DNA positive (9/12, 75%) samples than in all B19V DNA negative samples (50/145, 34.5%). Both comparisons were highly significant (p = 0.003 and 0.006). There were also higher rates of reticulocytopenia in viremic patients (20.0% vs. 14.3%) and samples (33.3% vs. 13.4%), respectively. Due to low number of samples with reticulocyte counts, the significance of these findings remains unclear.
## Case report of a primary b19v infection after pediatric liver transplantation
Patient PLTX_19, a three-year old boy suffering from progressive familial intrahepatic cholestasis, primary B19V infection occurred after liver transplantation. He underwent retransplantation after 143 days due to acute refractory rejection. B19V IgG was lacking despite IVIG transfusion after first transplantation. He received high doses of prednisolone (≥60 mg/m 2 /day) and a tacrolimus-based maintenance therapy in temporal relation to re-transplantation.
Parvovirus B19 DNA (<600 geq/mL) was detected for the first time three days after retransplantation. Unfortunately, neither the donor organ nor donor blood was available for testing, so it remains unknown if the organ was the source of infection. The patient developed an itching exanthema with target lesions and slapped cheeks two days after transplantation, consistent with the clinical manifestation of the fifth disease. The course of viremia and other markers are depicted in [fig_ref] Figure 1: Figure 1 [/fig_ref]. Course of patient PLTX_19. For more than three years after initial detection, B19V DNA was detectable with a maximal value of 10 5 geq/mL at month 7 after re-transplantation. During this period, the patient was B19V IgG negative or indeterminate and became transiently B19V IgM positive two years after re-transplantation. Anemia was present only two months before initial detection of B19V DNA with hemoglobin levels between 7.9 g/dL and 8. Course of patient PLTX_19. For more than three years after initial detection, B19V DNA was detectable with a maximal value of 10 5 geq/mL at month 7 after re-transplantation. During this period, the patient was B19V IgG negative or indeterminate and became transiently B19V IgM positive two years after re-transplantation. Anemia was present only two months before initial detection of B19V DNA with hemoglobin levels between 7.9 g/dL and 8.7 g/dL. Reticulocytopenia (6 )
## Comparison of pediatric and adult transplant recipients regarding clinical impact of b19v dna detection
The number of B19V DNA positive samples was significantly higher in pediatric than in adult liver transplant patients (10.3% vs. 2.7%, p < 0.001) [bib_ref] Low-level DNAemia of parvovirus B19 (genotypes 1-3) in adult transplant recipients is..., Plentz [/bib_ref]. Also, the prevalence of B19 viremia was higher in pediatric than adult liver transplant recipients (9.3% vs. 5.5%, not significant.).
In pediatric liver transplant patients, the overall rate of anemia was significantly lower than in adult liver transplant recipients (37.7% vs. 81.5%, p < 0.001). However, in pediatric patients, 15.3% (9/59) of anemic samples simultaneously showed B19Viremia, whereas in adult liver transplant patients this constellation was observed only in 2.6% (7/269) (p < 0.001).
# Discussion
In this first larger study on B19V in pediatric transplant patients, a B19V DNA prevalence of 9.3% was found, which is markedly higher than previously estimated on the basis of case reports [bib_ref] Parvovirus B19 infection in pediatric solid-organ and bone marrow transplantation, Broliden [/bib_ref]. There were higher B19V DNA loads in pediatric patients with most likely a recent primary infection pre-Tx [bib_ref] Quantitative evidence for persistence of human parvovirus B19 DNA in an immunocompetent..., Cassinotti [/bib_ref] [bib_ref] Human parvovirus B19, Heegaard [/bib_ref] [bib_ref] Parvovirus B19 clearance from peripheral blood after acute infection, Musiani [/bib_ref] than in adult patients with reactivation [bib_ref] Low-level DNAemia of parvovirus B19 (genotypes 1-3) in adult transplant recipients is..., Plentz [/bib_ref]. In all patients, the B19V genotype 1 was found, the most widely-spread genotype [bib_ref] Phylogenetic analysis of human parvovirus B19 sequences from eleven different countries confirms..., Hübschen [/bib_ref].Prolonged viremia after primary infection prior to transplantation had a wider impact on anemia than reactivations after transplantation [bib_ref] Low-level DNAemia of parvovirus B19 (genotypes 1-3) in adult transplant recipients is..., Plentz [/bib_ref]. The level of anemia seems to correlate with B19V DNA loads, since there is no anemia found in adult patients with low-level B19V DNAemia [bib_ref] Low-level DNAemia of parvovirus B19 (genotypes 1-3) in adult transplant recipients is..., Plentz [/bib_ref] [bib_ref] Frequent occurrence of parvovirus B19 DNAemia in the first year after kidney..., Porignaux [/bib_ref] , mild anemia in our pediatric collective with moderate viremia, and severe anemia in immunocompromised patients with high-level viremia [bib_ref] Long-term parvovirus B19 viremia associated with pure red cell aplasia after allogeneic..., Plentz [/bib_ref] [bib_ref] Recurrent high level parvovirus B19/genotype 2 viremia in a renal transplant recipient..., Liefeldt [/bib_ref] [bib_ref] Clinical implications of quantitative real time-polymerase chain reaction of parvovirus B19 in..., Park [/bib_ref] [bib_ref] Parvovirus B19 infection after transplantation: A review of 98 cases, Eid [/bib_ref] [bib_ref] Identification and characterization of acute infection with parvovirus B19 genotype 2 in..., Grabarczyk [/bib_ref] [bib_ref] How many times can parvovirus B19-related anemia recur in solid organ transplant..., Gosset [/bib_ref]. The overall rate of anemia in pediatric liver transplant recipients was distinctly lower than in adult transplant patients (37.7% vs. 84.2%) [bib_ref] Low-level DNAemia of parvovirus B19 (genotypes 1-3) in adult transplant recipients is..., Plentz [/bib_ref]. Lower rates of anemia in pediatric liver transplant patients are known [bib_ref] Risk factors for chronic anemia in pediatric orthotopic liver transplantation: analysis of..., Liem [/bib_ref] , the reasons remain unclear. It is remarkable that as many as 15.3% of samples with anemic hemoglobin levels were simultaneously B19V DNA positive, similar to 19.2% in anemic pediatric liver transplant recipients in a smaller collective [bib_ref] Parvovirus B19 infection in pediatric transplant patients, Nour [/bib_ref]. Thus, B19V viremia can be regarded a major risk factor for anemia in pediatric liver transplant recipients, in contrast to adults.
As many as one-third of all children with a former infection were still viremic at the time point of transplantation and thus at risk for anemia. IVIG was routinely transfused in CMV high-risk patients and in under-six-month-olds, which contributes to transient immunity [bib_ref] Immunoglobulin treatment in primary antibody deficiency, Maarschalk-Ellerbroek [/bib_ref] [bib_ref] Pharmacokinetics of viral antibodies after administration of intravenous immunoglobulin in patients with..., Thurmann [/bib_ref] [bib_ref] Pharmacokinetics of a new 10% intravenous immunoglobulin in patients receiving replacement therapy..., Wasserman [/bib_ref]. This raised the seroprevalence from 31.5% pre-to 70.4% post-Tx which is similar to that of adult transplant recipients [bib_ref] Low-level DNAemia of parvovirus B19 (genotypes 1-3) in adult transplant recipients is..., Plentz [/bib_ref] and led to a low incidence of primary infections after transplantation (1/54, 1.9%), comparable to that in adult transplant recipients (0.7%) [bib_ref] Low-level DNAemia of parvovirus B19 (genotypes 1-3) in adult transplant recipients is..., Plentz [/bib_ref].
The rate of B19V viremia in patients treated with tacrolimus-based regimens was significantly higher than in patients treated with cyclosporine-based regimens. Other case reports also show pure red-cell aplasia due to B19V infection after application of tacrolimus-in one case the infection remitted only after switching to cyclosporine [bib_ref] Parvovirus B19 infection causing red cell aplasia in renal transplantation on tacrolimus, Wong [/bib_ref] [bib_ref] Long-term remission of recurrent severe anemia as a result of parvovirus B19..., Shen [/bib_ref] [bib_ref] Pure red cell aplasia caused by Parvovirus B19 infection in solid organ..., Geetha [/bib_ref] as the immunosuppressive effect is lower [bib_ref] Cyclosporin versus tacrolimus for liver transplanted patients, Haddad [/bib_ref] [bib_ref] Tacrolimus versus cyclosporin as primary immunosuppression for kidney transplant recipients, Webster [/bib_ref]. Also BK virus infections occur more frequently in patients with tacrolimus-based immunosuppression [bib_ref] Immunosuppressive drugs for kidney transplantation, Halloran [/bib_ref]. High doses of prednisone also led to a significantly higher rate of B19V infections, consistent with a straight increase of CMV infection risk with cumulative methylprednisolone-dose [bib_ref] Interrelationships among quantity of human cytomegalovirus (HCMV) DNA in blood, donor-recipient serostatus,..., Cope [/bib_ref].
There was one unusual case of a primary B19V infection post-Tx with an unknown source of infection. Surprisingly, he presented with atypically low DNA-loads (<600-440,000 geq/mL), negative antibodies, and did not have anemia at all, but showed an exanthema consistent with fifth disease two days after transplantation. This manifestation is the result of immune complex formation [bib_ref] Experimental parvoviral infection in humans, Anderson [/bib_ref] , but in this case, viral load was much too low to build enough immune complexes, specific IgG and IgM were not detectable. As well, it remains unclear why high viral loads and prolonged pure red-cell aplasia did not occur, as described before in several case reports [bib_ref] Recurrent high level parvovirus B19/genotype 2 viremia in a renal transplant recipient..., Liefeldt [/bib_ref] [bib_ref] Anemia in lung transplant patient caused by parvovirus B19, Kariyawasam [/bib_ref] [bib_ref] Parvovirus-B19-associated complications in renal transplant recipients, Waldman [/bib_ref]. This case and another one in a heart transplanted adult [bib_ref] Low-level DNAemia of parvovirus B19 (genotypes 1-3) in adult transplant recipients is..., Plentz [/bib_ref] obviously show that not every primary infection necessarily leads to a severe course of infection.
In this study, parvovirus B19V viremia most often occurred as a prolonged shedding of virus after a primary infection prior to transplantation. It caused mild anemia. A primary infection post-Tx surprisingly did not result in a severe course. However, as 15% of anemic samples were B19Viremic, we recommend performing diagnostics for B19V DNA in anemic pediatric transplant recipients though routine screening after transplantation does not seem to be necessary.
[fig] Figure 1: Figure 1. Course of patient PLTX_19. For more than three years after initial detection, B19V DNA was detectable with a maximal value of 10 5 geq/mL at month 7 after re-transplantation. During this period, the patient was B19V IgG negative or indeterminate and became transiently B19V IgM positive two years after re-transplantation. Anemia was present only two months before initial detection of B19V DNA with hemoglobin levels between 7.9 g/dL and 8.7 g/dL. Reticulocytopenia (6‰) was detected only once in month 4 after second transplantation with low viral load at the same time. CSA-Cyclosporine A. M-Mycophenolat Mofetil. Sir -Sirolimus. IgM-immunoglobulin M. [/fig]
[fig] 7: g/dL. Reticulocytopenia (6‰) was detected only once in month 4 after second transplantation with low viral load at the same time. CSA-Cyclosporine A. M-Mycophenolat Mofetil. Sir -Sirolimus. IgM-immunoglobulin M. [/fig]
[table] Table 1: Characteristics of parvovirus B19 (B19V) DNA positive patients. [/table]
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s2orc_pubmed_articles
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Implementation and Analysis of a Wireless Sensor Network-Based Pet Location Monitoring System for Domestic Scenarios
The flexibility of new age wireless networks and the variety of sensors to measure a high number of variables, lead to new scenarios where anything can be monitored by small electronic devices, thereby implementing Wireless Sensor Networks (WSN). Thanks to ZigBee, RFID or WiFi networks the precise location of humans or animals as well as some biological parameters can be known in real-time. However, since wireless sensors must be attached to biological tissues and they are highly dispersive, propagation of electromagnetic waves must be studied to deploy an efficient and well-working network. The main goal of this work is to study the influence of wireless channel limitations in the operation of a specific pet monitoring system, validated at physical channel as well as at functional level. In this sense, radio wave propagation produced by ZigBee devices operating at the ISM 2.4 GHz band is studied through an in-house developed 3D Ray Launching simulation tool, in order to analyze coverage/capacity relations for the optimal system selection as well as deployment strategy in terms of number of transceivers and location. Furthermore, a simplified dog model is developed for simulation code, considering not only its morphology but also its dielectric properties. Relevant wireless channel information such as power distribution, power delay profile and delay spread graphs are obtained providing an extensive wireless channel analysis. A functional dog monitoring system is presented, operating over the implemented ZigBee network and providing real time information to Android based devices. The proposed system can be scaled in order to consider different types of domestic pets as well as new user based functionalities.In this sense, a high variety of person-oriented systems are being developed within the framework of e-health systems and therefore, most of these technologies are being extrapolated to farming and pet wellness applications. Monitoring of physiological parameters as ECG [1], pulse [2] or blood pressure [3], for patients, elderly people or athletes as well as their location[4], are the most common data obtained in this kind of systems.In relation to animal tracking and wellness, numerous identification systems have been developed over the years, specially for wildlife tracking and analysis, attaching radio transmitters to animals to monitor their location, behavior or migratory habits[5,6]. For farm animals or pets some identification systems have also been developed and deployed, although these systems are not based on wireless networks and therefore owners must extract the information indirectly.Nevertheless, novel farm animal monitoring systems based on WSN have been studied in recent years, in [7] a WSN is proposed for animal monitoring, in [8] the location of cows is monitored thanks to GPS location system and GSM telephony and in [9] a ZigBee-Based health monitoring system is presented, which takes data through rumination, heart rate, temperature and humidity sensors. Going one step further, in [10] a Livestock Monitoring System (LMS) is presented, where thanks to an integrated system for animal monitoring of their environment, production, growth and health, the production efficiency in farming is improved.When WSN are developed for pet-oriented applications, the main purpose of these systems is animal wellness and security. Thus, veterinary systems where photophethysmogram (PPG) and electrocardiogram (ECG) information are recorded wirelessly are presented in[11,12], a remote feeding system is developed in [13] and a system capable to recognize dog behavior thanks to an accelerometer attached to the canine is shown in[14]. Dogs also play a useful role in rescue operations and therefore some devices are attached to a canine in [15] to obtain not only useful information about dog health (ECG, PPG), but also environmental information about location or the presence of gas.Table 1presents a comparison of different wireless animal monitoring systems. It is worth noting that no specific application or system has been identified for pet location and monitoring applications within domestic indoor scenarios. The proposed system therefore provides a pet location and monitoring system, which provides an interactive context in order to retrieve parameters such as pet location, biomedical signal retrieval or behavior patterns, among others.
# Introduction
Nowadays, monitoring of objects as well as of living beings can be easily carried out thanks to a wide variety of transceivers working together with increasingly compact devices and the use of different wireless communication standards. These systems fall within the scope of the Internet of Things (IoT), where devices are connected to the internet and the information is sent without the interaction of human beings. Thus, a high variety of variables can be monitored in real time and in the case of living beings, their physiological parameters or location can be accurately measured.
Notwithstanding, these kind of systems must be deployed after an extensive radio-planning study to implement a robust and efficient Wireless Sensor Network (WSN), especially when the number of animals is high, they are in wide areas or they are inside complex places from the electromagnetic point of view.
In this work an indoor canine location and monitoring system for home environments is presented. An in-house 3D Ray Launching method is used to study indoor wireless channel performance inside a home when the Zigbee WSN is deployed and a receiver is attached to a dog. The main goal is the analysis of the influence of wireless propagation limitations in the implementation of a specific pet monitoring system, which has been implemented and tested. For this purpose, a simplified dog model has been developed which is compatible with the simulation tool accounting for its morphology and dielectric properties, considering that the wireless transceiver is inevitably attached to biological tissues and they are highly absorptive and dispersive. Radio planning analysis is performed, based on deterministic 3D Ray Launching code in order to analyze coverage/capacity relations, aiding in system election as well as on the number of required transceivers and their potential location within the scenario under analysis. Validation measurements have been carried out by attaching a Xbee device to a real dog to calibrate the simulation tool and compare obtained data with theoretical results. An android-based location application operating within the scenario is presented and tested in order to provide an interactive indoor pet location and monitoring system.
## Simulation scenario and simplified dog model
As previously mentioned, an in-house developed 3D Ray Launching (3D RL) code has been employed in order to perform radioplanning analysis and coverage/capacity estimations in order to provide system wireless connectivity, previously tested in a wide range of application [bib_ref] Analysis of Wireless Sensor Network Topology and Estimation of Optimal Network Deployment..., Aguirre [/bib_ref]. Moreover, the biological tissues can be considered, by means of a specific simplified human body model implemented for use within the 3D RL code [bib_ref] Evaluation of electromagnetic dosimetry of wireless systems in complex indoor scenarios within..., Aguirre [/bib_ref] [bib_ref] Characterization of UHF Radio Channels for Wireless Sensor Systems Embedded in Surfboards, Baghdadi [/bib_ref].
The 3D RL technique has been selected provided an adequate trade-off between accuracy and computational cost. Simulation techniques can be divided in two main groups, empirical and deterministic methods. Empirical methods [bib_ref] Empirical formula for propagation loss in land mobile radio services, Hata [/bib_ref] [bib_ref] Propagation factors controlling mean field strength on urban streets, Ikegami [/bib_ref] are site-specifi, measurement based and therefore, their results are strongly related to the framework scenario and original conditions. Thereofre, such techniques are time efficient but inaccurate when compared to deterministic techniques as FDTD, MOM, Ray Launching or Ray Tracing [bib_ref] Propagation prediction in and through buildings, Lee [/bib_ref] [bib_ref] A new metric to analyze propagation models, Prieto [/bib_ref]. On the other hand, deterministic techniques are based on numerical approaches to the resolution of Maxwell's equations and therefore, their computational complexity is high. However, Geometrical Optics based techniques (Ray tracing) show a good balance between both: computation time and accuracy.
The algorithm has been implemented in Matlab and is based on Geometrical Optics (GO) and Geometrical Theory of Diffraction (GTD). In order to enhance GO theory, the uniform extension of the GTD (UTD) is used with the diffracted rays, which are introduced to remove field discontinuities and to give proper field corrections, especially in the zero-field regions predicted by GO.
The input parameters in the algorithm are the angular and spatial resolution, which determine the accuracy of the model. A bundle of rays are considered in a finite sample of the possible directions of the propagation from the transmitter, and a ray is launched for each direction. When a ray impacts on an object, a reflecting ray is generated, and when a ray impacts an edge, a new family of diffracted rays is generated. Rays are launched at an elevation angle θ and with an azimuth angle Φ, as defined in the usual Cartesian coordinate system.
It is worth noting that a grid is defined in the simulation space, and all the parameters of the propagating rays are stored in each cuboid in the space. After that, with this stored parameters, simulation results such as received power or power delay profiles for each spatial point in the three dimensional scenario can be obtained.
Antenna patterns are taken into account in the algorithm to include the effects of antenna beamwidth in both azimuth and elevation. The material properties for all the elements within the Sensors 2016, scenario are also taken into account, given the dielectric constant and permittivity at the frequency range of operation of the system under analysis.
In this case a one story home [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] has been simulated, including furniture (chairs, tables, mirrors, beds, etc.) and taking under consideration not only the morphology of the scenario but also the dielectric properties of all the objects inside it. The scenario surface is 65 m 2 , divided in eight rooms. Three antennas have been placed within the scenario: one in the ceiling of living room, one in the main bedroom and one in the guest bedroom. In addition, another transceiver is placed simplified dog model. This configuration is chosen since the indoor canine tracking system requires at least three transmitters to carry out the triangulation process. Moreover, typical wireless motes can provide coverage levels, by means of experimental validation, in the order to 20-40 m 2 , as a function of object density. In this way, coverage conditions can be fulfilled, whilst enabling location capabilities. simulation results such as received power or power delay profiles for each spatial point in the three dimensional scenario can be obtained. Antenna patterns are taken into account in the algorithm to include the effects of antenna beamwidth in both azimuth and elevation. The material properties for all the elements within the scenario are also taken into account, given the dielectric constant and permittivity at the frequency range of operation of the system under analysis.
In this case a one story home [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] has been simulated, including furniture (chairs, tables, mirrors, beds, etc.) and taking under consideration not only the morphology of the scenario but also the dielectric properties of all the objects inside it. The scenario surface is 65 m 2 , divided in eight rooms. Three antennas have been placed within the scenario: one in the ceiling of living room, one in the main bedroom and one in the guest bedroom. In addition, another transceiver is placed simplified dog model. This configuration is chosen since the indoor canine tracking system requires at least three transmitters to carry out the triangulation process. Moreover, typical wireless motes can provide coverage levels, by means of experimental validation, in the order to 20-40 m 2 , as a function of object density. In this way, coverage conditions can be fulfilled, whilst enabling location capabilities. shows the employed simulation parameter, following conventional Xbee mote specifications (802.15, ZigBee device), the ones which will be used in measurement validations. ZigBee has been chosen as the base of the presented system considering its low energy consumption characteristics. The device attached to the dog must be as small as possible to provide high comfort levels and therefore the system must be energetically efficient to allow the use of small batteries. On the other hand, the ZigBee data rate is lower than, for example, 802.11 system based devices, which is not relevant in the proposed system, since the required transmitted information volume is potentially low. It is also worth noting that the final system design, after prototyping stage, can be more ergonomic, by including chip or conformal antennas if required. The implemented dog model is depicted in [fig_ref] Figure 2: Proportions of simplified dog model according with chosen height for medium size... [/fig_ref]. This model is compatible with the 3D RL simulation technique and includes the dielectric characteristics and proportions of a real dog, which have been extrapolated from measurement results related with cattle as well as characterization of human bodies, with dielectric constant values of the outer skin layer (in principle the most relevant) in order of εr ≈ 4 for the frequency of operation of the employed wireless communication system. Maintaining these proportions is not a trivial task, considering that every breed of dog has its own characteristics. However, most dogs can be classified in five different groups and therefore, the developed simplified canine model has been programmed to allow the generation of dogs of different proportions, from mini-dogs like Chihuahuas to giant-dogs like St. Bernards.
Once the kind of dog has been chosen in the simulation code, its height is defined and the dimensions of all elements of the dog are automatically calculated. In [fig_ref] Figure 2: Proportions of simplified dog model according with chosen height for medium size... [/fig_ref] proportions and its relation with the height of the dog are depicted for a medium size canine breed. shows the employed simulation parameter, following conventional Xbee mote specifications (802.15, ZigBee device), the ones which will be used in measurement validations. ZigBee has been chosen as the base of the presented system considering its low energy consumption characteristics. The device attached to the dog must be as small as possible to provide high comfort levels and therefore the system must be energetically efficient to allow the use of small batteries. On the other hand, the ZigBee data rate is lower than, for example, 802.11 system based devices, which is not relevant in the proposed system, since the required transmitted information volume is potentially low. It is also worth noting that the final system design, after prototyping stage, can be more ergonomic, by including chip or conformal antennas if required. The implemented dog model is depicted in [fig_ref] Figure 2: Proportions of simplified dog model according with chosen height for medium size... [/fig_ref]. This model is compatible with the 3D RL simulation technique and includes the dielectric characteristics and proportions of a real dog, which have been extrapolated from measurement results related with cattle as well as characterization of human bodies, with dielectric constant values of the outer skin layer (in principle the most relevant) in order of ε r ≈ 4 for the frequency of operation of the employed wireless communication system. Maintaining these proportions is not a trivial task, considering that every breed of dog has its own characteristics. However, most dogs can be classified in five different groups and therefore, the developed simplified canine model has been programmed to allow the generation of dogs of different proportions, from mini-dogs like Chihuahuas to giant-dogs like St. Bernards.
Once the kind of dog has been chosen in the simulation code, its height is defined and the dimensions of all elements of the dog are automatically calculated. In [fig_ref] Figure 2: Proportions of simplified dog model according with chosen height for medium size... [/fig_ref] proportions and its relation with the height of the dog are depicted for a medium size canine breed. The implemented simplified dog model is programmed to allow the consideration of not only different sizes but also different positions to be as adaptable as possible, considering the potential impact in propagation losses. As an example, in [fig_ref] Figure 3: Example of the simplified dog model in different position and with different... [/fig_ref] the dog model is depicted in different positions which can be considered within the simulation code. Since the communication system employed is a ZigBee Wireless Personal Area Network (WPAN), transceiver simulation parameters match with real device characteristics, corresponding to Xbee devices which have been used for validation measurements. In [fig_ref] Table 2: Simulation parameters used in the 3D Ray Launching code [/fig_ref] these parameters as well as resolution values used in simulation runs are shown. In any case, transceiver selection is in principle flexible, allowing if required other solutions based on 802.15 systems or even combined with 802.11 systems. The election of ZigBee in this case is based initially on the flexibility to implement low-cost and largely scalable networks, which can be easily connected via gateway to an external application server. The solution however can be adapted to other communication systems or even into a multi-system solution (combining for example ZigBee with WLAN connectivity to provide interaction between mobile devices and motes within the wireless sensor networks), depending on the functionalities to be developed in the future. The implemented simplified dog model is programmed to allow the consideration of not only different sizes but also different positions to be as adaptable as possible, considering the potential impact in propagation losses. As an example, in [fig_ref] Figure 3: Example of the simplified dog model in different position and with different... [/fig_ref] the dog model is depicted in different positions which can be considered within the simulation code. The implemented simplified dog model is programmed to allow the consideration of not only different sizes but also different positions to be as adaptable as possible, considering the potential impact in propagation losses. As an example, in [fig_ref] Figure 3: Example of the simplified dog model in different position and with different... [/fig_ref] the dog model is depicted in different positions which can be considered within the simulation code. Since the communication system employed is a ZigBee Wireless Personal Area Network (WPAN), transceiver simulation parameters match with real device characteristics, corresponding to Xbee devices which have been used for validation measurements. In [fig_ref] Table 2: Simulation parameters used in the 3D Ray Launching code [/fig_ref] these parameters as well as resolution values used in simulation runs are shown. In any case, transceiver selection is in principle flexible, allowing if required other solutions based on 802.15 systems or even combined with 802.11 systems. The election of ZigBee in this case is based initially on the flexibility to implement low-cost and largely scalable networks, which can be easily connected via gateway to an external application server. The solution however can be adapted to other communication systems or even into a multi-system solution (combining for example ZigBee with WLAN connectivity to provide interaction between mobile devices and motes within the wireless sensor networks), depending on the functionalities to be developed in the future. Since the communication system employed is a ZigBee Wireless Personal Area Network (WPAN), transceiver simulation parameters match with real device characteristics, corresponding to Xbee devices which have been used for validation measurements. In [fig_ref] Table 2: Simulation parameters used in the 3D Ray Launching code [/fig_ref] these parameters as well as resolution values used in simulation runs are shown. In any case, transceiver selection is in principle flexible, allowing if required other solutions based on 802.15 systems or even combined with 802.11 systems. The election of ZigBee in this case is based initially on the flexibility to implement low-cost and largely scalable networks, which can be easily connected via gateway to an external application server. The solution however can be adapted to other communication systems or even into a multi-system solution (combining for example ZigBee with WLAN connectivity to provide interaction between mobile devices and motes within the wireless sensor networks), depending on the functionalities to be developed in the future. In [fig_ref] Figure 4: Power distribution in the scenario when transmitter is placed in the living... [/fig_ref] power distribution results for the home floor when the living room is empty and when a dog is within the living room are presented. In this case the antenna has been placed in a lower height, over the glass table which is placed in the center of the living room. In both cases, when the transceiver is attached to the dog and when it is over the table the height is 0.3 m, although simulation results have been obtained for the complete scenario volume. Since the transmitter is placed in this room, most of the energy is confined in this area. However, power levels obtained in the rest of the scenario are compatible with usual Xbee receiver sensitivity thresholds in the range of −100 dBm. The difference between both situations is also noticeable, since more power is distributed when the room is empty and it is not absorbed by the dog. In any case, the difference between both images is more visible given that the transmitter is inside the same room as the dog and the presented power planes are at the dog height. As it can be seen from the estimation of received power levels within the scenario, an optimal configuration would require 2 transceivers within the scenario to satisfy coverage conditions for the domestic pet-static infrastructure links, implying an approximate node density of (1 node/30-40 m 2 ). These values are dependent on the specific configuration of the scenario, although the node density, for the case of large complexity (i.e., relevant number of walls and furniture elements), provides a radioplanning estimate to perform initial network deployment. This node density is one of the relevant outcomes of the radioplanning process, providing an initial network deployment estimation, which requires fine tuning as a function of site-specific details. In [fig_ref] Figure 4: Power distribution in the scenario when transmitter is placed in the living... [/fig_ref] power distribution results for the home floor when the living room is empty and when a dog is within the living room are presented. In this case the antenna has been placed in a lower height, over the glass table which is placed in the center of the living room. In both cases, when the transceiver is attached to the dog and when it is over the table the height is 0.3 m, although simulation results have been obtained for the complete scenario volume. Since the transmitter is placed in this room, most of the energy is confined in this area. However, power levels obtained in the rest of the scenario are compatible with usual Xbee receiver sensitivity thresholds in the range of −100 dBm. The difference between both situations is also noticeable, since more power is distributed when the room is empty and it is not absorbed by the dog. In any case, the difference between both images is more visible given that the transmitter is inside the same room as the dog and the presented power planes are at the dog height. As it can be seen from the estimation of received power levels within the scenario, an optimal configuration would require 2 transceivers within the scenario to satisfy coverage conditions for the domestic pet-static infrastructure links, implying an approximate node density of (1 node/30-40 m 2 ). These values are dependent on the specific configuration of the scenario, although the node density, for the case of large complexity (i.e., relevant number of walls and furniture elements), provides a radioplanning estimate to perform initial network deployment. This node density is one of the relevant outcomes of the radioplanning process, providing an initial network deployment estimation, which requires fine tuning as a function of site-specific details. In order to consider a more general case, the scenario has also been simulated introducing four human body models inside the home [fig_ref] Figure 5: Simulated scenario when four human bodies models are introduced in different locations [/fig_ref]. Two of them are placed in the living room next to the dog model and other two are distributed in different stances of the scenario. In [fig_ref] Figure 6: Comparison of receiver power when human body models are introduced in the... [/fig_ref] the difference between simulation results when only the dog is inside the home and when humans are introduced is depicted. The largest difference is shown in the furthest room of the home, reaching 30 dB values. This behavior is the consequence of introducing human bodies between transmitter area and this room, due to the fact that human bodies have absorbed and reflected the energy partially. In order to consider a more general case, the scenario has also been simulated introducing four human body models inside the home [fig_ref] Figure 5: Simulated scenario when four human bodies models are introduced in different locations [/fig_ref]. Two of them are placed in the living room next to the dog model and other two are distributed in different stances of the scenario. In [fig_ref] Figure 6: Comparison of receiver power when human body models are introduced in the... [/fig_ref] the difference between simulation results when only the dog is inside the home and when humans are introduced is depicted. The largest difference is shown in the furthest room of the home, reaching 30 dB values. This behavior is the consequence of introducing human bodies between transmitter area and this room, due to the fact that human bodies have absorbed and reflected the energy partially. In any case the influence of human bodies is low considering that the transmitter is not attached to them and that the number of people which usually can be found inside this kind of scenario is not large enough. Therefore, if a human monitoring system or an area of high human presence density were considered, the study of the presence of human bodies would be essential to determine the adequate performance of the communication system.
When a wireless sensor network is deployed the accuracy of data transmission must be assured to achieve the best possible user experience, provided by bit erro rate (BER) estimations. For the case of Quaternary Phase Shift Keying (QPSK) modulation (a typical modulation scheme employed in ZigBee as well as in 802.15 and 8021.11 systems), the approximate value of bit error probability is given by: In any case the influence of human bodies is low considering that the transmitter is not attached to them and that the number of people which usually can be found inside this kind of scenario is not large enough. Therefore, if a human monitoring system or an area of high human presence density were considered, the study of the presence of human bodies would be essential to determine the adequate performance of the communication system.
[formula] P b = Q(√ 2E b N 0 )(1) [/formula]
When a wireless sensor network is deployed the accuracy of data transmission must be assured to achieve the best possible user experience, provided by bit erro rate (BER) estimations. For the case of Quaternary Phase Shift Keying (QPSK) modulation (a typical modulation scheme employed in ZigBee as well as in 802.15 and 8021.11 systems), the approximate value of bit error probability is given by: In any case the influence of human bodies is low considering that the transmitter is not attached to them and that the number of people which usually can be found inside this kind of scenario is not large enough. Therefore, if a human monitoring system or an area of high human presence density were considered, the study of the presence of human bodies would be essential to determine the adequate performance of the communication system.
[formula] P b = Q(√ 2E b N 0 )(1) [/formula]
When a wireless sensor network is deployed the accuracy of data transmission must be assured to achieve the best possible user experience, provided by bit erro rate (BER) estimations. For the case of Quaternary Phase Shift Keying (QPSK) modulation (a typical modulation scheme employed in ZigBee as well as in 802.15 and 8021.11 systems), the approximate value of bit error probability is given by: where P b is the bit error probability, N 0 is the overall interference power spectral density (given by internal interference, external interference and thermal noise) in the scenario and E b is the energy per bit, given by:
[formula] P b = Q( 2E b N 0 )(1)E b = P Rx R b (2) [/formula]
where P Rx is the received power and R b is the transmission bit rate. Hence, BER strongly depends on the transmission speed, noise level in the scenario and received power. In [fig_ref] Figure 7: BER results considering different bit rates and noise levels for the antenna... [/fig_ref] BER results are depicted for different conditions when the transmitter is placed in the living room. Not only noise levels have been modified, but also bit rates and the presence of the dog have been taken under consideration. As expected, in any condition when noise level or transmission rate is reduced, low BER area is larger and therefore, longer range communication links can be established. Since one of the variables which determines BER is the received transmission power, energy lost by obstacles and multipath behavior of electromagnetic wave imply chenges in BER values all over the scenario. The presence of the dog decreases the overall power in the living room and low BER area is reduced when it is considered. The results depicted consider very high interference levels, which are not the usual case within a domestic environment. The usual practical case exhibits noise power spectral densities in the order of −120 dBm/Hz to −100 dBm/Hz. In this case, the main condition of operation is provided by coverage thresholds, which are a function of the requested bit rate and the sensititivy threshold. Taking into account the results for coverage level depicted in [fig_ref] Figure 4: Power distribution in the scenario when transmitter is placed in the living... [/fig_ref] , each one of the employed ZigBee transceivers provides coverage levels in an approximate area of 40 m 2 , as a function of the elements within the scenario. Therefore, in the domestic scenario under analysis, a network deployment of 1-3 transceivers per floor will satisfy initial coverage needs in order to enable bi-directional real time communication between the static transceivers and the wearable device located on the pet. Therefore, not only received power level thresholds are satisfied, but also requirements in terms of BER, leading to coverage/capacity relations that hold for the scenario under analysis, with a node density of (1 node/30-40 m 2 ) in order to satisfy both conditions for Xbee motes operating al 0 dBm transmission power levels. As in the previously derived node density values, these results provide relevant information related with the node radioplanning estimations.
## Experimental results and implemented application
Once the 3D Ray Launching simulation technique has been introduced, experimental validation results as well as the implementation of an dog indoor tracking application is presented. The goal is to validate the proposed simulation model of the pet (in this case, a parametrizable dog model), prior to testing the proposed monitoring application.
Experimental results are obtained by attaching to a dog a Xbee device working over an implemented Arduino software platform. In this case dog has been placed in the living room and simulation and measurement results have been obtained all over the home under analysis. Measurement results have been obtained with the aid of an Agilent FieldFox N9912A portable spectrum analyzer. In order to validate the results obtained from the deterministic 3D RL code, measurements have been performed with the aid of a portable spectrum analyzer, which is necessary due to the fact that measurement error is very small (in the order of 0.1 dB) with adequate bandwidth settings.
Received power levels can also be obtained (by means of Received Signal Strength Indication signals, RSSI) with the internal circuits of the ZigBee mote transceiver, but in this case, measurement errors are relatively high, within the order of 5 dB-8 dB. Therefore, wireless planning requires calibration in terms of precise spectral measurements and real operation can be estimated by considering a constant degradation off-set corresponding the RSSI detection circuits [bib_ref] Implementation of Context Aware e-Health Environments Based on Social Sensor Networks, Aguirre [/bib_ref]. Five measurement points are defined in different rooms of the home, which are depicted in. In addition, 14 measurements have been carried out placing the antenna in the living room, without the presence of the dog. In [fig_ref] Figure 9: Measurement vs simulation results when the Xbee prototype is attached to the... [/fig_ref] both, measurement and simulation results are compared for the first case, concluding that the simulation tool coupled with the simplified canine model, provides accurate results with a maximum error of 5 dB, a mean error of 2.52 dB and a standard deviation of 0.48 dB. In any case, it can be seen that the derived power value is far away from the sensitivity threshold and therefore, good performance of the ZigBee system can be expected, with the initial consideration of 1-3 static transceivers per household floor, as a function of the specific propagation losses of the considered scenario. In [fig_ref] Figure 9: Measurement vs simulation results when the Xbee prototype is attached to the... [/fig_ref] both, measurement and simulation results are compared for the first case, concluding that the simulation tool coupled with the simplified canine model, provides accurate results with a maximum error of 5 dB, a mean error of 2.52 dB and a standard deviation of 0.48 dB. In any case, it can be seen that the derived power value is far away from the sensitivity threshold and therefore, good performance of the ZigBee system can be expected, with the initial consideration of 1-3 static transceivers per household floor, as a function of the specific propagation losses of the considered scenario. In [fig_ref] Figure 9: Measurement vs simulation results when the Xbee prototype is attached to the... [/fig_ref] both, measurement and simulation results are compared for the first case, concluding that the simulation tool coupled with the simplified canine model, provides accurate results with a maximum error of 5 dB, a mean error of 2.52 dB and a standard deviation of 0.48 dB. In any case, it can be seen that the derived power value is far away from the sensitivity threshold and therefore, good performance of the ZigBee system can be expected, with the initial consideration of 1-3 static transceivers per household floor, as a function of the specific propagation losses of the considered scenario. In [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] measurement data compared with simulation results, when the transmitter is not attached to the dog, are depicted and in this case, the transmitter has been placed at a height of 0.8 m High accuracy is observed, with error values of 1.37 dB and a standard deviation of 2.55 dB. When two measurement results are compared, the influence of the dog is noticeable considering that received power values are in general higher within the considered stances when the home is empty. The influence of the furniture is also observable if the stances next to the living room are studied. As it can be seen in [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] , the received power level of point 11 is lower than the two points placed next to it (10 and 12) as a consequence of the power reflected by the mirror located between transmitter and the measurement point. For point 5 in [fig_ref] Figure 9: Measurement vs simulation results when the Xbee prototype is attached to the... [/fig_ref] and points 1, 2 and 3 in [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] , received power is lower in comparison with further points since most of the energy is reflected or absorbed by the furniture disposed in the middle wall of both stances. In [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] measurement data compared with simulation results, when the transmitter is not attached to the dog, are depicted and in this case, the transmitter has been placed at a height of 0.8 m High accuracy is observed, with error values of 1.37 dB and a standard deviation of 2.55 dB. When two measurement results are compared, the influence of the dog is noticeable considering that received power values are in general higher within the considered stances when the home is empty. The influence of the furniture is also observable if the stances next to the living room are studied. As it can be seen in [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] , the received power level of point 11 is lower than the two points placed next to it (10 and 12) as a consequence of the power reflected by the mirror located between transmitter and the measurement point. For point 5 in [fig_ref] Figure 9: Measurement vs simulation results when the Xbee prototype is attached to the... [/fig_ref] and points 1, 2 and 3 in [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] , received power is lower in comparison with further points since most of the energy is reflected or absorbed by the furniture disposed in the middle wall of both stances. As aforementioned, the dog location system works triangulating the received power of three transmitter or Zigbee Routers (ZB) distributed in different rooms of the home. Therefore, three different simulations have been launched considering those antennas to show the influence of furniture and the household topology. Simulation results for these configurations are depicted in [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref]. These results can be used to calibrate the location system since power distribution values within the complete scenario are obtained. Besides, the high influence of home distribution and furniture is noticeable. As an example, in main and guest rooms the influence of bed is evident considering that As aforementioned, the dog location system works triangulating the received power of three transmitter or Zigbee Routers (ZB) distributed in different rooms of the home. Therefore, three different simulations have been launched considering those antennas to show the influence of furniture and the household topology. Simulation results for these configurations are depicted in [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref]. In [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] measurement data compared with simulation results, when the transmitter is not attached to the dog, are depicted and in this case, the transmitter has been placed at a height of 0.8 m High accuracy is observed, with error values of 1.37 dB and a standard deviation of 2.55 dB. When two measurement results are compared, the influence of the dog is noticeable considering that received power values are in general higher within the considered stances when the home is empty. The influence of the furniture is also observable if the stances next to the living room are studied. As it can be seen in [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] , the received power level of point 11 is lower than the two points placed next to it (10 and 12) as a consequence of the power reflected by the mirror located between transmitter and the measurement point. For point 5 in [fig_ref] Figure 9: Measurement vs simulation results when the Xbee prototype is attached to the... [/fig_ref] and points 1, 2 and 3 in [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] , received power is lower in comparison with further points since most of the energy is reflected or absorbed by the furniture disposed in the middle wall of both stances. As aforementioned, the dog location system works triangulating the received power of three transmitter or Zigbee Routers (ZB) distributed in different rooms of the home. Therefore, three different simulations have been launched considering those antennas to show the influence of furniture and the household topology. Simulation results for these configurations are depicted in [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref]. These results can be used to calibrate the location system since power distribution values within the complete scenario are obtained. Besides, the high influence of home distribution and furniture is noticeable. As an example, in main and guest rooms the influence of bed is evident considering that These results can be used to calibrate the location system since power distribution values within the complete scenario are obtained. Besides, the high influence of home distribution and furniture is noticeable. As an example, in main and guest rooms the influence of bed is evident considering that when the situation of the bed is analyzed, a decrease in received power level is shown. Finally, it can be concluded that in the home the system will have enough power to carry out the triangulation process, considering that power in the complete area is higher than −100 dBm, above the typical receiver sensitivity value for ZigBee systems.
In order to provide pet monitoring functionalities, an in-house dog monitoring system is developed for android devices based on the deployed ZigBee network. A screenshot of implemented application is shown in [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] , where the distance walked by the dog can be easily known and the place where it is as well as the time that it has been in banned places can be monitored. The dog monitoring system consists on a set of wireless devices and a software component. The hardware includes a ZigBee device in charge of data communication and RSS (received signal strength) monitoring, a ZigBee sink in charge of data collection and command execution, an Android-based tablet in charge of data presentation, and a mini-PC, which includes the information system.
The dog carries in its harness the Arduino device, which includes a ZigBee communication module. The monitoring of the Received Signal Strength Indicator (RSSI) allows to determine the location of the dog. This value is obtained by the ZigBee transceiver each time a message is received. The message also includes the identifier of the ZigBee node. This information (ID + RSSI) is sent periodically or on demand to the sink node in order to provide the dog location. The Arduino device allows the operation on real-time connected mode or even, on unconnected mode. The first mode is used for real-time data transmission to the sink node, while the second-one is used for off-line dog tracking. The energy consumption is higher for the first mode, so an appropriate and limited use of this operation mode is recommended. The Arduino device includes a ZigBee communication module to minimize the energy consumption, since its consumption is lower than this of WiFi.illustrates the devices used. when the situation of the bed is analyzed, a decrease in received power level is shown. Finally, it can be concluded that in the home the system will have enough power to carry out the triangulation process, considering that power in the complete area is higher than −100 dBm, above the typical receiver sensitivity value for ZigBee systems. In order to provide pet monitoring functionalities, an in-house dog monitoring system is developed for android devices based on the deployed ZigBee network. A screenshot of implemented application is shown in [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] , where the distance walked by the dog can be easily known and the place where it is as well as the time that it has been in banned places can be monitored. The dog monitoring system consists on a set of wireless devices and a software component. The hardware includes a ZigBee device in charge of data communication and RSS (received signal strength) monitoring, a ZigBee sink in charge of data collection and command execution, an Android-based tablet in charge of data presentation, and a mini-PC, which includes the information system.
The dog carries in its harness the Arduino device, which includes a ZigBee communication module. The monitoring of the Received Signal Strength Indicator (RSSI) allows to determine the location of the dog. This value is obtained by the ZigBee transceiver each time a message is received. The message also includes the identifier of the ZigBee node. This information (ID + RSSI) is sent periodically or on demand to the sink node in order to provide the dog location. The Arduino device allows the operation on real-time connected mode or even, on unconnected mode. The first mode is used for real-time data transmission to the sink node, while the second-one is used for off-line dog tracking. The energy consumption is higher for the first mode, so an appropriate and limited use of this operation mode is recommended. The Arduino device includes a ZigBee communication module to minimize the energy consumption, since its consumption is lower than this of WiFi.illustrates the devices used. The infrastructure includes the mini-PC and the ZigBee sink. In this case, we have chosen a MeeGoPad T04 Windows 10 based mini-PC. It includes a quadcore Intel X5-Z8300 Cherry Trail processor and 2 GB of RAM memory, WiFi b/g/n and two USB ports. We connect a ZigBee sink to its USB 2.0 port and then we can interact with the Arduino device located at the dog's harness. We have developed a small and simple protocol to interact with the Arduino device. This protocol includes functionalities as the selection of the operation modes: location, tracking, rest; the battery level monitoring of the device; the firmware upload; the device diagnosis; and even more. The mini-PC runs a small relational database management system called SQLite (www.sqlite.org), which is embedded into the software. The C++−based software developed is a middleware in charge of data location storing into the database, data presentation on the graphical user interface GUI, and device management. [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] shows the hardware, while [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] shows the software architecture.
The middleware (see [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] includes at its lower layer the basic functionalities needed to communicate the Arduino device with the mini-PC (ZigBee communication), collect the sensed data (Data garbage) and the remote update of the firmware when needed (firmware update). The middle layer provides data storing into a relational database. In this case SQLite, that is absolutely embedded with the code in charge of data storing. The upper layer of the middleware provides the graphic user interface (GUI) functionalities. The WiFi communication module allows the communication with the Android device (tablet), allowing the interaction with the GUI module. Finally, the location and tracking of the pet is performed by the Location and Data monitoring modules. The location module applies a set of fuzzy rules to the data collected by the Arduino device, and stored into the database, and estimates the location of the dog. Data monitoring includes the tracking of the dog, and also the statistical analysis of its behavior.
Data presentation can be performed over the TV screen (the mini-PC has an HDMI connector) and a wireless keyboard/pointer device, or over an android-based tablet via WiFi communication. The GUI has been developed on HTML 5, since that allows its use on different devices. Given the simplicity of the GUI, and the low complexity of the functionalities required, HTML is a good choice for this app. The infrastructure includes the mini-PC and the ZigBee sink. In this case, we have chosen a MeeGoPad T04 Windows 10 based mini-PC. It includes a quadcore Intel X5-Z8300 Cherry Trail processor and 2 GB of RAM memory, WiFi b/g/n and two USB ports. We connect a ZigBee sink to its USB 2.0 port and then we can interact with the Arduino device located at the dog's harness. We have developed a small and simple protocol to interact with the Arduino device. This protocol includes functionalities as the selection of the operation modes: location, tracking, rest; the battery level monitoring of the device; the firmware upload; the device diagnosis; and even more. The mini-PC runs a small relational database management system called SQLite (www.sqlite.org), which is embedded into the software. The C++−based software developed is a middleware in charge of data location storing into the database, data presentation on the graphical user interface GUI, and device management. [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] shows the hardware, while [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] shows the software architecture.
The middleware (see [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] includes at its lower layer the basic functionalities needed to communicate the Arduino device with the mini-PC (ZigBee communication), collect the sensed data (Data garbage) and the remote update of the firmware when needed (firmware update). The middle layer provides data storing into a relational database. In this case SQLite, that is absolutely embedded with the code in charge of data storing. The upper layer of the middleware provides the graphic user interface (GUI) functionalities. The WiFi communication module allows the communication with the Android device (tablet), allowing the interaction with the GUI module. Finally, the location and tracking of the pet is performed by the Location and Data monitoring modules. The location module applies a set of fuzzy rules to the data collected by the Arduino device, and stored into the database, and estimates the location of the dog. Data monitoring includes the tracking of the dog, and also the statistical analysis of its behavior.
Data presentation can be performed over the TV screen (the mini-PC has an HDMI connector) and a wireless keyboard/pointer device, or over an android-based tablet via WiFi communication. The GUI has been developed on HTML 5, since that allows its use on different devices. Given the simplicity of the GUI, and the low complexity of the functionalities required, HTML is a good choice for this app. [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] shows a heat map, which displays the location of the dog along the time period selected by the user. The user can monitor the movement of the dog anywhere from the past week to the past hour, the distance traveled, as well as the specific time and number of times the dog has entered a forbidden zone. [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] shows a heat map, which displays the location of the dog along the time period selected by the user. The user can monitor the movement of the dog anywhere from the past week to the past hour, the distance traveled, as well as the specific time and number of times the dog has entered a forbidden zone. [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] shows a heat map, which displays the location of the dog along the time period selected by the user. The user can monitor the movement of the dog anywhere from the past week to the past hour, the distance traveled, as well as the specific time and number of times the dog has entered a forbidden zone. The location estimation is performed thanks to the ZigBee coverage. The ZigBee module of the Arduino device (mote) placed at the harness allows the measure of the RSSI values and then estimate the localization of the dog. Fuzzy location using RSSI values is a rough but reliable method that can be easily adapted to a given scenario, as we have previously experimented in [bib_ref] Fuzzy location and tracking on wireless networks, Astrain [/bib_ref]. We have chosen a fuzzy-based location estimator in order to determine the location of the dog according to the RSSI values measured by the Arduino device. The estimator is based on measuring the intensity of the received signal, something that can be done continuously, on a regular basis or on demand. Although many indoorg RF-based user location and tracking system, as RADAR [bib_ref] Radar: An in-building RF-based user location and tracking system, Bahl [/bib_ref] , have been widely considered, we consider more appropriated for the scenario here described the use of fuzzy logic. Fuzzy logic is used to handle the uncertainties that might be encountered and provide systems that do not require a specialized hardware or a great number of sensors or devices. As RSSI values can easily vary due to many reasons, for example due to the influence of the own body, we need to deal with inexact and uncertain information, so fuzzy methods [bib_ref] Fuzzy location and tracking on wireless networks, Astrain [/bib_ref] [bib_ref] A fuzzy logic-based system for indoor localization using WiFi in ambient intelligent..., Garcia-Valverde [/bib_ref] [bib_ref] Fuzzy Mobile-Robot Positioning in Intelligent Spaces Using Wireless Sensor Networks, Herrero [/bib_ref] are suitable for the scenario here described. The device measures the RSSI obtained from each ZigBee node of the flat (see [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] and transmits the values and the identifiers (IDs) of the nodes to the sink node, where the middleware located at the mini-PC stores the data collected into the database and estimates the location of the dog according to the signal strength map previously obtained. The comparison of the expected RSS values with those obtained allow to estimate the pet location. The radio propagation analysis presented in this paper allows obtaining the signal strength map in a simpler and less labor-intensive way than classical methods of signal measuring [bib_ref] Radar: An in-building RF-based user location and tracking system, Bahl [/bib_ref] [bib_ref] The horus WLAN location determination system, Youssef [/bib_ref] [bib_ref] Dynamic fingerprinting combination for improved mobile localization, Fang [/bib_ref]. The main restrictions of this method are the requisites of creating maps based on floor plans of indoors, of choosing the effective location of the (ZigBee) nodes used as beacons inside the building, and choosing the effective positioning technology and algorithms. Signal variation recommends using fuzzy techniques, since they perform better than crisp ones. Furthermore, the knowledge of the precise location often does not contribute to a better behavior of the location based systems. Fuzzy locating [bib_ref] Fuzzy location and tracking on wireless networks, Astrain [/bib_ref] determines the similarity (or even distance) between the RSS values obtained by the dog and the a priori knowledge with the operational ambience (the signal strength map of the flat). Tracking motion over time with the aid of a fuzzy automaton allows to increase the accuracy of the location estimator.
The location module has two main components, a set of fuzzy rules and a set of fuzzy automata. Rules are evaluated with the data collected and then location estimation is provided. An adaptive neuro-fuzzy inference system (ANFIS) has been used to obtain the rules. We have divided the flat in areas following a 8 × 10 mesh, and using the Matlab tool (fuzzy and neural network toolboxes) and the signal strength map previously obtained we have obtained a set of 63 rules as:
## S1 is high and s2 is verylow and s3 is high then location = entry
The obtention of the rules is simple, but the main problem is that the mechanism is totally dependent on the scenario considered. The cost of evaluation of fuzzy rules is very low since only a set of IF-THEN-ELSE sentences must be computed. Fuzzy automata require a bit more complicated calculus, since fuzzy operators are more complex, but not too much. That allows real-time operation. The fuzzy automata take advantage of the movement restriction, since the dog do not cross walls during its movement along the rooms of the flat. Thus, we define the potential travel routes that can be followed by the dog and we obtain a set of fuzzy automata. Each of the regions of the mesh previously defined is characterized as a symbol (character), and then a travel route is a string of symbols, where each symbol is the region where it has traveled. Tracking is performed by measuring the simuilarity between the string α representing the the path followed by the pet and the routes defined by the automata (ω i ). More information can be obtained in [bib_ref] Deformed fuzzy automata for correcting imperfect strings of fuzzy symbols, Garitagoitia [/bib_ref].
In order to validate the performance and accuracy of the system proposed by direct observation, we include a ground truth. We have fixed a forbidden area (see [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] and we have analyzed the behavior of the system during a day. Note the difficulty of the experimentation due to the limited cooperation of the dog. We have forced the dog to be placed in positions close to the forbidden room, but not entering it, either in front the door or the partitions. In the same way, we have forced the dog to enter the forbidden room and to sit and lie in different locations of this room. Results, depicted in [fig_ref] Table 3: Performance evaluation of the Location system [/fig_ref] , shows that the presence of the dog is incorrectly estimated 22.81% of the time, while it is succesfully determined the 77.19%. We analyze the presence (or not) of the dog in the forbidden area, determining the error/success rate of the estimations provided by the system. We define the success rate as the ratio between the number of times that the system has estimated that the dog was out of the room, when it was, and that the dog was in the room, when it was, and the total number of estimations (correct + incorrect estimations). In the same way, we define the error rate as the ratio between the number of incorrect estimations (the system has estimated that the dog was in the room, when it was out, and that the dog was out of the room, when it was in) and the total number of presence estimations. Incorrect estimations are due to both false positives (the dog has not entered the forbidden area, but the system so indicates) and false negatives (the dog has entered the forbidden area, but the system has not detected the intrusion), also addressed as type I and type II errors. The number of false positives and false negatives is quite similar, so any assumptions about them can be reached. As anticipated, the success rate obtained does not entirely satisfy the expected requirements, therefore we introduce the fuzzy automata. Fuzzy automata allow to discriminate impossible routes. Since the dog can only enter the room through the door (when opened) and the system permanently traces the location of the dog, the system corrects a great number of false positives and false negatives, resulting in a significantly higher success rate (close to 94.71%). [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] depicts the distribution grid used by the fuzzy automata to track the movement of the dog and then discriminate type I and type II errors. The dog's movement is modeled as a string of symbols, and the system builds a fuzzy automaton for each one of the possible sequences. Note that failing to pass through the walls significantly reduces the number of possible sequences. The string obtained is the concatenation of the grids traversed by the dog. Thus, the fuzzy automata compare the displacement followed by the dog with the set of all possible routes inside the flat and offers the value of similarity between them. The system selects the route that obtains a greater value of similarity allowing to correct many of the errors of type I and II.However, the error rate is not canceled (2.37% and 2.92%, respectively). The estimation errors concerning the door of the room are not discriminable. The automata are not able to determine whenever the door is opened or closed. succesfully determined the 77.19%. We analyze the presence (or not) of the dog in the forbidden area, determining the error/success rate of the estimations provided by the system. We define the success rate as the ratio between the number of times that the system has estimated that the dog was out of the room, when it was, and that the dog was in the room, when it was, and the total number of estimations (correct + incorrect estimations). In the same way, we define the error rate as the ratio between the number of incorrect estimations (the system has estimated that the dog was in the room, when it was out, and that the dog was out of the room, when it was in) and the total number of presence estimations. Incorrect estimations are due to both false positives (the dog has not entered the forbidden area, but the system so indicates) and false negatives (the dog has entered the forbidden area, but the system has not detected the intrusion), also addressed as type I and type II errors. The number of false positives and false negatives is quite similar, so any assumptions about them can be reached. As anticipated, the success rate obtained does not entirely satisfy the expected requirements, therefore we introduce the fuzzy automata. Fuzzy automata allow to discriminate impossible routes.
Since the dog can only enter the room through the door (when opened) and the system permanently traces the location of the dog, the system corrects a great number of false positives and false negatives, resulting in a significantly higher success rate (close to 94.71%). [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] depicts the distribution grid used by the fuzzy automata to track the movement of the dog and then discriminate type I and type II errors. The dog's movement is modeled as a string of symbols, and the system builds a fuzzy automaton for each one of the possible sequences. Note that failing to pass through the walls significantly reduces the number of possible sequences. The string obtained is the concatenation of the grids traversed by the dog. Thus, the fuzzy automata compare the displacement followed by the dog with the set of all possible routes inside the flat and offers the value of similarity between them.
The system selects the route that obtains a greater value of similarity allowing to correct many of the errors of type I and II.However, the error rate is not canceled (2.37% and 2.92%, respectively). The estimation errors concerning the door of the room are not discriminable. The automata are not able to determine whenever the door is opened or closed. When estimating the time the pet spends at each location, it should be noted that the system periodically calculates the location of the pet, and this is what determines the estimation accuracy. The measurement period determines the granularity of the time estimation. As the sampling period When estimating the time the pet spends at each location, it should be noted that the system periodically calculates the location of the pet, and this is what determines the estimation accuracy. The measurement period determines the granularity of the time estimation. As the sampling period is increased, the greater battery life of the ZigBee transceiver is achieved. Lengthening the sampling period involves a reduction on the accuracy of the estimation. If the dog moves between two consecutive observations, the system will not be aware of it. During the experimentation we have considered sampling periods of 5, 10, 30 and 60 s. Obviously, a sampling period of 5 s causes the transceiver battery runs out much earlier than in the case of considering a sample period of 60 s. A future work is to take advantage of the accelerometers located at motes to perform an intelligent choice of the sampling period. If the mote detects that our pet is not moving, it is not necessary to measure the RSSI level and this allows the ZigBee interface remains off longer, resulting in a lower battery consumption.
Pet monitoring, including the localization, tracking and time at forbidden areas of the pet (a dog in this case) has many similarities with the multiple existing proposals concerning human location tracking but the main difference occurs at the radio level. As it can be observed, the height and volume of the pet, which are significantly lower than those of people, motivates the RSSI values obtained. On one hand, the height plane in which lies the transceiver (in this case the transceiver is located in the dog's neck), and on the other hand, the way it affects the volume of the pet to the received signal. The ZigBee routers (ZR) are located on furniture that does not exceed one meter high, so that ZigBee signals bounce off many obstacles and penetrate a variety of close materials that cause varying effects. This is evidenced by a decrease in the value of RSSI observed. People tend to find above mentioned obstacles, and in general, the effect of these elements in the signal propagation is less relevant than in the case of pets. The custom that many pets have of spending long periods of time huddled in a position without moving too much is another matter to be considered. When pets are snuggled, they tend to tilt the neck to its body, which, depending on the orientation of the pet's transceiver to the ZR may cause observable changes in the RSSI values obtained. These variations, up to 5 dB, may be due to the directivity of the transceiver antenna, the ground effect, or even to the own body ot the pet. When the dog is snuggled, we observe average variations of RSSI values of 2.4 dBm and a maximum deviation of 5.1 dBm. Experimental results obtained do not allow to conclude that the animal's movement affects (impede or favor) its location. Something that seems reasonable, because the dimensions of the apartment do not allow the pet to run and achieve a significant speed.
# Discussion
In this work a novel indoor canine monitoring system based on WSN is presented. Thanks to this system, areas of the home that the dog has visited during the day can be easily known using any android based device, and furthermore, it can be monitored if the dog has entered into forbidden areas.
Since the system works considering the received power to carry out triangulation calculus, the deployment of the WSN network must be adequate and at least three routers must be visible for the device in any part of the home. In this work a 3D Ray Launching simulation technique is proposed for the proper deployment of the network and to calibrate application, since the power level in the different parts of the home for the three antennas must be registered in the application database.
Presented simulation tool can consider all parts of a complex scenario with the dielectric properties of all objects inside it and therefore, accurate results can be obtained if all parameters are correctly introduced. Besides, a canine simplified model compatible with the simulation code is developed for this work, which considering that the receiver is attached to the pet, is essential for the obtaining of accurate results. This model is flexible and allows the consideration of different kind of dogs and positions, taking under consideration all their properties.
BER results are also extracted from the simulation tool. This kind of results can help to find problems in the network as a consequence of interferences, since low error probability areas can be defined. These areas depends on the noise, transmission bit rate and the received power which depends on the transmitter antenna and the characteristics of the scenario. The radio planning analysis both in terms of path loss calculation as well as in terms of BER requirements show that for the type of scenario under analysis, mote deployment can be achieved with a node density of (1node/30-40 m 2 ), for the selected XBee transceivers. Other communication systems could be employed, as a function of future requirements such as required transmission speed or delay constraints derived by transmission of non-delay tolerant traffic.
In any case, simulation results are compared in [fig_ref] Figure 1: 3D view of the simulated measured scenario [/fig_ref] with a campaign of measurements carried out inside the scenario, validating the simulation tool as well as the simplified canine model. Those results allow using 3D ray launching simulation to design and develop pet indoor location systems minimizing the campaign of measurements carried out inside the scenario considered. The software development cycle is significantly reduced with the use of simulation data for the extraction of the location rules, and the definition of the fuzzy automata. The power distributions obtained from the 3D ray Launching model allow configuring the location module without requiring the use of expensive fingerprinting techniques. Since one of the main problems of indoor RF location systems is the need to rebuild the system whenever the working scenario changes, the simulation tool allows to recalculate the location system on a quick, easy and simple way. The location and monitoring system has been implemented and tested in order to validate location capabilities as well as connectivity of the system (and hence, interaction capabilities) for the scenario under analysis.
# Conclusions
In this work the viability of using a WSN network for dog monitoring is studied through a 3D Ray Launching simulation tool. In order to analyze the proposed system a simplified dog model has been developed considering different dog size proportions and dielectric properties of its biological tissues, in an automated way. A one-story home has been chosen as studied scenario considering its complete topology and characteristics. The main goal is the analysis of the influence of wireless propagation limitations in the implementation of a specific pet monitoring system, which has been implemented and tested.
A simplified parameterized dog model is implemented within the deterministic 3D RL code and introduced within the scenario under analysis in order to validate simulation results, proving to be an adequate approach in order to perform radioplanning tasks within the specific pet monitoring application. The estimations provide assessment in terms of required node density as well as on potential transceiver location, in order to comply with coverage/capacity requirements. Different measurement points are chosen and compared obtaining a maximum error of 5 dB in terms of RSSI detection error offset, demonstrating the accuracy of the 3D RL simulation method ane enabling node location tasks without previous measurement campaigns. In order to implement the pet monitoring system, an Android based indoor dog monitoring application has been coded in order to operate with XBee motes and a MeeGo mini PC, providing pet monitoring capabilities within the domestic household including a pet location algorithm based on fuzzy logic estimation. The proposed solution can be scaled in order to provide interactive communication with different pets and in different types of scenarios, with low deployment cost.
[fig] Figure 1: 3D view of the simulated measured scenario. [/fig]
[fig] Figure 2: Proportions of simplified dog model according with chosen height for medium size dog. [/fig]
[fig] Figure 3: Example of the simplified dog model in different position and with different sizes. [/fig]
[fig] Figure 4: Power distribution in the scenario when transmitter is placed in the living room for an empty living room (a) and when the dog is inside the lounge (b). [/fig]
[fig] Figure 5: Simulated scenario when four human bodies models are introduced in different locations. [/fig]
[fig] Figure 6: Comparison of receiver power when human body models are introduced in the home. [/fig]
[fig] Figure 7: BER results considering different bit rates and noise levels for the antenna placed in the living room. [/fig]
[fig] Figure 8: (a) Measurement points distributed around the home and the location of the dog; (b) Xbee device attached to the dog for measurements; (c) Measurement points distributed around the home when the dog is not considered. [/fig]
[fig] Figure 9: Measurement vs simulation results when the Xbee prototype is attached to the dog within the scenario under analysis. [/fig]
[table] Table 2: Simulation parameters used in the 3D Ray Launching code. [/table]
[table] Table 3: Performance evaluation of the Location system. [/table]
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10.1371/journal.pone.0025229
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CCBY
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3186794
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21991304
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s2orc_pubmed_articles
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Insights into the Internalization and Retrograde Trafficking of Dengue 2 Virus in BHK-21 Cells
Background: Dengue virus (DENV) enters cells via endocytosis, traffics to perinuclear (PN) region, the site of morphogenesis and exits by exocytosis. This study aims to understand the role of dynamin II, endosomes, microtubules (MT) and dynein in the early events of DENV replication.Findings: Using double immunoflourescence labelling of DENV-2 infected BHK-21 cells it was observed that the surface envelope (E) protein of the virion associated with dynamin II from 0-30 min post infection (p.i.). The sphincter like array of dynamin II supported its pinchase-like activity. The association with endosomes was observed from 0 min at cell periphery to 30 min in the perinuclear (PN) region, suggesting that internalization continued for 30 min. Association of E protein with alpha-tubulin was observed from 8 h indicating that it was the newly translated protein that trafficked on the MT. Dynein was found to associate with the E protein from 4 h in the cytoplasm to 48 h in the PN region and dissociate at 72 h. Association of E protein with dynein was confirmed by immunoprecipitation. Overexpression of dynamitin, which disrupts the dynein complex, resulted in loss of trafficking of viral E and core proteins. The findings corroborated with the growth kinetics assessed by quantitation of viral RNA in infected BHK-21 cells. The detection of E protein at 4 h-8 h correlated with detectable increase in viral RNA from 8 h. The detection of high concentrations of E protein in the PN region at 24-48 h coincided with release of virus into the supernatant starting from 36 h p.i. The dissociation of dynein from E protein by 72 h was coincident with maximum release of virus, hinting at a possible negative feedback for viral protein translation.
# Introduction
Dengue is by far the most devastating of all mosquito borne viral diseases, caused by dengue virus (DENV), a member of the family Flaviviridae. More than 3 billion humans live in dengue endemic regions of the world and currently more than 50 million infections occur annually with at least 500,000 individuals requiring hospitalization [bib_ref] Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem..., Gubler [/bib_ref] [bib_ref] Dengue: An update, Guzman [/bib_ref] [bib_ref] Emerging flaviviruses: The spread and resurgence of Japanese encephalitis, West Nile and..., Mackenzie [/bib_ref]. Development of antiviral agents has so far focused on inhibiting viral enzymes, i.e. nonstructural protein 3 (NS3) [bib_ref] Towards the design of antiviral inhibitors against flaviviruses: the case for the..., Lescar [/bib_ref]. An understanding of virus morphogenesis and proteinprotein interactions could provide new targets for intervention. DENV is an enveloped virus with a positive sense single-stranded ,11 kb RNA genome. Infection begins with attachment of virus particles to host receptors, several of which have been identified as putative receptors i.e. heparan sulphate [bib_ref] Flavivirus infection enhancement in macrophages: An electron microscopic study of viral cellular..., Gollins [/bib_ref] , heat shock proteins, Hsp70 and Hsp 90 [bib_ref] Heat Shock Protein 90 and Heat Shock Protein 70 Are Components of..., Vj [/bib_ref] , Glucose-regulated protein (GRP78/Bip) [bib_ref] Dengue virus entry into liver (HepG2) cells is independent of hsp90 and..., Cabrera-Hernandez [/bib_ref] , a 37-kDa/67-kDa high affinity laminin receptor [bib_ref] Dimensional VOPBA reveals laminin receptor (LAMR1) interaction with dengue virus serotypes 1,..., Phaik Hooi Tio [/bib_ref] [bib_ref] Growth and production of the dengue virus in C6/36 cells and identification..., Sakoonwatanyoo [/bib_ref] and Dendritic cell-specific intercellular adhesion molecule-3-Grabbing non-integrin (DC-SIGN) [bib_ref] DC-SIGN (CD209) Mediates Dengue Virus Infection of Human Dendritic Cells, Tassaneetrithep [/bib_ref]. After binding to the receptor DENV undergoes either clathrin mediated endocytosis [bib_ref] Rab 5 is required for the cellular entry of dengue and West..., Krishnan [/bib_ref] , or directly enters into the cytoplasm of cells by fusion at the plasma membrane (PM) [bib_ref] Flavivirus entry into cultured mosquito cells and human peripheral blood monocytes, Hase [/bib_ref] [bib_ref] A different mode of entry by dengue-2 neutralisation escape mutant virus, Lim [/bib_ref]. Following internalization, the virus envelope undergoes fusion with the endosomal membrane due to low pH, and the viral nucleocapsid is released into the host cytoplasm [bib_ref] Structure of the dengue virus envelope protein after membrane fusion, Modis [/bib_ref] [bib_ref] A structural perspective of the flavivirus life cycle, Mukhopadhyay [/bib_ref]. The viral RNA is then translated to yield structural and nonstructural viral proteins followed by transcription and replication of RNA, packaging and maturation in the perinuclear (PN) region [bib_ref] Ultrastructural aspects of the dengue virus (flavivirus) particle morphogenesis, Barth [/bib_ref] and egress by exocytosis [bib_ref] Proteolytic activation of tickborne encephalitis virus by furin, Stadler [/bib_ref] or budding at the PM [bib_ref] Flavivirus West Nile (Sarafend) egress at the plasma membrane, Ng [/bib_ref]. The entire process of morphogenesis is dependent on the movement of virus and viral components within the cell. The cytoskeleton is an integral component for intracellular trafficking [bib_ref] Viral strategies for intracellular trafficking: motors and microtubules, Leopold [/bib_ref] [bib_ref] A superhighway to virus infection, Greber [/bib_ref] with all 3 components, the microfilaments, the microtubules (MT) and the intermediate filaments, involved [bib_ref] Vimentin is required for dengue virus serotype 2 infection but microtubules are..., Chen [/bib_ref]. Dynein, is a microtubule associated motor protein and is responsible for the movement of cargo from cell periphery to cell centre, from the endoplasmic reticulum (ER) to the Golgi [bib_ref] Flavivirus West Nile (Sarafend) egress at the plasma membrane, Ng [/bib_ref]. A crucial role for dynein in the MT associated transport of viral proteins has been reported for hepatitis C [bib_ref] Hepatitis C Virus Core Protein Induces Lipid Droplet Redistribution in a Microtubule-and..., Boulant [/bib_ref] , African swine fever [bib_ref] African Swine Fever Virus Protein p54 Interacts with the Microtubular Motor Complex..., Alonso [/bib_ref] , Hanta [bib_ref] Dynein-Dependent Transport of the Hantaan Virus Nucleocapsid Protein to the Endoplasmic Reticulum-Golgi..., Ramanathan [/bib_ref] , polioand rabies [bib_ref] Interaction of the Rabies Virus P Protein with the LC8 Dynein Light..., Raux [/bib_ref] viruses. Although the major events in morphogenesis are broadly understood for DENV, the detailed events underlying, entry and retrograde trafficking have not been entirely delineated. The present study uses confocal microscopy to visualize internalization, endocytosis and early trafficking of DENV-2 proteins and shows for the first time association of dynamin II with the envelope (E) protein during entry and relevance of dynein in the retrograde trafficking of envelope and core (C) proteins.
# Materials and methods
## Cells and virus
BHK-21 (Baby Hamster Kidney) (C-13, American Type Culture Collection) cells were used as the cell line was suitable for both infection as well as transfection studies. The cells were maintained in Minimal Essential Medium (MEM) with 10% fetal bovine serum (FBS) at 37uC and 5% CO 2 , supplemented with 200 U/ml of penicillin/streptomycin and 1% glutamine. All cell culture reagents were procured from Gibco-BRL. DENV-2 (803347) was obtained from the Institute's Virus Repository and used in all the experiments.
## Antibodies and reagents
For immunofluorescence assay primary antibodies included mouse monoclonal antibody (MAb) against DENV-2 E glycoprotein and C protein (a kind gift from Dr. Askov, Queensland University of Technology, Australia), goat antibody against dynamin II, mouse MAb against dynein intermediate chain (Santa Cruz Biotechnology Inc., CA, USA), mouse MAb against alpha tubulin (Sigma, USA). Secondary antibodies used were goat antimouse IgG conjugated to FITC or donkey anti-mouse IgG conjugated to Alexa 488, donkey anti-goat IgG conjugated to Alexa 594 (Molecular Probes, Eugene, OR, USA) and rabbit antimouse IgG conjugated to TRITC (Sigma). Lysotracker Red (Molecular Probes) was used to stain endosomes.
## Immunofluorescence assay
BHK-21 cells were seeded one day prior to infection at a minimum concentration of 1610 5 cells/ml per 55 mm petri dish (Tarsons) containing 9622 mm cover slips. Cells were infected with DENV-2 at a multiplicity of infection (MOI) of 1 or 5. Adsorption was carried out for 1 h at 4uC to prevent virus entry and synchronize infection of cells. Cells were washed twice with cold medium to remove unbound virus and replenished with prewarmed medium. The cover slips were taken out of the culture at various time points post infection (p.i.) under sterile conditions, washed with 0.01 M phosphate buffer saline pH 7.2 (PBS) and fixed in ice-cold acetone for 10 min at 220uC for experiments with dynein. Cells labeled for dynamin II and endosomes were fixed with 3.7% paraformaldehyde for 30 min at room temperature (RT), followed by permeabilization with 0.1% Triton X-100 for 2 min and quenching with 0.01 M NH 4 Cl. The fixed cells were washed with PBS and blocked with 1% bovine serum albumin (BSA) in PBS for 30 min. For dual staining, viral E or C proteins were labeled with specific mouse MAbs and the cellular components with the specific antibodies as mentioned in [fig_ref] Table 1: Primary and secondary antibodies used for staining viral and cellular proteins [/fig_ref]. The primary antibodies to viral protein and cellular components were added simultaneously to the cells and incubated for 1 h followed by washing with PBS. This was followed by incubation with appropriate secondary antibodies conjugated to fluorescent dyes, FITC, TRITC, Alexa 488 or Alexa 594 [fig_ref] Table 1: Primary and secondary antibodies used for staining viral and cellular proteins [/fig_ref] added simultaneously. DAPI (49, 69 diamino-2-phenylindole) was used to stain nucleus in all experiments. At the end of the staining process, cover slips were washed and mounted onto slides using mounting medium MOWIOL (Calbiochem, CA, USA). Control cells, which were not infected but submitted to the same procedures, were included in all experiments as mock infected cultures.
## Transfection with gfp-dynamitin plasmid
The GFP-dynamitin expressing plasmid was a kind gift from Dr. Beate Sodeik, Hannover Medical School, Germany. BHK-21 cells, grown on coverslips in 6 well plates, were transfected with 2 ug GFP-dynamitin plasmid using Lipofectamine 2000 Plus (Invitrogen) according to manufacturers instructions. Briefly, 2 ug of plasmid was diluted in 250 ul of MEM mixed with 250 ul of MEM containing 10 ul of Lipofectamine and incubated for 30 min at RT. The DNA-liposome complex was then added to preformed monolayer of BHK-21 cells with 70% confluency in serum free medium and incubated at 37uC. Cells were then infected with DENV-2 (MOI of 1) at 6 h post transfection. The cells were fixed after 36 h p.i. and stained for DENV-2 E or C protein using mouse anti-E or anti-C MAb followed by anti-mouse IgG labeled with TRITC. For comparison mock-transfected cells were infected with DENV-2 and processed similarly.
## Image acquisition and image analysis
The images were acquired using Laser Scanning Microscope 510 META (Carl Zeiss, Germany) with 406and 636oil objective lens corrected for oil immersion. For dual color analysis, green and red emissions were recorded sequentially through appropriate filters (505-530 band-pass filters for Alexa 488/FITC and 560 nm long-pass filters for Alexa 594/TRITC). Post acquisition image analysis was done using the 3D projection option of LSM software, surface rendering option of Huygens Essential (Scientific Volume Imaging, The Netherlands) and maximum intensity projection using Imaris (Bitplane Scientific software). Three-dimensional (3D) images, which cover the entire depth of the cell, were derived from deconvolved images. All voxels (volume pixels) in the image with a given colocalization level were joined, forming a 3D surface. In the surface rendering option of the software, the surface is split into different closed unconnected parts enabling independent analysis.
## Coimmunoprecipitation assay
BHK-21 cells were infected with DENV-2 (MOI of 1). At 36 h p.i., the cells were washed with ice cold PBS (0.01 M), scraped and centrifuged at 2000 rpm. The cells were lysed in 5 pellet volumes of ice-cold lysis buffer (50 mM Tris-HCl,150 mM NaCl, 0.5% (v/ v) NP40, 0.1 mM NaF, 10 mM DTT and protease inhibitor cocktail). The clarified cell lysate containing approximately 500 ug of protein was incubated with 10 ul of equilibrated Sepharose protein A beads for one hour at 4uC for preclearing and centrifuged to recover lysate. The precleared lysate was incubated overnight at 4uC with human anti-DENV IgG (purified from a serum sample that had plaque reduction neutralization titre of 12,500) for formation of immune complexes. The immune complexes were captured by Sepharose protein A beads by incubating for 3 h at 4uC, centrifuged and washed three times with lysis buffer. The beads were resuspended in 30 ul of sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris, pH 6.8, 4% SDS, 20% glycerol), boiled for 5 min, centrifuged and the supernatant was loaded on 12.5% polyacrylamide gel under non reducing conditions. The proteins were transferred to nitrocellulose membrane for Western blot analysis. The blot was probed with anti-dynein antibody followed by goat anti-mouse IgG labeled with Horse radish peroxidase (HRP) (Sigma) and developed with diamino benzedene substrate. Mock infected BHK-21 cell lysate pulled down with Sepharose protein A beads served as negative control. Infected BHK-21 cells lysate was used as positive control.
## Real time pcr
Viral RNA was quantitated in the infected cultures using a two step real time RT-PCR test reported previously [bib_ref] Development of real time PCR for detection and quantitation of dengue viruses, Gurukumar [/bib_ref]. BHK-21 cells were infected at MOI of 1 and culture supernatant and cells were harvested at different time points starting from 0 h to 120 h p.i.
# Results
## Kinetics of denv-2 replication in bhk-21 cells
Prior to undertaking experiments on entry and early intracellular trafficking, the kinetics of viral RNA production in BHK-21 cells was determined. BHK-21 cells were infected with DENV-2 at MOI of 1 (,10 9 RNA molecules) and the cultures were assessed for cell-associated and cell free viral RNA at different time points p.i. by real time RT-PCR [fig_ref] Figure 1: Growth curve of DENV-2 in BHK-21 cells [/fig_ref]. Despite washing off the unadsorbed virus, viral RNA (10 5 molecules) was detected in cells as well as culture supernatant, from 0-6 h after infection. Similar levels of viral RNA at early time points after infection were shown in a previous report [bib_ref] Host gene expression profiling of dengue virus infection in cell lines and..., Fink [/bib_ref]. It is possible that virus particles, which are not internalized, remain loosely bound, and are detectable for a long time in culture. The inefficiency of the process of infection for flaviviruses with a PFU: particle ratio of 1:4000 has been reported before [bib_ref] Detection of yellow fever virus: a comparison of quantitative real-time PCR and..., Bae [/bib_ref] [bib_ref] Quantitative analysis of dengue-2 virus RNA during the extrinsic incubation period in..., Richardson [/bib_ref]. The cell-associated viral RNA levels showed a rise from 12 h onwards till 72 h (10 9 molecules) and then decreased to 10 7 particles by 120 h. Viral RNA in the supernatant started increasing from 36 h and peaked at 84 h (10 7 molecules) after which the level reached a plateau till 120 h. The cultures could not be tested after 120 h due to cell lysis.
## Association of dynamin ii with denv-2 e protein
Dynamin II, a MT associated GTPase protein is known to regulate clathrin-mediated endocytosis [bib_ref] Dynamin:GTP controls the formation of constricted coated pits, the rate limiting step..., Sever [/bib_ref] [bib_ref] Dynamin and its partners: a progress report, Schmid [/bib_ref]. The association of dynamin II with DENV-2 E protein and the duration of the association were determined to understand the kinetics of internalization. BHK-21 cells were infected with DENV-2 at MOI of 5 to ensure infection of most cells simultaneously. Virus adsorption was carried out on ice to synchronize internalization [bib_ref] Functional entry of dengue virus into Aedes albopictus mosquito cells is dependent..., Acosta [/bib_ref]. After adsorption virus was removed and the monolayer was washed with cold medium. Immediately after adding pre-warmed medium, cells were fixed and processed for staining after 0, 5, 10, 15, 30, 60 and 120 minutes. Cells were double stained for DENV-2 with mouse anti-E MAb and for dynamin II with goat antidynamin II antibody, followed by donkey anti-mouse IgG-Alexa 488 and donkey anti-goat IgG-Alexa 594. The E protein was presumed to represent the incoming virus, as it is the major surface protein on the virion. Colocalization of E protein and dynamin II was observed from 0 min to 30 min p.i. The colocalization foci were counted and analyzed in 20 fields at 0, 5, 15 and 30 min time points [fig_ref] Table 2: Characterization of foci of colocalization of E protein with dynamin II [/fig_ref]. The number of cells showing the presence of colocalization foci decreased from 50% at 0 min to 17% at 30 min. The size of the foci did not vary with time. The size of foci ranged from 0.75 um to 2 um, within which the spot of green fluorescence representing virus was 0.2-1 um (size of virus 50 nm) suggestive of each focus representing an aggregate of virus associating with dynamin II. In the area of colocalization the average fluorescence intensity for dyanmin II was 234 and did not change with time. The intensity of fluorescence for E protein decreased from 145 at 0 min to 84 at 15 min. The pattern of intensity reversed at points of no colocalization with average fluorescence intensity of 48 for dynamin II and 207 for E protein.
The colocalization of E protein with dynamin II is depicted in the split image [fig_ref] Figure 2: DENV-2 associates with dynamin II during internalization [/fig_ref]. The intensity of DENV E protein at a point of no colocalization [fig_ref] Figure 2: DENV-2 associates with dynamin II during internalization [/fig_ref] is higher than that observed in the area of colocalization [fig_ref] Figure 2: DENV-2 associates with dynamin II during internalization [/fig_ref] as shown by the intensity profiles. For a clearer depiction of the merging of virus and dynamin II, deconvolved images of the cell in [fig_ref] Figure 2: DENV-2 associates with dynamin II during internalization [/fig_ref] were generated using Huygens surface rendering [fig_ref] Figure 2: DENV-2 associates with dynamin II during internalization [/fig_ref] wherein free virus can be seen (green) around the focus of colocalization (yellow). To show that the complexes were present on the surface, z-stack images of the infected cells were acquired and analyzed by Maximum Intensity Projection (Imaris) [fig_ref] Figure 2: DENV-2 associates with dynamin II during internalization [/fig_ref] , which clearly shows the location of the aggregate on cell surface. Rotation of the image along its y-axis and 3D imaging using Zeiss software [fig_ref] Figure 2: DENV-2 associates with dynamin II during internalization [/fig_ref] further confirms the surface location of the complex. The colocalization between E protein and dynamin II decreased by 30 min p.i. [fig_ref] Figure 2: DENV-2 associates with dynamin II during internalization [/fig_ref]. At 60 min [fig_ref] Figure 2: DENV-2 associates with dynamin II during internalization [/fig_ref] there was no association between E protein and dynamin II. [fig_ref] Figure 2: DENV-2 associates with dynamin II during internalization [/fig_ref] shows the distribution of dynamin II in mock infected cells. The results illustrate the involvement of dynamin II in the process of virus internalization which peaked in the first 5 min of infection.
## Trafficking of virus within endosomes
Lysotracker Red dye was used to follow the kinetics of DENV-2 association within endosomes. Lysotracker red is an acidophilic dye which selectively stains low pH containing compartments and would therefore detect DENV-2 in both early and late endosomes Cells were infected with DENV-2 at 5 MOI, similar to the experiment with dynamin II and stained for E protein and endosomes at 0 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h post infection. Lysotracker red was added to cells 2 h prior to fixation and cells were labeled for E protein using mouse anti-E MAb followed by goat anti-mouse IgG FITC after fixing.
Tracking the movement of endosomes showed that the endosomes positive for E protein, were visible close to cell periphery at 0 min p.i. [fig_ref] Figure 3: DENV-2 E protein traffics within the endosomes post internalization [/fig_ref] , were present throughout the cytosol within 15 min and collected in the PN region by 30 min [fig_ref] Figure 3: DENV-2 E protein traffics within the endosomes post internalization [/fig_ref]. By 2 h [fig_ref] Figure 3: DENV-2 E protein traffics within the endosomes post internalization [/fig_ref] only a few cells showed association and by 4 h there was no colocalization [fig_ref] Figure 3: DENV-2 E protein traffics within the endosomes post internalization [/fig_ref]. The close association was further confirmed by the deconvolution analysis of the 30 min image [fig_ref] Figure 3: DENV-2 E protein traffics within the endosomes post internalization [/fig_ref]. The number of E-positive endosomes observed was higher than the number of dynamin II-E protein colocalization foci per cell, reflecting the transient nature of dynamin II-E protein complexes. The results indicate that DENV-2 traffics within endosomes and is delivered to the PN region within 30 min. Infected cells were labeled for the presence of viral C protein at 1 h, 2 h and 4 h p.i. to detect capsid released within the cells. Absence of positive signal indicated that the concentration of released capsid was below detectable levels. The decrease in E protein-positive endosomes by 2 h indicates end of entry events.
## Association of denv-2 e protein with microtubule (mt)
The microtubule network is the major highway along which there is movement of endocytosed material. Colocalization of the E protein with alpha tubulin was therefore examined in BHK-21 cells infected at 1 MOI. Infected cells were fixed at different time points, 1 h, 2 h, 4 h, 8 h and 24 h p.i. The fixed cells were labeled first with mouse anti-E MAb followed by goat anti-mouse IgG FITC. The labeled cells were saturated with unlabeled goat antimouse IgG, then stained with mouse anti-alpha tubulin MAb followed by rabbit anti-mouse IgG TRITC. Close association between the two proteins was observed from 8 h [fig_ref] Figure 4: DENV-2 E protein traffics on microtubules [/fig_ref] and maximized at 24 h [fig_ref] Figure 4: DENV-2 E protein traffics on microtubules [/fig_ref] especially in the PN region, representative of the microtubule organization centre (MTOC). Mock infected cells are shown in [fig_ref] Figure 4: DENV-2 E protein traffics on microtubules [/fig_ref]. No colocalization was observed at early time points prior to 8 h p.i. Although confocal microscopy has its limitations, the observation of E protein associating with microtubules from 8 h onwards and not prior to that suggested that microtubules are intimately involved in trafficking of newly synthesized E protein and not the entering virions.
## Dynein dependent retrograde trafficking of denv-2 e protein
Dynein is a minus-end directed motor protein complex, which is implicated in the trafficking of viral proteins towards the PN region [bib_ref] Dynein-Dependent Transport of the Hantaan Virus Nucleocapsid Protein to the Endoplasmic Reticulum-Golgi..., Ramanathan [/bib_ref] [bib_ref] The role of the cytoskeleton during viral infection, Dohner [/bib_ref]. DENV morphogenesis is known to occur in the PN region in the endoplasmic reticulum (ER) and Golgi apparatus [bib_ref] Ultrastructural aspects of the dengue virus (flavivirus) particle morphogenesis, Barth [/bib_ref] [bib_ref] Composition and three-dimensional architecture of the dengue virus replication and assembly sites, Welsch [/bib_ref]. To determine whether dynein was the motor protein involved in MT based trafficking of DENV-2 E protein, infected cells were fixed and doubled stained for dynein intermediate chain and viral E protein at different time points from 2 h to 72 h p.i. For E protein and dynein, both the primary antibodies were mouse derived therefore the labeling was done sequentially with an intervening step of blocking. After reacting the cells with mouse anti-E MAb followed by rabbit anti-mouse IgG TRITC, the cells were blocked with unlabeled goat anti-mouse IgG. Cells were then incubated with mouse anti-dynein MAb followed by donkey antimouse IgG Alexa 488. There was no E protein visible at 2 h (not shown). Panel 5A shows the pattern of E-dynein association at 4 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h with the intensity profiles for both fluorochromes in the marked region of colocalization. Dynein in uninfected cells was distributed evenly in the entire cell [fig_ref] Figure 5: DENV-2 E protein associates with dynein during retrograde trafficking [/fig_ref]. In the infected cell, intensity of dynein increased around the E protein. At 4 h, the E protein was present at very low concentration and colocalized with dynein. As time progressed, the concentration of E protein increased and by 12 h, a discrete bead like pattern of E-dynein complex, was seen which corresponded to the distribution of ER suggestive of trafficking to and from ER [bib_ref] Immunofluorescent sites in Vero cells infected with the flavivirus Kunjin, Ng [/bib_ref]. At 24-48 h maximum concentration of the dynein-E complex was observed in the PN region. Association of viral protein in the Golgi apparatus has been reported before [bib_ref] Markers for trans-Golgi membranes and the intermediate compartment localize to induced membranes..., Mackenzie [/bib_ref]. At 60 h, colocalization was weak and by 72 h, dissociation between dynein and E protein was observed. Another facet of infection was revealed by the intensity profiles accompanying each split image. In infected cells the concentration of E protein and dynein were inversely proportional to each other as infection progressed indicating the requirement of dynein for early trafficking of E (intensity profiles in [fig_ref] Figure 5: DENV-2 E protein associates with dynein during retrograde trafficking [/fig_ref]. Deconvolved images were used to generate 3D reconstruction of E-dynein at 4 h [fig_ref] Figure 5: DENV-2 E protein associates with dynein during retrograde trafficking [/fig_ref] , 48 h [fig_ref] Figure 5: DENV-2 E protein associates with dynein during retrograde trafficking [/fig_ref] and 72 h [fig_ref] Figure 5: DENV-2 E protein associates with dynein during retrograde trafficking [/fig_ref] , which depicted the association followed by dissociation of dynein with E protein as infection progressed. The kinetics of association of dynein with E protein suggests that the newly synthesized E protein uses dynein for retrograde trafficking to the site of assembly and dissociates from it at 72 h when maximum virus is present in the cell supernatant [fig_ref] Figure 1: Growth curve of DENV-2 in BHK-21 cells [/fig_ref].
## Co-immunoprecipitation of dynein with denv-2 e protein
Co-immunoprecipitation was used to prove that the colocalization between E protein and dynein observed by confocal microscopy represented formation of E-dynein complex. BHK-21 cells were infected with DENV-2 and lysed at 36 h p.i., a time point at which maximum colocalization was observed. IgG, purified from the serum of a dengue immune individual was used to immunoprecipitate the proteins from infected and uninfected cell lysates. The immune complexes resolved by SDS-polyacrylamide gel electrophoresis (PAGE) under non reducing condition followed by Western blot were identified with antibody directed against the intermediate chain of dynein [fig_ref] Figure 5: DENV-2 E protein associates with dynein during retrograde trafficking [/fig_ref]. The reactivity of anti-dynein antibodies with cell-associated dynein is shown in Lane 1. The presence of dynein in the immune complex coprecipitated with human anti-dengue IgG coated sepharose A beads is visible in Lane 2. There was no signal in the immunoprecipitated uninfected BHK-21 lysate in Lane3. The results of the co-immunoprecipitation assay validated the colocalization of E protein-dynein visible in infected cells by confocal microscopy.
## In silico docking of e protein with dynein
The light chain of dynein (LC8) bound to residues 128-138 of intermediate chain (IC74) is reported to contain the cargo binding site [bib_ref] Structure and dynamics of LC8 complexes with KXTQT-motif peptides: swallow and dynein..., Benison [/bib_ref] of the MT-dependent motor protein complex. Hex (Version 5.1), which is a molecular superimposition and docking program based on 8 Dimensional fast Fourier transform (FFT), was used to carry out docking between the E protein of DENV-2 and dynein using structures downloaded from the Protein Data Bank (PDB). DENV-2 E monomer (PDB:1TG8) was docked with dynein (PDB:2P2T). The model with the lowest energy was selected for further analysis using Accelrys Discovery Studio visualizer (version 2.5). Five residues on the dynein molecule were found to interact with five residues on the E protein [fig_ref] Figure 6: Docking of DENV-2 E protein with dynein [/fig_ref]. The five residues identified in the E protein [fig_ref] Figure 6: Docking of DENV-2 E protein with dynein [/fig_ref] were conserved in 87 other strains of DENV-2 (protein sequences downloaded from Genbank) when analyzed by Clustal W2 (MEGA 5.03). All five residues were located in domain I of the E protein but were discontinuous. Whether the residues are actually involved in binding will require further studies.
## Loss of dynein motor activity by over expression of dynamitin
Dynein along with its cofactor dynactin, mediates MTdependent retrograde trafficking. Over expression of p50/ dynamitin, a subunit of dynactin acts as a dominant-negative inhibitor of dynein-dynactin interaction which blocks dynein mediated transport [bib_ref] Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent..., Burkhardt [/bib_ref] [bib_ref] Molecular characterization of the 50 Kd subunit of dynactin reveals function for..., Echeverri [/bib_ref]. The effect of dynamitin over expression on DENV-2 E protein trafficking was investigated. Cells were transfected with GFP-dynamitin and infected with DENV-2 at 6 h post transfection. The infected cells were stained for E protein at 36 h p.i., the time point at which maximum colocalization of E protein with dynein had been observed. In control nontransfected infected cultures, .90% of the cells were infected and positive for E protein which accumulated in the PN region [fig_ref] Figure 7: Over expression of Dynamitin inhibits trafficking of DENV-2 E and core proteins [/fig_ref]. In transfected infected cultures GFP-dynamitin positive cells showed low expression of E protein in the cytosol and not in the PN region as seen in adjacent dynamitin-negative cells [fig_ref] Figure 7: Over expression of Dynamitin inhibits trafficking of DENV-2 E and core proteins [/fig_ref]. Therefore there was inhibition of expression of E protein and its trafficking to the PN region. The effect of over-expression of dynamitin on the transport of C protein was also investigated. DENV C protein is known to contain nuclear localization signals, and is transported to the nucleus [bib_ref] Intracellular localization and determination of a nuclear localization signal of the core..., Wang [/bib_ref]. In the infected mock transfected cells C protein was observed in the cytosol and in the nucleus [fig_ref] Figure 7: Over expression of Dynamitin inhibits trafficking of DENV-2 E and core proteins [/fig_ref]. In comparison in GFP-dynamitin transfected infected cells, the C protein was expressed but restricted to the cytosol [fig_ref] Figure 7: Over expression of Dynamitin inhibits trafficking of DENV-2 E and core proteins [/fig_ref]. Cells showing high intensity of GFP-dynamitin (white arrowhead) had no detectable C protein. A similar pattern was also seen for the E protein (not shown). The results thus unequivocally proved the role of dynein in trafficking of DENV-2 E and C proteins.
# Discussion
The current study was undertaken to determine the interactions of cellular proteins with E protein during the internalization and trafficking of DENV-2 in mammalian fibroblast cells (BHK-21) using confocal microscopy. DENV has been shown to gain entry into different cell types using different modes of internalisation, i.e. clathrin/caveolae dependent endocytosis [bib_ref] Rab 5 is required for the cellular entry of dengue and West..., Krishnan [/bib_ref] and fusion at PM [bib_ref] A different mode of entry by dengue-2 neutralisation escape mutant virus, Lim [/bib_ref]. We studied the dynamin II dependency of virus entry. Dynamin II belongs to a family of GTPases associated with formation of nascent vesicles and promotion of vesicle fission [bib_ref] Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae, Pelkmans [/bib_ref] and is closely associated with clathrin-mediated endocytosis. We were successful in showing a strong association between DENV-2 E protein and dynamin II using confocal microscopy. The E protein is the major surface glycoprotein of DENV, which is responsible for engaging the receptor [bib_ref] Dengue virus infectivity depends on envelope protein binding to target cell heparin..., Chen [/bib_ref] [bib_ref] The entry machinery of flaviviruses, Heinz [/bib_ref]. Therefore visual- ising the E protein in the early steps of virus entry was considered representative of tracking the virion. We used the E-dynamin II association to determine the kinetics of DENV-2 entry. Colocalization of DENV-2 E protein with dynamin II within seconds was in concordance with the function of dynamin II, which during clathrin mediated endocytosis aggregates around the vesicle and serves as pinchase-like mechanoenzyme to facilitate the formation of endocytic vesicles by severing nascent endocytic pits from the PM [bib_ref] Dynamin: switch or pinchase?, Thompson [/bib_ref]. The higher intensity of dynamin II at the foci of colocalization was perhaps a reflection of dynamin II aggregation at the site of endocytosis [bib_ref] Dynamin and its role in membrane fission, Hinshaw [/bib_ref]. The observation of lower intensity of E protein in the foci of colocalization compared to the points of no colocalization could be due to lower accessibility of the E protein to the antibody once the virus had entered the process of endocytosis. The presence of large amounts free virions on the cell surface was supported by the detection of substantial viral RNA at 0-6 h p.i. by real time RT-PCR. This kind of visualization of the endocytosis process is being shown for the first time. The maximum internalization occurs within the first 15 min of infection, however the E-dynamin II complex could be visualized at the cell surface till 30 min. This indicates that DENV-2 internalization is a process that goes on till 30 min p.i. The kinetics with confocal microscopy was in agreement with the internalization kinetics reported earlier by electron microscopy [bib_ref] Flavivirus infection enhancement in macrophages: An electron microscopic study of viral cellular..., Gollins [/bib_ref] and estimation of internalized infectious virus by PFU assay [bib_ref] Analysis of the Steps Involved in Dengue Virus Entry into Host Cells, Hung [/bib_ref] , where maximum internalization of virus occurs within the first few minutes of infection.
Labelling for endosomes and E protein revealed that DENV-2 localised in endosomes within 0 min p.i. and by 30 min the E protein positive endosomes were observed mostly in the PN region. Therefore internalization and the process of virus trafficking to the PN region via endosomes continue for 30 min. Association of dengue virus with early and late endosomes has been shown at 3 min and 17 min post infection respectively by live cell imaging [bib_ref] Analysis of the endocytic pathway mediating the infectious entry of mosquito-borne flavivirus..., Chu [/bib_ref] [bib_ref] Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in..., Van Der Schaar [/bib_ref]. The absence of endosome free E protein in the cytoplasm during this period indicated that virus travels to the site of replication protected in the endocytic vesicles and uncoating occurs directly at the site of replication. Following endocytosis the low pH of the endosomes results in trimerization of the envelope protein [bib_ref] Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic..., Allison [/bib_ref] [bib_ref] Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E..., Heinz [/bib_ref] followed by fusion and uncoating of nucleocapsid. It has been reported for other flaviviruses that most of the steps in replication occur in the PN region [bib_ref] Assembly and Maturation of the Flavivirus Kunjin Virus Appear To Occur in..., Mackenzie [/bib_ref]. Intracellular shuttling of both proteins and vesicles requires cytoskeletal filaments and molecular motor proteins [bib_ref] Motor protein receptors: moonlighting on other jobs, Klopfenstein [/bib_ref] [bib_ref] Membrane motors, Allan [/bib_ref]. Staining for MT and DENV-2 E protein showed close association between the two from 8 h onwards, with viral protein accumulating in the PN region. Cytoplasmic dynein transports cargo toward the microtubule minus end. Co staining with dynein revealed colocalization of DENV-2 E protein with dynein from 4 h p.i. Before this time point the E protein could not be seen either in association with dynein or independent. The last sighting of E protein was at 2 h within endosomes. Transcription of viral RNA has been reported to occur by 2 h [bib_ref] Analysis of the endocytic pathway mediating the infectious entry of mosquito-borne flavivirus..., Chu [/bib_ref]. It follows therefore that uncoating and the first round of translation of genomic RNA should be occurring before 2 h. The fact that the E protein could not be observed in the cells till 4 h indicated that the concentration of the initially translated protein is too low to be detected by immunofluorescence assay. Therefore the E protein was seen colocalized with dynein only after the de novo synthesis of viral proteins had reached a substantial concentration. By 12 h most of the dynein was engaged by the viral E protein. The E-dynein complex aggregated to the PN region by 36 h, which is now known to be the region of high viral activity [bib_ref] Ultrastructural aspects of the dengue virus (flavivirus) particle morphogenesis, Barth [/bib_ref] [bib_ref] Assembly and Maturation of the Flavivirus Kunjin Virus Appear To Occur in..., Mackenzie [/bib_ref]. Further proof of binding of E protein to dynein was provided by co-immunoprecipitation at 36 h post infection and also by protein-protein docking analysis. A putative site of interaction comprising of five residues was identified on the E protein. Three of the five residues are present in the recognition sequence on dynein-binding cargo proteins. Dynein mediated transport of the E protein on the microtubules is perhaps involved in the movement of E protein to the ER and from the ER to the Golgi apparatus, the major players in DENV morphogenesis. The loss of association by 60-72 h was co-incidental with maximum release of virus into the supernatant of infected cultures as proved by real time RT-PCR data. Loss of association with dynein indirectly suggests reduction in synthesis of E protein. Therefore, it is possible that when there is peak virus production, there is shutdown of viral protein synthesis.
To prove that dynein mediated trafficking was essential for DENV-2 replication, dynein motor activity was disrupted by over expression of dynamitin and its effect was seen on the expression of viral E protein. Dynamitin, a subunit of dynein dynactin complex, is required for cargo transport [bib_ref] Molecular characterization of the 50 Kd subunit of dynactin reveals function for..., Echeverri [/bib_ref]. Over expression of dynamitin resulted in lower concentrations of viral E and C proteins and inhibited their translocation to target sites. Higher expression of dynamitin resulted in total inhibition of E and C protein expression, which was indicative of inhibition of virus replication. Thus dynein was crucial to the trafficking of newly synthesized DENV structural proteins, E and C.
In conclusion the virions gain entry via dynamin II assisted endocytosis. They are translocated from cell periphery to PN region within endosomes. The newly translated DENV-2 protein binds to dynein and traffics on MT to PN region which is known to be the site of assembly. The association of dynein in the intracellular transport of DENV-2 proteins is being shown for the first time.
[fig] Figure 1: Growth curve of DENV-2 in BHK-21 cells. BHK-21 cells were infected with DENV-2 (803447) at 1 MOI and culture supernatants and cell lysates were collected at regular intervals from 0-120 h p.i. Viral RNA was quantitated by real time RT-PCR. doi:10.1371/journal.pone.0025229.g001 [/fig]
[fig] Figure 2: DENV-2 associates with dynamin II during internalization. BHK-21 cells were infected at MOI of 5. The cells were fixed at different time intervals and stained for DENV-2 with mouse anti-E MAb and for dynamin II with goat anti-dynamin II antibody, followed by donkey anti-mouse IgG Alexa 488 and donkey anti-goat IgG Alexa 594. The images of the E-dynamin complex analyzed using different programs are shown; A] split images with magnified insets at 0 min, B] intensity profile at 0 min showing points of colocalization and C] no colocalization D] 3D deconvolved image of optical sections of 2.5 mm using Huygen's essential software E] Maximum Intensity projection (MIP) highlighting the surface location of the complex (Imaris 7.0) F] 3 D projections at different angles (i) 60u, ii) 90u, iii) 100u, iv)110u, v) 120u and vi) 130u using Carl Zeiss AIM 4.0. G] split image at 30 min and H] no association at 1 h (Imaris 7.0). I] Mock infected cells showing the presence of dynamin II. doi:10.1371/journal.pone.0025229.g002 [/fig]
[fig] Figure 3: DENV-2 E protein traffics within the endosomes post internalization. BHK-21 cells were infected at MOI of 5. Cells were stained for endosomes by adding lysotracker red to the cells 2 h prior to fixation. The cells were stained for DENV-2 E protein using mouse anti-E MAb followed by goat anti-mouse IgG FITC. Split images show DENV-2 E protein associating with endosomes at A] 0 min B] 30 min C] 2 h and D] 4 h. E] 3D reconstruction of the z stacked (thickness 3.30 mm) deconvolved image for the 30 min time point. doi:10.1371/journal.pone.0025229.g003 [/fig]
[fig] Figure 4: DENV-2 E protein traffics on microtubules. BHK-21 cells infected at 1 MOI were fixed at different time points and stained for DENV-2 E protein and anti-alpha tubulin. The E protein was labelled with mouse anti-E MAb followed by goat anti-mouse IgG FITC conjugate. Cells were saturated with unlabeled goat anti-mouse IgG and then stained with mouse anti-alpha tubulin MAb followed by rabbit anti-mouse IgG TRITC. Infected cells showing colocalization of DENV E protein and alpha-tubulin, A] at 8 h and B] at 24 h. C] Mock infected cells showing distribution of alpha-tubulin. doi:10.1371/journal.pone.0025229.g004 [/fig]
[fig] Figure 5: DENV-2 E protein associates with dynein during retrograde trafficking. BHK-21 cells infected with DENV-2 at 1 MOI were fixed at different time points p.i. DENV-2 E protein was stained using mouse anti-E MAb followed by rabbit anti-mouse IgG TRITC, saturated with goat antimouse IgG and then stained for dynein using mouse anti-dynein MAb followed by donkey anti-mouse IgG Alexa 488. A] The split images and the intensity profiles showing the colocalization are displayed for each of the time points; 4 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h. B] Mock infected control cells stained for DENV-2 E protein and dynein. C] The 3D reconstruction of the z stacked deconvolved images showing the beginnings of [/fig]
[fig] Figure 6: Docking of DENV-2 E protein with dynein. The E protein monomer [PDB:1TG8] was docked with dynein light chain (LC8) [PDB:2P2T] using Hex 5.1 version. A] The carbon chain backbone models of the two proteins docked together, E protein (pink) and dynein (orange). B] The interacting residues of E protein (blue) and dynein (green) C] list of interacting residues on E protein and dynein. doi:10.1371/journal.pone.0025229.g006 [/fig]
[fig] Figure 7: Over expression of Dynamitin inhibits trafficking of DENV-2 E and core proteins. BHK-21 cells transfected with GFPdynamitin expressing plasmid were infected with DENV-2 at 1 MOI. The cells were fixed after 36 h p.i. and stained for DENV-2 E protein (A, B) or C protein (C, D) with mouse anti-E or anti-core MAb followed by antimouse IgG TRITC. A] Control non-transfected infected cells B] Cells transfected with GFP-dynamitin, and infected 6 h post transfection showed low levels of E protein (white arrows) in dynamitin expressing cells compared to the high intensity of E protein (white arrowheads) in dynamitin negative cells. C] Non transfected infected cell showing nuclear localization of core protein (white arrows). D] Cells showing low levels of core protein in cytoplasm (white arrows) in GFP-dynamitin expressing cells. One cell with high expression of GFP-dynamitin shows complete absence of infection (white arrowhead). doi:10.1371/journal.pone.. Lysates of DENV-2 infected or uninfected BHK-21 cells were co-immunoprecipitated with anti-DENV IgG antibody and the blot was probed with anti-dynein antibody followed by goat anti-mouse HRP. Lane 1 -DENV-2 infected. BHK-21 cell lysate, Lane 2 -Immunoprecipitated DENV-2 infected BHK-21 cells, Lane 3 -Immunoprecipitated uninfected BHK-21 cells. doi:10.1371/journal.pone.0025229.g005 [/fig]
[table] Table 1: Primary and secondary antibodies used for staining viral and cellular proteins. [/table]
[table] Table 2: Characterization of foci of colocalization of E protein with dynamin II. Average of the peak intensity observed in the focus of colocalization. doi:10.1371/journal.pone.0025229.t002 [/table]
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Synthesis, Photoisomerization, Antioxidant Activity, and Lipid-Lowering Effect of Ferulic Acid and Feruloyl Amides
Citation: Lambruschini, C.; Demori, I.; El Rashed, Z.; Rovegno, L.; Canessa, E.; Cortese, K.; Grasselli, E.; Moni, L. Synthesis, Photoisomerization, Antioxidant Activity, and Lipid-Lowering Effect of Ferulic Acid and Feruloyl Amides. Molecules 2021, 26, 89. https://dx.
# Introduction
Ferulic acid (FA) belongs to the phenolic acid group commonly present in plant tissues. It is widely found in fruits, vegetables, whole grains, cereal seeds, coffee, beer [bib_ref] Antioxidant Properties of Ferulic Acid and Its Possible Application, Zduńska [/bib_ref] , as well as in numerous non-edible bio-resources such as bagasse, wheat bran, and beetroot pulp, etherified to lignin or esterified to hemicelluloses. FA displays low toxicity, quite efficient intestinal absorption and high stability [bib_ref] The Bioavailability of Ferulic Acid Is Governed Primarily by the Food Matrix..., Adam [/bib_ref]. These properties, together with the powerful antioxidant activity, render FA a suitable candidate to be considered for the treatment of several oxidative stress-based pathologies such as cancer, cardiomyopathy, skin disorders, brain disorders, viral infections, diabetes, etc. [bib_ref] New insights into the ameliorative effects of ferulic acid in pathophysiological conditions, Ghosh [/bib_ref]. Some molecular mechanisms underlying FA antioxidant effects have been clarified. FA induces the translocation of NF-E2-related factor (Nrf2) into the nucleus, where it hetero-dimerizes with musculoaponeurotic fibrosarcoma protein (Maf) and binds to antioxidant response element (ARE), thus promoting the transcription of oxidative stress-responsive genes [bib_ref] Increased cell migration and plasticity in Nrf2-deficient cancer cell lines, Rachakonda [/bib_ref]. FA also induces an increase in the expression of superoxide dismutase and catalase in a p38-mediated manner, thus exerting a pro-survival role [bib_ref] Porras, A. p38α Mediates Cell Survival in Response to Oxidative Stress via..., Gutiérrez-Uzquiza [/bib_ref]. Moreover, FA has been proven to modulate proteins belonging to matrix metalloproteinase (MMP) family, which are involved in several disorders [bib_ref] New insights into the ameliorative effects of ferulic acid in pathophysiological conditions, Ghosh [/bib_ref] , and to inhibit mammalian target of rapamycin (mTOR), the master key regulator of autophagy process, controlling several survival or death signaling pathways that may commit the fate of cancer cells. As a member of the family of natural phenols, FA is able to act as an antioxidant intercepting and reacting with free radicals faster than target biomolecules like lipids, fats, and proteins. Several studies have been performed to investigate the mechanism followed by phenolic antioxidants [bib_ref] Predicting the Activity of Phenolic Antioxidants: Theoretical Method, Analysis of Substituent Effects,..., Wright [/bib_ref]. Generally, two major mechanisms are involved in the antioxidant activity: H-atom abstraction (HAT) and single-electron transfer (SET) followed by deprotonation; in both cases, a relatively stable phenoxyl radical is formed. In principle, all the phenol compounds can be considered antioxidants and act with both mechanisms, but their efficiency is strongly dependent on the substituents present on the aromatic ring. FA contains a methoxy group and a propenoic acid moiety at the ortho and para positions of phenol, respectively. The electron-donating methoxy group might be responsible for the increased antioxidant activity of FA with respect to 4-hydroxycinnamic acid, while the E double bond of the propenoic group might play a responsible role in the stabilization of the phenoxyl radical by resonance [bib_ref] Computational and experimental validation of antioxidant properties of synthesized bioactive ferulic acid..., Adeyemi [/bib_ref]. Although several natural phenols containing a catechol, or a pyrogallol type rings, present high radical scavenging activity in vitro, their high susceptibility to oxidation makes their half-life in the body strongly reduced and therefore their applicability as active pharmaceutical ingredients limited [bib_ref] Oxidized epigallocatechin gallate inhibited lysozyme fibrillation more strongly than the native form, An [/bib_ref] [bib_ref] Inhibition of amyloid fibrillation of lysozyme by phenolic compounds involves quinoprotein formation, Feng [/bib_ref]. On the contrary, FA is much more stable to oxidation under physiological conditions and more promising from a pharmacokinetic point of view.
However, FA suffers a relatively low solubility in hydrophobic media, which limits its ability to cross lipid-rich cell membranes and its application as an inhibitor of lipids and fats autoxidation [bib_ref] Computational and experimental validation of antioxidant properties of synthesized bioactive ferulic acid..., Adeyemi [/bib_ref]. For this reason, the synthesis of feruloyl derivatives with antioxidant activity and physicochemical tuned properties is highly desirable. Recently, we published a very fast and efficient synthesis of a series of polyphenols containing from 2 to 4 hydroxy-substituted aryl groups, most of them derived from renewable sources such as natural phenols [bib_ref] Multicomponent Synthesis of Polyphenols and their in vitro Evaluation as Potential β-Amyloid..., Galante [/bib_ref] [bib_ref] Multicomponent, fragment-based synthesis of polyphenol-containing peptidomimetics and their inhibiting activity on beta-amyloid..., Lambruschini [/bib_ref] [bib_ref] Biophysical and in Vivo Studies Identify a New Natural-Based Polyphenol, Counteracting Aβ..., Tomaselli [/bib_ref]. Applying our short fragment-based approach, a series of tertiary feruloyl amides were synthesized. We thought that this convergent approach, based on an Ugi multicomponent reaction of phenol-containing simple substrates, represents a convenient tool for the generation of feruloyl derivatives with the aim of modulating the pharmacokinetic properties and biostability and maintaining the antioxidant effect.
Herein, we describe the revisited synthesis of feruloyl amide trans-FEF77 (t-FEF77). FEF77 is a peptoid-like compound containing 2 additional hydroxy-substituted aryl groups, derived from renewable sources, as tyramine and 4-hydroxybenzaldehyde. Among our in-house library of feruloyl amides, we selected FEF77 as a "proof-of-concept compound" to demonstrate if this class of FA derivatives maintains the anti-oxidant and biological properties of FA. Moreover, FEF77 contains an additional monophenolic group and we evaluated its effect. Chemical assays and in vitro cellular models were used to perform a comparative analysis of FA and t-FEF77 antioxidant potentials. In addition, the influence of the configuration of the double bond on the antioxidant activity was investigated thanks to the complete light-mediated isomerization of t-FEF77 into cis-FEF77 (c-FEF77). Finally, the lipid-lowering effect of FA and t-FEF77 was evaluated in a validated in vitro model of hepatic steatosis.
# Results
## Synthesis of fef77
Based on our standard and optimized procedure [bib_ref] Multicomponent Synthesis of Polyphenols and their in vitro Evaluation as Potential β-Amyloid..., Galante [/bib_ref] [bib_ref] Multicomponent, fragment-based synthesis of polyphenol-containing peptidomimetics and their inhibiting activity on beta-amyloid..., Lambruschini [/bib_ref] peptoid-like polyphenols can be generated in a three-steps process, involving: (1) Ugi reaction employing phenolic building blocks protected as allyl ethers; (2) removal of the allyl protecting groups and acetylation of the crude; (3) final solvolysis of polyacetates. This general strategy solved the problems that emerged during our preliminary investigation, where, in several cases, free phenolic groups had a negative effect on the yield and cleanliness of the Ugi reaction. In this work, we tried to access our target compounds FEF77 in just one step by employing components containing the free phenols in the Ugi reaction (Scheme 1). Using standard reaction conditions, we could isolate trans-FEF77 after column chromatography in good yield and high HPLC purity. free phenolic groups had a negative effect on the yield and cleanliness of the Ugi reaction.
In this work, we tried to access our target compounds FEF77 in just one step by employing components containing the free phenols in the Ugi reaction (Scheme 1). Using standard reaction conditions, we could isolate trans-FEF77 after column chromatography in good yield and high HPLC purity. Scheme 1. One-step synthesis of feruloyl amide trans-FEF77 employing ferulic acid, 4-hydroybenzaldeyde, tyramine, and methyl isocyanide.
## Study of stability of caffeic acid, fa, and fef77 in buffer solutions
To prove the superior stability of FA and amide-derivative t-FEF77, we monitored them and their catechol analog (caffeic acid) by HPLC. As shown in [fig_ref] Figure 1: Cont. [/fig_ref] , after 24 h at room temperature in phosphate-buffered saline (PBS), FA is perfectly stable and even after seven days the amount is ~90% with respect to the starting value. On the other hand, caffeic acid (CA) was proven to be highly unstable; actually, after 24 h, a clear decrease of the HPLC area was observed and after one week, the amount of CA still present in solution was only 30%. During the HPLC analysis of t-FEF77, we identified a minor peak, which indicated the presence of a small amount of cis-isomer. Moreover, we found that the amount of cis isomer increased over time if the sample was simply kept on the bench exposed to the natural light of the laboratory, until it became the majority. In any case, the stability of t-FEF77 was completely restored, keeping the sample in the dark [fig_ref] Figure 1: Cont. [/fig_ref].
## Study of stability of caffeic acid, fa, and fef77 in buffer solutions
To prove the superior stability of FA and amide-derivative t-FEF77, we monitored them and their catechol analog (caffeic acid) by HPLC. As shown in [fig_ref] Figure 1: Cont. [/fig_ref] , after 24 h at room temperature in phosphate-buffered saline (PBS), FA is perfectly stable and even after seven days the amount is~90% with respect to the starting value. On the other hand, caffeic acid (CA) was proven to be highly unstable; actually, after 24 h, a clear decrease of the HPLC area was observed and after one week, the amount of CA still present in solution was only 30%. During the HPLC analysis of t-FEF77, we identified a minor peak, which indicated the presence of a small amount of cis-isomer. Moreover, we found that the amount of cis isomer increased over time if the sample was simply kept on the bench exposed to the natural light of the laboratory, until it became the majority. In any case, the stability of t-FEF77 was completely restored, keeping the sample in the dark [fig_ref] Figure 1: Cont. [/fig_ref]. free phenolic groups had a negative effect on the yield and cleanliness of the Ugi reaction.
In this work, we tried to access our target compounds FEF77 in just one step by employing components containing the free phenols in the Ugi reaction (Scheme 1). Using standard reaction conditions, we could isolate trans-FEF77 after column chromatography in good yield and high HPLC purity. Scheme 1. One-step synthesis of feruloyl amide trans-FEF77 employing ferulic acid, 4-hydroybenzaldeyde, tyramine, and methyl isocyanide.
## Study of stability of caffeic acid, fa, and fef77 in buffer solutions
To prove the superior stability of FA and amide-derivative t-FEF77, we monitored them and their catechol analog (caffeic acid) by HPLC. As shown in [fig_ref] Figure 1: Cont. [/fig_ref] , after 24 h at room temperature in phosphate-buffered saline (PBS), FA is perfectly stable and even after seven days the amount is ~90% with respect to the starting value. On the other hand, caffeic acid (CA) was proven to be highly unstable; actually, after 24 h, a clear decrease of the HPLC area was observed and after one week, the amount of CA still present in solution was only 30%. During the HPLC analysis of t-FEF77, we identified a minor peak, which indicated the presence of a small amount of cis-isomer. Moreover, we found that the amount of cis isomer increased over time if the sample was simply kept on the bench exposed to the natural light of the laboratory, until it became the majority. In any case, the stability of t-FEF77 was completely restored, keeping the sample in the dark [fig_ref] Figure 1: Cont. [/fig_ref]. Although trans-cis photoisomerization of cinnamic acids has been previously documented in the literature [bib_ref] Photoisomerization of ethyl ferulate: A solution phase transient absorption study, Horbury [/bib_ref] [bib_ref] Trans-cis-equilibrium of hydroxycinnamic acids during irradiation of aqueous solutions at different pH, Kahnt [/bib_ref] , the process is usually promoted by UV light, which is unlikely to be present inside a laboratory environment. Moreover, FA and CA did not show this behavior when treated in the same conditions. These data led us to deeper investigate the photoisomerization of FEF77.
## Light conversion study of fef77
The photoisomerization studies were carried out using three different light sources: 300 nm, 350 nm, and direct sunlight. First, 70 mM solutions of FA and t-FEF77 in CD3OD were exposed to the selected sources and NMR spectra were registered over time. The use of NMR allowed us to confirm the structure of the cis isomer for the newly formed molecules and to calculate the trans/cis ratio [fig_ref] Figure 2: Photoisomerization of FA and FEF77 over time [/fig_ref]. Both compounds exhibited the tendency to isomerize, but we observed substantial differences; actually, FEF77 reaches higher conversion than FA when the photostationary state is achieved. At 350 nm, after 60 min, the isomerization of t-FEF77 was complete (94.2% of c-FEF77), while the amount of cis-FA was only 45%. At 300 nm, the conversion of t-FEF77 was lower (83%), whilst that of FA was higher (59%). When the samples were exposed to direct sunlight, we observed a similar behavior to 350 nm, and the complete conversion of FEF77 was achieved after 60 min, while for FA, the amount of cis was only 40% after 120 min. Having in hand pure c-FEF77, we were able to compare the antioxidant activity of the three compounds. To the best of our knowledge, it is the first time that a complete isomerization of a FA derivative has been observed. Although trans-cis photoisomerization of cinnamic acids has been previously documented in the literature [bib_ref] Photoisomerization of ethyl ferulate: A solution phase transient absorption study, Horbury [/bib_ref] [bib_ref] Trans-cis-equilibrium of hydroxycinnamic acids during irradiation of aqueous solutions at different pH, Kahnt [/bib_ref] , the process is usually promoted by UV light, which is unlikely to be present inside a laboratory environment. Moreover, FA and CA did not show this behavior when treated in the same conditions. These data led us to deeper investigate the photoisomerization of FEF77.
## Light conversion study of fef77
The photoisomerization studies were carried out using three different light sources: 300 nm, 350 nm, and direct sunlight. First, 70 mM solutions of FA and t-FEF77 in CD 3 OD were exposed to the selected sources and NMR spectra were registered over time. The use of NMR allowed us to confirm the structure of the cis isomer for the newly formed molecules and to calculate the trans/cis ratio [fig_ref] Figure 2: Photoisomerization of FA and FEF77 over time [/fig_ref]. Both compounds exhibited the tendency to isomerize, but we observed substantial differences; actually, FEF77 reaches higher conversion than FA when the photostationary state is achieved. At 350 nm, after 60 min, the isomerization of t-FEF77 was complete (94.2% of c-FEF77), while the amount of cis-FA was only 45%. At 300 nm, the conversion of t-FEF77 was lower (83%), whilst that of FA was higher (59%). When the samples were exposed to direct sunlight, we observed a similar behavior to 350 nm, and the complete conversion of FEF77 was achieved after 60 min, while for FA, the amount of cis was only 40% after 120 min. Although trans-cis photoisomerization of cinnamic acids has been previously documented in the literature [bib_ref] Photoisomerization of ethyl ferulate: A solution phase transient absorption study, Horbury [/bib_ref] [bib_ref] Trans-cis-equilibrium of hydroxycinnamic acids during irradiation of aqueous solutions at different pH, Kahnt [/bib_ref] , the process is usually promoted by UV light, which is unlikely to be present inside a laboratory environment. Moreover, FA and CA did not show this behavior when treated in the same conditions. These data led us to deeper investigate the photoisomerization of FEF77.
## Light conversion study of fef77
The photoisomerization studies were carried out using three different light sources: 300 nm, 350 nm, and direct sunlight. First, 70 mM solutions of FA and t-FEF77 in CD3OD were exposed to the selected sources and NMR spectra were registered over time. The use of NMR allowed us to confirm the structure of the cis isomer for the newly formed molecules and to calculate the trans/cis ratio [fig_ref] Figure 2: Photoisomerization of FA and FEF77 over time [/fig_ref]. Both compounds exhibited the tendency to isomerize, but we observed substantial differences; actually, FEF77 reaches higher conversion than FA when the photostationary state is achieved. At 350 nm, after 60 min, the isomerization of t-FEF77 was complete (94.2% of c-FEF77), while the amount of cis-FA was only 45%. At 300 nm, the conversion of t-FEF77 was lower (83%), whilst that of FA was higher (59%). When the samples were exposed to direct sunlight, we observed a similar behavior to 350 nm, and the complete conversion of FEF77 was achieved after 60 min, while for FA, the amount of cis was only 40% after 120 min. Having in hand pure c-FEF77, we were able to compare the antioxidant activity of the three compounds. To the best of our knowledge, it is the first time that a complete isomerization of a FA derivative has been observed. Having in hand pure c-FEF77, we were able to compare the antioxidant activity of the three compounds. To the best of our knowledge, it is the first time that a complete isomerization of a FA derivative has been observed. To evaluate the antioxidant activity of FA, t-FEF77, and c-FEF77, we first performed the standard 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay. DPPH assay is conducted mon-itoring the decrease of the absorbance at 515 nm of a solution of DPPH radical in the presence of different concentrations of antioxidant. The molar ratio (MR) and the percentage of radical scavenging activities (RSA) are easily determined as already reported in the literature [bib_ref] Conjugation of Hydroxytyrosol with Other Natural Phenolic Fragments: From Waste to Antioxidants..., Tassano [/bib_ref]. Moreover, the half-maximal efficient concentrations (EC 50 ) are determined when the system reaches a steady-state (after 240 min) and reported as molar ratio [fig_ref] Table 1: Results of DPPH assays on compounds FA, t-FEF77, and c-FEF77 [/fig_ref].
As expected, the RSA depends on time and on the concentration of the antioxidant. The first experiments were performed monitoring the absorbance at 515 nm of DPPH in the presence of different concentrations of FA and t-FEF77 for 15 min. The results showed in [fig_ref] Table 1: Results of DPPH assays on compounds FA, t-FEF77, and c-FEF77 [/fig_ref] revealed that both phenolic compounds presented appreciable DPPH radical scavenging potential, which appeared concentration-dependent. Clearly, the RSA(1) values are slightly lower for t-FEF77. This result is consistent with previous studies, where it has been shown that FA was more effective than the urethane amide derivative [bib_ref] Computational and experimental validation of antioxidant properties of synthesized bioactive ferulic acid..., Adeyemi [/bib_ref] or a tyrosine-amide derivative [bib_ref] N-Hydroxycinnamoyl amides of fluorinated amino acids: Synthesis, anti-tyrosinase and DPPH scavenging activities, Chochkova [/bib_ref] in scavenging DPPH radicals. Since the lower antioxidant efficiency of monophenol rings has been well established in literature [bib_ref] Benzoic and Cinnamic Acid Derivatives as Antioxidants: Structure−Activity Relation, Natella [/bib_ref] , we decided to carry out the experiments for a longer time, in order to evaluate the contribution of the additional monophenolic groups present in FEF77. Hence, based on RSA (2) values, FA and t-FEF77 showed a similar profile. The molar EC 50 was even higher for t-FEF77 than for FA, which shows that the presence of two additional monophenolic rings and a tertiary amide at the end of the propenoic side, do not affect the antioxidant performance of FA. On the other hand, c-FEF77 showed a slightly lower radical scavenging activity, reaching just 47% of RSA(2) at the highest concentration examined.
## Antioxidant activity in hecv cells
Preliminarily, the effects of FA, t-FEF77, and c-FEF77 on human endothelial cord vein (HECV) cell viability was checked by MTT cell viability assay [bib_ref] Excess fructose and fatty acids trigger a model of nonalcoholic fatty liver..., Grasselli [/bib_ref]. Starting from 50 µM, a concentration at which a decrease in cell viability has been reported for FA [bib_ref] Redox cycling of endogenous copper by thymoquinone leads to ROS-mediated DNA breakage..., Zubair [/bib_ref] , we halved the doses until reaching 3.125 µM. As shown in , none of the three molecules exerted a cytotoxic effect at any of the concentrations tested in the range 3.125-50 µM. Therefore, the intermediate dose of 12.5 µM was selected for further experiments.
Secondly, we tested HECV cell viability after a 2 h treatment with H 2 O 2 ranging from 0.25 to 1.5 mM. As shown in [fig_ref] 2. 4: Antioxidant Activity of FA, t-FEF77, and c-FEF77 2 [/fig_ref] , at concentrations ≤0.75 mM, H 2 O 2 significantly reduced cell viability by ≥50% (p ≤ 0.001 with respect to control). Therefore, 0.75 mM H 2 O 2 was the concentration selected for further experiments, in which HECV cells were pretreated with 12.5 µM polyphenols (FA, t-FEF77, and c-FEF77) for 24 h and subsequently with H 2 O 2 for 2 h. As shown in [fig_ref] 2. 4: Antioxidant Activity of FA, t-FEF77, and c-FEF77 2 [/fig_ref] , polyphenol pretreatment significantly prevented the decrease in cell viability induced by H 2 O 2 (+25% for both FA and t-FEF77 and +28% for c-FEF77; p ≤ 0.001 for all with respect to H 2 O 2 0.75 mM, [fig_ref] 2. 4: Antioxidant Activity of FA, t-FEF77, and c-FEF77 2 [/fig_ref].
The protective properties of FA, t-FEF77, and c-FEF77 against oxidative stress were confirmed by 2 ,7 -dichlorofluorescin diacetate (DCF-DA) stain for reactive oxygen species (ROS) detection in HECV cells.
Molecules 2021, 26, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/molecules concentration at which a decrease in cell viability has been reported for FA [bib_ref] Redox cycling of endogenous copper by thymoquinone leads to ROS-mediated DNA breakage..., Zubair [/bib_ref] , we halved the doses until reaching 3.125 μM. As shown in , none of the three molecules exerted a cytotoxic effect at any of the concentrations tested in the range 3.125-50 μM. Therefore, the intermediate dose of 12.5 μM was selected for further experiments. Secondly, we tested HECV cell viability after a 2 h treatment with H2O2 ranging from 0.25 to 1.5 mM. As shown in [fig_ref] 2. 4: Antioxidant Activity of FA, t-FEF77, and c-FEF77 2 [/fig_ref] , at concentrations ≤0.75 mM, H2O2 significantly reduced cell viability by ≥50% (p ≤ 0.001 with respect to control). Therefore, 0.75 mM H2O2 was the concentration selected for further experiments, in which HECV cells were pretreated with 12.5 μM polyphenols (FA, t-FEF77, and c-FEF77) for 24 h and subsequently with H2O2 for 2 h. As shown in [fig_ref] 2. 4: Antioxidant Activity of FA, t-FEF77, and c-FEF77 2 [/fig_ref] , polyphenol pretreatment significantly prevented the decrease in cell viability induced by H2O2 (+25% for both FA and t-FEF77 and +28% for c-FEF77; p ≤ 0.001 for all with respect to H2O2 0.75 mM, [fig_ref] 2. 4: Antioxidant Activity of FA, t-FEF77, and c-FEF77 2 [/fig_ref].
The protective properties of FA, t-FEF77, and c-FEF77 against oxidative stress were confirmed by 2′,7′-dichlorofluorescin diacetate (DCF-DA) stain for reactive oxygen species (ROS) detection in HECV cells. In control cells, FA, t-FEF77, and c-FEF77 treatments did not affect intracellular ROS content (not shown), but prevented the oxidative damage induced by subsequent treatment with H 2 O 2 . These results are demonstrated by the evident decrease in fluorescence depicted in the representative images of [fig_ref] 2. 4: Antioxidant Activity of FA, t-FEF77, and c-FEF77 2 [/fig_ref]. concentration at which a decrease in cell viability has been reported for FA [bib_ref] Redox cycling of endogenous copper by thymoquinone leads to ROS-mediated DNA breakage..., Zubair [/bib_ref] , we halved the doses until reaching 3.125 μM. As shown in , none of the three molecules exerted a cytotoxic effect at any of the concentrations tested in the range 3.125-50 μM. Therefore, the intermediate dose of 12.5 μM was selected for further experiments. Secondly, we tested HECV cell viability after a 2 h treatment with H2O2 ranging from 0.25 to 1.5 mM. As shown in [fig_ref] 2. 4: Antioxidant Activity of FA, t-FEF77, and c-FEF77 2 [/fig_ref] , at concentrations ≤0.75 mM, H2O2 significantly reduced cell viability by ≥50% (p ≤ 0.001 with respect to control). Therefore, 0.75 mM H2O2 was the concentration selected for further experiments, in which HECV cells were pretreated with 12.5 μM polyphenols (FA, t-FEF77, and c-FEF77) for 24 h and subsequently with H2O2 for 2 h. As shown in [fig_ref] 2. 4: Antioxidant Activity of FA, t-FEF77, and c-FEF77 2 [/fig_ref] , polyphenol pretreatment significantly prevented the decrease in cell viability induced by H2O2 (+25% for both FA and t-FEF77 and +28% for c-FEF77; p ≤ 0.001 for all with respect to H2O2 0.75 mM, [fig_ref] 2. 4: Antioxidant Activity of FA, t-FEF77, and c-FEF77 2 [/fig_ref].
The protective properties of FA, t-FEF77, and c-FEF77 against oxidative stress were confirmed by 2′,7′-dichlorofluorescin diacetate (DCF-DA) stain for reactive oxygen species (ROS) detection in HECV cells.
## Lipid-lowering effect of fa and t-fef77
We tested the lipid-lowering effects of FA and FEF77 in a validated in vitro model of hepatic steatosis consisting of FaO rat hepatoma cells incubated for 3 h with oleate/palmitate mixture (O/P; 2:1 molar ratio, final concentration 0.75 mM) [bib_ref] Models of non-Alcoholic Fatty Liver Disease and Potential Translational Value: The Effects..., Grasselli [/bib_ref]. To this aim, we used only t-FEF77, since the trans and cis isomers exhibited a comparable antioxidant ability both in DPPH assay and mostly in the HECV cellular model. In preliminary experiments, we checked the effects of FA and t-FEF77 on FaO cell viability. Neither FA nor t-FEF77 induced any changes in cell viability both in the absence and presence of O/P, as measured by MTT assay [fig_ref] Figure 5: Effects of FA and t-FEF77 in FaO cells treated in the absence... [/fig_ref].
The effects of FA and t-FEF77 on triglyceride (TAG) accumulation in O/P FaO cells were then evaluated. As shown in [fig_ref] Figure 5: Effects of FA and t-FEF77 in FaO cells treated in the absence... [/fig_ref] , cell exposure to O/P for 3 h resulted in significant TAG accumulation (+105%; p ≤ 0.001 with respect to control), as previously demonstrated [bib_ref] Non-receptor-mediated actions are responsible for the lipid-lowering effects of iodothyronines in FaO..., Grasselli [/bib_ref] [bib_ref] 3,5-diiodo-L-thyronine modifies the lipid droplet composition in a model of hepatosteatosis, Grasselli [/bib_ref]. FA or t-FEF77 per se did not alter lipid content in control cells (data not shown), but the treatment of O/P cell with FA or t-FEF77 12.5 μM for 24 h significantly reduced intracellular TAG content with respect to O/P (−26%; p ≤ 0.01). These results were confirmed by fluorescence microscopy visualization of intracellular lipid droplets stained
## Lipid-lowering effect of fa and t-fef77
We tested the lipid-lowering effects of FA and FEF77 in a validated in vitro model of hepatic steatosis consisting of FaO rat hepatoma cells incubated for 3 h with oleate/palmitate mixture (O/P; 2:1 molar ratio, final concentration 0.75 mM) [bib_ref] Models of non-Alcoholic Fatty Liver Disease and Potential Translational Value: The Effects..., Grasselli [/bib_ref]. To this aim, we used only t-FEF77, since the trans and cis isomers exhibited a comparable antioxidant ability both in DPPH assay and mostly in the HECV cellular model. In preliminary experiments, we checked the effects of FA and t-FEF77 on FaO cell viability. Neither FA nor t-FEF77 induced any changes in cell viability both in the absence and presence of O/P, as measured by MTT assay [fig_ref] Figure 5: Effects of FA and t-FEF77 in FaO cells treated in the absence... [/fig_ref]. [fig_ref] Figure 5: Effects of FA and t-FEF77 in FaO cells treated in the absence... [/fig_ref] , only a few green droplets are detectable in control FaO cells, whereas their number increases after O/P treatment. Upon a 24 h incubation with FA or t-FEF77, fewer lipid droplets are detectable as compared to O/P cells. The effects of FA and t-FEF77 on triglyceride (TAG) accumulation in O/P FaO cells were then evaluated. As shown in [fig_ref] Figure 5: Effects of FA and t-FEF77 in FaO cells treated in the absence... [/fig_ref] , cell exposure to O/P for 3 h resulted in significant TAG accumulation (+105%; p ≤ 0.001 with respect to control), as previously demonstrated [bib_ref] Non-receptor-mediated actions are responsible for the lipid-lowering effects of iodothyronines in FaO..., Grasselli [/bib_ref] [bib_ref] 3,5-diiodo-L-thyronine modifies the lipid droplet composition in a model of hepatosteatosis, Grasselli [/bib_ref]. FA or t-FEF77 per se did not alter lipid content in control cells (data not shown), but the treatment of O/P cell with FA or t-FEF77 12.5 µM for 24 h significantly reduced intracellular TAG content with respect to O/P (−26%; p ≤ 0.01). These results were confirmed by fluorescence microscopy visualization of intracellular lipid droplets stained with BODIPY 493/503. As shown in [fig_ref] Figure 5: Effects of FA and t-FEF77 in FaO cells treated in the absence... [/fig_ref] , only a few green droplets are detectable in control FaO cells, whereas their number increases after O/P treatment. Upon a 24 h incubation with FA or t-FEF77, fewer lipid droplets are detectable as compared to O/P cells.
# Discussion
The trans-cis photoisomerization of cinnamic acids is usually promoted by UV light and generally produces a mixture of isomers. During the light-mediated isomerization, we were able to fully convert t-FEF77 in c-FEF77, which could be isolated in pure form and characterized. Thanks to this unprecedented result, the UV-Vis spectra were registered and compared, showing a general decrease of intensity for c-FEF77. Trans-FEF77 has a maximum at 330 nm that is not present in c-FEF77 and there is an overall blue-shift of the peaks for c-FEF77 in the 250-350 nm region [fig_ref] Figure 1: Cont. [/fig_ref] in Supplementary Materials). Our hypothesis for these observations is a partial distortion from planarity for c-FEF77 due to steric hindrance of the tertiary amide and therefore the extended conjugation is no longer present. Based on the UV-Vis spectra, we can also explain the different amounts of c-FEF77 at 350 and 300 nm, since the trans/cis ratio at the photostationary state depends on the relative molar attenuation coefficient (ε) of the two isomers and on the relative quantum yield (Φ) of their photochemical conversion [bib_ref] Cis-trans photoisomerization of azobenzene. Solvent and triplet donors effects, Bortolus [/bib_ref]. At 300 nm, both isomers absorb, although t-FEF77 has a higher molar attenuation coefficient and at the photostationary state, a 17:83 ratio is observed. On the other hand, at 350 nm, only t-FEF77 absorbs and therefore a complete isomerization can occur.
Similarly, the distortion from planarity caused by the steric hindrance of the tertiary amide might cause the slightly lower radical scavenging activity of c-FEF77. Actually, the conjugated double bond in the side chain produces a stabilizing effect by resonance on the phenoxyl radical, thus enhancing the antioxidant activity of FA and t-FEF77 with respect to c-FEF77.
As the relative scavenging activity is influenced by analytical conditions [bib_ref] Structure−DPPH• Scavenging Activity Relationships: Parallel Study of Catechol and Guaiacol Acid Derivatives, Ordoudi [/bib_ref] , other assays have been performed by using biological systems represented by HECV cells [bib_ref] Polyphenolic extract attenuates fatty acid-induced steatosis and oxidative stress in hepatic and..., Vergani [/bib_ref] in which oxidative damage was induced by incubation with 0.75 mM H 2 O 2 . FA, t-FEF77, and c-FEF77, all significantly prevented H 2 O 2 injury without differences among these three molecules. Taken together, our results suggest that the different antioxidant activities of FA, t-FEF77, and c-FEF77 as measured by DPPH assay are not appreciable when tested in biological systems, and that all the three compounds exhibit a comparable protective action against oxidative damage in a cellular model.
Oxidative stress and ROS overproduction are considered a basic mechanism underlying the development of different pathologies such as cancer, cardiovascular events, neurodegenerative diseases, and metabolic impairments. Some of us developed a long-time experience in the use of experimental models of non-alcoholic fatty liver disease (NAFLD). NAFLD is a physio-pathological condition affecting 25% of the population worldwide and capable of worsening towards more severe pathologies such as steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma [bib_ref] Lipid metabolism and liver inflammation. I. Hepatic fatty acid uptake: Possible role..., Bradbury [/bib_ref]. The pathogenesis of NAFLD is complex, encompassing different mechanisms and multiple coexisting "hits", among which oxidative stress can be considered as the starting point of the hepatic damage [bib_ref] Nonalcoholic steatosis and steatohepatitis. III. Peroxisomal beta-oxidation, PPAR alpha, and steatohepatitis, Reddy [/bib_ref]. As a matter of fact, natural anti-oxidant compounds such as vitamin E and plant-derived polyphenols have been investigated and proved to exert beneficial effects in experimental models of NAFLD [bib_ref] Beneficial effects of the Mediterranean spices and aromas on non-alcoholic fatty liver..., Baselga-Escudero [/bib_ref].
FA and methyl FA have been proven to exert hepatoprotective effects against acetaminophen or methotrexate hepatotoxicity, and to reduce hepatic steatosis in alcohol and high fat diet-fed animals [bib_ref] New insights into the ameliorative effects of ferulic acid in pathophysiological conditions, Ghosh [/bib_ref] [bib_ref] Methyl ferulic acid attenuates ethanol-induced hepatic steatosis by regulating AMPK and FoxO1..., Cheng [/bib_ref] [bib_ref] Ferulic acid prevents oxidative stress, inflammation, and liver injury via upregulation of..., Mahmoud [/bib_ref]. These observations are in line with our results, demonstrating a comparable lipid-lowering action of both FA and FEF77 in a validated in vitro model of NAFLD. Moreover, we demonstrated a significant protective capacity of FA and FEF77 against the oxidative damage of endothelial cells (HECV). Taken together, these data could be considered beneficial in NAFLD, since sinusoidal endothelium dysfunction is involved in the development of fibrosis and worsening of liver damage. In vitro models of hepatic steatosis are recognized valuable tools to study NAFLD mechanisms directly at the cellular level, keeping the translational value of the observed results, without exposing humans to unnecessary risks. Undoubtedly, information in this field needs to be confirmed by clinical studies. Nevertheless, from a therapeutic perspective, we consider our results of interest, since the development of synthetic products maintaining similar properties to those of the corresponding natural compounds is a difficult but desirable goal, taking into account that no specific pharmacological treatment has been approved for NAFLD so far.
# Materials and methods
## General remarks
NMR spectra were taken at r.t. in CD 3 OD at 300 MHz ( 1 H), and 75 MHz ( 13 C), using as an internal standard, the central peak of CD 3 OD ( 1 H-NMR in CD 3 OD; 3.310 ppm) or the central peak of CD 3 OD ( 13 C in CD 3 OD; 49.00 ppm). Chemical shifts are reported in ppm (δ scale). Peak assignments were made with the aid of gCOSY and gHSQC experiments. In the ABX system, the proton A is considered upfield and B downfield. IR spectra were recorded as solid, oil, or foamy samples, with the ATR (attenuated total reflectance) technique. TLC analyses were carried out on silica gel plates and viewed at UV (λ = 254 nm or 360 nm) and developed with Hanessian stain (dipping into a solution of (NH 4 ) 4 MoO 4 ·4H 2 O (21 g) and Ce(SO 4 ) 2 ·4H 2 O (1 g) in H 2 SO 4 (31 mL) and H 2 O (469 mL) and warming). R f values were measured after an elution of 7-9 cm. Column chromatography was done with the "flash" methodology by using 220-400 mesh silica. Petroleum ether (40-60 - C) is abbreviated PE. All reactions employing dry solvents were carried out under nitrogen. Extractions were always repeated three times and organic extracts were always dried over Na 2 SO 4 and filtered before evaporation to dryness. Caffeic acid, ferulic acid, and DPPH radical were purchased from Sigma Aldrich Chemical; methanol and acetonitrile (HPLC grade) were hypergrade for LC-MS from Merk or Carlo Erba Reagents.
Photoinduced isomerization of FEF77 were performed with sunlight or a Southern New England Ultraviolet Company Rayonet ® apparatus equipped with 8 Iles Optical lamps (300 or 350 nm). The lamps used were cylindrical with a length of 26.5 cm and a power of 8 watts. The sample was fixed at a 10 cm distance from the lamp.
UV-Vis spectra and DPPH assay were performed using Varian Cary ® 50 UV-Vis Spectrophotometer.
HPLC analysis was performed on Agilent HP 1100 equipped with a DAD detector (220 nm) and column Phenomenex Gemini 3u C6-Phenyl 110A (4 µm, 3 × 150 mm 2 ). Mobile phases were (A) H 2 O + 0.1% TFA and (B) CH 3 CN + 0.1% TFA, gradient from 10% to 100% of B in 15 min, flow 0.34 mL/min, and 26 - C. Mass analysis was performed on Microsaic 4000 MiD ® mass spectrometer.
## Synthesis of t-fef77
A solution of 4-hydroxybenzaldehyde (122 mg, 1.00 mmol) in dry methanol (1 mL) was treated with tyramine (138 mg, 1.00 mmol) under nitrogen atmosphere. After 15 min, the solution was treated at room temperature with trans-ferulic acid (194 mg, 1.00 mmol) and methyl isocyanide (64 µL, 1.10 mmol). After 16 h, the mixture was concentrated and the residue was filtered on a short column of silica gel with EtOAc + 3% MeOH to give t-FEF77 (317 mg, 66%), as white foam. R f = 0.29 (EtOAc); [bib_ref] Multicomponent, fragment-based synthesis of polyphenol-containing peptidomimetics and their inhibiting activity on beta-amyloid..., Lambruschini [/bib_ref]
## Synthesis of c-fef77
A solution of t-FEF77 (25 mg) in MeOH (3 mL) in a test tube was exposed to direct sun light. After 3 h, the isomerization is complete and the solvent was removed under vacuum.
## Stability assays
Stock solutions at 25 mM in DMSO of FA, CA, and t-FEF77 were prepared. Work solutions at 0.25 mM in PBS pH 7.4 were made by diluting stock solutions, in such a manner that the final solutions contain 1% of DMSO. HPLC analysis were run at 0, 24, 48, 96 h and 7 days, and meanwhile, the work solutions were kept at room temperature. The stability was evaluated by measuring the integral area of the peak and the integrals were expressed as a percentage of the value at 0 h, which was imposed equal to 100.
## Photoisomerization studies
First, 70 mM solutions of FA and t-FEF77 in CD 3 OD were exposed to different light sources (300, 350 nm, and direct sunlight) and 1 H-NMR spectra were registered over time until the steady-state was reached [bib_ref] Photoisomerization of ethyl ferulate: A solution phase transient absorption study, Horbury [/bib_ref] [bib_ref] Nonalcoholic steatosis and steatohepatitis. III. Peroxisomal beta-oxidation, PPAR alpha, and steatohepatitis, Reddy [/bib_ref] , and 120 min). The trans/cis ratio was determined by integration.
## Dpph assays
Method A: a 50 µM solution of DPPH (2.90 mL, 145 nmol) in methanol was placed in a cuvette. A solution of FA or t-FEF (with a known concentration from 200 to 800 µM; 100 µL, from 20 to 80 nmol) in methanol was added, and the UV absorbance was monitored versus time at λ = 515 nm with a UV/Vis spectrophotometer. Absorbance values were taken every 0.1 min for 15 min. Experiments were performed at five different concentrations of the antioxidant. [fig_ref] Table 1: Results of DPPH assays on compounds FA, t-FEF77, and c-FEF77 [/fig_ref] reports only selected concentrations.
Method B: a 50 µM solution of DPPH (2.90 mL, 145 nmol) in methanol was placed in a cuvette. A solution of FA, t-FEF77, or c-FEF77 (with a known concentration from 200 to 800 µM; 100 µL, from 20 to 80 nmol) in methanol was added, and the UV absorbance was monitored versus time at λ = 515 nm with a UV/Vis spectrophotometer. Absorbance values were taken at 30, 60, 120, 240 min. Experiments were performed at four different concentrations of the antioxidant.
## Chemicals
All chemicals, unless otherwise indicated, were supplied by Sigma-Aldrich Corp. (Milan, Italy).
## Cell culture and treatments
Human endothelial cord vein (HECV) cells (Cell Bank and Culture-GMP-IST-Genoa, Italy) are a human endothelial cell line isolated from the umbilical vein; they were grown in a humidified atmosphere with 5% CO 2 at 37 - C in Dulbecco's modified Eagle's medium high glucose (D-MEM) supplemented with L-glutamine and 10% fetal calf serum (FCS).
FaO cells (European Collection of Authenticated Cell Cultures, Sigma-Aldrich) are a rat hepatoma cell line maintaining hepatocyte-specific markers [bib_ref] Coordinate roles of insulin and glucose on the growth of hepatoma cells..., Lauris [/bib_ref]. Cells were grown in a humidified atmosphere with 5% CO 2 at 37 - C in Coon's modified Ham's F12 medium supplemented with L-glutamine and 10% FCS.
For cell treatments, stock solutions of FA, t-FEF77, and c-FEF77 were made in DMSO at the concentration of 25 mM and store at −20 - C. Stock solutions were serially diluted to working solutions in culture medium. The final concentration of DMSO in working solutions was 0.1% maximum.
Oxidative stress was induced by incubating confluent HECV cells with increasing concentrations of H 2 O 2 (0.25-1.5 mM) for 2 h. The effects of polyphenols were studied after incubation of sub-confluent HECV cells with FA, t-FEF77, or c-FEF77 at different concentrations (3.125-50 µM) for 24 h.
To induce intracellular lipid accumulation and obtain an in vitro model of hepatic steatosis, sub-confluent FaO cells were treated for 3 h with a mixture of oleate/palmitate (O/P) at a final concentration of 0.75 mM (2:1 molar ratio) [bib_ref] Models of non-Alcoholic Fatty Liver Disease and Potential Translational Value: The Effects..., Grasselli [/bib_ref]. Thereafter, 'steatotic' O/P cells were incubated for 24 h with FA or t-FEF77 12.5 µM.
## Mtt assay for determination of cell viability
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is a measure of the effects of treatments on cellular populations. This colorimetric assay is based on the reduction of the yellow tetrazolium MTT into purple formazan crystals by metabolically active and alive cells. MTT was dissolved in PBS at a concentration of 5 mg/mL and filtered through 0.22 µm pores. The working solution was diluted in culture medium at the final concentration of 0.5 mg/mL. The cells were then incubated for 3 h in a humidified atmosphere with 5% CO 2 at 37 - C. Precipitated formazan was then dissolved in acid-alcohol (0.04 N HCl in 2-propanol) solution and read at 570 nm in a Varian Cary-50Bio spectrophotometer (Agilent, Milan, Italy) [bib_ref] Excess fructose and fatty acids trigger a model of nonalcoholic fatty liver..., Grasselli [/bib_ref].
## Intracellular ros visualization by fluorescence microscopy
The oxidation of the cell-permeant 2 -7 dichlorofluorescin diacetate (DCF-DA, Fluka, Germany) to 2 -7 dichlorofluorescein (DCF) is used for determining in situ the production of H 2 O 2 and other ROS. A stock solution of DCF-DA (10 mM in DMSO) was prepared and stored at −20 - C in the dark. At the end of the different treatments, cells were loaded with 10 µM DCF-DA in PBS for 30 min at 37 - C in the dark. Then, the cells were washed several times with PBS. Images were acquired in PBS by using an inverted Olympus IX53 microscope (Olympus, Milano, Italy) equipped with a CCD UC30 camera and a digital image acquisition software (cellSens Entry) at 10× magnification.
## Intracellular triglyceride content
FaO cells were scraped in PBS and lysed. Lipids were extracted using the chloroform/methanol (2:1) method. Triglyceride (TAG) content was measured by using "Triglycerides liquid" kit (Sentinel, Milan, Italy) and quantified spectrophotometrically in a Varian Cary-50Bio spectrophotometer (Agilent, Milan, Italy). Values were normalized to protein content, as measured by Bradford assay [bib_ref] A rapid and sensitive method for the quantitation of microgram quantities of..., Bradford [/bib_ref]. Data are expressed as percent TAG content relative to controls [bib_ref] Ethanol and fatty acids impair lipid homeostasis in an in vitro model..., Vecchione [/bib_ref].
## Lipid droplet visualization by fluorescence microscopy
Cells grown on coverslips were rinsed with PBS (pH 7.4) and fixed with 4% paraformaldehyde for 20 min at room temperature. Neutral lipids were stained by incubation with 1 µg/mL BODIPY 493/503 (Molecular Probes, Life technologies, Monza, Italy) in PBS for 30 min [bib_ref] Fluorescent high-content imaging allows the discrimination and quantitation of E-LDL-induced lipid droplets..., Grandl [/bib_ref]. After washing, nuclei were stained and slides were mounted with 4 ,6-diamidino-2-phenylindole (DAPI, 5 µg/mL) (ProLong Gold medium with DAPI; Invitrogen). Mounted slides were examined by Nikon Eclipse E80i light microscope (Nikon, Tokyo, Japan) equipped with the standard epifluorescence filter set up. Images were captured under oil with a 100× plan apochromatic objective. Analyses were performed on two independent experiments measuring at least 40 cells for each treatment using the ImageJ software [bib_ref] Thyromimetic actions of tetrabromobisphenol A (TBBPA) in steatotic FaO rat hepatoma cells, Grasselli [/bib_ref].
# Conclusions
Oxidative stress rules a plethora of pathological processes, thus research on polyphenolic compounds is of utmost importance in the hope of discovering new therapeutic agents against such threats to human health. Data presented in this study have shown that tertiary feruloyl amides as trans-FEF77 possess similar antioxidant activity and lipidlowering effect compared to natural ferulic acid. This finding opens the way for studying specific structural modifications of FA obtaining a fine-tuning of physicochemical properties. Moreover, to the best of our knowledge, this paper has reported the first complete light-mediated isomerization of a ferulic derivative.
Supplementary Materials: The following are available online, [fig_ref] Figure 1: Cont. [/fig_ref] : Copies of UV-Vis spectra of t-FEF77 and c-FEF77 in methanol, [fig_ref] Figure 2: Photoisomerization of FA and FEF77 over time [/fig_ref] : Copies of 1 H-and 13 C-NMR spectra of t-FEF77 and c-FEF77.
[fig] Figure 1: Cont. [/fig]
[fig] Figure 2: Photoisomerization of FA and FEF77 over time. [/fig]
[fig] 2. 4: Antioxidant Activity of FA, t-FEF77, and c-FEF77 2.4.1. DPPH Assay and Radical Scavenging Activity [/fig]
[fig] Figure 3, Figure 3: Viability of human endothelial cord vein (HECV) cells treated with: (A) FA, (B) t-FEF77, or (C) c-FEF77 at 3.125, 6.25, 12.5, 25, and 50 μM for 24 h. Data are expressed as percent vs. control (0). Values are mean ± S.D from at least three independent experiments. In control cells, FA, t-FEF77, and c-FEF77 treatments did not affect intracellular ROS content (not shown), but prevented the oxidative damage induced by subsequent treatment with H2O2. These results are demonstrated by the evident decrease in fluorescence depicted in the representative images of Figure 4C. Viability of human endothelial cord vein (HECV) cells treated with: (A) FA, (B) t-FEF77, or (C) c-FEF77 at 3.125, 6.25, 12.5, 25, and 50 µM for 24 h. Data are expressed as percent vs. control (0). Values are mean ± S.D from at least three independent experiments. [/fig]
[fig] Figure 3 Molecules 2021 ,Figure 4: Viability of human endothelial cord vein (HECV) cells treated with: (A) FA, (B) t-FEF77, or (C) c-FEF77 at 3.125, 6.25, 12.5, 25, and 50 μM for 24 h. Data are expressed as percent vs. control (0). Values are mean ± S.D from at least three independent experiments.In control cells, FA, t-FEF77, and c-FEF77 treatments did not affect intracellular ROS content (not shown), but prevented the oxidative damage induced by subsequent treatment with H2O2. These results are demonstrated by the evident decrease in fluorescence depicted in the representative images ofFigure 4C.26, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/molecules B) Effects of FA, t-FEF77, and c-FEF77 on oxidative stress in HECV cells. (a) HECV cell viability after a 2 h treatment with H2O2; (b) Viability of HECV cells pretreated with 12.5 μM polyphenols (FA, t-FEF77, and c-FEF77) for 24 h and subsequently with H2O2 for 2 h. (c) Reactive oxygen species (ROS) in HECV cells were underlined by 2′,7′dichlorofluorescin diacetate (DCF-DA) stain in the lower panel (upper panel shows the corresponding bright-field; bar: 50 μm). Data are expressed as percent vs. control (0). Values are mean ± S.D from at least three independent experiments. Significant differences are denoted by symbols on bars (*** p ≤ 0.001 vs control; ### p ≤ 0.001 vs H2O2). [/fig]
[fig] Figure 4: Effects of FA, t-FEF77, and c-FEF77 on oxidative stress in HECV cells. (A) HECV cell viability after a 2 h treatment with H 2 O 2 ; (B) Viability of HECV cells pretreated with 12.5 µM polyphenols (FA, t-FEF77, and c-FEF77) for 24 h and subsequently with H 2 O 2 for 2 h. (C) Reactive oxygen species (ROS) in HECV cells were underlined by 2 ,7 -dichlorofluorescin diacetate (DCF-DA) stain in the lower panel (upper panel shows the corresponding bright-field; bar: 50 µm). Data are expressed as percent vs. control (0). Values are mean ± S.D from at least three independent experiments. Significant differences are denoted by symbols on bars (*** p ≤ 0.001 vs. control; ### p ≤ 0.001 vs. H 2 O 2 ). [/fig]
[fig] Figure 5: Effects of FA and t-FEF77 in FaO cells treated in the absence or presence of O/P. (a) FaO rat hepatoma cell viability after a 3 h treatment with or without O/P and subsequent treatment with 12.5 μM polyphenols (FA or t-FEF77); (b) Intracellular TAG content in steatotic FaO treated or not with 12.5 μM polyphenols (FA or t-FEF77). (c) Fluorescence microscopy visualization of neutral lipids by BODIPY 493/503 fluorescence (green). Nuclei are visualized by DAPI fluorescence (blue) (magnification 63X; scale bars: 10 μm). Data are expressed as percent vs. control (0). Values are mean ± S.D from at least three independent experiments. Significant differences are denoted by symbols on bars (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs control; ## p ≤ 0.01 vs O/P). [/fig]
[fig] Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. [/fig]
[table] Table 1: Results of DPPH assays on compounds FA, t-FEF77, and c-FEF77. [/table]
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10.7554/eLife.26487
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CCBY
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5779228
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28826473
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s2orc_pubmed_articles
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Pirating conserved phage mechanisms promotes promiscuous staphylococcal pathogenicity island transfer
Targeting conserved and essential processes is a successful strategy to combat enemies. Remarkably, the clinically important Staphylococcus aureus pathogenicity islands (SaPIs) use this tactic to spread in nature. SaPIs reside passively in the host chromosome, under the control of the SaPI-encoded master repressor, Stl. It has been assumed that SaPI de-repression is effected by specific phage proteins that bind to Stl, initiating the SaPI cycle. Different SaPIs encode different Stl repressors, so each targets a specific phage protein for its de-repression. Broadening this narrow vision, we report here that SaPIs ensure their promiscuous transfer by targeting conserved phage mechanisms. This is accomplished because the SaPI Stl repressors have acquired different domains to interact with unrelated proteins, encoded by different phages, but in all cases performing the same conserved function. This elegant strategy allows intra-and inter-generic SaPI transfer, highlighting these elements as one of nature's most fascinating subcellular parasites.
# Introduction
The Staphylococcus aureus pathogenicity islands (SaPIs) are the prototypical members of an extremely common and recently identified family of mobile genetic elements, the phage-inducible chromosomal islands (PICIs) [bib_ref] Phage-inducible islands in the Gram-positive cocci, Martínez-Rubio [/bib_ref]. The SaPIs are clinically relevant because they carry and disseminate superantigen genes, especially those for toxic shock toxin and enterotoxin B. They are very widespread among the staphylococci and are exclusively responsible for menstrual toxic shock, a rare but important human disease. In the absence of a helper phage they reside passively in the host chromosome, under the control of a global SaPI-coded repressor, Stl, a DNA-binding protein whose sequence is rather poorly conserved among the different members of the SaPI family .
Following infection by a helper phage or induction of a helper prophage, they excise, replicate extensively, and are packaged in phage-like particles composed of phage virion proteins, leading to very high frequencies of inter-as well as intrageneric transfer . In previous work we demonstrated that SaPI de-repression is effected by specific phage proteins that bind to Stl, disrupting the Stl-DNA complex and thereby initiating the excisionreplication-packaging (ERP) cycle of the islands . Different SaPIs encode different Stl repressors, so each SaPI targets a different phage protein for its de-repression. Thus, the inducers for SaPIbov1, SaPIbov2 and SaPI1 correspond to the phage trimeric dUTPase (Dut), 80a ORF15 and Sri, respectively . Since SaPIs require phage proteins to be packaged, this strategy couples the SaPI and phage cycles, but imposes a significant transmission cost on the helper phages [bib_ref] Virus satellites drive viral evolution and ecology, Frígols [/bib_ref]. Importantly, although phages carrying mutations in the genes encoding the aforementioned SaPI inducers can be propagated in the lab, these mutations have a fitness cost when the mutant phages compete with the wild-type phages in the same conditions [bib_ref] Virus satellites drive viral evolution and ecology, Frígols [/bib_ref] , which indicates that the phage coded SaPI inducers provide an important function for the phages in nature.
We recently proposed that phages could easily overcome this SaPI imposed cost using two complementary strategies that result in phages with reduced or null capacity to induce the islands [bib_ref] Virus satellites drive viral evolution and ecology, Frígols [/bib_ref]. On the one hand, phages can encode allelic variants of the SaPI inducers with reduced affinity for the SaPI coded Stl repressor. On the other hand, some phages seem to overcome SaPI induction by replacing the phage-encoded SaPI inducing gene by another one encoding an analogous protein (an unrelated protein that performs the same biological function). Although experiments performed in the laboratory suggest that in response to these strategies SaPIs can antagonistically coevolve by inactivating their Stl repressors, this strategy superimposes a high cost for the bacteria, associated with an uncontrolled SaPI replication [bib_ref] Virus satellites drive viral evolution and ecology, Frígols [/bib_ref] , so it is unlikely that this occurs in nature.
A recent study, however, questioned the idea that phages could overcome the SaPI tyranny by replacing the SaPI inducing gene by another one encoding a functionally related protein. While all eLife digest Many harmful microbes can produce different molecules that make them more effective in causing and spreading diseases. These molecules can also be obtained from 'mobile genetic elements' that can be transferred between bacteria within a population. Pathogenicity islands are one such type of mobile genetic element and are very common among bacteria known as staphylococci. They spread toxin-encoding genes between bacteria, including one that can lead to a condition called toxic shock syndrome in humans.
Pathogenicity islands are normally found within the DNA of the bacteria, where they are deactivated by specific repressor proteins. However, in the presence of another type of mobile genetic element -the bacteriophages -the repressor proteins start to interact with specific proteins encoded by the bacteriophages. This allows the pathogenicity islands to become active and spread to other bacteria.
Previous research has shown that in the bacterium known as Staphylococcus aureus, different pathogenicity islands have different repressors. Scientists therefore assumed that the repressors are only able to interact with certain bacteriophage proteins. However, since pathogenicity islands are widespread in nature, it could be possible that they use other ways to hijack the bacteriophage machinery to ensure their transfer.
To test this hypothesis, Bowring et al. studied two types of pathogenicity islands in S. aureus and revealed that their two different repressors did not interact with specific bacteriophage proteins as previously hypothesized. Instead, each repressor could interact with multiple bacteriophage proteins that had a variety of different structures, including proteins from completely different bacteriophages.
Bowring et al. also discovered that each of the analyzed repressor proteins did not actually recognize any specific shared structural features on the bacteriophage proteins, but rather evolved to target proteins that play the same role in various bacteriophages. This suggests the repressors target a specific process rather than a single protein. This strategy allows them to be transferred within the same species, but also between different ones.
A next step will be to better understand how a repressor can recognize structurally unrelated proteins, and establish what evolutionary forces are driving this phenomenon. A deeper knowledge of how pathogenicity islands spread between staphylococci is vital to understand how these bacteria can become resistant to treatments such as antibiotics. the staphylococcal S. aureus phages encode Duts; some encode dimeric and others trimeric Duts, never both [bib_ref] Virus satellites drive viral evolution and ecology, Frígols [/bib_ref]. Importantly, dimeric and trimeric Duts are completely unrelated both in sequence and structure, representing a nice example of convergent evolution . While the 80a and f11 phage-encoded trimeric Duts were initially described as the SaPIbov1 inducers , the dimeric Dut from phage fNM1 also induces SaPIbov1 [bib_ref] The type 2 dUTPase of bacteriophage fNM1 initiates mobilization of Staphylococcus aureus..., Hill [/bib_ref] [bib_ref] Derepression of SaPIbov1 is independent of jNM1 type 2 dUTPase activity and..., Hill [/bib_ref]. The fact that both dimeric and trimeric Duts induce SaPIbov1 raised the interesting possibility that the Stl repressors could target different phage proteins, significantly increasing the capacity of the SaPIs to be induced and transferred. This result also raised other interesting questions about the SaPIs: is this phenomenon exclusive of SaPI-bov1 or are other SaPIs also induced by unrelated proteins? If that was the case for specific SaPIs, are these unrelated proteins always performing the same function for the phages or conversely it is possible that a specific SaPI repressor interacts with proteins performing unrelated functions? And finally, what is the molecular mechanism by which the SaPI-encoded Stl repressors interact with different proteins?
Here we set out to answer all these questions and have demonstrated that it is more complicated than expected for the phages to overcome the SaPIs superimposed tyranny. Our results provide evidence of inter-species PICI transfer in nature. We have also deciphered the molecular mechanism used by the SaPIs to hijack the helper phage machinery in order to get high intra-and inter-generic transference: instead of interacting with specific partners, SaPIs have evolved a fascinating strategy that promotes their high transfer by pirating conserved phage mechanisms.
# Results
The SaPIbov1 Stl repressor interacts with the fO11 dimeric Dut protein What is the mechanism by which the SaPIbov1 repressor interacts with apparently unrelated proteins? Obviously, and since the trimeric and dimeric Duts perform the same biological function, the most likely scenario would be the existence of a conserved domain in the phage-encoded proteins that would be recognised by the SaPIbov1 coded Stl repressor. The structure of the phage 80a and f11 coded Duts has recently been solved [bib_ref] Structure and enzymatic mechanism of a moonlighting dUTPase, Leveles [/bib_ref]. Moreover, in-depth structural, genetic and biochemical studies have demonstrated that the trimeric Dut domains IV, V and VI are involved in SaPIbov1 Stl recognition [bib_ref] Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity..., Maiques [/bib_ref]. To know whether similar domains are present in the dimeric Duts, we initially addressed the following question: does the SaPIbov1 Stl interact just with the fNM1 dimeric Dut or can it interact with other phage coded dimeric Duts? To solve this question, we analysed the fO11 dimeric Dut. As occurred with the trimeric Duts , the dimeric fNM1 and fO11 Duts are basically identical except in a divergent central region (Figure 1-figure supplement 1). Interestingly, the fO11 dimeric Dut also induces the SaPIbov1 and SaPIbov5 cycles [fig_ref] Figure 1: Induction of SaPIbov1 and SaPIbov5 by the fO11 dimeric Dut [/fig_ref]. Note that SaPIbov5 was also included in these studies because it encodes the same Stl repressor as SaPIbov1, with both islands being induced by the same helper phages [bib_ref] Convergent evolution of pathogenicity islands in helper cos phage interference, Carpena [/bib_ref] [bib_ref] Adaptation of Staphylococcus aureus to ruminant and equine hosts involves SaPI-carried variants..., Viana [/bib_ref]. Expression of the fO11 dimeric Dut (from the Pcad promoter in expression vector pCN51) in a SaPIbov1 or SaPIbov5 positive strain demonstrated that this protein is sufficient to induce the SaPI cycles. Thus, when overexpressed, the cloned fO11 dimeric dut induced SaPIbov1 and SaPIbov5 excision and replication [fig_ref] Figure 1: Induction of SaPIbov1 and SaPIbov5 by the fO11 dimeric Dut [/fig_ref].
In all 3 characterised SaPIs (SaPI1, SaPIbov1 and SaPIbov2), Stl blocks SaPI induction by binding to the SaPI stl-str divergent region, blocking transcription of most of the SaPI genes. SaPI de-repression occurs after a direct protein-protein interaction between the cognate phage inducer and the SaPI coded Stl repressor . To test if the mechanism involving the fO11 dimeric Dut in SaPIbov1 induction matches with that previously reported for the other SaPIs, we first demonstrated that fO11 Dut induces xis expression, which normally is repressed by Stl. This was confirmed using plasmid pJP674, which carries a b-lactamase reporter gene fused to xis, downstream of str and the Stl SaPIbov1 -repressed str promoter, and also encodes Stl SaPIbov1 (see [fig_ref] Figure 1: Induction of SaPIbov1 and SaPIbov5 by the fO11 dimeric Dut [/fig_ref]. The cloned fO11 dut gene was introduced on vector pCN51 and expression was tested in the presence or absence of an inducing concentration of CdCl 2 . Induction of fO11 dut strongly increased blactamase expression from the str promoter [fig_ref] Figure 1: Induction of SaPIbov1 and SaPIbov5 by the fO11 dimeric Dut [/fig_ref]. Moreover, the predicted protein-protein interaction between the fO11 Dut and the Stl SaPIbov1 repressor was confirmed by co-expression and and SaPIbov5 respectively, were complemented with a plasmid expressing the 3xFLAG-tagged fO11 dimeric Dut. Samples were isolated at 0' or 3 hr after induction with 0.5 mM CdCl 2 and Southern blots were performed using a probe for the SaPIbov1/SaPIbov5 integrase. The upper band is 'bulk' DNA, including chromosomal, phage, and replicating SaPI. CCC indicates covalently closed circular SaPI DNA. In these experiments, as no helper phage was present, the excised and replicating SaPI DNA appears as part of the bulk DNA or as CCC molecules, rather than the linear monomers that are seen following helper phage-mediated induction and packaging. (B) Derepression of str transcription by fO11 Dut expression. The diagram represents a schematic of a blaZ transcriptional fusion generated in pJP674. b-lactamase assays were performed on strains containing pJP674 together with a pCN51derived plasmid expressing the fO11 Dut (JP14818) or the empty pCN51 control (JP15105). Samples were taken after 5 hr in the absence or following induction with 5 mM Cadmium. All data is the result of five independent experiments. Error bars represent SEM. A 2-way ANOVA with Sidak's multiple comparisons test was performed to compare mean differences within rows. Adjusted p values were as follows: fO11 = 0.0004 *** , pCN51 = 0.9579 ns . ns, not significant. (C) Affinity chromatography of the fO11 Dut for the His-tagged SaPIbov1 Stl. Strains were induced with 10 mM isopropyl-b-d-thiogalactoside (IPTG) and samples taken at 3 hr. Cells were disrupted and expressing proteins were applied to a Ni 2+ column and eluted. Lane 2, elution fraction for His 6 -Stl SaPIbov1 and Dut FO11 (JP14832). Lane 1, corresponding elution fraction for Stl SaPIbov1 and Dut FO11 (JP14833, no His 6 -tag). Proteins were confirmed by Mass Spectrometry analysis. DOI: https://doi.org/10.7554/eLife.26487.003
The following source data and figure supplement are available for figure 1:
Source data 1. b-lactamase assay data and statistical analysis for the dimeric FO11 Dut. DOI: https://doi.org/10.7554/eLife.26487.005 [fig_ref] Figure 1: Induction of SaPIbov1 and SaPIbov5 by the fO11 dimeric Dut [/fig_ref] continued on next page affinity purification of His 6 -Stl SaPIbov1 and untagged fO11 Dut proteins. It was possible to co-purify a complex between His 6 -Stl SaPIbov1 and fO11 Dut [fig_ref] Figure 1: Induction of SaPIbov1 and SaPIbov5 by the fO11 dimeric Dut [/fig_ref]. The identity of each of these bands was confirmed by amino acid sequencing and mass spectrometry. We conclude from these results that dimeric fO11 Dut induces the SaPI cycle using the same mechanism described for the unrelated trimeric Dut proteins. Moreover, and although this is not the scope of this study, these results also involve the dimeric Duts in SaPI signalling.
## The sapibov1 stl repressor has different interacting domains
It is predicted that dimeric and trimeric Duts acquire a completely unrelated fold . However, since both dimeric and trimeric Duts perform the same enzymatic activity, we hypothesised that these proteins could have conserved domains responsible for the interaction with Stl. To test this hypothesis, and since the structure of the staphylococcal phage-encoded dimeric Duts remains unsolved, the structure of the dimeric fO11 Dut in complex with the nonhydrolyzable dUTP analog a,b-imido-dUTP (dUPNPP) and Mg 2+ was determined at 2.1 Å resolution (Supplementary file 1). The crystal structure showed 2 molecules in the asymmetric unit organized as a homodimer [fig_ref] Figure 2 -: figure supplement 1 [/fig_ref]. The structure shows that fO11 is an all-helix protein composed of only seven a-helices (a1, residues 7-23; a2, 29-47; a3 61-82; a4, 86-98; a5, 104-108; a6, 110-121 and a7, 127-141) per protomer. Interestingly, the fO11 dimeric Dut has a 'compact' conformation compared to counterparts in other organisms (which encompass ten or more a-helices) [fig_ref] Figure 2 -: figure supplement 1 [/fig_ref] and [fig_ref] Figure 2 -: figure supplement 1 [/fig_ref] -figure supplement 1). Nevertheless, the fO11 protomer presents the characteristic structural core of dimeric Duts composed of four helices (a1-a3 and a7 in fO11) that conforms the active centre where the nucleotide binds [fig_ref] Figure 2 -: figure supplement 1 [/fig_ref]. In the fO11 dimer both active centres are oriented towards the same molecule face, forming a long channel that accommodates two molecules of dUPNPP. The rest of the protomer is placed on the opposite molecule face (residues 83-138), which corresponds to the divergent region in phage-encoded dimeric Duts but also adopts a helical fold (helices a4-a6 in fO11) [fig_ref] Figure 2 -: figure supplement 1 [/fig_ref] and Since in the trimeric Duts the motifs IV, V and VI are essential for interaction with the Stl repressor [bib_ref] Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity..., Maiques [/bib_ref] , we looked for the presence of structural elements with similar topology in the dimeric Dut. As could be anticipated by the difference in folding between the trimeric (all-beta) and dimeric (all-alpha) proteins, none of these motifs are present in the fO11 Dut [fig_ref] Figure 2 -: figure supplement 1 [/fig_ref]. In the trimeric Duts, these three motifs place together surrounding the nucleotide in the active centre [fig_ref] Figure 3: Dimeric and trimeric S [/fig_ref] , thus we wondered whether the Stl recognition site was generated spatially by the disposition of specific residues provided by these three motifs rather than by the motifs themselves. To check this possibility we spatially compared the active sites of both types of Duts by superimposing the nucleotide-binding sites of the trimeric 80a and dimeric fO11 phagic Duts [fig_ref] Figure 3: Dimeric and trimeric S [/fig_ref]. As was previously observed in the comparison of the active centres from other dimeric and trimeric Duts [bib_ref] The crystal structure of Trypanosoma cruzi dUTPase reveals a novel dUTP/dUDP binding..., Harkiolaki [/bib_ref] , the way of dUTP recognition and binding is completely different in both Dut types, not only in the orientation of the plane of the uracil moiety, which showed a relative rotation of more than 75˚, but also in the disposition of the phosphates. In trimeric Duts, the a-phosphates acquire a gauche catalytic-competent geometry (Kovári et al., 2008) meanwhile a trans conformation is observed in the dimeric fO11 Dut [fig_ref] Figure 3: Dimeric and trimeric S [/fig_ref]. Furthermore, the b and g phosphates differ in their relative disposition, chelating a single divalent metal in the trimerics, versus two in the dimerics [bib_ref] On the catalytic mechanism of dimeric dUTPases, Hemsworth [/bib_ref] [fig_ref] Figure 2 -: figure supplement 1 [/fig_ref]. Therefore, the active centres in both types of enzymes show divergent architecture and, consequently, the spatial disposition of the residues surrounding the nucleotides, including those provided by motif IV, V and VI, is completely different. Taken together, these results strongly suggest that the SaPIbov1 Stl repressor has different interacting domains/ways to recognise the unrelated trimeric and dimeric Duts.
To go further with these analyses, we generated a set of deletional mutants in the SaPIbov1 Stl repressor, with the idea that some of these mutants would specifically affect the interaction of the SaPIbov1 Stl repressor with one of the Dut types under study, but not with the other. Sequence analysis and in silico modelling indicates that SaPIbov1 Stl is mainly an a-helical protein composed of a N-terminal HTH DNA-binding domain (residues 1-80) and C-terminal portion of unknown function that seems to be conformed of two domains connected by a region of low complexity (residues 167-179) (Figure 4-figure supplement 1 and Supplementary file 2; [bib_ref] Evidence-based structural model of the staphylococcal repressor protein: separation of functions into..., Nyíri [/bib_ref]. Thus, we generated Stl deletional variants lacking the N-terminal DNA binding domain (residues 1-86; StlD HTH ) or the most C-terminal subdomain (residues 176-267; StlD Cter ) (Figure 4-figure supplement 1). Unfortunately, these mutants couldn't be analysed in vivo, since the generated Stl mutant repressors had lost the capacity to block the SaPI cycle. To solve that problem, we expressed the different Stl mutants in E. coli, and analysed in vitro their capacity to interact with the different Duts. Interestingly, deletion of the N-terminal DNA-binding domain abolished the interaction with the trimeric f11 but not with the dimeric fO11 Dut. Conversely, the elimination of the C-terminal subdomain impairs the binding to the dimeric but not to the trimeric Dut . Moreover, it has been shown the interaction with the Stl repressor inhibits the dUTPase activity of both dimeric and trimeric Duts [bib_ref] The type 2 dUTPase of bacteriophage fNM1 initiates mobilization of Staphylococcus aureus..., Hill [/bib_ref] [bib_ref] Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel..., Szabó [/bib_ref]. Here we have confirmed this inhibitory activity for the f11 and fO11 Duts with the full-length Stl protein . Furthermore, and in agreement with the binding capacity shown by the Stl deletional variants, StlD HTH inhibits the dUT-Pase activity of dimeric but not trimeric Duts, while StlD Cter has the opposite capacity . The fact that the SaPIbov1 Stl has particular regions for interacting with the trimeric and dimeric Duts supports the idea that the SaPIbov1 Stl repressor has evolved distinct ways to specifically interact with the dimeric or trimeric Duts.
## The phage 80a encoded sak recombinase is the inducer for sapi2
We next addressed the question of whether the previous phenomenon was exclusive to SaPIbov1. To do that, we initially tried to identify the phage 80a inducer for SaPI2, a SaPI frequently responsible for the clinically relevant menstrual toxic shock syndrome (TSS; [bib_ref] Sequence analysis reveals genetic exchanges and intraspecific spread of SaPI2, a pathogenicity..., Subedi [/bib_ref]. Since SaPIs severely interfere with helper phage reproduction, a classical strategy used to identify non-essential SaPI inducers is to generate spontaneous phage mutants that are able to form plaques in the presence of the SaPIs. This strategy selects for phage mutants that have lost the ability to mobilise the islands because of mutations they carry in the SaPI inducer genes. These mutations usually generate non-functional proteins that have also lost their capacity to relieve Stl-mediated repression [bib_ref] Virus satellites drive viral evolution and ecology, Frígols [/bib_ref]. After many attempts, we obtained only a single spontaneous 80a phage mutant which was able to form plaques on S. aureus strain RN4220 containing SaPI2, suggesting that the SaPI2 inducer is absolutely essential for the phage cycle even in laboratory conditions. In this mutant the 3' region of the 80a ORF16 has been lost. Translation of this mutated gene generates a chimeric protein fused with the single strand binding protein (Ssb; 80a ORF17; -figure supplement 1). Since in this mutant phage the ssb gene (including its ribosomal binding site) is unaffected and can be transcribed and translated independently of the chimeric structure, this result suggests that ORF16 is the SaPI2 inducer.
The 80a ORF16 protein belongs to the Sak family of single strand annealing proteins (SSAP, also called recombinases) involved in homologous recombination [bib_ref] Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5..., Lopes [/bib_ref] [bib_ref] Lactococcal phage p2 ORF35-Sak3 is an ATPase involved in DNA recombination and..., Scaltriti [/bib_ref]. Although for many of these proteins their role in the phage cycle has not been established yet, we have recently demonstrated that this protein is essential for 80a phage replication [bib_ref] Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus, Neamah [/bib_ref]. Note, however, that the chimeric Sak-Ssb protein is still functional for the phage, as demonstrated by the fact that the mutant phage encoding this protein still replicates and forms plaques in a sensitive recipient strain.
Expression of the 80a sak (ORF16) gene (from the Pcad promoter in expression vector pCN51) in a SaPI2 positive strain demonstrated that Sak is sufficient to induce this SaPI. Thus, when overexpressed, the cloned sak (but not the chimeric Sak-Ssb protein) induced SaPI2 excision and replication . As the protein levels produced from these constructs are comparable , this result clearly shows that although expressed, the chimeric protein has lost its capacity to induce SaPI2. Moreover, and to confirm that the mechanism involving Sak in SaPI2 induction matches with that previously reported for the other SaPIs, we demonstrated that 80a Sak induces expression of showing the difference in folding between dimeric (all-alpha) and trimeric (all-beta) Duts. The dUPNPP molecules in the active centres are represented in stick and the Mg ions as spheres. For clarity only one dUPNPP molecule is shown in the trimeric structure. The structural motifs implicated in Stl recognition for trimeric Duts [bib_ref] Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity..., Maiques [/bib_ref] are labelled and coloured in cyan, magenta and red for motif IV, V and VI, respectively. (B) Superimposition of dUPNPP molecules in the active centres of fO11 (green tones) and 80a (orange tones) shows that the bound nucleotide molecules acquire different conformations (stick representation), including the disposition of the Mg ions (sphere representation), and that the spatial arrangement of the structural elements conforming each active centre is essentially different. No structural element equivalent to the Stl binding motifs of 80a (coloured as in A) is observed in fO11. DOI: https://doi.org/10.7554/eLife.26487.008 the SaPI2 Stl repressed genes. This was confirmed using plasmid pJP1977, which carries a b-lactamase reporter gene fused to xis, downstream of str and the Stl SaPI2 -repressed str promoter, and also encodes Stl SaPI2 (see [fig_ref] Figure 6: Phage SSAPs bind SaPI2 Stl protein [/fig_ref]. The cloned sak gene was introduced on vector pCN51 and expression was tested in the presence or absence of an inducing concentration of CdCl 2 . Induction of sak, but not the chimeric sak-ssb, strongly increased b-lactamase expression from the str promoter [fig_ref] Figure 6: Phage SSAPs bind SaPI2 Stl protein [/fig_ref]. Moreover, the predicted protein-protein interaction between Sak and the Stl SaPI2 repressor was confirmed by co-expression and affinity purification of His 6 -Stl SaPI2 and untagged Sak proteins. It was possible to co-purify a complex between His 6 -Stl SaPI2 and Sak [fig_ref] Figure 6: Phage SSAPs bind SaPI2 Stl protein [/fig_ref] , whereas untagged Sak alone did not bind to the resin. The chimeric Sak-Ssb, which does not derepress SaPI2
[formula] φ!!"" #" $" #" $" #" $" #" %&'" $" #" #" $" $" $" $" %&'∆ (&)* "" $" $" $" #" #" $" $" %&'∆ +,+ "" $" $" $" $" $" #" #" φ-!!" #" $" #" $" #" $" #" %&'" $" #" #" $" $" $" $" %&'∆ (&)* "" $" $" $" #" #" $" $" %&'∆ +,+ "" $" $" $" $" $" #" #" φO11 φO11 + Stl φO11 + Stl∆ Cter φO11 + Stl∆ HTH φ11 φ11 + Stl φ11 + Stl∆ Cter φ11 + Stl∆ HTH φ!!"" [/formula]
." [fig_ref] Figure 6: Phage SSAPs bind SaPI2 Stl protein [/fig_ref] , did not co-purify with His 6 -Stl SaPI2 , confirming the specificity of the His 6 -Stl SaPI2 ::Sak interaction. The identity of each of these bands was confirmed by amino acid sequencing and mass spectrometry.
The Sak4 recombinase also induces SaPI2
Next, and based on the fact that both dimeric and trimeric Duts induce SaPIbov1, we explored the possibility that the SaPI2 Stl repressor could also target different phage proteins, significantly increasing the capacity of SaPI2 to be induced and transferred. Interestingly, phages f80 and f52A can also induce the SaPI2 cycle [bib_ref] Precisely modulated pathogenicity island interference with late phage gene transcription, Ram [/bib_ref] , although none of them encodes a 80a Sak protein. To test the possibility that SaPI2 was targeting another protein, we tried to identify the SaPI2 Cloned genes Top, schematic representation of the blaZ transcriptional fusion generated in plasmid pJP1977. Bottom, strains containing pJP1977-and pCN51-derivative plasmids expressing the different SSAPs under study were assayed for b-lactamase activity in the absence of or 3 hr after induction with 5 mM CdCl 2 . Samples were normalized for total cell mass. Experiment data is in triplicate. Error bars represent SEM. A 2-way ANOVA with Sidak's multiple comparisons test was performed to compare mean differences within rows. Adjusted p values were as follows: Sak = 0.0001 *** , Sak4 = 0.0001 *** , Erf = 0.0001 *** , Redb=0.0001 *** , chimera = 0.999 ns . ns, not significant. (B) Affinity chromatography of 80a Sak (ORF16) using His 6 -Stl SaPI2 . E. coli strain expressing the pair was isopropyl-b-Dthiogalactoside (IPTG)-induced and, after disruption of the cells, the expressed proteins were applied to a Ni 2+ agarose column and eluted. The presence of the different proteins was monitored in the elute fraction (E) by Coomassie staining. M: molecular weight marker. (C) Bacterial adenylate cyclase-based two-hybrid (BACTH) analysis. Spots in each row represent three independent colonies. Plasmid combinations are indicated in the right columns. Bottom, quantification of the BACTH analysis after 2 hr of IPTG (5 mM) induction. Experiment data is in triplicate. Error bars represent SEM. A 1-way ANOVA with Sidak's multiple comparisons test was performed to compare mean differences within rows. Adjusted p values were as follows; Sak = 0.0221*, Sak4 = 0.0030**, Erf = 0.0158*, Redb=0.0014**, chimera (Chim) = 0.1980 ns . ns, not significant. [fig_ref] Figure 6: Phage SSAPs bind SaPI2 Stl protein [/fig_ref] continued on next page inducer in phages f80 and f52A by generating spontaneous phage mutants that can grow in the presence of the island. After many attempts, we did not get any phage mutants capable of forming plaques in a SaPI2 positive strain, suggesting that the SaPI2 inducers are also essential for the biology of these phages, even in laboratory conditions. In view of this result, and bearing in mind that both the dimeric and trimeric Duts have the same biological (enzymatic) function for the phage, we hypothesised that the f80 or f52A SaPI2 inducers would be functionally related to the 80a Sak protein. Since the S. aureus phages display synteny, we speculated that the genes located in the same genome position as the 80a sak gene would be essential for the phage, would have a recombinase function, and would encode for the SaPI2 inducer. While phages f80 and f52A do not contain an orf homologue to 80a sak, all three phages encode identifiable ssb genes, which in the case of the 80a phage is located downstream of the sak gene [fig_ref] Figure 2 -: figure supplement 1 [/fig_ref]. Thus, we analysed the possibility that the genes upstream of ssb were the SaPI2 inducers. Both f80 and f52A phages carried an identical gene, named ORF13 in phage f80 and ORF16 in phage f52A, which encodes a non-related protein to the 80a Sak [fig_ref] Figure 3: Dimeric and trimeric S [/fig_ref]. This protein belongs to a distinct family of SSAPs, Sak4 [bib_ref] Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5..., Lopes [/bib_ref]. While Sak4 and Sak are not homologous in sequence [fig_ref] Figure 3: Dimeric and trimeric S [/fig_ref] , we have recently demonstrated that they are both SSAPs (recombinases) performing a similar function in their cognate phages [bib_ref] Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus, Neamah [/bib_ref].
[formula] sak (80α) stl (SaPI2) stl (SaPI2) stl (SaPI2) stl (SaPI2) sak4 (ϕ52A) erf (ϕSLT) chimera (80α) Positive control Negative control (empty plasmids) C stl (SaPI2) redβ (ϕN315) Sak Sak4 Erf Redβ Chim NC )) ) ) [/formula]
The results above support the hypothesis proposing that unrelated proteins performing the same function for the phages could all act as inducers for a specific SaPI. Thus, expression of the f80 and f52A Sak4 proteins in a SaPI2 positive strain demonstrated that they are sufficient to induce the SaPI2 cycle . Moreover, expression of the sak4 genes strongly increased b-lactamase expression from the Stl-repressed str promoter [fig_ref] Figure 6: Phage SSAPs bind SaPI2 Stl protein [/fig_ref]. Since expression of the f52A Sak4 protein in E. coli generated an insoluble protein which aggregates, we couldn't co-purify a complex between His 6 -Stl SaPI2 and untagged f52A Sak4. However, a two-hybrid assay confirmed the strong interaction between both the f52A Sak4 recombinase and the SaPI2 Stl repressor and between the 80a Sak protein and the SaPI2 Stl pair (used here as a control; [fig_ref] Figure 6: Phage SSAPs bind SaPI2 Stl protein [/fig_ref] , confirming that the phage Sak4 protein is a bona fide SaPI2 inducer.
## Unrelated phage-encoded recombinases induce sapi2
Since SaPI2 superimposes a high cost for the phage, it could be possible that staphylococcal phages would initially avoid this interference by encoding additional SSAPs, unrelated to Sak or Sak4. In turn, and if the hypothesis we propose here is correct, it could also be possible that the SaPI repressor would evolve to target these new phage encoded recombinase proteins. In silico scrutiny looking at the genes located upstream of the ssb genes revealed that staphylococcal phages encode at least 4 distant SSAP families, including Erf, Redb, and the aforementioned Sak and Sak4 [fig_ref] Figure 3: Dimeric and trimeric S [/fig_ref]. All the staphylococcal phages encode one SSAP, in accordance with the fact that these proteins are essential for the phage [bib_ref] Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus, Neamah [/bib_ref]. To test the possibility that these other unrelated recombinases also induced SaPI2, we characterised in detail those present in phages fSLT (ORF 17) and fN315 (SA1794), which belong to the Erf and Redb families of SSAPs, respectively, and have completely different sequences . We selected phage fSLT because it is clinically relevant, encoding the Panton-Valentine leukocidin (PVL) toxin. Applying the same methodology and strategies previously used to characterise Sak and Sak4, our results confirm that: (i) the expression of the fSLT Erf and fN315 Redb proteins is sufficient to induce the SaPI2 cycle ; (ii) expression of these recombinases prevents Stl from binding to the SaPI2 stl-str divergent region [fig_ref] Figure 6: Phage SSAPs bind SaPI2 Stl protein [/fig_ref] and (iii) the two-hybrid assay confirmed the interaction between the SaPI2 Stl repressor and the Erf and Redb recombinases [fig_ref] Figure 6: Phage SSAPs bind SaPI2 Stl protein [/fig_ref]. The following source data is available for figure 6:
Source data 1. b-lactamase assay data and statistical analysis for the recombinases. DOI: https://doi.org/10.7554/eLife.26487.017 Source data 2. BACTH analysis data and statistical analysis for the recombinases. DOI: https://doi.org/10.7554/eLife.26487.018
Finally, since the existence of different interacting domains in the Stl repressor explains why both the dimeric and trimeric proteins can induce SaPIbov1, we wondered if a similar mechanism was employed by the SaPI2 island. Structure-based modelling of Sak, Sak4, Erf and Redb suggested they are unrelated, although Sak, Erf and Redb can be connected through remote homology relationships [bib_ref] Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5..., Lopes [/bib_ref]. Thus, it has been proposed that Sak, Erf and Redb belong to a large superfamily adopting a shortcut Rad52-like fold [bib_ref] Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5..., Lopes [/bib_ref]. However, structural models produced with I-Tasser [bib_ref] The I-TASSER Suite: protein structure and function prediction, Yang [/bib_ref] and Phyre2 [bib_ref] The Phyre2 web portal for protein modeling, prediction and analysis, Kelley [/bib_ref] servers for Sak (phage 80a), Erf (fSLT) and Redb (fN315) only proposed the Rad52 fold for Sak, whereas for Erf and Redb alternating foldings, non-related with Rad52 recombinases, were proposed with low confidence [fig_ref] .Figure 7: Interestingly, the f52A coded Sak4 recombinase induced SaPI2 and ScCIUMC-CNS-990, while the... [/fig_ref] and Supplementary files 4A and B). By contrast, remote homologs to Sak4 are predicted to adopt a shortcut Rad51/RecA fold [bib_ref] Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5..., Lopes [/bib_ref] and models obtained from I-Tasser and Phyre2 servers proposed this fold for the f52A Sak4 recombinase with good confidence [fig_ref] .Figure 7: Interestingly, the f52A coded Sak4 recombinase induced SaPI2 and ScCIUMC-CNS-990, while the... [/fig_ref] and Supplementary files 4A and B). Taken together, these results suggest that the most likely scenario explaining why the SaPI2 Stl repressor can interact with different recombinases is the existence of different interacting domains in the repressor.
## Pirating conserved phage processes supports inter-specific sapi transfer
Both we and others have previously demonstrated that the SaPIs can be inter-and intra-generically transferred [bib_ref] Phage-mediated intergeneric transfer of toxin genes, Chen [/bib_ref] [bib_ref] Role of staphylococcal phage and SaPI integrase in intra-and interspecies SaPI transfer, Maiques [/bib_ref]. Although this process occurs at astonishingly high frequencies in the lab, its impact in nature remains unsolved. The fact that the mechanisms involved in the life cycle of the phages are conserved among species raised an interesting possibility: by targeting conserved phage processes SaPI-like elements would be successfully spread and maintained in nature. To test if these Stl repressors interact with the SaPIbov1 or SaPI2 inducers, we used the aforementioned strategy to generate a set of plasmids in which the divergent str/str-xis region of the PICIs was fused to a b-lactamase reporter gene. These derivatives were generated for the PICIs encoding the most distantly related Stl repressors: ShoCI794_SEPI (S. hominis) and ShaCI51-48 (S. haemolyticus), encoding a SaPIbov1 Stl homolog, and ShaCI137133 (S. haemolyticus), SeCI-NIHLM095 (S. epidermidis) and SsCIUMC-CNS-990 (S. simulans) carrying a SaPI2 Stl homolog. Next, the capacity of the dimeric fO11 or trimeric f11 Duts (for the SaPIbov1-like Stl repressors), or the ability of the different SSAPs (for the SaPI2-like Stl repressors) to induce the PICI cycle was tested by introducing the pCN51 derivatives expressing the different SaPI inducers in the strains carrying the reporter plasmids. Remarkably, both the dimeric fO11 and trimeric f11 Duts induced b-lactamase expression from the Stl-repressed str promoters present in the ShoCI794_SEPI and ShaCI51-48 islands [fig_ref] Table 1: Dimeric and trimeric dUTPases induce PICIs from other species encoding SaPIbov1-like Stl... [/fig_ref] , suggesting that the Stl repressors encoded in all these islands have a common origin.
Even more interesting were the results obtained with the SaPI2 Stl homologs [fig_ref] Table 2: Unrelated SSAPs differentially induce PICIs from other species encoding SaPI2-like Stl repressors... [/fig_ref]. All the islands were induced by at least one of the recombinases, although the distribution was not as uniform as with the Duts. Thus, the 80a coded Sak induced SaPI2, SeCINIHLM095 and ShaCI137133, but not ScCIUMC-CNS-990 [fig_ref] Table 2: Unrelated SSAPs differentially induce PICIs from other species encoding SaPI2-like Stl repressors... [/fig_ref]. The chimeric 80a Sak-Ssb protein induced none, supporting that the different PICI coded Stl repressors are structurally related [fig_ref] Table 2: Unrelated SSAPs differentially induce PICIs from other species encoding SaPI2-like Stl repressors... [/fig_ref] induced SaPI2 and SeCINIHLM095. Finally, the fN315 coded Redb recombinase uniquely induced SaPI2, but not the other islands [fig_ref] Table 2: Unrelated SSAPs differentially induce PICIs from other species encoding SaPI2-like Stl repressors... [/fig_ref]. Taken together, including the previous results with the SaPIbov1 Stl mutants, these results strongly support the idea that although originally related, the different Stl repressors have evolved different domains to interact with the phage-coded inducers. It is striking how the SaPIs have evolved an elegant tactic to be highly transferred both intra-and inter-generically. However, and in the case of the inter-generic transfer of the elements, to be completely effective this strategy requires that the phages infecting the new SaPI-recipient species encode the conserved SaPI inducers. To test this, we analysed the presence of SaPIbov1 or SaPI2 inducing genes in a subset of staphylococcal phages infecting species other than S. aureus. As shown in Supplementary file 6, we were able to identify homologs to the previously characterised SaPIbov1 or SaPI2 inducer in all the analysed phages, although with different degrees of identity among the members of the distinct families. Next, and to support the idea that once the inter-species transfer occurs the PICI can be maintained in the new recipient species, we tested whether the Dut encoded in the S. epidermidis phage IPLA6, or the recombinase encoded in S. epidermidis phage PH15, were capable of inducing the cycle of the different PICI elements encoding SaPIbov1 or SaPI2 Stl homologs. That was the case, and the behaviour of the S. epidermidis IPLA6 trimeric Dut was identical to that observed for the trimeric f11 Dut [fig_ref] Table 1: Dimeric and trimeric dUTPases induce PICIs from other species encoding SaPIbov1-like Stl... [/fig_ref] , while the functioning of the fPH15 Sak recombinase was indistinguishable from that observed with the homologue S. aureus 80a coded Sak [fig_ref] Table 2: Unrelated SSAPs differentially induce PICIs from other species encoding SaPI2-like Stl repressors... [/fig_ref]. In summary, our results confirm the idea that the PICIs have established a fascinating parasitic strategy that may allow their promiscuous transfer and widespread maintenance in nature.
## Inter-species pici transfer occurs in nature
The fact that some islands present in different species encode identical proteins (including not just the Stl repressors as demonstrated here but also other PICI proteins) strongly supports the idea that The following source data available for [fig_ref] Table 1: Dimeric and trimeric dUTPases induce PICIs from other species encoding SaPIbov1-like Stl... [/fig_ref] :
Source data 1. b-lactamase assay data and statistical analysis for the SaPIbov1 Stl homologues. DOI: https://doi.org/10.7554/eLife.26487.021 some ancestral elements were horizontally transferred among species. In the different species these elements probably evolved independently, trying to adapt to the new cognate host. Our previous results, however, suggest that by pirating conserved phage mechanisms this inter-species transit probably occurs constantly in nature. To test that possibility, we scrutinised the genome databases looking for identical PICIs present in different staphylococcal species. We initiated this analysis by comparing the genomes of those PICIs encoding identical Stl proteins. Highlighting the versatility of these elements and the successful strategy they use to spread in nature, our analysis revealed that the ScCIM23864:W1 (S. caprae) and SlCIFDAARGOS_141 (S. lugdunensis) elements are identical (just 3 mismatches over 13,847 nt; Supplementary file 7).
# Discussion
The manner by which related SaPIs have acquired the ability to exploit conserved phage processes by targeting structurally unrelated proteins as antirepressors represents a remarkable evolutionary adaptation. Our results suggest that the most likely scenario explaining why the SaPI/PICI Stl repressors can interact with different phage coded inducers is the existence of different interacting domains in the SaPI Stl repressors. The presence of these different domains highlights the co-evolutionary and constant battle established between the helper phages, trying to avoid PICIs induction, and the parasitic PICIs, trying to interact with non-inducing phages [bib_ref] Virus satellites drive viral evolution and ecology, Frígols [/bib_ref]. This mechanism could also be responsible, at least in part, for the widespread distribution of PICIs in nature.
Note that we have recently demonstrated the existence of these elements in many Gram-positive cocci [bib_ref] Phage-inducible islands in the Gram-positive cocci, Martínez-Rubio [/bib_ref].
We hypothesised that at the beginning of the SaPI-phage war, a single phage protein may have been originally targeted; to escape from SaPI de-repression, because SaPIs interfere with phage maturation, substitution of the gene encoding this protein to one expressing a non-related, but functionally similar protein could have had a selective advantage for the phage. A second stage in SaPI evolution could have involved divergence of the SaPI repressor, enabling it to complex with structurally non-related phage proteins. The fact that the Stl repressors interact with structurally unrelated proteins performing the same function makes this strategy unique in nature and extremely effective. Note that in terms of increasing their transferability, a more simple strategy for the SaPIs could have been to select for Stl repressors that can interact with proteins performing different functions for the The following source data available for phage. However, since phages have mosaicism, encoding multiple versions of unrelated proteins performing the same function (as also demonstrated here), this strategy would select for phages insensitive to the SaPIs that encode the correct combination of non-inducing proteins. By contrast, and since the processes targeted by the SaPIs are extremely well conserved in the staphylococcal phages, the fact that the SaPIs target different versions of proteins involved in the same biological processes limits the capacity of the phages to overcome SaPI parasitism, ensuring the transferability of these elements. Thus, our results show that SaPI-phage interactions represent a remarkable microcosm within the bacterial intracellular universe, highlighting SaPIs as one of the most fascinating and effective subcellular parasites. However, our results raise an interesting question. Why do some repressors interact just with one inducer, limiting their capacity to be transferred, while others seem to have a broader spectrum of inducers? Our hypothesis is that although all the analysed phages encode putative SaPI inducers, these are different in sequence (see Supplementary file 6), so the repressors present in the different PICIs have evolved to increase their interaction with the specific inducers encoded in the cognate phages infecting these bacterial species. This also would explain the divergence in sequence observed in related Stl repressors. This hypothesis is currently under study.
Lactococcus lactis encodes a plasmid with an abortive infection mechanism, AbiK [bib_ref] Lactococcal phage genes involved in sensitivity to AbiK and their relation to..., Bouchard [/bib_ref]. As occurs with SaPI2, the proteins targeted by the AbiK system are the different phage encoded SSAPs involved in homologous recombination [bib_ref] Lactococcal phage genes involved in sensitivity to AbiK and their relation to..., Bouchard [/bib_ref]. Although the mechanism by which AbiK blocks phage reproduction remains unclear, it does not seem to involve the formation of a complex between the AbiK protein and the recombinases, as occurs with SaPI2 [bib_ref] Lactococcal phage genes involved in sensitivity to AbiK and their relation to..., Bouchard [/bib_ref] [bib_ref] A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in..., Wang [/bib_ref].
Since the discovery of the SaPIs, it has gradually become apparent that prophages and PICIs have evolved in much more interesting ways than has generally been realised. PICIs are sophisticated, elegant and extremely effective parasites. They have incorporated an impressive arsenal of effective strategies to interfere with helper phages, ensuring their presence in nature . We anticipate here that novel and unexpected mechanisms of PICI-mediated phage interference will soon be characterised, which will highlight the fascinating biology of these subcellular creatures and their cognate helper phages.
# Materials and methods
## Bacterial strains and growth conditions
The bacterial strains used in this study are listed in Supplementary file 8A. S. aureus was grown in Tryptic soy broth (TSB) or on Tryptic soy agar plates. E. coli was grown in LB broth or on LB agar plates. Antibiotic selection was used where appropriate. Preparation and analysis of phage lysates was performed essentially as previously described [bib_ref] SaPI mutations affecting replication and transfer and enabling autonomous replication in the..., Ubeda [/bib_ref].
# Dna methods
General DNA manipulations were performed using standard procedures. Plasmid constructs used in this study (Supplementary file 8B) were generated by cloning PCR products obtained with oligonucleotide primers, listed in Supplementary file 8C.
Detection probes for SaPI DNA in Southern blots were generated by PCR using primers SaPI-bov1-112mE and SaPIbov1-113cB (SaPIbov1 and SaPIbov5) or Tet-1m and Tet-2c (SaPI2), listed in Supplementary file 8C. Probe labelling and DNA hybridization were performed following the protocol provided with the PCR-DIG DNA-labelling and chemiluminescent detection kit (Roche). Southern blot experiments were performed as previously described .
fO11 dut was cloned into pET28a vector (Novagen) using primers listed in Supplementary file 8C. Plasmids pETNKI-StlD HTH and pETNKI-StlD Cter for expression of Stl deletional variants were produced using plasmid pETNKI-Stl as template [bib_ref] Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity..., Maiques [/bib_ref]. pETNKI-StlD Cter plasmid expressing Stl residues from 1 to 176 was generated by site direct mutagenesis introducing a stop codon in pETNKI-Stl after Lys176 using the Stl_M1-K176_Fw and Stl_M1-K176_Rv primers and Q5 Site-Directed Mutagenesis Kit (NEB). pETNKI-StlD HTH plasmid expressing Stl residues from 87 to 267 was generated by PCR-amplifying the encoding region with the primers Stl_T87-N267_Fw and Stl_T87-N267_Rv. The Ligation-Independent Cloning (LIC) system [bib_ref] High-throughput production of human proteins for crystallization: the SGC experience, Savitsky [/bib_ref] was used to clone the PCR fragment into the pETNKI-his-SUMO3-LIC plasmid (kindly supplied by Patrick Celie, NKI Protein facility) previously digested with a KpnI (Fermentas). All clones were sequenced at the IBV Core Sequencing facility or by Eurofins MWG Operon.
## Southern and western blot sample preparation
Samples were taken at times 0' and 3 hr following plasmid induction and pelleted. The samples were re-suspended in 50 ml lysis buffer (47.5 ml TES-Sucrose and 2.5 ml lysostaphin [12.5 mg ml -1 ]) and incubated at 37˚C for 1 hr. For the Southern blot analysis, 55 ml of SDS 2% proteinase K buffer (47.25 ml H2O, 5.25 ml SDS 20%, 2.5 ml proteinase K [20 mg ml -1 ]) was added before incubation at 55˚C for 30 min. Samples were vortexed for 1 hr with 11 ml of 10x loading dye. Cycles of incubation in dry ice and ethanol, then at 65˚C were performed. Samples were run on 0.7% agarose gel at 25V overnight. DNA was transferred to a membrane and exposed using a DIG-labelled probe and anti-DIG antibody, before washing and visualisation.
Preparation of S. aureus samples for western blot was performed by re-suspending pellets in 200 ml digestion/lysis buffer (50 mM Tris-HCl, 20 mM MgCl2, 30% w/v raffinose) plus 1 ml of lysostaphin (12.5 mg ml -1 ), mixed briefly, and incubated at 37˚C for 1 hr. 2X Laemmli sample buffer (Bio-Rad, 2-mercaptoethanol added) was added to the samples, which were heated at 95˚C for 10 min, put on ice for 5 min and fast touch centrifuged. Samples were run on SDS-PAGE gels (15% Acrylamide, Bio-Rad 30% Acrylamide/Bis Solution) before transferring to a PVDF transfer membrane (Thermo Scientific, 0.2 mM) using standard methods. Western blot assays were performed using anti-Flag antibody probes (Monoclonal ANTI-FLAG M2-Peroxidase (HRP) antibody, Sigma-Aldrich) as per the protocol supplied by the manufacturer.
## Two-hybrid assay
The two-hybrid assay for protein-protein interaction was done as described previously [bib_ref] The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli, Battesti [/bib_ref] using two compatible plasmids; pUT18c expressing T18 fusion with the individual recombinases, and pKNT25 expressing the T25 fusion with the Stl SaPI2 . Both plasmids were co-transformed into E. coli BTH101 for the Bacterial Adenylate Cyclase Two Hybrid (BACTH) system and plated on LB +Ampicillin and Kanamycin + X gal as an indicator. After incubation at 30˚C for 24 hr (early reaction) and 48 hr (late reaction), the protein-protein interaction was detected by a colour change. Blue colonies represent an interaction between the two clones, while white/yellow colonies are negative for any interaction.
For quantification of the BACTH analysis, overnight cultures were diluted 1/100 and grown to mid-log before induction with 5 mM IPTG. After 2 hr, 2 ml of culture was sampled and pelleted, before resuspension in the same volume of Z buffer (0.06M Na 2 HPO 4 .7H 2 O, 0.04M NaH 2 PO 4 .H 2 O, 0.01M KCl, 0.001M MgSO 4 , 0.05M b-mercaptoethanol, pH 7.0). The OD 600 was recorded and cells were permeabilized with chloroform and 0.1% SDS. The assay reaction was started using ONPG (onitrophenyl-b-D-galactoside, 4 mg/ml), and vortexed and incubated at 30˚C until yellow. The reaction was stopped using Na 2 CO 3 and the reaction time recorded. Samples were spun down and the OD 420 and OD 550 were recorded. Miller Units were calculated as follows, where T is time of reaction (minutes) and V is the volume of culture used in the assay (ml): Miller Units = 1000 x (OD 420 -1.75 x OD 550 ) / (T xV x OD 600 ).
## Enzyme assays
For the b-Lactamase assays, cells were obtained at 0.2-0.3 OD 540 and at 5 hr post-induction with/ without 5 mM CdCl 2 . b-Lactamase assays, using nitrocefin as substrate, were performed as described , using a ELx808 microplate reader (BioTek). An adjustment was made in reading time, with plates read every 20 s for 30 mins. b-Lactamase units/ml are defined as [(slope) (Vd)]/[(Em)(l)(s)]. Slope is the Dabsorbance/hour, V is the volume of the reaction, d is the dilution factor, Em is the millimolar extinction coefficient for the nitrocefin (20,500 M À1 cm À1 at 486 nm), l is the path length (cm), and s is the sample amount. dUTPase activity was measured by Malachite Green assay as previously described [bib_ref] Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity..., Maiques [/bib_ref]. Briefly, Dut (30 nM) alone or in presence of a 5X molar ratio (monomer) of Stl (full length or truncated versions) was incubated overnight at 4˚C in Stl buffer (400 mM NaCl; 75 mM Hepes7.5; 5 mM MgCl 2 ). The experiment was carried out at 25˚C and started by the addition of dUTP (10 mM final concentration).
## Statistical analyses
As indicated in the figure legends either a two-way ANOVA comparison with Sidak's adjustment for multiple comparisons was conducted or a one-way ANOVA, as appropriate. All analysis was done using Graphpad Prism 6 software.
## Protein expression and purification
Trimeric f11 Dut and Stl were expressed and purified as previously described [bib_ref] Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity..., Maiques [/bib_ref]. StlD Cter was purified following an identical protocol as for the Stl full-length protein. StlD HTH was produced from E. coli BL21 (DE3) (Novagen) cultures harbouring the pETNKI-StlD HTH plasmid. The culture was grown at 37˚C in LB medium supplemented with 33 mg/ml kanamycin up to an OD 600 of 0.5-0.6, and then protein expression was induced with 0.1 mM isopropyl-b-D thiogalactopyranoside (IPTG) at 20˚C for 16 hr. After induced cells were harvested by centrifugation at 4˚C for 30 min at 3500 Â g, the cell pellet was resuspended in buffer A (75 mM HEPES pH 7.5, 400 mM NaCl and 5 mM MgCl 2 ) supplemented with 1 mM PMSF and sonicated. A soluble fraction was obtained after centrifugation at 16 000 Â g for 40 min, and it was loaded on a pre-equilibrated His Trap HP column (GE Healthcare). After washing with 10 column volumes of buffer A, the protein was eluted by adding buffer A supplemented with 500 mM imidazole. The eluted protein was digested for His-SUMO3 tag removal using SENP2 protease at a molar ratio 1:50 (protease:eluted protein) for 16 hr at 4˚C with slow shaking. After digestion, the sample was loaded one more time into the preequilibrated His Trap HP column to remove the His-SUMO3 tag and SENP2 protease from the Stl protein. Fractions were analysed by SDS-PAGE and those fractions with purest digested Stl protein were selected, concentrated and stored at À80˚C.
His-tagged dimeric fO11 Dut was overexpressed in E. coli BL21 (DE3) (Novagen) harbouring the pJP1938 plasmid. The cells were grown to exponential phase at 37˚C in LB medium supplemented with 33 mg/ml kanamycin, and then protein expression was induced by the addition of 1 mM IPTG for 3 hr. After induction, cells were harvested by centrifugation, re-suspended in buffer A supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF) and lysed by sonication. The lysate was clarified by centrifugation and the soluble fraction was loaded on a His Trap HP column pre-equilibrated with buffer A. The column was washed with the same buffer supplemented with 10 mM imidazole and proteins were eluted with buffer A supplemented with 500 mM imidazole. The eluted proteins were concentrated and loaded onto a Superdex S200 (GE Healthcare) equilibrated with buffer B (75 mM HEPES pH 7.5, 250 mM NaCl and 5 mM MgCl 2 ) for size exclusion chromatography. The fractions were analysed by SDS-PAGE and those fractions showing purest protein were selected, concentrated and stored at À80˚C.
Mass Spectrometry analyses were performed at the proteomics facility of SCSIE, University of Valencia.
fO11 Dut crystallization and data collection fO11 Dut protein in complex with dUPNPP protein was crystallized using the sitting drop method in the Crystallogenesis facility of IBV. fO11 (at 10 mg/ml) was incubated with 0.5 mM dUPNPP (2-Deoxyuridine-5-[(a,b)-imido]triphosphate; Jena Biosciences) and 5 mM MgCl 2 during 8 hr at 4˚C and sitting drops were set up at 21˚C. The best crystals were obtained using 0.2 M magnesium chloride, 0.1 M Tris-HCl pH8.5, 20% PEG 8000 as liquor mother. Crystals were frozen in liquid nitrogen respecting crystallization condition, increasing the cryobuffer to 35% PEG 8000 concentration for the diffraction process. Diffraction data was collected from single crystals at 100 K on ALBA (Barcelona, Spain) and DLS (Didcot, UK) synchrotrons and processed and reduced with Mosflm [bib_ref] Autoindexing diffraction images with iMosflm, Powell [/bib_ref] and Aimless [bib_ref] How good are my data and what is the resolution?, Evans [/bib_ref] programs from the CCP4 suite [bib_ref] Overview of the CCP4 suite and current developments, Winn [/bib_ref]. The data-collection statistics for the best data sets used in structure determination are shown in Supplementary file 3.
fO11 Dut-dUPNPP structure determination Protein structure was solved by molecular replacement with Phaser [bib_ref] Phaser crystallographic software, Mccoy [/bib_ref] and an edited PDB of the dimeric Dut from phage fDI as a model (5MYD). Based on sequence homology between fO11 and fDI Duts (70% identity), we excluded amino acids 82-140, corresponding to the divergent regions present in the phage dimeric Duts, from the starting model. This decision was made in order to reduce the imposition of any initial structural conformation to this variable region. Iterative refinement, rebuilding and validation steps were done using programs Coot [bib_ref] Features and development of Coot, Emsley [/bib_ref] and Phenix [bib_ref] PHENIX: a comprehensive Python-based system for macromolecular structure solution, Adams [/bib_ref]. The final model includes two Dut molecules (amino acids sequence 4-160 and 3-158) forming one dimer in an asymmetric unit with one dUPNPP molecule and two Mg ions bound at each of the two active centres. The final structure has good geometry as indicated by the Ramachandran plots (any residue in the disallowed region). A summary of structure refinement statistics is shown in Supplementary file 3.
## Native gel mobility shift assay
Purified proteins were mixed at 40 mM 1:1 molar ratio in a buffer A (final volume 18 ml) and incubated at 4˚C overnight. Samples were loaded into an 8% polyacrylamide gel and electrophoresis was performed at 4˚C. Native gels were stained with coomassie brilliant blue.
## In silico protein modelling and structure comparison
The 3D homology model of 80a, fSLT, 52A and fN315 SSAPs, and SaPIbov1 Stl were constructed using I-Tasser (default mode) [bib_ref] The I-TASSER Suite: protein structure and function prediction, Yang [/bib_ref] and Phyre2 (intensive mode) [bib_ref] The Phyre2 web portal for protein modeling, prediction and analysis, Kelley [/bib_ref] servers [fig_ref] Figure 2 -: figure supplement 1 [/fig_ref]. Intrinsic protein disorder was predicted by the metaserver Metadisorder [bib_ref] MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins, Kozlowski [/bib_ref].
[fig] Figure 1: Induction of SaPIbov1 and SaPIbov5 by the fO11 dimeric Dut. (A) SaPIbov1 and SaPIbov5 excision and replication following induction of the cloned fO11 dut gene. Strains JP6774 and JP11634, containing SaPIbov1 [/fig]
[fig] Figure 2 -: figure supplement 1). [/fig]
[fig] Figure 3: Dimeric and trimeric S. aureus phagic Duts present completely different folding. (A) Cartoon representation of fO11 dimeric Dut (protomers coloured in blue and yellow) and 80a trimeric Dut (PDB 3ZEZ; protomers coloured in blue, yellow and pink) [/fig]
[fig] Figure 4, Figure supplement 1: SaPIbov1 Stl has different regions to interact with the trimeric and dimeric Duts. (A) Native gel mobility shift assays were used to test the binding capacity of fO11 dimeric and f11 trimeric Duts with full-length and truncated versions of Stl. The appearance of bands with alternated migration with respect to the individual proteins (labelled by asterisk) indicates formation of a complex. (B) dUTPase activity for fO11 and f11 was measured by malachite green assay in the presence and the absence of Stl variants. The reaction time-course is represented as the development of green colour (measured at 630 nm). Results are representative of three independent experiments. DOI: https://doi.org/10.7554/eLife.26487.009 The following figure supplement is available for figure 4: Stl models and constructs design. DOI: https://doi.org/10.7554/eLife.26487.010 [/fig]
[fig] Figure 5, Figure supplement 1 013, Figure supplement 3 014, Figure supplement 4: Induction of SaPI2 by different phage-encoded SSAPs (recombinases). A non-lysogenic derivative of strain RN4220 Dspa carrying SaPI2 was complemented with plasmids expressing different 3xFLAG-tagged SSAP proteins. One millilitre of each culture (optical density (OD) 540nm =0.3) was collected 3 hr after treatment with 5 mM CdCl 2 and used to prepare standard mini-lysates, which were resolved on a 0.7% agarose gel, Southern blotted and probed for SaPI2 DNA. The upper band is 'bulk' DNA, including chromosomal, phage, and replicating SaPI. CCC indicates covalently closed circular SaPI2 DNA. In these experiments, as no helper phage was present, the excised and replicating SaPI DNA appears as part of the bulk DNA or as CCC molecules, rather than the linear monomers that are seen following helper phage-mediated induction and packaging. The lower panel is a western blot probed with antibody to the FLAG-tag carried by the SSAP proteins. sak: 80a ORF16; sak4: f52A ORF16; erf: fSLT ORF17; redb: fN315 ORF SA1794; chi: chimeric 80a sak-ssb; pCN51: empty vector.DOI: https://doi.org/10.7554/eLife.26487.011 The following figure supplements are available for figure 5: Sequence of the chimeric ORF16-17 protein. DOI: https://doi.org/10.7554/eLife.26487.012 Figure supplement 2. Localisation of the recombinase and ssb genes in different staphylococcal phage genomes. DOI: https://doi.org/10.7554/eLife.26487.Alignment of predicted Sak (80a) and Sak4 (f52A) staphylococcal phage SSAPs. DOI: https://doi.org/10.7554/eLife.26487.Alignment of predicted staphylococcal phage SSAPs. DOI: https://doi.org/10.7554/eLife. [/fig]
[fig] Figure 6: Phage SSAPs bind SaPI2 Stl protein. (A) Derepression of str transcription by ssap expression. [/fig]
[fig] .Figure 7: Interestingly, the f52A coded Sak4 recombinase induced SaPI2 and ScCIUMC-CNS-990, while the fSLT Erf recombinase 3D models for 80a, fSLT, f52A and fN315 SSAPs. Cartoon representation of 3D structures of 80a, fSLT, f52A and fN315 phage recombinases generated by I-Tasser and Phyre2. Alpha helices, beta strands and loops are coloured in cyan, magenta and orange, respectively. The experimental structures of RecA from E. coli (PDB 2REC) proposed as structural homolog of the f52A recombinase, and the human Rad52 recombinases (1H2I) proposed as structural homolog of 80a, fSLT, and fN315 recombinases are also presented for folding comparison. [/fig]
[table] Table 1: Dimeric and trimeric dUTPases induce PICIs from other species encoding SaPIbov1-like Stl repressors a . [/table]
[table] Table 2: Unrelated SSAPs differentially induce PICIs from other species encoding SaPI2-like Stl repressors a . [/table]
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Glucose Oxidase/Egg White Protein Microparticles with a Redox Mediator for Glucose Biosensors on a Screen-Printed Electrode and a Decomposable Electrode
Citation: Rasitanon, N.; Rattanapan, P.; Kaewpradub, K.; Buranachai, C.; Jeerapan, I. Glucose Oxidase/Egg White Protein Microparticles with a Redox Mediator for Glucose Biosensors on a Screen-Printed Electrode and a Decomposable Electrode. Biosensors 2023, 13, 772.Abstract: Glucose oxidase (GOx) is a typical model enzyme used to create biosensors. Exploring a strategy to prepare ready-to-use functional enzymatic microparticles combining GOx and food-based proteins offers compelling advantages. However, no reports exist on the integration of egg white materials to synthesize functional biorecognition particles with glucose oxidation catalytic functions for electrochemical biosensors. Here, we demonstrate functional microparticles combining egg white proteins, GOx, and 9,10-phenanthrenequinone (PQ). The egg white proteins crosslink to form three-dimensional scaffolds to accommodate GOx and redox molecules. The PQ mediator enhances electron transfer between the electrode surface and the GOx enzyme's flavin adenine dinucleotides. The functional microparticles are directly applied to the printed electrode. The performance of these microparticles is evaluated using a screen-printed carbon nanotube (CNT)-modified electrode coated with GOx/PQ/egg white protein microparticles. The analytical performance of the system exhibits a linear range of 0.125−40 mM, with a maximum current ( I max ) and a Michaelis-Menten constant (K m ) being 0.2 µA and 4.6 mM, respectively. Additionally, a decomposable electrode composed of CNTs and edible oil conjugated with functional enzyme microparticles is shown to undergo degradation under gastric conditions. Utilizing food-based proteins to accommodate enzymes and to create redox-active microparticles for catalyzing glucose oxidation offers advantages in developing affordable and degradable bioelectrodes. This concept holds promise for advancing biocompatible electrodes in biosensor and bioelectronics applications.
# Introduction
Food-based materials have become increasingly popular in biomedical applications, sensors, and environmentally friendly "green" applications. This is primarily due to their biocompatibility, biodegradability, and low toxicity [bib_ref] Disposable sensors in diagnostics, food, and environmental monitoring, Dincer [/bib_ref]. An example of this "green" strategy involved utilizing edible materials, such as carbon composites as conductors and olive oil as binders, to create disposable electrochemical sensors [bib_ref] Edible electrochemistry: Food materials based electrochemical sensors, Kim [/bib_ref]. These sensors are capable of measuring uric acid, ascorbic acid, and dopamine, which are commonly found in saliva, gastric fluids, or intestinal fluids. Another example is the use of biodegradable electrodes that incorporate interconnected three-dimensional conductive nanocomposites (e.g., single-wall carbon nanotubes (SWCNTs)), graphene, and conductive polymers within edible starch-chitosan substrates [bib_ref] Biodegradable transparent substrate based on edible starch-chitosan embedded with natureinspired three-dimensionally interconnected..., Miao [/bib_ref]. The starch and chitosan components were sourced Biosensors 2023, 13, 772 2 of 18 from potatoes and crab shells. These electrodes, based on the starch-chitosan substrate, exhibited rapid biodegradation in a lysozyme solution at room temperature, leaving no toxic residues behind. Additionally, another composite paste relying on biodegradable conductive oleogel was demonstrated [bib_ref] An electrically conductive oleogel paste for edible electronics, Cataldi [/bib_ref]. The materials were composed of food-grade beeswax, vegetable oil, and activated carbon conductive fillers. Biodegradable conductive oleogel composites showed antibacterial and hydrophobic properties, making them suitable for contact with food and ensuring the prevention of contamination while providing food's sensor capabilities. The promising performance of recently developed sensors, composed of food-based materials, suggests significant potential for advancing environmentally friendly electronics or diagnostic tools designed for biochemical monitoring purposes.
In addition to utilizing food-based materials, it is essential to explore strategies for immobilizing enzymes in affordable materials. This exploration is crucial for enabling practical biosensing applications because an important process to create a biosensor is enzyme immobilization, which involves attaching enzymes to solid supports through physical adsorption, entrapment within a matrix or encapsulation, or covalent bonding [bib_ref] Encapsulation of glucose oxidase microparticles within a nanoscale layer-by-layer film: Immobilization and..., Trau [/bib_ref]. The immobilization of enzymes offers several advantages, including the repeatable usage of biomolecules and improving their stability [bib_ref] Enzyme immobilization and its applications in food processing: A review, Bashir [/bib_ref]. The delicate nature of enzymes poses a significant challenge in producing functional enzyme-based materials. Enzymes are inherently fragile and prone to losing their catalytic activity if mishandled or exposed to extreme environments. Various materials, including lignin [bib_ref] Lignin Nanoparticles Support Lipase-Tyrosinase Enzymatic Cascade in the Synthesis of Lipophilic Hydroxytyrosol..., Tomaino [/bib_ref] , chitosan [bib_ref] Immobilization of α-amylase on chitosan-montmorillonite nanocomposite beads, Mardani [/bib_ref] , and egg white proteins [bib_ref] The effect of porosity and stiffness of glutaraldehyde cross-linked egg white scaffold..., Guo [/bib_ref] , have been utilized to enhance enzyme immobilization and to improve their stability. The immobilized enzymes demonstrated higher activity when compared with free enzymes. In addition to stability, it is necessary to devise a strategy for synthesizing readily available functional enzymatic materials that can be utilized as a biocatalytic layer on desired electrodes. The development of such a method simplifies fabrication processes, saves time, and enhances accessibility for customization according to specific requirements. Consequently, considerable efforts have been directed towards the development of effective strategies for immobilizing enzymes using solid supports made from biomaterials.
Biomaterial microparticles (MPs) are of great interest in various applications, including encapsulation, tissue engineering, and biosensing, due to their advantageous structural, environmental protection, and functional abilities as a carrier system [bib_ref] Electrospray Deposition of Polyvinylidene Fluoride (PVDF) Microparticles: Impact of Solvents and Flow..., Joaquim [/bib_ref] [bib_ref] Review on nanoparticles and nanostructured materials: Bioimaging, biosensing, drug delivery, tissue engineering,..., Harish [/bib_ref]. While the majority of MPs are made from synthetic polymers (such as poly(glycolic acid), poly(lactic acid), poly(p-dioxanone), and copolymers of trimethylene carbonate and glycolide) [bib_ref] Biodegradable synthetic polymers for tissue engineering, Gunatillake [/bib_ref] , exploring the utilization of inexpensive biomaterials is crucial in expanding the possibilities for developing biocompatible MPs with functional properties. An example of mesoporous silica (SiO 2 ) MPs derived from CNTs was synthesized using a one-pot spray pyrolysis process, providing efficient support for the enzyme immobilization [bib_ref] SiO2 microparticles with carbon nanotube-derived mesopores as an efficient support for enzyme..., Kumar [/bib_ref]. The resulting porous SiO 2 MPs, functionalized with glutaraldehyde, were utilized for immobilizing lipase. This immobilization process led to improved biochemical properties (higher activity and stability at a wide range of pH values, and harsh organic solvent concentrations) and enhanced thermostability of the lipase. The mesoporous SiO 2 -bound lipase was used to construct a lipase biosensor for triglyceride. However, this work does not integrate foodbased materials as degradable electrodes, and the method of synthesis described in this approach requires a high temperature, as high as 500 - C. Importantly, naturally derived materials such as cellulose, chitosan, and silk are suitable for "green" strategies. One example relied on the use of cellulose and chitosan conjugated with magnetic microspheres, which were prepared using the sol-gel transition method, with ionic liquids serving as the solvent for cellulose and chitosan dissolution and regeneration [bib_ref] Biocompatible magnetic cellulose-chitosan hybrid gel microspheres reconstituted from ionic liquids for enzyme..., Liu [/bib_ref]. These composite microspheres were also activated with glutaraldehyde to immobilize glucose oxidase (GOx). The resulting GOx-immobilized microspheres exhibited excellent catalytic activity for glucose oxidation. However, their potential applications in electrochemical sensors were not investigated. Another example is the use of silk coatings on poly(lactic-coglycolic acid) and alginate microspheres for encapsulating horseradish peroxidase and tetramethylrhodamine-conjugated bovine serum albumin (BSA) [bib_ref] Silk coatings on PLGA and alginate microspheres for protein delivery, Wang [/bib_ref]. Another strategy involves the emulsification-internal gelation process, which can immobilize GOx onto the surface of alginate microspheres [bib_ref] Immobilization of Glucose Oxidase on Sodium Alginate Microspheres, Stadolnikova [/bib_ref]. Free carboxyl groups present in alginate microspheres activate carbodiimide and N-hydroxysuccinimide, enabling the covalent immobilization of GOx. This process leads to the strong binding of the GOx to the alginate microsphere's surface through the formation of amide bonds. Although this study described the synthesis of sodium alginate microspheres, the size distribution of particles varied. No efforts have been made yet to develop electrochemical glucose sensor applications.
In addition to those approaches, the application of a calcium carbonate (CaCO 3 ) template for preparing protein MPs has been reported. For example, one fabrication method used coprecipitation to entrap protein molecules such as BSA inside spherical CaCO 3 templates [bib_ref] Bioinspired protein microparticles fabrication by peptide mediated disulfide interchange, Lai [/bib_ref]. This concept relied on the intracellular disulfide interchange reactions at physiological conditions mediated by glutathione to initiate disulfide interchange reactions between the BSA molecules. After the glutathione and CaCO 3 templates were removed, the entrapped BSA molecules form new intermolecular disulfide bonds with neighboring molecules, resulting in BSA MPs. Afterward, BSA-horseradish peroxidase MPs were prepared to show the colorimetric reaction between horseradish peroxidase and 3,3',5,5'-tetramethylbenzidine in the presence of H 2 O 2 (an oxidizing agent). This existing report focused on colorimetric and fluorescence techniques, excluding electrochemical applications. In addition to the use of BSA, the use of hen eggs has previously been reported to synthesize hollow MPs [bib_ref] Hollow protein microparticles formed through cross-linking by an Au3+ initiated redox reaction, Schijven [/bib_ref]. It can be fabricated using high-density lipoproteins in egg yolk and sacrificial CaCO 3 templates. This work utilized a redox reaction between Au 3+ ions and proteins, which resulted in the formation of a protein gel network. However, to the best of our knowledge, no efforts have been made to demonstrate the use of food-based particles with enzymatic functions for degradable and electrochemical biosensors. Therefore, further research is required to explore food-based materials with enzymatic particles and to advance the technology towards environmentally friendly synthesis, inexpensive production, and practical implementation in biodegradable electronics.
This work presents the first demonstration of GOx/(9,10-phenanthrenequinone, PQ)/ egg white protein MPs, which can be used to develop glucose sensors [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref]. The egg white protein MPs were crosslinked with glutaraldehyde to generate a 3D scaffold structure, which increases the active site availability for GOx as the enzyme immobilization and enhances the analytical performance. Note that egg white proteins are inexpensive, costing 0.003 USD per 1 g, in contrast to the price of other common proteins, such as BSA (25 USD per 1 g). In this work, the egg white protein MPs were bound with PQ as a mediator, facilitating electron transfer between an electrode and the GOx enzyme. Additionally, the PQ/egg white protein MPs were immobilized with GOx to develop glucose sensors. We demonstrated a proof-of-concept model of GOx coupled with food-based materials in the form of MPs. This is because a glucose sensor plays a crucial role in monitoring glucose levels, especially in the context of diabetes management. It provides individuals with valuable information to make informed decisions regarding their diet, medication, and overall well-being [bib_ref] Materials for diabetes therapeutics, Bratlie [/bib_ref]. Impedance and amperometry techniques were utilized to demonstrate the capability of glucose-sensitive MPs (i.e., GOx/PQ/egg white protein MPs). Additionally, the electrochemical properties of degradable electrodes fabricated using carbon materials mixed with edible olive oil and coated with GOx/PQ/egg white protein MPs were investigated toward glucose detection. These types of MP and electrode exhibit the potential for degradation. Moreover, these sensors prove to be cost-effective and environmentally friendly biomaterials. These advancements open up promising future applications in various fields, such as healthcare, biotechnology, and personal wellness.
# Materials and methods
# Chemicals and materials
Multiwalled CNTs (95% purity, diameter = 5-15 nm, length = 10-30 µm) were from Luoyang Advanced Material Co., Ltd., Shanghai, China. D−(+)-glucose anhydrous was from Fluka, Neu-Ulm, Germany. GOx (from Aspergillus niger, type VII, 248,878 units g −1 ), L-ascorbic acid, L-lactic acid, uric acid, creatinine, chitosan (from shrimp shell, ≥75%), potassium hexacyanoferrate (III) and glutaraldehyde solution (grade II, 25% in H2O) were from Sigma-Aldrich (Saint Louis, MO, USA
# Materials and methods
# Chemicals and materials
Multiwalled CNTs (95% purity, diameter = 5-15 nm, length = 10-30 µm) were from Luoyang Advanced Material Co., Ltd., Shanghai, China. D−(+)-glucose anhydrous was from Fluka, Neu-Ulm, Germany. GOx (from Aspergillus niger, type VII, 248,878 units g −1 ), L-ascorbic acid, L-lactic acid, uric acid, creatinine, chitosan (from shrimp shell, ≥75%), potassium hexacyanoferrate (III) and glutaraldehyde solution All chemical solutions were prepared using deionized water (18.2 MΩ cm) from a Milli Q Merck system (Darmstadt, Germany). A 0.1 M potassium phosphate-buffered solution (PBS) with a pH of 7.0 was prepared using KH 2 PO 4 and K 2 HPO 4 , and it was employed as the supporting electrolyte. All electrochemical performance was studied in a 0.1 M PBS solution with a pH of 7.0, unless stated otherwise. The artificial gastric fluid preparation was achieved by using 1 g of NaCl with 4 mL of HCl and by adjusting the volume to 500 mL using water [bib_ref] A smart drug delivery system based on biodegradable chitosan/poly (allylamine hydrochloride) blend..., Sarwar [/bib_ref].
## Instruments and electrochemical measurement
A µStat-I 400 (Metrohm Dropsens, S.L., Asturias, Spain) controlled via DropView8400 software version 3.78 was used to examine electrochemical performances, including cyclic voltammetry (CV) and amperometry. Electrochemical impedance spectroscopy (EIS) was performed using µAutolab Type III FRA 2, Metrohm controlled via NOVA software version 1.11. The EIS characterizations were conducted at glucose concentrations of 0, 0.5, 5, and 10 mM at a potential of 0.2 V, employing a frequency range of 10 0 -10 4 Hz with 50 sample points in between in a logarithmic scale and an amplitude of 0.005 V. Working electrodes (such as screen-printing electrodes coated with GOx/PQ/egg white protein MPs) were evaluated with Pt counter and Ag/AgCl (3.0 M KCl) reference electrodes during the three-electrode-system analysis. The amperometry response was measured, after 30 s of immersion in the test solution, at constant potential at 0.2 V (vs. Ag/AgCl) for 30 s. Calibration plots were obtained by increasing the glucose concentration in 0.1 M PBS, pH 7.0. Infrared spectra (IR) were measured using an FTS165 FTIR spectrometer. Fluorescence measurements were conducted using an F-2700 fluorometer from Hitachi High-Tech Science Corporation, Tokyo, Japan. Optical microscopy images were captured using an Olympus IX70 microscope from Japan.
## Preparation of gox/pq/egg white protein mps
To prepare the desired functional biocatalytic MPs, porous, biocompatible, and decomposable CaCO 3 MPs were co-synthesized with egg white proteins. This initial step aimed to encapsulate the egg white proteins, which would later be transformed into a biofriendly three-dimensional (3D) scaffold capable of accommodating active components. The CaCO 3 MPs served as a hard microtemplate to ensure a uniform shape for the synthesized functional particles, as illustrated in [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref]. First, 1 mL of egg white proteins was mixed with 1.5 mL of 1 M Na 2 CO 3 solution and rapidly stirred at 600 rpm for 2 min using a magnetic stirrer, while 1.5 mL of 1 M CaCl 2 solution was added into the mixture [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref] (Aa)). The egg white proteins were extracted directly and slowly using a 24-gauge syringe needle with a length of 38 mm from a fresh egg. The resulting white precipitate (CaCO 3 MPs loaded with egg white proteins) was subsequently washed with 2-propanol, ethanol, and water. After each washing step, the cleaned CaCO 3 MPs loaded with egg white proteins were collected via centrifugation at 3000 rpm for 1 min at room temperature. Subsequently, the CaCO 3 MPs loaded with egg white proteins were washed three times with 400 µL of 1.5 M NaCl and centrifuged at 3000 rpm for 5 min at room temperature (25 - C). Next, the CaCO 3 templates loaded with egg white proteins were incubated with 1 mL of 0.5% v/v glutaraldehyde for 6 h using a tube plate rotator [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref] (Ab)). After that, precipitates were washed with water through centrifugation at 3000 rpm for 1 min at room temperature (25 - C). Following this step, the CaCO 3 template was removed by adjusting the pH of the suspended egg white protein MPs/CaCO 3 with the addition 400 µL of 0.2 M HCl solution [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref] (Ac)). The resulting egg white protein MPs were collected via centrifugation at 3000 rpm for 1 min at room temperature and washed with water and butan-1-ol. Then, 400 µL of butan-1-ol and vortex was added. After that, egg white protein MPs were centrifuged at 3000 rpm for 5 min at room temperature (25 - C) to collect the precipitate of crosslinked egg white protein MPs. To introduce a redox mediator into the egg white protein MPs, 750 µL of 5 mM PQ was added to incubate it with egg white protein MPs for 2 h and using a tube plate rotator [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref] (Ad)). The excess PQ was removed through centrifugation at 3000 rpm for 1 min, and the resulting mixture was redispersed with butan-1-ol for washing. After that, the supernatant was removed, and the PQ/egg white protein MP precipitants were collected. Subsequently, the PQ-egg white protein MPs were incubated with 750 µL of 0.5% v/v glutaraldehyde for 30 min using a tube plate rotator and removing the excess glutaraldehyde through centrifugation at 3000 rpm for 1 min. Finally, GOx solution (10 mg mL −1 GOx dissolved in 10% v/v of egg white in 0.05 M PBS, pH 7.0) was then added to the PQ/egg white protein MPs and incubated for 2 h to immobilize the enzyme in the MPs using a tube plate rotator [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref] (Ae)). The excess of GOx was removed through centrifugation at 3000 rpm for 1 min and redispersed with 750 µL of 0.05 mM PBS pH 7.0. In this process, GOx/PQ/egg white protein MPs were obtained, which were dispersed in 0.05 M PBS at pH 7.0. These MPs were ready to use for electrode modification.
## Preparation of cnt-modified carbon ink
To prepare a CNT-modified ink, 60 mg of CNTs was dispersed in 490 µL of toluene using a homogenizer probe (model AR-0975) level 1 for 3 min. Next, 7 g of the carbon conductive ink (Guangzhou Print Area Technology Co. Ltd., Guangzhou, China) was mixed with the mixture using a mixing machine (Shashin Kagaku, Kyoto, Japan, Kakuhunter, SK-300SII) for 30 min at 2000 rpm. The resulting CNT-modified carbon ink was printed on a polyethylene terephthalate sheet as a working electrode. The working area of the working electrode was 0.3 cm × 0.5 cm. The electrode was electrochemically cleaned by applying a constant voltage of 1.50 V for 60 s in 1.0 M Na 2 CO 3 .
## Preparation of biosensors on degradable electrodes
The use of olive oil in constructing a carbon paste electrode has been reported [bib_ref] Edible electrochemistry: Food materials based electrochemical sensors, Kim [/bib_ref]. In this work, the degradable electrodes for glucose determination were prepared using CNT as the conductive filler material and edible olive oil as a binder. To prepare a degradable electrode, 30 mg of CNT and 30 µL of olive oil were thoroughly hand-mixed using a mortar and pestle. A portion of the resulting mixed paste was then packed into a tube with a diameter of 0.4 cm and a length of 0.5 cm. For electrochemical measurements, electrical contacts were established by inserting a conductive stainless-steel wire into the top side of the packed paste. To functionalize the biosensor, 10 µL of the GOx/PQ/egg white protein MPs was dropped on the modified electrode and dried. After that, 10 µL of 1.5% wt/v chitosan in 0.25 M acetic acid was dropped onto the electrode and dried at room temperature.
# Results and discussion
## Principle of preparation gox/pq/egg white protein mps
Encapsulating egg white proteins with a CaCO 3 template involved coprecipitation, where CaCO 3 MPs were formed simultaneously with egg white proteins. In this strategy, egg white proteins were initially dissolved in an aqueous solution containing calcium ions (Ca 2+ ). Following that, carbonate ions (CO [bib_ref] Biodegradable transparent substrate based on edible starch-chitosan embedded with natureinspired three-dimensionally interconnected..., Miao [/bib_ref] 2− ) were added to bind with Ca 2+ and to form solid CaCO 3 particles. These solid particles acted as a trap, encapsulating the egg white protein molecules within them [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref] (Aa)). After the egg white proteins were entrapped inside the CaCO 3 particles, the loaded particles were crosslinked using glutaraldehyde [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref]. This process involved treating proteins, mainly ovalbumin, with glutaraldehyde, which formed strong covalent bonds between the amino groups of the protein and the aldehyde groups of glutaraldehyde, resulting in a cross-linked network. The protein in egg white underwent a conformational transition, changing from a watersoluble form to a water-insoluble form. The crosslinked ovalbumin could serve as a scaffold for the subsequent immobilization of enzymes. Subsequently, the sacrificial CaCO 3 MPs were dissolved using an acidic solution, resulting in the formation of egg white protein MPs that maintained the spherical shape of the original CaCO 3 particles [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref] (Ac)). During this step, the CaCO 3 particles broke down, and Ca 2+ ions were released along with carbon dioxide. This process is widely employed in various applications, including material synthesis, because an acid solution can selectively dissolve and remove CaCO 3 without affecting other materials or surfaces. It is an inexpensive and easy-to-implement method [bib_ref] Microparticulate biomolecules by mild CaCO 3 templating, Schmidt [/bib_ref].
To showcase an application of the food-based protein MPs, a glucose biosensor was presented as an example. The subsequent functionalization of essential components, including redox molecules and GOx, into the egg white protein MPs, was demonstrated.
After achieving crosslinked egg white protein MPs, the particles were incubated with PQ [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref]. PQ is an organic compound that serves as a redox mediator, facilitating electron transfer between the electrode surface and the enzyme. It consists of a phenanthrene ring fused with a quinone moiety [bib_ref] Electrochemical deposition and investigation of poly-9, 10-phenanthrenequinone layer, Genys [/bib_ref]. Quinones belong to a class of organic compounds characterized by a conjugated cyclic system with two carbonyl groups [bib_ref] Chapter 25-Analysis of quinonoids, Niaz [/bib_ref]. They can undergo reversible reductions, both one-electron and two-electronbased, resulting in the generation of a semiquinone radical or a quinone, respectively [bib_ref] Molecular properties of 9, 10-phenanthrenequinone and benzil, Hanif [/bib_ref]. The mediated function of a quinone compound plays a critical role in enhancing the efficiency and sensitivity of biosensing applications. It facilitates the transfer of electrons involved in electrochemical reactions. Moreover, a mediator-based glucose biosensor improves electron transfer, reduces interference effects, and mitigates the impact of oxygen compared with the first generation of enzymatic glucose biosensors [bib_ref] Electrochemical glucose sensors in diabetes management: An updated review (2010-2020), Teymourian [/bib_ref]. Therefore, our sensor utilized the second generation of enzymatic glucose sensors with PQ as the mediator.
To create glucose-sensitive MPs, we immobilized PQ/egg white protein MPs with GOx, an enzyme that catalyzes the oxidation of glucose [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref]. This immobilization technique improved the stability of the enzyme, making it well-suited for glucose biosensors. GOx is composed of two identical 80 kDa subunits and is associated with two non-covalently bound flavin adenine dinucleotides. The flavin adenine dinucleotides coenzyme serves as an electron carrier and facilitates the oxidation of D-glucose. This reaction yields D-gluconolactone and H 2 O 2 as products, while the mediator PQ facilitates electron transfer between the enzyme and the electrode surface [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref]. Once the PQ/GOx/egg white protein MPs were obtained, they were ready to be applied in glucose biosensor applications.
## Characterization and morphology
Optical microscopy was employed to examine the morphologies of GOx/PQ/egg white protein MPs once the CaCO 3 template was eliminated. This method enabled visual observation and analysis of the physical structures and shapes of the MPs. [fig_ref] Figure 2: Characterization of materials [/fig_ref] shows that egg white protein MPs exhibit a uniform spherical shape with a narrow size distribution, characterized by an average diameter of 5 µm. Additionally, the corresponding CaCO 3 MPs loaded with egg white proteins before template removal demonstrate a similar size distribution, with an average diameter of 5 µm [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref].
The morphologies of egg white protein MPs loaded with PQ and GOx were examined using scanning electron microscopy (SEM) [fig_ref] Figure 2: Characterization of materials [/fig_ref]. The GOx/PQ/egg white protein MPs exhibited a spherical structure. During the coprecipitation process involving CaCl 2 and Na 2 CO 3 , nucleation led to the formation of granules, which subsequently assembled into interconnected spherical structures with pores. This arrangement facilitated the encapsulation of many molecules of egg white proteins within the template. The uniform shape and small size of these spherical MPs offer advantages such as a high surface-to-volume ratio, enabling enhanced contact with target substances and electrode surfaces. Additionally, their homogeneity ensures consistent performance.
By studying changes in the fluorescence emission spectrum with excitation wavelength at 300 nm, it is possible to examine molecular interactions such as binding to a target molecule or undergoing conformational changes [fig_ref] Figure 2: Characterization of materials [/fig_ref]. We investigated the fluorescence emission spectra of raw egg white, egg white protein MPs, PQ/egg white protein MPs, and GOx/PQ/egg white protein MPs, under an emission wavelength range of 315-550 nm. The fluorescence emission spectra reveal that crosslinking with glutaralde-hyde did not significantly alter the spectral shape of fluorescence emitted from egg white proteins [fig_ref] Figure 2: Characterization of materials [/fig_ref] (Ca)), compared with crosslinked egg white protein MPs [fig_ref] Figure 2: Characterization of materials [/fig_ref]. The morphologies of egg white protein MPs loaded with PQ and GOx were examined using scanning electron microscopy (SEM) [fig_ref] Figure 2: Characterization of materials [/fig_ref]. The GOx/PQ/egg white protein MPs exhibited a spherical structure. During the coprecipitation process involving CaCl2 and Na2CO3, nucleation led to the formation of granules, which subsequently assembled into interconnected spherical structures with pores. This arrangement facilitated the encapsulation of many molecules of egg white proteins within the template. The uniform shape and small size of these spherical MPs offer advantages such as a high surfaceto-volume ratio, enabling enhanced contact with target substances and electrode surfaces. Additionally, their homogeneity ensures consistent performance.
By studying changes in the fluorescence emission spectrum with excitation wavelength at 300 nm, it is possible to examine molecular interactions such as binding to a target molecule or undergoing conformational changes [fig_ref] Figure 2: Characterization of materials [/fig_ref]. We investigated the fluorescence emission spectra of raw egg white, egg white protein MPs, PQ/egg white protein MPs, and GOx/PQ/egg white protein MPs, under an emission wavelength range of 315-550 nm. The fluorescence emission spectra reveal that crosslinking with glutaraldehyde did not significantly alter the spectral shape of fluorescence emitted from egg white proteins [fig_ref] Figure 2: Characterization of materials [/fig_ref] , compared with crosslinked egg white protein MPs [fig_ref] Figure 2: Characterization of materials [/fig_ref] (Cb)). [fig_ref] Figure 2: Characterization of materials [/fig_ref] (Cc) shows the shift in the PQ/egg white protein MP signal to a lower wavelength position compared with the peak signal of PQ alone [fig_ref] Figure 2: Characterization of materials [/fig_ref]. Note that a peak on the shoulder around 380 nm could be observed in [fig_ref] Figure 2: Characterization of materials [/fig_ref] (Cc), while a peak at 420 nm was observed in free PQ [fig_ref] Figure 2: Characterization of materials [/fig_ref]. This observation suggests that PQ molecules, located within the protein scaffold, may be sensitive to the environment provided by the egg white protein MPs, resulting in the observed hypsochromic shift. It also indicates that the PQ molecules were securely loaded within the 3D crosslinked structure of the egg white proteins. The emission peak of the GOx/PQ/egg white protein MPs was observed to undergo almost no shift [fig_ref] Figure 2: Characterization of materials [/fig_ref] (Cd)) when compared with the spectrum of PQ/egg [fig_ref] Figure 2: Characterization of materials [/fig_ref]. Note that a peak on the shoulder around 380 nm could be observed in [fig_ref] Figure 2: Characterization of materials [/fig_ref] (Cc), while a peak at 420 nm was observed in free PQ [fig_ref] Figure 2: Characterization of materials [/fig_ref]. This observation suggests that PQ molecules, located within the protein scaffold, may be sensitive to the environment provided by the egg white protein MPs, resulting in the observed hypsochromic shift. It also indicates that the PQ molecules were securely loaded within the 3D crosslinked structure of the egg white proteins. The emission peak of the GOx/PQ/egg white protein MPs was observed to undergo almost no shift [fig_ref] Figure 2: Characterization of materials [/fig_ref] (Cd)) when compared with the spectrum of PQ/egg white protein MPs, likely because of the dominance of fluorescence emission from GOx that overwhelms the fluorescence emission from PQ/egg white protein MPs.
Furthermore, we employed Fourier transform infrared spectroscopy (FTIR) to confirm the formation of GOx/PQ/egg white protein MPs, which provided information about functional groups and various bonds. Specifically, the IR spectra of egg white proteins prior to crosslinking with glutaraldehyde showed a broad peak at 3278 cm −1 , corresponding to O-H stretching and N-H stretching, which are characteristic functional groups of proteins [fig_ref] Figure 2: Characterization of materials [/fig_ref]. Moreover, it showed a peak of C=O stretching (at around 1633 cm −1 ) and N-H bending (at around 1548 cm −1 ). After the crosslinking step using glutaraldehyde, the intensity of these two peaks decreased (at 1635 and 3346 cm −1 ) [fig_ref] Figure 2: Characterization of materials [/fig_ref]. Additionally, the C-H stretching (at around 2933-2959 cm −1 ), N-H stretching (at around 2874 cm -1 ), and C-H bending (at around 1380-1465 cm -1 ) were prominent when crosslinking with egg white proteins. This information suggested a successful crosslinking reaction between the egg white proteins and glutaraldehyde. The IR spectrum of PQ/egg white protein MPs showed a O-H stretching peak at around 3323 cm −1 [fig_ref] Figure 2: Characterization of materials [/fig_ref]. Furthermore, these PQ/egg white protein MPs showed increasing intensity of the C-H stretching peak at around 2873-2958 cm −1 . Moreover, the C-H bending peak (at around 1380-1465 cm −1 ) was still present with a slight increase in intensity. This suggests successful loading of PQ into the egg white protein MPs. After functionalizing the GOx (prepared in egg white proteins) in PQ/egg white protein MPs, the FTIR analysis revealed two distinct peaks: a O-H stretching peak and C=O stretching peaks at around 3280 and 1640 cm −1 , respectively [fig_ref] Figure 2: Characterization of materials [/fig_ref]. These two peaks were more prominent due to the addition of GOx along with additional egg white proteins. Therefore, the addition of GOx and egg white proteins enhanced the intensity of the O-H stretching and C=O stretching peaks, attributable to the functional groups present in GOx and egg white proteins. Additionally, this large addition obscured other peaks associated with those particular functional groups.
## Electrochemical characterizations
To assess the electrochemical performance of the egg white protein MPs and PQcontaining MPs, CV was employed. Three distinct working electrodes were investigated: (a) a bare screen-printed electrode, (b) an electrode coated with egg white protein MPs, and (c) an electrode coated with PQ/egg white protein MPs. The CV curve in [fig_ref] Figure 3: Electrochemical studies [/fig_ref] , obtained at a scan rate of 10 mV s −1 , reveals the background current of the screen-printed electrode when evaluating in 0.1 M PBS at pH 7.0, serving as the supporting electrolyte (indicated by (a)). This indicates the stability of the screen-printed electrode within a potential window ranging from −0.40 to 0.10 V. The observed increase in cathodic current may be attributed to the oxygen reduction reaction occurring at a higher cathodic potential (initiating around −0.20 V).
The impact of loading PQ molecules in MPs on the electrochemical properties was investigated. The PQ is a redox mediator as it can undergo reversible oxidation and reduction reactions, facilitating electron transfer during electrochemical processes [bib_ref] Electrochemical deposition and investigation of poly-9, 10-phenanthrenequinone layer, Genys [/bib_ref]. The inclusion of PQ in egg white protein MPs resulted in the emergence of distinctive redox peaks in the voltammogram, corresponding to the oxidation and reduction of PQ (represented by the blue line, curve c in [fig_ref] Figure 3: Electrochemical studies [/fig_ref] , in contrast to the screen-printed electrode and egg white-protein-MP-modified electrodes (curves a and b). The oxidation and reductions peak were observed at −0.14 V and −0.20 V. This notable increase in peak currents could be attributed to the redox activity of PQ. The well-defined shapes of the redox peaks in [fig_ref] Figure 3: Electrochemical studies [/fig_ref] , curve c, indicate the two-electron reduction and oxidation process of PQ at the electrode modified with the PQ-containing MPs [bib_ref] 10-phenanthrenequinone: Role of reactive oxygen species and redox signaling, Yang [/bib_ref]. Note that PQ molecules contain high aromaticity, leading to π-π stacking interactions between PQ and the screen-printed carbon nanomaterials, particularly at sp 2 carbon in CNTs, ensuring a strong and effective interaction. This suggests the successful immobilization of PQ on egg white protein MPs and the redox performance of the modified electrode using the synthesized MPs.
Furthermore, we estimated the capacitance of the electrodes by considering the cyclic voltammogram (CVs). For more details, refer to Equation (S1), Equation (S2), and Supporting Note S1. This evaluation enables us to determine the amount of charge involved in the electrochemical reaction. The specific capacitance values of screen-printed CNT-modified electrode, egg white protein MPs, and PQ-containing MPs were 0.2, 0.2, and 1.5 mF cm −2 , respectively. This result demonstrates that the attachment of PQ enhances the charge storage capabilities through successful reversible redox reactions on the electrode. [fig_ref] Figure 3: Electrochemical studies [/fig_ref] illustrates the CVs of screen-printed electrode coated with PQ-containing MPs, with a scan rate ranging from 2.5 to 200 mV s −1 in 0.1 M PBS pH 7.0. These plots reveal the presence of quasi-reversible PQ redox couples, and the anodic/cathodic peak current ratio (I c /I a ratio) approaches unity, ranging from 1.0 to 1.1. The CV shapes remain well preserved, indicating that the screen-printed electrode coated with PQ-containing MPs exhibits good rate performance. Furthermore, as depicted in [fig_ref] Figure 3: Electrochemical studies [/fig_ref] , the resulting anodic and cathodic peak currents show a proportional relationship to the square root of the scan rate. This behavior aligns with the Randles-Sevcik equation [bib_ref] A practical beginner's guide to cyclic voltammetry, Elgrishi [/bib_ref] and suggests that the processes occurring at the PQ/egg white-protein-MP-modified electrode are diffusioncontrolled, consistent with previous studies conducted on a PQ-modified electrode [bib_ref] Sol-gel-derived carbon ceramic electrode containing 9, 10-phenanthrenequinone, and its electrocatalytic activity toward..., Yang [/bib_ref].
Furthermore, we estimated the capacitance of the electrodes by considering the cyclic voltammogram (CVs). For more details, refer to Equation (S1), Equation (S2), and Supporting Note S1. This evaluation enables us to determine the amount of charge involved in the electrochemical reaction. The specific capacitance values of screen-printed CNTmodified electrode, egg white protein MPs, and PQ-containing MPs were 0.2, 0.2, and 1.5 mF cm −2 , respectively. This result demonstrates that the attachment of PQ enhances the charge storage capabilities through successful reversible redox reactions on the electrode. [fig_ref] Figure 3: Electrochemical studies [/fig_ref] illustrates the CVs of screen-printed electrode coated with PQ-containing MPs, with a scan rate ranging from 2.5 to 200 mV s −1 in 0.1 M PBS pH 7.0. These plots reveal the presence of quasi-reversible PQ redox couples, and the anodic/cathodic peak current ratio (Ic/Ia ratio) approaches unity, ranging from 1.0 to 1.1. The CV shapes remain well preserved, indicating that the screen-printed electrode coated with PQ-containing MPs exhibits good rate performance. Furthermore, as depicted in [fig_ref] Figure 3: Electrochemical studies [/fig_ref] , the resulting anodic and cathodic peak currents show a proportional relationship to the square root of the scan rate. This behavior aligns with the Randles-Sevcik equation [bib_ref] A practical beginner's guide to cyclic voltammetry, Elgrishi [/bib_ref] and suggests that the processes occurring at the PQ/egg white-protein-MP-modified electrode are diffusion-controlled, consistent with previous studies conducted on a PQ-modified electrode [bib_ref] Sol-gel-derived carbon ceramic electrode containing 9, 10-phenanthrenequinone, and its electrocatalytic activity toward..., Yang [/bib_ref]. Additionally, we studied the effect of scan rate on the surface capacitance by considering the area under the cyclic voltammogram, with PQ serving as the redox-active molecules attached to the electrode [fig_ref] Figure 3: Electrochemical studies [/fig_ref]. We observed a decrease in capacitance from 2.64 to 0.56 mF cm −2 when the scan rate increased from 2.5 to 200 mV s −1 . Faster scan rates during CV resulted in a decrease in capacitance because of the limited time available for redox reactions to occur at the electrode surface.
Additionally, we measured CVs using the bare electrode, egg white protein MPs, and PQ-containing MPs in 0.1 M KCl, with and without an additional external redox. In the KCl electrolyte, the PQ-containing MPs exhibited a higher redox peak current compared with the bare electrode and egg white protein MPs. This difference is due to the presence of PQ as a redox mediator (see inset [fig_ref] Figure 6: Degradable biosensors [/fig_ref]. When the solution contained an external redox probe, we observed a relatively smaller redox peak current when using the electrode modified with egg white protein MPs and PQ-containing MPs, compared with the bare electrode [fig_ref] Figure 6: Degradable biosensors [/fig_ref]. This decrease can be attributed to the insulating nature of egg white protein and the GOx enzyme, which restricts the accessibility of the external redox probe in reaching the electrode. Furthermore, we compared the active surface area of the pristine commercial conductive ink with a screen-printed CNT-modified electrode (see [fig_ref] Figure 6: Degradable biosensors [/fig_ref] and Supporting Note S2). The redox peak of the screen-printed electrode was higher than that of the commercial conductive electrode. The active area of the screen-printed electrode was 0.12 cm −2 , while that of the commercial electrode was 0.09 cm −2 . This indicates that the modification can increase the active area by approximately 25% compared with the pristine electrode. Additionally, the EIS measurements showed that our ink composed of CNT exhibited a lower resistance value than the commercial ink (see . This enhancement can be attributed to improved electron transfer.
To evaluate the bioelectrocatalytic characteristics of the GOx/PQ-based MPs, while minimizing the contribution of capacitive current resulting from coated materials and the large electroactive area of the CNT-based electrode, the LSV technique was performed at a low scan rate of 5 mV s −1 . The LSV technique was used to vary the potential of the screenprinted electrode coated with GOx/PQ-based MPs linearly with time while measuring the resulting current. The voltammograms shown in [fig_ref] Figure 3: Electrochemical studies [/fig_ref] (dashed line) display an anodic peak around 0.15 V, indicating the oxidation of PQ molecules in the absence of glucose. This voltammogram reflects the oxidation behavior of PQ. In the presence of glucose, the oxidation peak current increased [fig_ref] Figure 3: Electrochemical studies [/fig_ref]. The anodic peak observed during LSV was higher compared with the absence of glucose, indicating the effective catalytic action of GOx, functionalized in the synthesized MPs. The presence of glucose led to an increased anodic peak in the LSV, enabling quantitative measurement of the glucose levels. Amperometry was further employed to demonstrate glucose biosensors using these GOx/PQ-based MPs (Section 3.4 Amperometry). This observation confirms the successful electrochemical behavior of a screen-printed electrode coated with GOx/PQ-based MPs upon the addition of glucose. This is a result of effective enzyme immobilization and PQ mediation within the microspheres.
In addition to DC techniques, EIS is one of the techniques that has been widely used, particularly in chemical and biosensing applications [bib_ref] Potential of printed electrodes for electrochemical impedance spectroscopy (EIS): Toward membrane fouling..., Goh [/bib_ref]. In this study, EIS was conducted to evaluate the bioelectrochemical performance of the synthesized GOx-based MPs towards changes in glucose concentration. This evaluation was performed by comparing a bare electrode [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref] to a screen-printing electrode coated with GOx/PQ/egg white protein MPs [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref]. Phase angle plots, as shown in [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref] (Aa,Ba), were observed across a frequency range of 10 0 to 10 4 Hz and with varying glucose concentrations ranging from 0.5 to 10 mM. The phase angle values were found to be around 50-75 - over a frequency range of 10 0 to 5 × 10 1 Hz, which is lower than the expected 90 - for ideal capacitive behavior [bib_ref] Heterogeneous electron transfer kinetics and electrocatalytic behaviour of mixed self-assembled ferrocenes and..., Nkosi [/bib_ref]. [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref] (Ab) displays a Bode plot including the total impedance (log Z) as a function of the logarithm of the frequency depicting different glucose concentrations. The slope of the bare screen-printed electrode was determined to be −0.8 based on data at a frequency of 10 0 -10 2 Hz. Additionally, the inset calibration graph in [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref] (Ab) illustrates the relationship between log Z at a frequency of 10 4 Hz and glucose concentration. The inset plot shows that the impedance signal obtained from the bare electrode does not depend on changes in glucose concentration due to the absence of glucose-sensitive particles.
When comparing the performance of a screen-printed CNT-modified electrode to an electrode modified with GOx/PQ/egg white protein MPs, the impedance signal increased with increasing glucose concentration from 0.5 to 10 mM. The slopes of the log Z vs. log f graph obtained for screen-printing electrodes coated with GOx/PQ-based MPs were determined to be −0.7 (over a range of 10 0 to 10 2 Hz) [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref] , indicating pseudocapacitive behavior in the mid-frequency region. At high-frequency regions (over around 300 Hz), the slopes approached zero, indicating resistive behavior at those frequencies [bib_ref] Heterogeneous electron transfer kinetics and electrocatalytic behaviour of mixed self-assembled ferrocenes and..., Nkosi [/bib_ref]. Furthermore, the calibration graph (shown in the inset in [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref] (Bb)), which depicts the correlation between the logarithm of impedance (log Z) recorded at a high frequency of 10 4 Hz and glucose concentration, exhibited an upward trend in the impedance signal as the glucose concentration increased. When using GOx/PQ/egg white protein MPs, the sensitivity for glucose detection (measured in mM −1 unit) was enhanced by approximately 43 times compared with the bare electrode. These results confirm the glucose-sensitive behavior of the synthesized GOx-based protein MPs. Additionally, these findings highlight the beneficial effects of electrode modification in improving the electrochemical performance of glucose sensing applications. Additionally, we compared the Bode plot generated from experimental data with the fit of the EIS data [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref]. The Randles circuit was applied to simulate the EIS data [fig_ref] Figure 5: Study of electrochemical enzyme kinetics and selectivity [/fig_ref]. The circuit consists of a resistor (R el ) representing the electrolyte resistance, a constant phase element (CPE dl ), or a capacitor (C) for the double layer for non-ideal behavior at the electrode-electrolyte interface, and a Warburg element (W D ) describing the semi-infinite diffusion of the species in the electrolyte towards the electrode surface.
Biosensors 2023, 13, x FOR PEER REVIEW 12 of 18 a frequency of 10 0 -10 2 Hz. Additionally, the inset calibration graph in [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref] (Ab) illustrates the relationship between log at a frequency of 10 4 Hz and glucose concentration. The inset plot shows that the impedance signal obtained from the bare electrode does not depend on changes in glucose concentration due to the absence of glucose-sensitive particles. When comparing the performance of a screen-printed CNT-modified electrode to an electrode modified with GOx/PQ/egg white protein MPs, the impedance signal increased with increasing glucose concentration from 0.5 to 10 mM. The slopes of the log vs. log f graph obtained for screen-printing electrodes coated with GOx/PQ-based MPs were determined to be −0.7 (over a range of 10 0 to 10 2 Hz) [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref] , indicating pseudocapacitive behavior in the mid-frequency region. At high-frequency regions (over around 300 Hz), the slopes approached zero, indicating resistive behavior at those frequencies [bib_ref] Heterogeneous electron transfer kinetics and electrocatalytic behaviour of mixed self-assembled ferrocenes and..., Nkosi [/bib_ref]. Furthermore, the calibration graph (shown in the inset in [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref] (Bb)), which depicts the correlation between the logarithm of impedance (log Z) recorded at a high frequency of 10 4 Hz and glucose concentration, exhibited an upward trend in the impedance signal as the glucose concentration increased. When using GOx/PQ/egg white protein MPs, the sensitivity for glucose detection (measured in mM −1 unit) was enhanced by approximately 43 times compared with the bare electrode. These results confirm the glucose-sensitive behavior of the synthesized GOx-based protein MPs. Additionally, these findings highlight the beneficial effects of electrode modification in improving the electrochemical performance of glucose sensing applications. Additionally, we compared the Bode plot generated from experimental data with the fit of the EIS data [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref]. The Randles circuit was applied to simulate the EIS data [fig_ref] Figure 5: Study of electrochemical enzyme kinetics and selectivity [/fig_ref]. The circuit consists of a resistor ( el)
## Amperometry
Investigating the electrochemical enzyme kinetics of redox-mediated GOx reactions is crucial as it helps determine the enzyme's affinity for glucose and its maximum catalytic efficiency. In our study using a heterogeneous system, we employed an amperometric technique to examine the interaction between GOx and glucose (substrate) and to measure the rate of electron generation over time [fig_ref] Figure 5: Study of electrochemical enzyme kinetics and selectivity [/fig_ref]. By incrementally adding glucose concentrations ranging from 0.125 to 40 mM while maintaining a potential of 0.20 V vs. Ag/AgCl, we correlated the current responses with the glucose concentration [fig_ref] Figure 5: Study of electrochemical enzyme kinetics and selectivity [/fig_ref]. Moreover, the calibration in the inset of [fig_ref] Figure 5: Study of electrochemical enzyme kinetics and selectivity [/fig_ref] shows a linear range of glucose concentration from 0.125 to 10 mM with a sensitivity of 0.008 µA mM −1 . The Michaelis-Menten constant (K m ) serves as an important indicator of enzyme-substrate kinetics. The results showed that K m was 4.6 mM, indicating that the immobilized GOx exhibited high enzymatic activity and that the proposed electrode had a strong affinity for glucose. The current responses exhibited the characteristic features of the Michaelis-Menten kinetic mechanism (Equation (1)). In enzymes that follow the Michaelis-Menten mechanism, increasing substrate concentrations in the initial phase led to a rapid increase in the current, followed by a gradual increase as the enzyme approaches its maximum activity. At high substrate concentrations, the enzyme becomes saturated, and the maximum achieved current reflects the enzyme-substrate complex being fully formed. Thus, the resulting curve represents the kinetic parameters defining the upper and lower boundaries of substrate concentration. Therefore, K m serves as an important parameter reflecting the enzyme-substrate affinity and influences the reaction velocity at various glucose concentrations.
[formula] I = I max [glucose] K m + [glucose](1) [/formula]
where I is the steady-state current after the addition of glucose, I max is the maximum current obtained from saturated glucose concentrations, [glucose] is the glucose concentration, and K m is the Michaelis-Menten constant. Investigating the selectivity of the new biosensor for glucose is crucial due to the presence of various physiologically significant interferences in real samples, such as a saliva sample that contains glucose, uric acid, lactate, ascorbic acid, and creatinine. To determine the biosensor's selectivity, [fig_ref] Figure 5: Study of electrochemical enzyme kinetics and selectivity [/fig_ref] ,D were used to assess the impact of different potential interferences on the response to 2 mM uric acid [bib_ref] Electrochemical sensors targeting salivary biomarkers: A comprehensive review, Mani [/bib_ref] , 12.5 mM lactate [bib_ref] Electrochemical sensors targeting salivary biomarkers: A comprehensive review, Mani [/bib_ref] , 0.5 mM creatinine [bib_ref] Chemical analysis in saliva and the search for salivary biomarkers-a tutorial review, Ngamchuea [/bib_ref] , and 100 mM ascorbic acid [bib_ref] Interference of ascorbic acid with chemical analytes, Meng [/bib_ref]. These used concentrations were around five times as high as those informed in the literature. The selectivity test results indicate that the presence of coexisting compounds has little effect on the glucose response. For this experiment, the GOx/PQ/egg white protein MPs, based on a screenprinted electrode, could measure glucose using amperometry techniques at a low applied voltage in electrochemical analysis, resulting in reduced electrical input and power consumption. Utilizing low applied voltages can help prevent electrode degradation and enhance electrode stability, as high voltages may induce side reactions [bib_ref] High-Voltage Deprotonation of Layered-Type Materials as Newly Identified Cause of Electrode Degradation, Yang [/bib_ref]. Additionally, low voltages can enable better control over the desired reaction and enhance process selectivity, as certain reactions may exhibit different mechanisms or pathways at high voltages, leading to unwanted by products or decreased selectivity due to interference effect from substances, e.g., ascorbic acid and uric acid [bib_ref] High-sensitivity ion detection at low voltages with current-driven organic electrochemical transistors, Ghittorelli [/bib_ref]. Furthermore, functionalizing enzymes in MP environments allows for achieving a high level of specificity [bib_ref] Mesoporous materials for encapsulating enzymes, Lee [/bib_ref]. When enzymes are immobilized or encapsulated within MPs, the enzymatic reactions occur in a To validate our results and to obtain a deeper comprehension of the enzymatic properties, we compared the K m value calculated for our biosensor with values reported in the literature for similar enzyme-substrate systems on amperometric biosensors. For example, in the comparison of K m values, the encapsulation of GOx within spindle-like copper hydroxysulfate nanocrystals facilitated the precipitation Cu 2+ ions and biomimetic mineralization of brochantite [bib_ref] Highly active, stable and self-antimicrobial enzyme catalysts prepared by biomimetic mineralization of..., Li [/bib_ref]. The K m of the free enzyme was compared with that of GOx@copper hydroxysulfate nanocrystals, yielding K m values of 19.01 mM and 14.39 mM, respectively. Another example, a CNT-chitosan-nanowire electrode was immobilized with GOx, resulting in tight immobilization within the matrix through adsorption [bib_ref] Multiwalled carbon nanotubes grafted chitosan nanobiocomposite: A prosperous functional nanomaterials for glucose..., Gomathi [/bib_ref]. The enzyme electrode exhibited a low K m value of 7.1 mM, which can be attributed to synergistic augmentation from CNT, chitosan, and the GOx matrix. The K m value of these papers were higher than that in our work, which obtained a weaker binding affinity between the enzyme and substrate, requiring higher substrate concentrations to reach half of the maximum reaction velocity. Additionally, a glucose sensor was developed using zinc oxide nanowires immobilized with GOx, which led to a K m value of 4.1 mM [bib_ref] Tailoring zinc oxide nanowires for high performance amperometric glucose sensor, Zang [/bib_ref]. The observation of similar K m values with our work in different enzyme-substrate systems suggests comparable affinities between immobilized GOx and glucose.
Investigating the selectivity of the new biosensor for glucose is crucial due to the presence of various physiologically significant interferences in real samples, such as a saliva sample that contains glucose, uric acid, lactate, ascorbic acid, and creatinine. To determine the biosensor's selectivity, [fig_ref] Figure 5: Study of electrochemical enzyme kinetics and selectivity [/fig_ref] ,D were used to assess the impact of different potential interferences on the response to 2 mM uric acid [bib_ref] Electrochemical sensors targeting salivary biomarkers: A comprehensive review, Mani [/bib_ref] , 12.5 mM lactate [bib_ref] Electrochemical sensors targeting salivary biomarkers: A comprehensive review, Mani [/bib_ref] , 0.5 mM creatinine [bib_ref] Chemical analysis in saliva and the search for salivary biomarkers-a tutorial review, Ngamchuea [/bib_ref] , and 100 mM ascorbic acid [bib_ref] Interference of ascorbic acid with chemical analytes, Meng [/bib_ref]. These used concentrations were around five times as high as those informed in the literature. The selectivity test results indicate that the presence of coexisting compounds has little effect on the glucose response. For this experiment, the GOx/PQ/egg white protein MPs, based on a screen-printed electrode, could measure glucose using amperometry techniques at a low applied voltage in electrochemical analysis, resulting in reduced electrical input and power consumption. Utilizing low applied voltages can help prevent electrode degradation and enhance electrode stability, as high voltages may induce side reactions [bib_ref] High-Voltage Deprotonation of Layered-Type Materials as Newly Identified Cause of Electrode Degradation, Yang [/bib_ref]. Additionally, low voltages can enable better control over the desired reaction and enhance process selectivity, as certain reactions may exhibit different mechanisms or pathways at high voltages, leading to unwanted by products or decreased selectivity due to interference effect from substances, e.g., ascorbic acid and uric acid [bib_ref] High-sensitivity ion detection at low voltages with current-driven organic electrochemical transistors, Ghittorelli [/bib_ref]. Furthermore, functionalizing enzymes in MP environments allows for achieving a high level of specificity [bib_ref] Mesoporous materials for encapsulating enzymes, Lee [/bib_ref]. When enzymes are immobilized or encapsulated within MPs, the enzymatic reactions occur in a controlled and confined environment. This microenvironment enhances the enzyme's specificity by facilitating substrate recognition and catalytic activity. The MP environment provides protection to the enzymes from harsh conditions or interference from other molecules, thereby increasing their stability and preserving their specificity. Therefore, the use of MP environments offers significant advantages in attaining and maintaining high enzyme specificity across glucose biosensor applications.
## Glucose biosensors on degradable electrodes
Degradable electrodes are gaining interest due to their environmentally friendly and inexpensive nature [bib_ref] An electrically conductive oleogel paste for edible electronics, Cataldi [/bib_ref]. They allow for the recycling of nanomaterials, approaching the concept of zero waste, and offer great exploration potential for the future. It is intriguing to further investigate materials that can support the development of decomposable electrodes. In this regard, we attempted to incorporate the use of food materials. Our degradable carbon electrode was constructed using CNT and edible olive oil to enhance biodegradability and ecological safety. Furthermore, the decomposable CNT/vegetable oil combination was modified with biodegradable functional MPs. In this study, our aim was to develop an electrode coupled with glucose-sensitive MPs for glucose detection. The materials used in all components were designed to dissociate when exposed to acidic conditions and agitation, which mimic environments found in the human stomach [fig_ref] Figure 6: Degradable biosensors [/fig_ref].
The biosensing performance of the presented degradable biosensors was demonstrated through chronoamperometric measurements to monitor glucose levels with an applied potential of 0.6 V [fig_ref] Figure 6: Degradable biosensors [/fig_ref]. A linear relationship between the measured oxidation current and the glucose concentration was observed across the range of 0-40 mM [fig_ref] Figure 6: Degradable biosensors [/fig_ref]. To demonstrate the degradable capability of the electrode material, which refers to its ability to undergo controlled degradation or dissolution over time in a specific environment, we conducted experiments on the dissociation of GOx/PQ-based MPs and carbon electrodes. The dissociations of materials were observed at 60 min for GOx/PQ-based MPs [fig_ref] Figure 6: Degradable biosensors [/fig_ref] and 30 min for the degradable carbon electrode [fig_ref] Figure 6: Degradable biosensors [/fig_ref]. Biosensors designed for degradation over time could experience dissociation in acidic conditions, such as the gastric environment, due to chemical alterations or structural degradation. Both the electrodes and protein materials are susceptible to degradation in this acidic and mechanically agitated environment. These demonstrations represent the potential of degradable electrodes to reduce environmental problems from undecomposed materials and the successful development of a biodegradable glucose biosensor.
concept of zero waste, and offer great exploration potential for the future. It is intriguing to further investigate materials that can support the development of decomposable electrodes. In this regard, we attempted to incorporate the use of food materials. Our degradable carbon electrode was constructed using CNT and edible olive oil to enhance biodegradability and ecological safety. Furthermore, the decomposable CNT/vegetable oil combination was modified with biodegradable functional MPs. In this study, our aim was to develop an electrode coupled with glucose-sensitive MPs for glucose detection. The materials used in all components were designed to dissociate when exposed to acidic conditions and agitation, which mimic environments found in the human stomach [fig_ref] Figure 6: Degradable biosensors [/fig_ref]. The biosensing performance of the presented degradable biosensors was demonstrated through chronoamperometric measurements to monitor glucose levels with an applied potential of 0.6 V [fig_ref] Figure 6: Degradable biosensors [/fig_ref]. A linear relationship between the measured oxidation current and the glucose concentration was observed across the range of 0-40 mM [fig_ref] Figure 6: Degradable biosensors [/fig_ref]. To demonstrate the degradable capability of the electrode material, which refers to its ability to undergo controlled degradation or dissolution over time in a specific environment, we conducted experiments on the dissociation of GOx/PQ-based MPs and carbon electrodes. The dissociations of materials were observed at 60 min for GOx/PQ-based MPs [fig_ref] Figure 6: Degradable biosensors [/fig_ref] and 30 min for the degradable carbon electrode [fig_ref] Figure 6: Degradable biosensors [/fig_ref]. Biosensors designed for degradation over time could experience dissociation in acidic conditions, such as the gastric environment, due to chemical alterations or structural degradation. Both the electrodes and protein materials are susceptible to degradation in this acidic and mechanically agitated environment. These demonstrations represent the potential of degradable electrodes to reduce environmental problems from undecomposed materials and the successful development of a biodegradable glucose biosensor.
# Conclusions
We synthesized new food-based protein MPs and functional GOx/PQ/egg white protein MPs, using CaCO 3 templates. This approach of utilizing food-based proteins to form homogeneous microspheres showed promising potential for glucose biosensor applications. The egg white protein MPs provided a biocompatible and supportive matrix for incorporating the enzyme and redox molecules. By incorporating PQ as a redox mediator, the biosensor enhanced the electrochemical response and sensitivity for glucose detection. We combined GOx with egg white protein MPs to create sensitive biorecognition layers, as demonstrated by impedance AC mode and amperometry DC mode. These functional protein MPs were integrated onto screen-printed CNT-modified electrodes and decomposable edible olive oil/carbon electrodes. This combination offered benefits, including a simple and cost-effective manufacturing method for the biosensor. Additionally, the integration of GOx/PQ-based MPs and olive oil-based carbon electrodes enabled degradation under simulated gastric conditions, emphasizing the environmentally friendly nature and "green" applications of our materials. For future directions, it would be interesting to address the remaining challenges associated with the use of enzymatic materials in complex situations. Additionally, it is important to investigate a large population of real matrices in biofluids. By developing cheap, biodegradable, biocompatible materials; improving enzyme immobilization techniques; and exploring practical applications, it is conceivable that such concepts can unlock new potential for these materials in various applications.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/bios13080772/s1, the supplementary figures and sections in the research study focus on the characterization and electrochemical studies of the materials used in the glucose sensor. [fig_ref] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with... [/fig_ref]. optical microscopic images of different materials. [fig_ref] Figure 2: Characterization of materials [/fig_ref]. the fluorescence emission spectrum of PQ alone. [fig_ref] Figure 3: Electrochemical studies [/fig_ref]. the apparent capacitance profiles at various scan rates. [fig_ref] Figure 4: EIS studies of glucose sensor on [/fig_ref]. demonstrates EIS studies of the glucose sensor. [fig_ref] Figure 5: Study of electrochemical enzyme kinetics and selectivity [/fig_ref]. the equivalent circuit of EIS for the screen-printed electrode. [fig_ref] Figure 6: Degradable biosensors [/fig_ref]. electrochemical studies of the different electrodes. . EIS studies of screen-printed CNT-modified and commercial conductive carbon electrodes. Supporting Note S1. The areal capacitance calculation. Supporting Note S2. The electroactive surface area of the electrode.
[fig] Figure 1: Schematic diagram of glucose biosensors based on GOx/egg white protein MPs with a redox mediator. (A) Illustration of synthesis of a GOx/PQ/egg white protein MP: (a) Loading egg white proteins in a CaCO3 template. (b) Crosslinking egg white protein MPs. (c) Removing the CaCO3 template. (d) Functionalizing a PQ mediator. (e) Immobilizing GOx to obtain a PQ/egg white protein MP. (B) The components of a screen-printed electrode functionalized with GOx/PQ/egg white protein MPs along with the redox reactions occurring on its surface. [/fig]
[fig] Figure 2: Characterization of materials. (A) The optical microscopic image of GOx/PQ/egg white protein MPs. (B) SEM image of a screen-printed electrode coated with GOx/PQ/egg white protein MPs. (C) Fluorescence emission spectrum of (a) egg white proteins, (b) egg white protein MPs, (c) PQ/egg white protein MPs, and (d) GOx/PQ/egg white protein MPs at emission wavelengths from 315 to 550 nm. (D) FTIR spectra of (a) egg white proteins, (b) egg white protein MPs, (c) PQ/egg white protein MPs, and (d) GOx/PQ/egg white protein MPs. [/fig]
[fig] Figure 3: Electrochemical studies. (A) CVs obtained at scan rate 10 mV s −1 from (a,black) screenprinted CNT-modified electrode, (b,red) screen-printed electrode coated with egg white protein MPs, and (c,blue) screen-printed electrode coated with PQ-containing MPs. (B) CVs obtained from screen-printed electrode coated with PQ-containing MPs at different scan rates ranging from 2.5 to 200 mV s −1 . The scan rates (1-11) used were 2.5, 5, 10, 25, 50, 75, 100, 125, 150, 175 and 200 mV s −1 . (C) Plots of anodic peak current in function with square root of the scan rate obtained on a screenprinted electrode coated with PQ-containing MPs. (D) Linear sweep voltammetry (LSV) obtained from screen-printed electrode coated with GOx/PQ-based MPs at 0 and 20 mM glucose with a scan rate of 5 mV s −1 . [/fig]
[fig] Figure 4: EIS studies of glucose sensor on (A) a screen-printed CNT-modified electrode compared with (B) screen-printed electrode coated with GOx/PQ/egg white protein MPs at glucose concentrations of 0, 0.5, 5, and 10 mM in 0.1 M PBS with a pH of 7.0, using a frequency range of 10 0 -10 4 Hz, an amplitude of 5 mV, and 0.2 V DC; (a) phase angle vs. log f at frequency between 10 0 and 10 4 Hz. (b) Bode plots at frequency 10 0 -10 4 Hz. The insets present a glucose calibration plot using log obtained at a frequency of 10 4 Hz. [/fig]
[fig] Biosensors 2023 , 18, Figure 5: 13, x FOR PEER REVIEW 14 of Study of electrochemical enzyme kinetics and selectivity. (A) Amperograms obtained from the screen-printed electrode coated with GOx/PQ/egg white protein MPs with an applied potential of 0.2 V vs. Ag/AgCl; glucose concentrations at 0, 0.125, 0.5, 1.25, 2.5, 5, 7.5, 10, 20, 30, and 40 mM. (B) The calibration plot between 0.125 and 40 mM and inset calibration in the range 1.25−10 mM. (C) Interference study in (1) blank and in the presence of (2) 2 mM uric acid; (3) 12.5 mM lactate; (4) 0.5 mM creatinine; (5) 100 mM ascorbic acid; (6-9) 0.5, 2.5, 5.0, and 10.0 mM glucose; potential step to 0.2 V (vs. Ag/AgCl). (D) The chart displays the responses towards glucose and other substances. [/fig]
[fig] Figure 5: Study of electrochemical enzyme kinetics and selectivity. (A) Amperograms obtained from the screen-printed electrode coated with GOx/PQ/egg white protein MPs with an applied potential of 0.2 V vs. Ag/AgCl; glucose concentrations at 0, 0.125, 0.5, 1.25, 2.5, 5, 7.5, 10, 20, 30, and 40 mM. (B) The calibration plot between 0.125 and 40 mM and inset calibration in the range 1.25−10 mM. (C) Interference study in (1) blank and in the presence of (2) 2 mM uric acid; (3) 12.5 mM lactate; (4) 0.5 mM creatinine; (5) 100 mM ascorbic acid; (6-9) 0.5, 2.5, 5.0, and 10.0 mM glucose; potential step to 0.2 V (vs. Ag/AgCl). (D) The chart displays the responses towards glucose and other substances. [/fig]
[fig] Figure 6: Degradable biosensors. (A) A schematic diagram illustrating GOx/PQ/egg white protein MPs based on degradable biosensors. (B) Amperograms of the degradable electrode coated with the GOx/PQ/egg white protein MPs were recorded for a duration of 30 s, using an applied potential of 0.6 V vs. Ag/AgCl. The recordings were taken for various glucose concentrations at 0, 5, 10, 20, 30, and 40 mM. (C) The calibration linear range plots at glucose concentrations 5-40 mM and error bars (n = 3). (D) The degradability of the GOx/PQ/egg white protein MPs in a simulated gastric environment, assessed at 0 and 60 min. (E) The degradability of the CNT/olive oil electrode coated with glucose-sensitive MPs and chitosan in a simulated gastric environment, assessed at 0 and 30 min. [/fig]
[fig] Author: Contributions: Conceptualization, I.J.; Methodology, N.R. and I.J.; Validation, N.R., P.R., K.K. and I.J.; Formal Analysis, N.R., P.R. and I.J.; Investigation, N.R. and P.R.; Resources, C.B. and I.J.; Data Curation, N.R. and I.J.; Writing-Original Draft Preparation, N.R., P.R., K.K., C.B. and I.J.; Writing-Review and Editing, N.R., P.R., K.K., C.B. and I.J.; Visualization, N.R., P.R., K.K. and I.J.; Supervision, I.J.; Project Administration, I.J.; Funding Acquisition, I.J. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This research was funded by the National Research Council of Thailand (NRCT), grant number N41A640129, and Prince of Songkla University, Hat Yai, Thailand. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data are contained within the article and the Supplementary Materials. [/fig]
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10.1089/ars.2019.7803
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CCBY
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7247050
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31218894
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s2orc_pubmed_articles
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Metabolic Responses to Reductive Stress
Significance: Reducing equivalents (NAD(P)H and glutathione [GSH]) are essential for maintaining cellular redox homeostasis and for modulating cellular metabolism. Reductive stress induced by excessive levels of reduced NAD + (NADH), reduced NADP + (NADPH), and GSH is as harmful as oxidative stress and is implicated in many pathological processes. Recent Advances: Reductive stress broadens our view of the importance of cellular redox homeostasis and the influences of an imbalanced redox niche on biological functions, including cell metabolism. Critical Issues: The distribution of cellular NAD(H), NADP(H), and GSH/GSH disulfide is highly compartmentalized. Understanding how cells coordinate different pools of redox couples under unstressed and stressed conditions is critical for a comprehensive view of redox homeostasis and stress. It is also critical to explore the underlying mechanisms of reductive stress and its biological consequences, including effects on energy metabolism. Future Directions: Future studies are needed to investigate how reductive stress affects cell metabolism and how cells adapt their metabolism to reductive stress. Whether or not NADH shuttles and mitochondrial nicotinamide nucleotide transhydrogenase enzyme can regulate hypoxia-induced reductive stress is also a worthy pursuit. Developing strategies (e.g., antireductant approaches) to counteract reductive stress and its related adverse biological consequences also requires extensive future efforts. Antioxid. Redox Signal. 32, 1330-1347.
# Introduction
M ammalian cells depend on a series of oxidation and reduction (redox) reactions to generate energy (e.g., ATP) and to synthesize essential cellular components (e.g., nucleic acids) from nutrients in support of their biological functions. In a redox reaction, electrons flow from reducing agents (reductants) to oxidizing agents (oxidants). Cellular redox couples, mainly nicotinamide adenine dinucleotide (NAD + )/reduced NAD + (NADH), phosphorylated NAD + (NADP + )/reduced NADP + (NADPH), and reduced glutathione (GSH)/GSH disulfide (GSSG), are responsible for the bulk of cellular electron transfer [bib_ref] Redox environment of the cell as viewed through the redox state of..., Schafer [/bib_ref] [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref]. These redox couples serve as cofactors or substrates for enzymatic or nonenzymatic neutralization of reactive oxygen species (ROS) to sustain a relatively reducing environment in cells.
Importantly, these redox couples also link the cellular redox environment with cellular energetics. For instance, NAD + serves as an electron sink to support glycolysis; NADH provides electrons for mitochondrial oxidative phosphorylation (OXPHOS); and NADPH is a major electron source for reductive biosynthesis of fatty acids and nucleic acids [bib_ref] NAD+ metabolism: bioenergetics, signaling and manipulation for therapy, Yang [/bib_ref] [bib_ref] NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological..., Ying [/bib_ref]. Given that these reducing equivalents are indispensable for cellular redox homeostasis and energy metabolism, an imbalance of the redox state of these molecules is implicated in a variety of pathological conditions, such as cardiovascular diseases, neurodegenerative diseases, cancer, and aging [bib_ref] NAD(+) Metabolism and the control of energy homeostasis: a balancing act between..., Canto [/bib_ref] [bib_ref] NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological..., Ying [/bib_ref].
In this review, we first introduce the cellular redox network and redox stress, and then delineate the metabolic sources and cellular distribution of the NAD + /NADH, NADP + /NADPH, and GSH/GSSG redox couples. This information is followed by an explanation of how perturbations in these ratios lead to reductive stress. Finally, we discuss metabolic responses to reductive stress and their impact on cell function.
## Cellular redox network and redox stress
## Redox network in mammalian cells
The cellular redox niche is balanced by pro-oxidants and antioxidants. Cellular pro-oxidants include ROS (e.g., superoxide anion [O [bib_ref] Glutathione: new roles in redox signaling for an old antioxidant, Aquilano [/bib_ref] -] and hydrogen peroxide [H 2 O 2 ]), reactive nitrogen species (RNS; e.g., nitric oxide [NO ]), and their derivatives (e.g., peroxynitrite [ONOO -]), which are primarily produced by enzymes, such as NAD(P)H oxidases (NOXs) and uncoupled nitric oxide synthases, or by the mitochondrial electron transport chain as functional by-products [bib_ref] Redox control of the cell cycle in health and disease, Sarsour [/bib_ref]. To counteract these oxidants, cells have developed antioxidant systems, including antioxidant enzymes, for example, superoxide dismutases (SOD1-3), catalase, glutathione peroxidases (GPx1-8), thioredoxins (Trx1-2), and peroxiredoxins (Prx1-6); and nonenzymatic small molecules, for example, GSH, a-tocopherol, and ascorbate [bib_ref] Redox control of the cell cycle in health and disease, Sarsour [/bib_ref].
Of note, the production of cellular oxidants is compartment specific, but their targets can be local or/and remote from the source compartment. For example, mitochondria-generated H 2 O 2 can function as a signaling molecule in mitochondria (local) or/and in cytosol (remote) (26). In the vasculature, endothelial cell-derived NO can diffuse from the endothelium to adjacent smooth muscle cells leading to vasodilation . Unlike oxidants, antioxidant enzymes exercise their functions depending on their specific subcellular localization. For example, manganese SOD (SOD2), a mitochondrial matrix-localized enzyme, can only convert O 2 into H 2 O 2 in mitochondria, where H 2 O 2 is subsequently reduced to water by mitochondrial peroxidases (such as catalase, GPx1, GPx4, and Prx2).
The GSH/GSSG couple and the NADP + /NADPH couple play crucial roles in the two-electron reduction of peroxides. GSH is a cosubstrate of GPxs for H 2 O 2 removal. In this context, two molecules of GSH donate two electrons to reduce H 2 O 2 to water and themselves are oxidized to GSSG, which is then recycled back to GSH by glutathione reductase (GR) using NADPH as an electron donor [bib_ref] Quantitative redox biology: an approach to understand the role of reactive species..., Buettner [/bib_ref]. It is interesting to note that glutaredoxins (Grx1-2) also utilize GSH to reduce protein intra/inter-disulfides (PS 2 or proteinglutathione disulfide [PSSG]) into protein sulfhydryl groups (-SH) [bib_ref] Quantitative redox biology: an approach to understand the role of reactive species..., Buettner [/bib_ref]. In addition to GR, NADPH is also an FIG. 1. Cellular redox network. Cellular redox homeostasis is maintained by a delicate balance between pro-oxidant and antioxidant systems. Cellular pro-oxidants primarily include O 2 and H 2 O 2 , which are generated by enzymes or/and mitochondrial respiration. SODs convert O 2 to H 2 O 2 , which is further neutralized to water by catalase, GPxs, and Prxs. Catalase has very low affinity for H 2 O 2 (but with a high turnover and high catalytic efficiency) and can reduce H 2 O 2 to water when its levels reach the millimolar range. GPxs require two molecules of reduced GSH for H 2 O 2 reduction and GSH is oxidized to GSSG in this reaction. GSH can also be used by glutaredoxins [Grx-(SH) 2 ] to reduce protein intra/inter-disulfide (PS 2 or PSSG) into reduced protein cysteinyl residues (P[SH] 2 or PSH + GSH). The recycling of GSSG to GSH is catalyzed by GR, which utilizes NADPH as an electron donor. Prxs extract electrons from reduced thioredoxins [Trx-(SH) 2 ] to reduce H 2 O 2 or organic hydroperoxides (ROOH) to H 2 O or/and alcohols (ROH), respectively; and Trx-(SH) 2 is simultaneously oxidized into Trx-S 2 . Like GSH, Trx-(SH) [bib_ref] Glutathione: new roles in redox signaling for an old antioxidant, Aquilano [/bib_ref] can also reduce protein disulfides into protein cysteinyls. The recycling of Trx-S 2 is catalyzed by TRs using NADPH as an electron donor. NADPH is generated by G6PD in the PPP of glucose metabolism. Therefore, the cellular redox state and cell metabolism are closely linked through NADPH. Adapted from references [bib_ref] Quantitative redox biology: an approach to understand the role of reactive species..., Buettner [/bib_ref] with permission. 6-PG, 6-phosphogluconate; G-6-P, glucose-6-phosphate; G6PD, glucose-6-phosphate dehydrogenase; GPxs, glutathione peroxidases; GR, glutathione reductase; Grx, glutaredoxin; GSH, glutathione; GSSG, GSH disulfide; H 2 O 2 , hydrogen peroxide; NADPH, reduced NADP + ; O 2 -, superoxide anion; PPP, pentose phosphate pathway; Prxs, peroxiredoxins; PSSG, protein-glutathione disulfide; SODs, superoxide dismutases; TRs, thioredoxin reductases; Trx, thioredoxin.
indispensable cofactor for thioredoxin reductases (TRs). Likewise, NADPH provides two electrons for TRs to reduce oxidized Trx (Trx-S 2 ) to its active dithiol form Trx-(SH) 2 . The reduced form Trx-(SH) 2 serves as an electron source for regeneration of Prxs, which reduce H 2 O 2 and other organic hydroperoxides to water and alcohols, respectively (7, 8) . Notably, intracellular total Trx content was estimated to be 1-10 lM in bovine tissues (34), which is 2-3 orders of magnitude lower than total GSH levels (mM range).
## Redox stress
Under physiological conditions, cellular pro-oxidants are maintained at steady-state levels required for many biological processes, such as cell signaling, proliferation, and differentiation (30, 87). However, when the balance between prooxidants and antioxidants is adversely perturbed, redox stress, including oxidative stress and reductive stress, occurs .
The concept of oxidative stress was first introduced by Paniker et al. in 1970 in studies of the GSH/GSSG couple in H 2 O 2 -stimulated normal and GR-deficient human erythrocytes [bib_ref] Effect of glutathione reductase deficiency on the stimulation of hexose monophosphate shunt..., Paniker [/bib_ref]. This term is now defined as the imbalance between cellular pro-oxidant levels and antioxidant capacity in favor of the former (30, [bib_ref] Adaptions to hypoxia and redox stress essential concepts confounded by misleading terminology, Loscalzo [/bib_ref] [bib_ref] Redox control of the cell cycle in health and disease, Sarsour [/bib_ref] , implying that an excess in oxidant levels leads to oxidative stress . It is commonly ac-cepted that oxidative stress induced by excess ROS/RNS levels is detrimental to biological functions and is involved in the development of many pathological conditions, such as cardiovascular diseases, neurodegenerative diseases, aging, and cancer (30, [bib_ref] ROS function in redox signaling and oxidative stress, Schieber [/bib_ref] [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref] ; however, it is important to note that these oxidants (e.g., O 2 and H 2 O 2 ) are capable of either oxidizing or reducing other molecules depending on the standard redox potential (E°¢) of two reactants [fig_ref] Table 1: Standard Reduction Potentials [/fig_ref] [bib_ref] Redox state and redox environment in biology, Schafer [/bib_ref]. For example, in the Fenton reaction, O 2 is oxidized to O 2 and H 2 O 2 is reduced to hydroxyl radical (HO ), which is the most oxidizing species (E°¢ = 2310 mV).
[formula] O 2 -+ Fe 3+ / O 2 + Fe 2+ (1) H 2 O 2 + Fe 2+ / Fe 3+ + HO + HO -(2) O 2 -+ H 2 O 2 / O 2 + HO + HO -(Net reaction) [/formula]
Likewise, H 2 O 2 can reduce ferryl hemoglobin and oxidize methionine; and itself is oxidized to O 2 and reduced to water, respectively [bib_ref] Adaptions to hypoxia and redox stress essential concepts confounded by misleading terminology, Loscalzo [/bib_ref]. Therefore, increases in O 2 and H 2 O 2 levels can be viewed as creating oxidative or reductive stress depending on the relative abundance of redox-coupled species.
Unlike oxidative stress, reductive stress is not as wellcharacterized a concept. This term was first described by , the experiments in which the authors mimicked anoxia by treating rat hepatocytes with chemicals to block mitochondrial respiration and ATP production (socalled chemical hypoxia) (27). The authors proposed that electron carriers became reduced due to limited oxygen availability (e.g., hypoxia) and were reoxidized during reoxygenation (e.g., reperfusion) leading to a burst of ROS generation, termed ''reductive stress'' (27). This term currently has a more generalized definition, that is, reductive stress is an imbalance between cellular pro-oxidant levels and reducing capacity in favor of the latter (30, [bib_ref] Adaptions to hypoxia and redox stress essential concepts confounded by misleading terminology, Loscalzo [/bib_ref] [bib_ref] Redox control of the cell cycle in health and disease, Sarsour [/bib_ref]. In the context of this review, we define reductive stress as an excess accumulation of reducing equivalents (specifically NADH, NADPH, and GSH), exceeding the capacity of endogenous oxidoreductases.
Under physiological conditions, cellular redox buffers [GSH/GSSG and NAD(P)H/NAD(P) + ] have sufficient capacity (termed basal redox buffer capacity [ReBC]) to maintain cellular oxidants and reductants at physiological levels (ROS as signaling molecules) . When cells sustain oxidative or reductive insults, redox buffers increase to a certain level (termed compensatory ReBC) to counteract these redox stresses and restore redox homeostasis. Under these circumstances, cellular oxidants and reductants are still maintained within physiological ranges. However, when this compensatory response reaches a maximum, the ReBC is exceeded and oxidative or reductive stress occurs . Importantly, reductive stress diminishes cellular ROS levels to below their physiological levels and thus perturbs their signaling functions. From a different viewpoint, reductive stress can also promote ROS production (e.g., by partially reducing oxygen) and thus is proposed to promote ''oxidative stress'' in essence, depending on the redox couples in which these ROS are engaged [bib_ref] Increased reactive oxygen species production during reductive stress: the roles of mitochondrial..., Korge [/bib_ref] [bib_ref] Glutathione redox state regulates mitochondrial reactive oxygen production, Shen [/bib_ref] [bib_ref] Pathogenesis of chronic hyperglycemia: from reductive stress to oxidative stress, Yan [/bib_ref].
Like oxidative stress, reductive stress also impairs cellular functions (31). For example, the endoplasmic reticulum (ER) has a relatively oxidizing environment, which is required for structural disulfide formation of membrane and secretory proteins. However, under reductive stress, protein disulfide bonds inappropriately form, resulting in activation of the unfolded FIG. 2. Redox stress. The NADH/NAD + , NADPH/ NADP + , and GSH/GSSG redox couples are the major cellular redox buffers. Under unstressed conditions, these three redox couples have adequate capacity to maintain redox homeostasis, termed basal ReBC (X-axis; green box under red line). When cellular ROS levels increase (Left Y-axis; red line), these redox buffers are also able to respond by elevating basal ReBC to a certain level, termed the compensatory ReBC (X-axis; gray area under red line). Under both circumstances, cellular ROS levels are maintained at physiological levels to ensure normal biological function, such as signaling molecule adequacy (Right Y-axis; blue line). However, when this compensatory response continues beyond a certain threshold at which the maximal ReBC is exceeded by cellular reducing of ROS levels (X-axis, black triangle), reductive stress can occur (red line). By contrast, oxidative stress occurs when cellular ReBC decreases (Xaxis, black triangle), or/and cellular oxidation of ROS production is overwhelming. Both reductive stress and oxidative stress (collectively named as redox stress) can promote ROS production leading to oxidative damages to macromolecules and perturbations of cellular functions. NAD + , nicotinamide adenine dinucleotide; NADH, reduced NAD + ; NADP + , phosphorylated NAD + ; ReBC, redox buffer capacity; ROS, reactive oxygen species.
protein response and ER stress [bib_ref] Oxidative homeostasis regulates the response to reductive endoplasmic reticulum stress through translation..., Maity [/bib_ref]. In line with this concept, we and others showed that reductive stress perturbs protein disulfide formation leading to ER stress in yeast and intracellular retention of membrane proteins in human cells (32, [bib_ref] Oxidative homeostasis regulates the response to reductive endoplasmic reticulum stress through translation..., Maity [/bib_ref] [bib_ref] Thioredoxins are required for protection against a reductive stress in the yeast..., Trotter [/bib_ref] [bib_ref] Regulation of the protein disulfide proteome by mitochondria in mammalian cells, Yang [/bib_ref]. Furthermore, mice with cardiac-specific overexpression of mutated human aB-crystallin (hR120GCryAB) displayed increases in GSH levels and in activities of GR, glucose-6phosphate dehydrogenase (G6PD), catalase, and GPx (indicative of reductive stress), which led to the development of cardiac hypertrophy and heart failure in these mice [bib_ref] Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in..., Rajasekaran [/bib_ref] [bib_ref] Sustained activation of nuclear erythroid 2-related factor 2/ antioxidant response element signaling..., Rajasekaran [/bib_ref]. Elevated cytosolic NADH/NAD + and its resulting reductive stress were observed in early diabetic animals owing to increased oxidation of substrates (e.g., sorbitol, nonesterified fatty acids, and glucuronic acid pathway metabolites), which was coupled with the reduction of NAD + to NADH [bib_ref] Cytosolic NADH/ NAD+, free radicals, and vascular dysfunction in early diabetes mellitus, Ido [/bib_ref]. Thus, reductive stress is deleterious to cells and has been linked to many pathological conditions, including cardiovascular diseases and diabetes mellitus (31, 51, 105).
## Metabolic sources and cellular distribution of the nad(h) and nadp(h) couples
NAD + , a key cofactor for many enzymes including metabolic enzymes, is critical for various biological processes [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref]. Mammalian cells can synthesize NAD + de novo from its precursors and recycle NAD + from NADH. Depending on availability of its precursors, NAD + can be synthesized via three pathways: the de novo pathway using tryptophan as a precursor, the Preiss-Handler pathway using nicotinic acid as a precursor, and the salvage pathway using nicotinamide (NAM) and nicotinamide riboside (NR) as precursors [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref]. NAD + can be phosphorylated to NADP + via NAD + kinases (NADKs); the oxidized forms of NAD + and NADP + can be reduced to NADH and NADPH by their corresponding de-hydrogenases [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref]. We and others have extensively reviewed the enzymatic steps and the regulatory factors of all three NAD + biosynthetic pathways [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref] [bib_ref] NAD+ metabolism: bioenergetics, signaling and manipulation for therapy, Yang [/bib_ref] [bib_ref] NAD+ and NADH in cellular functions and cell death, Ying [/bib_ref]. In this review, we primarily focus on the interconversion of NAD(H) and NADP(H) via metabolic enzymes.
## Metabolic sources of the nad(h) and nadp(h) couples
The interconversion of NAD(P) + and NAD(P)H is mediated by metabolic enzymes involved in glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle (Figs. 3 and 4). Glycolysis and the TCA cycle are the two major sources of cytosolic and mitochondrial NAD(H), respectively . In the cytosol, the interconversion of NAD + and NADH is catalyzed by two enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) [bib_ref] Aerobic glycolysis: meeting the metabolic requirements of cell proliferation, Sy [/bib_ref] [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref]. GAPDH catalyzes the reversible conversion of glyceraldehyde-3-phosphate (G-3-P) to 1,3-bisphosphoglycerate in conjunction with the corresponding interconversion of the NAD + /NADH couple. LDH reduces pyruvate to lactate coupled with the oxidization of NADH to NAD + in glycolysis. Of note, LDH can also catalyze the reverse reaction by reducing NAD + to NADH and oxidizing lactate to pyruvate when lactate is utilized as a fuel source for the TCA cycle [bib_ref] Glucose feeds the TCA cycle via circulating lactate, Hui [/bib_ref] [bib_ref] Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice, Sonveaux [/bib_ref]. Additional sources of cytosolic NAD(H) include the alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases [bib_ref] Alcohol metabolism, Cederbaum [/bib_ref].
In the mitochondria, eight molecules of NADH can be generated by per molecule of glucose through the TCA cycle under well-oxygenated conditions [bib_ref] NAD+ metabolism: bioenergetics, signaling and manipulation for therapy, Yang [/bib_ref]. The pyruvate dehydrogenase (PDH) complex reduces NAD + to NADH in conjunction with decarboxylating pyruvate to acetyl-CoA. The latter enters the TCA cycle, where NADH is produced by isocitrate dehydrogenase 3 (IDH3), a-ketoglutarate dehydrogenase (KGDH), and malate dehydrogenase 2 (MDH2) (56, 106) . Moreover, NAD + -linked malic enzyme (ME2) and glutamate dehydrogenases (GLUD1-2) also contribute to the mitochondrial NADH pool (81, 106) . Cytosolic NADPH is primarily generated from the PPP by G6PD and 6-phosphogluconate dehydrogenase (6PGD) [bib_ref] Aerobic glycolysis: meeting the metabolic requirements of cell proliferation, Sy [/bib_ref] [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref] [bib_ref] NAD+ metabolism: bioenergetics, signaling and manipulation for therapy, Yang [/bib_ref]. G6PD converts glucose-6-phosphate (G-6-P) to 6-phosphogluconate, which is further metabolized into ribulose-5-phosphate by 6PGD. Both reactions are coupled to the reduction of NADP + to NADPH . Of note, ribulose-5-phosphate and its derivative ribose-5-phosphate (R-5-P) can be converted into fructose-6-phosphate (F-6-P) and G-3-P by transaldolase/transketolase through a series of nonoxidative reactions and thereby be diverted into glycolysis [bib_ref] Aerobic glycolysis: meeting the metabolic requirements of cell proliferation, Sy [/bib_ref].
In addition, other enzymes also contribute to the cytosolic NADPH pool, such as IDHs, MEs, methylene-tetrahydrofolate dehydrogenases (MTHFDs), and 10-formyl-tetrahydrofolate dehydrogenases (FTHFDHs) [bib_ref] Aerobic glycolysis: meeting the metabolic requirements of cell proliferation, Sy [/bib_ref] [bib_ref] Serine and one-carbon metabolism in cancer, Vousden [/bib_ref] , all of which have both cytosolic and mitochondrial isozymes. For example, cytosolic IDH (IDH1) and mitochondrial IDH2 catalyze the oxidation of isocitrate to a-ketoglutarate (a-KG) and the reduction of NADP + to NADPH. Cytosolic malic enzyme (ME) 1 decarboxylates malate to pyruvate coupled with the reduction of NADP + to NADPH. Moreover, two enzymes in folate metabolite also generate NADPH; cytosolic MTHFD (MTHFD1) catalyzes 5,10-methylenetetrahydrofolate (5,10-methylene-THF) to 5,10-methenyl-THF; and cytosolic FTHFDH catalyzes the recycling of 10-formyl-THF to THF [bib_ref] Serine and one-carbon metabolism in cancer, Vousden [/bib_ref].
Mitochondrial NADPH can be produced by the mitochondrial enzymes IDH2, ME3, MTHFD2, and FTHFDH using the same reaction as their corresponding cytosolic isozymes [bib_ref] Aerobic glycolysis: meeting the metabolic requirements of cell proliferation, Sy [/bib_ref] [bib_ref] Serine and one-carbon metabolism in cancer, Vousden [/bib_ref]. NADP + -dependent GLUDs can also generate NADPH through the conversion of glutamate to a-KG (81). Of note, another significant source of mitochondrial NADPH is nicotinamide nucleotide transhydrogenase (NNT). NNT resides in the mitochondrial inner membrane and obtains electrons from NADH to reduce NADP + into NADPH utilizing the proton gradient across mitochondrial inner membrane as a driving force: NADH + NADP + ) / NAD + + NADPH (35, 84). It has been estimated that IDH2, NNT, and ME3 contributed 70%, 22%, and 8%, respectively, of mitochondrial NADPH production in cardiac tissue of C57BL/6N mice [bib_ref] Reversal of mitochondrial transhydrogenase causes oxidative stress in heart failure, Nickel [/bib_ref].
## Compartmental distribution of cellular nad(h) and nadp(h)
The intracellular distribution of NAD(H) and NADP(H) is highly compartmentalized owing to specific localization of their biosynthetic enzymes and membrane permeability to these dinucleotides [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref]. For example, mitochondrial nicotinamide mononucleotide adenylyltransferase and mitochondrial NADK support NAD + and NADP + synthesis, respectively, in this organelle [bib_ref] NAD(+) Metabolism and the control of energy homeostasis: a balancing act between..., Canto [/bib_ref] [bib_ref] Identification and characterization of a human mitochondrial NAD kinase, Ohashi [/bib_ref] [bib_ref] MNADK, a novel liver-enriched mitochondrionlocalized NAD kinase, Zhang [/bib_ref]. While NAD(P) + can freely pass through the nuclear membrane, none of these dinucleotides can diffuse across the mitochondrial inner membrane (35, 114). Of note, different compartmental NAD(H) and NADP(H) pools differentially respond to
## Fig. 3. metabolic sources of nad(h).
In the cytosol, the interconversion of NAD + and NADH is mediated by two enzymes GAPDH and LDH and by the alcohol metabolism enzymes ADH and ALDH. In the mitochondrial matrix, PDH, ME2, GLUD, and TCA cycle enzymes (IDH3, KGDH, and MDH2) contribute to NAD(H) production. 1,3-BPG, 1,3bisphosphoglycerate; a-KG, a-ketoglutarate; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; G-3-P, glyceraldehyde-3-phosphate; GAPDH, glyceraldehyde-3phosphate dehydrogenase; GLUD, glutamate dehydrogenase; GLUT, glucose transporter; IDH, isocitrate dehydrogenase; KGDH, aketoglutarate dehydrogenase; LDH, lactate dehydrogenase; MDH2, malate dehydrogenase 2; ME, malic enzyme; OAA, oxaloacetate; PDH, pyruvate dehydrogenase; TCA, tricarboxylic acid. exogenous stimuli. Typically, cytoplasmic and nuclear NAD(H) pools are more sensitive to changes in redox stress than the mitochondrial NAD(H) pool, which is stably maintained and required for cell survival under redox insults [bib_ref] Nutrientsensitive mitochondrial NAD+ levels dictate cell survival, Yang [/bib_ref] [bib_ref] Genetically encoded fluorescent sensors for intracellular NADH detection, Zhao [/bib_ref].
Although cytosolic, nuclear, and mitochondrial NAD(H) and NAD(P)H pools often function in their respective compartments, these compartmental dinucleotide pools also exchange via shuttle mechanisms to maintain the overall cellular redox environment and redox-dependent functions. Cytosolic NADH can freely diffuse through the porous mitochondrial outer membrane; however, it is unable to pass through the mitochondrial inner membrane [bib_ref] NAD(+) Metabolism and the control of energy homeostasis: a balancing act between..., Canto [/bib_ref] [bib_ref] NAD kinase levels control the NADPH concentration in human cells, Pollak [/bib_ref] [bib_ref] NAD+ metabolism: bioenergetics, signaling and manipulation for therapy, Yang [/bib_ref]. To circumvent this limitation, cells developed two NADH shuttles: the malate/aspartate shuttle and the glycerol-3phosphate shuttle (35, [bib_ref] Neuronal and astrocytic shuttle mechanisms for cytosolic-mitochondrial transfer of reducing equivalents: current..., Mckenna [/bib_ref] [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref].
In the first shuttle, cytosolic NADH is oxidized to NAD + by MDH1 in conjunction with the reduction of oxaloacetate (OAA) to malate; whereas in the mitochondria, NAD + is reduced to NADH by MDH2 coupled with the oxidation of malate back to OAA in the TCA cycle [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref]. This shuttle also requires two transporters, the a-KG/malate antiporter (encoded by SLC25A11 gene), which transfers cytosolic malate into the mitochondrial matrix; and the aspartate/glutamate antiporter (encoded by SLC25A13 gene), which transfers mitochondrial aspartate into the cytosol [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref]. Unlike the malate/aspartate shuttle, the glycerol-3-phosphate shuttle is irreversible and needs only one enzyme, glycerol-3phosphate dehydrogenase (GPDH) [bib_ref] Neuronal and astrocytic shuttle mechanisms for cytosolic-mitochondrial transfer of reducing equivalents: current..., Mckenna [/bib_ref]. Cytosolic GPDH oxidizes NADH to NAD + in conjunction with the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3phosphate, whereas mitochondrial GPDH catalyzes the reverse reaction that oxidizes glycerol-3-phosphate into DHAP and reduces FAD into FADH 2 . Therefore, such shuttle mechanisms enable cells to maintain adequate NAD + in the cytosol in support of glycolysis and sufficient NADH in the mitochondria in support of OXPHOS.
Like NADH, NADP(H) cannot diffuse across the mitochondrial inner membrane [bib_ref] NAD(+) Metabolism and the control of energy homeostasis: a balancing act between..., Canto [/bib_ref] [bib_ref] NAD kinase levels control the NADPH concentration in human cells, Pollak [/bib_ref] [bib_ref] NAD+ metabolism: bioenergetics, signaling and manipulation for therapy, Yang [/bib_ref]. The exchange of cytosolic and mitochondrial NADP(H) pools is, therefore,
## Fig. 4. metabolic sources of nadp(h) and the nadph shuttle.
In the cytosol, NADPH is primarily produced by G6PD and 6PGD in the PPP, where the products R-5-P and Xu-5-P can be diverted into glycolysis by transaldolase and transketolase. ME1 and IDH1 also contribute to cytosolic NADP(H) production. In addition, two enzymes in folate metabolism, MTHFD1 and FTHFDH, are also the sources of cytosolic NADP(H). Mitochondrial NADP(H) is generated by NADP + -dependent IDH2, NNT, and ME3. The cytosolic and mitochondrial NADP(H) pools are exchanged through the isocitrate-a-KG shuttle, where cytosolic IDH1 and mitochondrial IDH2 catalyze the interconversion of isocitrate and a-KG in conjunction with the interconversion of NADP + and NADPH. The citrate carrier protein (encoded by SLC25A1 gene) and the a-KG/malate antiporter (encoded by SLC25A11 gene) mediate the transport of isocitrate and a-KG between the cytosol and mitochondria, respectively. 6PGD, 6-phosphogluconate dehydrogenase; FTHFDH, 10-formyl-tetrahydrofolate dehydrogenase; MTHFD, methylene-tetrahydrofolate dehydrogenase; NNT, nicotinamide nucleotide transhydrogenase; R-5-P, ribose-5-phosphate; Ru-5-P, ribulose-5-phosphate; SCL25A1, solute carrier family 25 member 1; SCL25A11, solute carrier family 25 member 11; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate; Xu-5-P, xylulose-5-phosphate.
carried out by the isocitrate-a-KG shuttle through IDH1 and IDH2 isozymes (35, 73, 106) . Cytosolic IDH1 and mitochondrial IDH2 catalyze the reversible interconversion between isocitrate and a-KG in conjunction with the interconversion of NADP + and NADPH [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref]. This shuttle also requires two carrier proteins, the citrate carrier protein (encoded by SLC25A1 gene), which exports mitochondrial isocitrate into the cytosol in exchange for malate; and the a-KG/ malate antiporter, which is the same carrier as in the malate/ aspartate shuttle [bib_ref] NAD(H) and NADP(H) redox couples and cellular energy metabolism, Xiao [/bib_ref]. Thus, the isocitrate-a-KG shuttle plays a pivotal role in maintaining cellular NADPH levels. Taken together, these shuttle mechanisms enable cells to maintain redox and energy homeostasis under a wide range of physiopathological conditions.
## Biosynthesis and cellular distribution of gsh
GSH is the most abundant thiol antioxidant in mammalian cells. It serves as a substrate for H 2 O 2 removal by GPxs and itself is oxidized to GSSG [bib_ref] Glutathione synthesis, Lu [/bib_ref] [bib_ref] Redox environment of the cell as viewed through the redox state of..., Schafer [/bib_ref]. The recycling of GSSG to GSH is catalyzed by GR using NADPH as an electron donor. In addition to its antioxidant function, GSH is also involved in modulation of cellular redox signaling (e.g., protein S-glutathionylation), regulation of cell proliferation and cell death, and detoxification of xenobiotics and their metabolites [bib_ref] Gene expression profiling reveals a signaling role of glutathione in redox regulation, Fratelli [/bib_ref] [bib_ref] Glutathione synthesis, Lu [/bib_ref].
## Gsh biosynthesis
GSH is a tripeptide and is synthesized from precursor amino acids, l-glutamate, l-cysteine, and glycine, in the cytosol, through two ATP-dependent steps . In the first step, glutamate reacts with cysteine to form cglutamylcysteine under the catalysis of the rate-limiting enzyme, c-glutamylcysteine ligase (GCL). The GCL enzyme is composed of two functional subunits: catalytic (GCLC) and modifier (GCLM). In the following step, GSH synthetase (GS) catalyzes the formation of GSH by adding glycine to cglutamylcysteine [bib_ref] Glutathione synthesis, Lu [/bib_ref] [bib_ref] Redox environment of the cell as viewed through the redox state of..., Schafer [/bib_ref]. Notably, glutamate and cysteine are linked by an unconventional c-linkage peptide bond, which can be hydrolyzed only by c-glutamyl transpeptidase (GGT) [bib_ref] Glutathione synthesis, Lu [/bib_ref]. Since GGT is expressed on the exofacial surface of specific cells (e.g., the liver, lung, vascular endothelium, kidney tubular cells, and biliary epithelial cells) [bib_ref] Gamma-glutamyl transferase: a novel cardiovascular risk biomarker, Mason [/bib_ref] , degradation of GSH takes place only in the extracellular space; intracellular GSH is resistant to degradation [bib_ref] Glutathione synthesis, Lu [/bib_ref].
The regulation of GSH synthesis is determined by the availability of its constituent amino acids, especially cysteine and glutamate, and the enzymatic activity of GCL. An excellent review by Lu in 2013 systematically summarized the mechanisms involved in modulations of cysteine availability and GCL activity [bib_ref] Glutathione synthesis, Lu [/bib_ref]. In the context of this review, we discuss recently identified mechanisms regulating cellular GSH levels.
Hypoxia-inducible factor 1a (HIF-1a) is a central transcriptional regulator of cellular metabolism under hypoxic conditions. Ample evidence supports that (pseudo)hypoxia increases cellular GSH levels via both HIF-1a-dependent and HIF-1a-independent mechanisms. Lu et al. reported that the GCLM subunit and the cystine transporter (xCT, encoded by SLC7A11 gene) are transcriptional targets of HIF-1a in triple negative breast cancer cells under hypoxia [bib_ref] Chemotherapy triggers HIF-1-dependent glutathione synthesis and copper chelation that induces the breast..., Lu [/bib_ref]. Treating these cells with chemotherapeutic drugs stimulated the mRNA and protein expression of these two genes and increased intracellular GSH levels through an HIF-1adependent mechanism [bib_ref] Chemotherapy triggers HIF-1-dependent glutathione synthesis and copper chelation that induces the breast..., Lu [/bib_ref]. Knockout of prolyl hydroxylase domain-containing protein 2 (PHD2) stabilized HIF-1a protein and activated HIF-1a signaling, which transcriptionally upregulated glutaminase 1 (GLS1) expression leading to a decrease in ROS production and increases in cellular GSH levels, GSH/GSSG, and cell survival in murine periosteal progenitor cells, suggesting that HIF-1a signaling promotes glutaminolysis to provide glutamate for GSH synthesis [bib_ref] HIF-1alpha Promotes glutamine-mediated redox homeostasis and glycogendependent bioenergetics to support postimplantation bone..., Stegen [/bib_ref].
Interestingly, HIF-1a-independent regulation of GSH synthesis has also been reported under hypoxia. Recent work showed that hypoxia upregulated xCT SLC7A11 expression and increased intracellular cystine levels and GSH synthesis in multiple types of cancer cells [bib_ref] Stress-induced inhibition of nonsense-mediated RNA decay regulates intracellular cystine transport and intracellular..., Gardner [/bib_ref]. Mechanistically, SLC7A11 upregulation was mediated by activation of the protein kinase RNA-like ER kinase/eukaryotic initiation FIG. 5. The biosynthesis and cellular distribution of GSH. Glu, Cys, and Gly are three precursor amino acids of GSH. GSH synthesis requires two successive steps catalyzed by two ATP-dependent enzymes: GCL (the ratelimiting enzyme) and GS. Glu can be replenished by endogenous and exogenous Gln via GLS-mediated glutaminolysis. Cys can be generated by reduction of endogenous and exogenous cystine. GSH levels in the cytosol and nucleus are within the 1-11 mM range with a half-cell reduction potential (E hc ) of -290 mV. By contrast, the ER has a more oxidizing environment with GSH/GSSG of 1-3:1 and an E hc of -175 to -185 mV. Cytosolic GSH can be transported into mitochondria through three potential transporters, KGC, DIC, and TTC. Mitochondrial GSH levels and the E hc are comparable with that of cytosolic and nuclear pools. In addition, GSH and GSSG can also be secreted into the extracellular compartment; however, their levels are very low comparatively (within micromolar range). Extracellular GSH can be degraded by the cell surface enzyme GGT to form Glu and Cys-Gly, further metabolized into free Cys and Gly by DP. The resulting amino acids can be reused by cells for intracellular GSH synthesis. Cys, cysteine; Cys-Gly, cysteinyl-glycine; DIC, dicarboxylate carrier; DP, dipeptidases; ER, endoplasmic reticulum; GCL, cglutamylcysteine ligase; GGT, c-glutamyl transpeptidase; GLS, glutaminase; Glu, glutamate; Gly, glycine; GS, GSH synthetase; KGC, a-KG carrier; TTC, tricarboxylate carrier; xCT, cystine transporter.
## 1336
XIAO AND LOSCALZO factor 2a (eIF2a) branch of the ER stress response but not HIF-1a signaling. Activation of transcription factor eIF2a could transcriptionally upregulate SLC7A11 expression or posttranscriptionally stabilize SLC7A11 mRNA in a cell type-specific manner [bib_ref] Stress-induced inhibition of nonsense-mediated RNA decay regulates intracellular cystine transport and intracellular..., Gardner [/bib_ref]. In addition, p53 can transcriptionally upregulate mitochondrial glutaminase (GLS2) expression in several cancer cells under unstressed and stressed conditions (e.g., ionizing radiation and H 2 O 2 ) (36). Enhanced expression of GLS2 by p53 activation or by ectopic overexpression promoted glutaminolysis leading to increases in cellular glutamate and GSH levels [bib_ref] Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant..., Hu [/bib_ref]. The metabolites lactate and fumarate are also regulators of GSH synthesis. For example, blockade of lactate export by inhibition of monocarboxylate transporter 1 increased intracellular lactate levels and decreased cellular c-glutamylcysteine and GSH levels in human Raji Burkitt lymphoma cells [bib_ref] Blocking lactate export by inhibiting the Myc target MCT1 Disables glycolysis and..., Doherty [/bib_ref]. Mechanistically, lactate accumulation inhibited glucose uptake and glycolysis leading to depletion of cellular ATP levels. Since both GSH synthesis enzymes GCL and GS require ATP for activity, depletion of ATP by lactate suppressed activity and consequently GSH synthesis [bib_ref] Blocking lactate export by inhibiting the Myc target MCT1 Disables glycolysis and..., Doherty [/bib_ref].
In addition, chronic accumulation of fumarate by deletion of fumarate hydratase (FH) led to the formation of succinic GSH, a covalent adduct of fumarate and GSH, which correlated with persistent oxidative stress and cellular senescence in renal cells [bib_ref] Address correspondence to: Dr. Joseph Loscalzo Division of Cardiovascular Medicine Department of..., Zheng [/bib_ref]. Interestingly, loss of FH also triggered a compensatory rise in GSH levels by upregulation of cystine transport SLC7A11 expression and increases in cystine uptake and glutaminolysis-dependent GSH biosynthesis [bib_ref] Address correspondence to: Dr. Joseph Loscalzo Division of Cardiovascular Medicine Department of..., Zheng [/bib_ref]. Furthermore, evidence from diabetic rats and cultured mature adipocytes demonstrated that fumarate as an electrophile can directly react with GSH and cysteine residues in proteins via a Michael addition reaction forming S-(2-succinyl)-cysteine (succination), supporting the view that fumarate can irreversibly modify GSH [bib_ref] Succination of protein thiols during adipocyte maturation: a biomarker of mitochondrial stress, Nagai [/bib_ref]. Taken together, these lines of evidence highlight that GSH biosynthesis is influenced by several interrelated mechanisms, including transcriptional, posttranscriptional, and metabolic regulation.
## Cellular distribution of the gsh/gssg couple
The GSH/GSSG couple is the principal redox buffer of a cell owing to its high concentration compared with other redox couples [bib_ref] Redox environment of the cell as viewed through the redox state of..., Schafer [/bib_ref]. Intracellular GSH distributes in different compartments . The majority of intracellular GSH (>80%) is in the cytosol with an estimated concentration of 1-11 mM; mitochondrial GSH accounts for 10%-15% of the GSH, with its concentration estimated to be 5-11 mM. Five to 10% of total intracellular GSH is present in the nucleus with a concentration of the same or greater than cytosolic GSH; only a small percentage of GSH is in the ER [bib_ref] Glutathione synthesis, Lu [/bib_ref] [bib_ref] Redox environment of the cell as viewed through the redox state of..., Schafer [/bib_ref]. Intracellular GSH is predominantly in its reduced form (>98%). The GSH/ GSSG ratio in the cytosol is typically ‡30:1-100:1 with a redox potential of -290 mV; this ratio in the mitochondria is estimated to be >100:1 with a redox potential of £ -300 mV. By contrast, this ratio appears to be 1:1 to 3:1 in the ER with a redox potential of -175 to -185 mV, which accounts for the relatively oxidizing environment of the ER [bib_ref] Reductive stress in inflammation-associated diseases and the pro-oxidant effect of antioxidant agents, Perez-Torres [/bib_ref] [bib_ref] Redox environment of the cell as viewed through the redox state of..., Schafer [/bib_ref].
It is notable that cytosolic GSH can exchange with other intracellular compartments. This compartmental exchange mechanism is essential for oxidative defense in the mitochondrion since it cannot synthesize GSH [bib_ref] Regulation of hepatic glutathione synthesis: current concepts and controversies, Lu [/bib_ref]. GSH is a negatively charged molecule and cannot freely traverse mitochondrial membranes. Thus, the relocalization of cytosolic GSH to mitochondria requires specific carriers . The a-KG carrier (encoded by SLC25A11) and the dicarboxylate transport mechanisms (dicarboxylate carrier, encoded by SLC25A10) are two potential candidates for importing cytosolic GSH to the mitochondria, mainly in the kidney and liver; the tricarboxylate carrier (encoded by SLC25A1) is a potential GSH transporter in the brain and astrocytes [bib_ref] Glutathione and mitochondria, Ribas [/bib_ref]. By contrast, some organelles, such as the nucleus, have their own GSH pools, which are independent of cytosolic GSH [bib_ref] Redox environment of the cell as viewed through the redox state of..., Schafer [/bib_ref]. This interesting phenomenon implies that cells likely preserve nuclear GSH to maintain a reducing environment and to prevent potential oxidative DNA damage. Thus, the distribution of intracellular GSH is organelle specific and exchange among different (but not all) pools is essential for maintenance of GSH homeostasis.
Mammalian cells can continuously secret GSH and GSSG into the extracellular compartment. In vivo, the liver is the major source of GSH in the circulation [bib_ref] Glutathione synthesis, Lu [/bib_ref] [bib_ref] Redox environment of the cell as viewed through the redox state of..., Schafer [/bib_ref]. GSH levels in human plasma of healthy adults were estimated to be 2-5 lM and the GSSG concentration was found to be much lower (0.14 lM) [bib_ref] Redox state of glutathione in human plasma, Jones [/bib_ref] [bib_ref] Blood and plasma glutathione measured in healthy subjects by HPLC: relation to..., Michelet [/bib_ref] [bib_ref] Quantification of glutathione in plasma samples by HPLC using 4-fluoro-7-nitrobenzofurazan as a..., Wang [/bib_ref]. Intriguingly, plasma GSH levels declined in the elderly and in diabetic patients with a corresponding rise in GSSG levels [bib_ref] Glutathione in human plasma: decline in association with aging, agerelated macular degeneration,..., Samiec [/bib_ref] , indicating a shift to a more oxidizing state in aging and diabetes. As mentioned above, extracellular GSH can be catabolized only in the extracellular space of certain cell types that express the GGT enzyme [bib_ref] Glutathione: new roles in redox signaling for an old antioxidant, Aquilano [/bib_ref]. GGT hydrolyzes GSH into glutamate and cysteinyl-glycine, which is further catabolized into cysteine and glycine by dipeptidases at the cell surface (2, 102) . The resulting amino acids can be taken up by cells for intracellular GSH synthesis. Therefore, this extra/intercellular communication mechanism ensures cellular GSH levels are maintained optimally in the steady state.
## Reductive stress induced by nad(p)h and gsh
As discussed in the Cellular Redox Network and Redox Stress section, NAD(H) can modulate energy metabolism; and NAD(P)H and GSH are indispensable reducing equivalents in response to oxidative stress. Paradoxically, excessive accumulation of cellular NAD(P)H and/or GSH leads to reductive stress and cellular dysfunction.
## Nadh and reductive stress
An excess of NADH levels leads to reductive stress and ultimately ROS production [fig_ref] FIG. 6: Excess in NADH levels induces reductive stress [/fig_ref]. Mitochondrial NADH is oxidized to NAD + at mitochondrial respiratory complex I (NADH dehydrogenase). Emerging evidence supports that NADH oxidation at complex I is inhibited under hypoxia, which leads to a buildup of NADH and consequent reductive stress. Berney et al. reported that deletion of two [NiFe]hydrogenases, Hyd2 and Hyd3, increased the NADH/NAD + ratio twofold under hypoxic conditions, resulting in reductive stress and cell death in oxygen-obligate mycobacteria [bib_ref] An obligately aerobic soil bacterium activates fermentative hydrogen production to survive reductive..., Berney [/bib_ref]. Likewise, hypoxia increased cytosolic NADH/NAD + (indicated by an increase in lactate/pyruvate) and induced reductive stress, which correlated with ATP depletion and cytotoxicity in primary rat hepatocytes [bib_ref] Modulating hypoxia-induced hepatocyte injury by affecting intracellular redox state, Khan [/bib_ref]. Under hypoxia, accumulation of NADH is believed to provide more electrons for one-electron reduction of oxygen to O 2 - . Indeed, increases in cytosolic NADH/NAD + and mitochondrial NAD(P)H levels were coupled with elevated mitochondrial ROS production in hypoxia-challenged bovine coronary artery smooth muscle cells (24). Recently, we reported that hypoxia triggered reductive stress and enhanced mitochondrial ROS generation in primary human lung fibroblasts [bib_ref] Hypoxia-mediated increases in L-2-hydroxyglutarate coordinate the metabolic response to reductive stress, Oldham [/bib_ref].
In agreement with these in vitro studies, NADH-induced reductive stress and elevated mitochondrial ROS production were also observed in a cardiac ischemia/reperfusion (IR) injury mouse model. During ischemia, high NADH levels triggered a reversal of succinate dehydrogenase (SDH; complex II) activity leading to accumulation of succinate in various tissues, including the heart. After reperfusion, the accumulated succinate was rapidly reoxidized by SDH leading to ROS generation at complex I through a reverse electron transport mode and thereby cardiac IR injury [bib_ref] Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS, Chouchani [/bib_ref]. Blockade of ischemic succinate accumulation by inhibition of SDH attenuated mitochondrial ROS production and cardiac IR injury [bib_ref] Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS, Chouchani [/bib_ref]. Therefore, this evidence from various sources indicates that hypoxia increases the NADH pool leading to ROS generation and reductive stress.
In addition, reductive stress resulting from high NADH is also associated with elevated ROS production under ambient oxygen tensions. For example, addition of exogenous complex I substrates, such as glutamate plus malate, or a-KG, significantly augmented NADH levels and mitochondrial membrane potential, which stimulated H 2 O 2 production by *10-fold in isolated rat brain mitochondria [bib_ref] Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox..., Starkov [/bib_ref]. Interestingly, this increase in H 2 O 2 production was diminished by over 50% when NADH oxidation and mitochondrial respiration were enhanced by subsequent addition of ADP or the respiratory uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Furthermore, subsequent addition of the complex I inhibitor rotenone entirely blocked NADH oxidation and caused a potentiation in H 2 O 2 production after mitochondrial membrane potential was collapsed by addition of FCCP [bib_ref] Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox..., Starkov [/bib_ref] , indicating that a high NADH level is crucial for ROS production at complex I.
Likewise, in isolated bovine heart mitochondria, addition of NADH (1-3 lM) promoted electron transfer from reduced flavin to O 2 forming O 2 at complex I within seconds (100-200 sec) [bib_ref] The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine..., Kussmaul [/bib_ref]. Treating rat L6 myoblasts with the antioxidant N-acetyl-l-cysteine (NAC; 1 mM for 1 h) induced reductive stress by increasing the NADH/NAD + ratio, mitochondrial H 2 O 2 levels, and free radical leak [bib_ref] Reductive stress impairs myoblasts mitochondrial function and triggers mitochondrial hormesis, Singh [/bib_ref]. In addition, insulin stimulation increased cellular lactate production and lactate/ pyruvate (proportional to NADH/NAD + ), which promoted cellular O 2 production by NADH-dependent NOX enzymes in cultured SD rat thoracic aortic smooth muscle cells [bib_ref] Insulin-stimulated NADH/NAD+ redox state increases NAD(P)H oxidase activity in cultured rat vascular..., Kahn [/bib_ref]. Pretreatment with pyruvate or OAA significantly mitigated insulin-induced reductive stress in these cells. These cell-free and cell culture studies suggest that a high NADH/NAD + promotes ROS production and induces reductive stress.
The mitochondrial NNT enzyme catalyzes reversible interconversion of the NADH/NAD + and NADPH/NADP + redox couples. Mounting evidence demonstrates that dysfunction of NNT leads to reductive stress. The NNT enzyme has a loss-offunction variant in C57BL/6J mice owing to a spontaneous mutation at the first exon and a multiple exon deletion (exon 7-11) in the NNT gene [bib_ref] A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J..., Toye [/bib_ref]. Compared with NNT wild-type mice (C57BL/6N mice), isolated liver mitochondria from C57BL/6J mice had higher NADH/total NAD(H) and lower NADPH/total NADP(H) on ADP stimulation, which correlated with a reduced rate of removal of exogenous organic peroxides (33, [bib_ref] The contribution of nicotinamide nucleotide transhydrogenase to peroxide detoxification is dependent on..., Ronchi [/bib_ref].
Similarly, in isolated gastrocnemius muscle fibers of C57BL/ 6J mice, addition of pyruvate increased NADH production by the PDH complex, which correlated with a twofold elevation in H 2 O 2 levels (17). The production of H 2 O 2 was further augmented by fivefold when PDH complex flux was enhanced by the combination of pyruvate and carnitine [bib_ref] Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy-consuming redox circuit, Fisher-Wellman [/bib_ref]. By contrast, H 2 O 2 production was not affected in isolated muscle fibers from C57BL/6N mice (functional NNT) when flux from the PDH complex was stimulated by the same substrates [bib_ref] Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy-consuming redox circuit, Fisher-Wellman [/bib_ref]. These results suggest that NNT loss-of-function leads to reductive stress through increasing NADH accumulation and decreasing NADPH production, and that NNT can integrate with the PDH complex to form a redox circuit regulating H 2 O 2 production. In addition, knockdown of NNT increased cellular and mitochondrial NADH/total NAD(H) in human SK-Hep1 cells under basal and ADP-stimulated conditions compared with control shRNA-transfected cells, whereas cellular and mitochondrial NADPH/total NADP(H) was much lower in NNT-silenced cells, which correlated with an increase in mitochondrial ROS generation (33). These findings suggest that NNT loss-offunction blocks the interconversion of NADH to NADPH leading to NADH accumulation and reductive stress.
These observations were further supported by an in vivo study [bib_ref] Reversal of mitochondrial transhydrogenase causes oxidative stress in heart failure, Nickel [/bib_ref]. showed that under pathological cardiac pressure overload induced by transverse aortic constriction, NNT wild-type C57BL/6N mice displayed more severe heart failure and higher mortality compared with C57BL/6J mice lacking functional NNT activity [bib_ref] Reversal of mitochondrial transhydrogenase causes oxidative stress in heart failure, Nickel [/bib_ref]. Enhanced failure in the myocardium of wild-type mice coincided with a decline in NADPH levels and an increase in NADH, H 2 O 2 production, and oxidative damage [bib_ref] Reversal of mitochondrial transhydrogenase causes oxidative stress in heart failure, Nickel [/bib_ref]. These findings suggest that pathological cardiac pressure overload induces NNT reverse functional mode to generate NADH at the expense of NADPH resulting in a high NADH/NAD + and reductive stress, further promoting ROS at complex I leading to oxidative injury and cell death [fig_ref] FIG. 6: Excess in NADH levels induces reductive stress [/fig_ref].
Of note, the NADH/NAD + imbalance-induced reductive stress is a common phenomenon in diabetes and diabetic syndromes. The central hypothesis is that diabetic hyperglycemia causes a pseudohypoxic increase in the NADH/ Under stressed states, such as exogenous addition of Cx I substrates, hypoxia, NNT reversal, NNT inactivation, and RET, mitochondrial NADH/NAD + increases leading to oneelectron reduction of oxygen to O 2 or/and two-electron reduction of oxygen into H 2 O 2 at respiratory Cx I. Extensive reductive ROS levels result in reductive stress, which is detrimental to cellular function. Cx I, complex I; RET, reverse electron transfer.
NAD + ratio and reductive stress [bib_ref] Hyperglycemic stress and carbon stress in diabetic glucotoxicity, Luo [/bib_ref] [bib_ref] Hyperglycemic pseudohypoxia and diabetic complications, Williamson [/bib_ref]. Specifically, during chronic hyperglycemia, NADH production is enhanced through glycolysis and the polyol pathways, whereas NAD + levels are depleted owing to overactivation of NAD +consuming enzymes (e.g., poly ADP ribose polymerase [PARP]), together leading to an increase in the NADH/NAD + ratio and promoting ROS production by mitochondrial respiratory complex I due to NADH overload, eventually resulting in reductive stress [bib_ref] Cytosolic NADH/ NAD+, free radicals, and vascular dysfunction in early diabetes mellitus, Ido [/bib_ref] [bib_ref] Sources and implications of NADH/NAD(+) redox imbalance in diabetes and its complications, Wu [/bib_ref] [bib_ref] Pathogenesis of chronic hyperglycemia: from reductive stress to oxidative stress, Yan [/bib_ref]. For example, using a streptozotocin-induced diabetic rat model, Wu et al. found activation of the polyol pathway and an upregulation of PARP-1 in the lungs of diabetic rats compared with nondiabetic animals, which correlated with increases in the NADH/NAD + ratio, the activities of mitochondrial respiratory complex I-IV, and cellular ROS production [bib_ref] Redox imbalance and mitochondrial abnormalities in the diabetic lung, Wu [/bib_ref] , supporting the view that reductive stress occurs in the development of diabetic complications. Taken together, these lines of in vitro and in vivo evidence highlight that excess NADH induces reductive stress and ultimately promotes ROS generation.
## Nadph/gsh and reductive stress
NADPH and GSH are essential for oxidative stress defense; and NADPH is indispensable for GSH recycling by GR (see the Cellular Redox Network and Redox Stress section). However, excessive levels of cellular GSH and/or NADPH also lead to reductive stress .
NADPH is a substrate for NOX enzymes to produce H 2 O 2 and O 2 -(5). As expected, cardiac-specific overexpression of wild-type NOX4 (WT-NOX4) decreased NADPH/NADP + and GSH/GSSG, which correlated with enhanced ROS production and cardiac dysfunction in mice under IR challenge [bib_ref] Elimination of NADPH oxidase activity promotes reductive stress and sensitizes the heart..., Yu [/bib_ref]. By contrast, overexpression of a dominant negative mutant of NOX4 (DN-NOX4; loss of NOX4 activity) increased the ratios of these two redox couples in the myocardium. Paradoxically, elevated ROS levels and cardiac dysfunction were also observed in DN-NOX4 mice under IR challenge, indicating that high NADPH and GSH levels also enhance ROS generation and induce reductive stress by mechanisms yet to be determined [bib_ref] Elimination of NADPH oxidase activity promotes reductive stress and sensitizes the heart..., Yu [/bib_ref].
G6PD is the major source of the cytosolic NADPH pool (see the Metabolic Sources and Cellular Distribution of the NAD(H) and NADP(H) Couples section). Overexpression of G6PD increased cellular NADPH levels and upregulated the expression of NOX cofactors, which potentiated ROS production and oxidative damage in mouse pancreatic b cells and thymic lymphoma cells [bib_ref] G6PD up-regulation promotes pancreatic beta-cell dysfunction, Lee [/bib_ref] [bib_ref] Glucose 6-phosphate dehydrogenase overexpression models glucose deprivation and sensitizes lymphoma cells to..., Tome [/bib_ref]. By contrast, loss of G6PD activity was found to be beneficial in many disease models by limiting generation of high NADPH levels and reductive stress. For example, G6PD deficiency reduced NADPH levels and significantly inhibited angiotensin II (Ang II)-induced O 2 production and thickening of the medial layer of the aorta [bib_ref] Glucose-6 phosphate dehydrogenase deficiency decreases the vascular response to angiotensin II, Matsui [/bib_ref]. Similar effects were observed in pacing-induced heart failure where inhibition of G6PD activity abrogated elevations in NADPH levels and ROS production in failing hearts (28).
Furthermore, mice with cardiac-specific overexpression of a mutated human aB-crystallin (R120G mutant) developed protein aggregation cardiomyopathy and exhibited reductive stress in the heart as evidenced by increases in GSH levels, GSH/GSSG, and the activities of GR, G6PD, catalase, and GPx [bib_ref] Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in..., Rajasekaran [/bib_ref]. Notably, these pathological alternations and reductive stress were significantly normalized by replacing the wild-type G6PD with a hypomorphic G6PD mutant in the R120G mice [bib_ref] Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in..., Rajasekaran [/bib_ref] , suggesting that the G6PD-mediated reductive stress contributes to the development of this disease. Mechanistically, the reductive stress observed in R120G mutant mice was attributed to activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF-2) signaling pathway, which transcriptionally upregulated the mRNA or/and protein expression of key antioxidant enzymes (e.g., GR, G6PD, GPx1, and catalase) and GSH synthetic enzymes (e.g., GCLC and GCLM), leading to increases in the GSH content and GSH/GSSG ratio, and to a decrease in ROS levels in heart tissue [bib_ref] Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in..., Rajasekaran [/bib_ref] [bib_ref] Constitutive activation of Nrf2 induces a stable reductive state in the mouse..., Shanmugam [/bib_ref]. Importantly, knockout of NRF2 significantly aborted the upregulated gene expression, normalized reductive stress, and reduced cardiac hypertrophy in R120G mutant mice [bib_ref] Nrf2 deficiency prevents reductive stress-induced hypertrophic cardiomyopathy, Kannan [/bib_ref] [bib_ref] Sustained activation of nuclear erythroid 2-related factor 2/ antioxidant response element signaling..., Rajasekaran [/bib_ref].
Likewise, a lamin C mutation in muscle tissues of Drosophila or human muscular dystrophy patients led to reductive stress demonstrated by increases in GSH and NADPH levels FIG. 7. Excess in NADPH or/and GSH levels triggers reductive stress. Increases in NADPH/NADP + or/and GSH/ GSSG due to increases in their production (e.g., G6PD overexpression, NRF-2 activation, Hsp27 overexpression, GCL overexpression, and lamin C mutation) or decreases in their consumption (e.g., overexpression of DN-NOX4) lead to reductive stress. DN-NOX4, dominant negative NADPH oxidase 4; Hsp27, heat shock protein 27; IR, ischemia/reperfusion; NRF-2, nuclear factor (erythroid-derived 2)-like 2. and activation of the NRF-2 pathway [bib_ref] Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway, Dialynas [/bib_ref]. Mechanistically, the increase in NADPH levels was contributed by elevated IDH activity but was independent of G6PD and 6PGD activity in this model [bib_ref] Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway, Dialynas [/bib_ref]. Unlike under oxidative stress where NRF-2 activation is mediated by oxidative inactivation of its sequester protein Keap-1, its activation under reductive stress was mediated by upregulation of an autophagy adaptor p62/ SQSTM1, which also binds Keap-1 and thus enables nuclear translocation of NRF-2 and activation of its target genes [bib_ref] Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway, Dialynas [/bib_ref].
In addition, transgenic mice with cardiac-specific heat shock protein 27 (Hsp27) overexpression developed cardiac hypertrophy and dysfunction, which correlated with increases in GSH levels, GSH/GSSG, and GPx1 activity and with a decrease in total ROS levels in the heart (119). Inhibition of GPx1 activity partially normalized the cardiomyopathy in Hsp27 transgenic mice, suggesting that reductive stress induced by excess GSH levels and GPx1 activity contributes to cardiac dysfunction in this model [bib_ref] Involvement of reductive stress in the cardiomyopathy in transgenic mice with cardiac-specific..., Zhang [/bib_ref]. Furthermore, increases in cellular GSH levels by either supplementing with the thiol-donor NAC or overexpressing GSH synthetic enzyme GCLC or/and GCLM subunit(s) diminished cellular ROS levels and reduced cellular 2GSH/GSSG redox potential by 7-12 mV [bib_ref] Glutathione-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity, Zhang [/bib_ref]. Paradoxically, this more reducing redox environment correlated with increases in mitochondrial oxidation and cell death, suggesting that elevated GSH levels result in reductive stress and cytotoxicity [bib_ref] Glutathione-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity, Zhang [/bib_ref]. Taken together, these lines of evidence clearly support that excess NADPH or/and GSH levels induce reductive stress.
## Metabolic responses to reductive stress
The NAD(H), NADP(H), and GSH/GSSG redox couples are critical regulators of cellular metabolism. Specifically, glycolysis and the TCA cycle produce NADH, which provides electrons for mitochondrial OXPHOS and ATP production [bib_ref] Aerobic glycolysis: meeting the metabolic requirements of cell proliferation, Sy [/bib_ref]. NADP + supports the PPP to generate NADPH that is indispensable for reductive biosynthesis of nucleotides, amino acids, and lipids [bib_ref] Aerobic glycolysis: meeting the metabolic requirements of cell proliferation, Sy [/bib_ref]. GSH links amino acid metabolism with cellular redox status. Thus, reductive stress induced by altered ratios of these three redox couples affects cellular metabolism and vice versa.
## Metabolic coordination under nad(p)h-induced reductive stress
Metabolic adaption to hypoxia is a vital survival mechanism for metazoa. Under hypoxia, cell metabolism shifts away from mitochondrial OXPHOS to glycolysis for energy production, which is primarily mediated by activation of HIF-1a signaling. As mentioned in the Reductive Stress Induced by NAD(P)H and GSH section, hypoxia also induces reductive stress by NADH accumulation. Whether or not there is a link between reductive stress and metabolic reprogramming had not been well understood until recently.
We and others showed that l-2-hydroxyglutarate (L2HG), a reduced metabolite of a-KG by MDH1/2 or LDHA, selectively accumulated as a fundamental response to hypoxia in various primary and cancer cells [bib_ref] Hypoxia Induces Production of L-2-Hydroxyglutarate, Intlekofer [/bib_ref] [bib_ref] Hypoxia-mediated increases in L-2-hydroxyglutarate coordinate the metabolic response to reductive stress, Oldham [/bib_ref]. The accumulation of L2HG levels correlated with increases in cellular NADH/NAD + and mitochondrial ROS production indicative of reductive stress under hypoxia [bib_ref] Hypoxia-mediated increases in L-2-hydroxyglutarate coordinate the metabolic response to reductive stress, Oldham [/bib_ref]. Strikingly, increasing cellular L2HG levels by knockdown of L2HG dehydrogenase (the only known enzyme that oxidizes L2HG back to a-KG) decreased mitochondrial oxygen consumption and lactate production, which indicates that L2HG could inhibit both mitochondrial respiration and glycolysis to blunt their production of NADH and to mitigate attendant reductive stress [bib_ref] Hypoxia-mediated increases in L-2-hydroxyglutarate coordinate the metabolic response to reductive stress, Oldham [/bib_ref].
The mitochondrial NNT enzyme is an important source of mitochondrial NADPH levels (see the Metabolic Sources and Cellular Distribution of the NAD(H) and NADP(H) Couples section). Overexpression of NNT increased NADPH/NADP + resulting in reductive stress in melanoma cells (23). Intriguingly, NADPH-dependent reductive stress stimulated glutaminolysis to generate a-KG, which was then converted to succinyl-CoA by oxidative decarboxylation in the TCA cycle or to citrate via IDH2-mediated reductive carboxylation (23). By contrast, NNT silencing decreased NADH/NAD + and NADPH/NADP + , which correlated with a decline in glutamine catabolism and a rise in the flux of glucose oxidation into the TCA cycle and energy production thereby (23). These results suggest that melanoma cells switch their energy source from glucose to glutamine under conditions of reductive stress.
A very recent study also reported reductive stress and metabolic reprogramming in human SK-Hep1 cells with NNT knockdown (33). Specifically, knockdown of NNT resulted in elevations in cellular and mitochondrial NADH/total NAD(H) and decreases in cellular and mitochondrial NADPH/total NADP(H) with a concomitant increase in mitochondrial O 2 production, indicating that NNT silencing induces reductive stress in these cells (33). Intriguingly, NNT silencing also promoted mitochondrial membrane hyperpolarization, increased oxygen consumption and ATP production, and diminished lactate production, implying a metabolic shift from glycolysis to mitochondrial OXPHOS to correct the accumulation of NADH in these cells. This metabolic shift led these cells being more vulnerable to the cytotoxic effects of the mitochondrial OXPHOS inhibitor rotenone but more resistant to the glycolysis inhibitor 3-bromopyruvate (33). Moreover, enhanced glutaminolysis and reductive carboxylation were also observed in NNT-silenced cells, leading to accumulation of a-KG and its derivative metabolites (e.g., citrate, fumarate, and malate). Mechanistically, this inhibition of glycolytic activity was attributable to destabilization of HIF-1a protein, possibly through a-KG-dependent PHD enzyme-mediated hydroxylation and enhanced degradation of HIF-1a protein (33).
In addition, primary mouse aortic endothelial cells isolated from C57BL/6J mice consumed less oxygen, had lower GPx activity, and produced more O 2 when stimulated by Ang II compared with cells from C57BL/6N mice, suggesting that loss of NNT function impairs mitochondrial function [bib_ref] Nicotinamide nucleotide transhydrogenase activity impacts mitochondrial redox balance and the development of..., Leskov [/bib_ref]. Furthermore, increasing evidence strongly supports that reductive stress observed in C57BL/6J mice lacking functional NNT correlated with systemic metabolic alterations demonstrated by glucose intolerance, reductions in glucose-induced insulin secretion and energy expenditure, and an increase in susceptibility to high-fat diet-induced obesity [bib_ref] Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy-consuming redox circuit, Fisher-Wellman [/bib_ref] [bib_ref] Diet-induced obesity in two C57BL/6 substrains with intact or mutant nicotinamide nucleotide..., Nicholson [/bib_ref] [bib_ref] Alterations of pancreatic islet structure, metabolism and gene expression in dietinduced obese..., Roat [/bib_ref].
Of note, NNT can also operate in a reverse mode to generate NADH from NADPH and NAD + , resulting in elevated NADH/NAD + , reductive stress, and metabolic adaption. For example, in C57BL/6N mice challenged with pressure overload using transverse aortic constriction, NNT operated through its reverse enzymatic mode, which promoted NADH production at the expense of NADPH and its antioxidant capacity [bib_ref] Reversal of mitochondrial transhydrogenase causes oxidative stress in heart failure, Nickel [/bib_ref]. Increased NADH production accelerated mitochondrial respiration by fueling to mitochondrial OX-PHOS, which combined with the compromised antioxidant capacity of NADPH resulted in increases in mitochondrial ROS production and oxidative damage, and ultimately cardiac dysfunction [bib_ref] Reversal of mitochondrial transhydrogenase causes oxidative stress in heart failure, Nickel [/bib_ref]. These findings indicate that cardiomyocytes correct NADH-induced reductive stress by stimulating its oxidation through mitochondrial respiration.
In isolated pancreatic islets of C57BL/6N mice, glucose stimulation increased the NADPH/total NADP(H), which correlated with a reduction in mitochondrial GSH oxidation [bib_ref] NNT reverse mode of operation mediates glucose control of mitochondrial NADPH and..., Santos [/bib_ref]. Interestingly, the increased ratio was caused by a suppression of the reverse operation of NNT enzyme rather than a stimulation of its forward operation since NADH/total NAD(H) was also elevated by the identical challenge. The rise in NAD(P)H levels correlated with enhancement of glucose-stimulated insulin secretion, glycolysis (as demonstrated by elevated levels of G-6-P and F-6-P), and mitochondrial respiration (as demonstrated by increases in glucose oxidation, oxygen consumption, ATP production, and the TCA cycle intermediate metabolites citrate, malate, and fumarate) [bib_ref] NNT reverse mode of operation mediates glucose control of mitochondrial NADPH and..., Santos [/bib_ref]. Collectively, NNT is therefore a key enzyme bridging cell metabolism and reductive stress.
As discussed in the Metabolic Sources and Cellular Distribution of the NAD(H) and NADP(H) Couples section, the malate/aspartate shuttle is a pivotal mechanism for compartmental exchange of NADH and NAD + to maintain high NAD + levels in the cytosol and high NADH levels in mitochondria. Disruption of this exchange mechanism could perturb NADH/NAD + in both compartments and thus could affect cellular redox homeostasis and energy metabolism [fig_ref] FIG. 8: Inhibition of the malate/aspartate NADH shuttle induces reductive stress and metabolic reprogramming [/fig_ref]. For example, knockdown of mitochondrial glutamate-OAA transaminase (GOT2), a key enzyme in the malate/ aspartate shuttle, increased cytosolic NADH levels and concurrently reduced mitochondrial NADH levels, which were concomitant with decreased NADPH/NADP + and an increase in cellular ROS production in PANC-1 cells [bib_ref] SIRT3-dependent GOT2 acetylation status affects the malateaspartate NADH shuttle activity and pancreatic..., Yang [/bib_ref]. GOT2 silencing also significantly inhibited cellular ATP production and cell proliferation, indicating suppression of mitochondrial respiration [bib_ref] SIRT3-dependent GOT2 acetylation status affects the malateaspartate NADH shuttle activity and pancreatic..., Yang [/bib_ref]. Interestingly, recovery of GOT2 activity by re-expressing an acetylation-mimetic GOT2 in these cells inhibited cellular ROS production and restored mitochondrial NADH levels, cellular ATP production, and cell proliferation [bib_ref] SIRT3-dependent GOT2 acetylation status affects the malateaspartate NADH shuttle activity and pancreatic..., Yang [/bib_ref].
Moreover, inhibition of the malate/aspartate shuttle with aminooxyacetic acid (AOA, a potent inhibitor of aspartate aminotransferase) augmented cytosolic NADH/NAD + reflected by elevated lactate/pyruvate and glycerol-3-phosphate/DHAP resulting in a more reductive environment in the cytoplasmic compartment of primary porcine aortic smooth muscle cells [bib_ref] NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle, Barron [/bib_ref]. Notably, AOA treatment also attenuated glucose oxidation and oxygen consumption but increased glycolytic activity and lactate production, indicating a metabolic shift toward glycolysis [bib_ref] NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle, Barron [/bib_ref]. These results suggest that perturbation of the malate/aspartate shuttle results in cytosolic retention of NADH and reductive stress, decreasing NADH availability in mitochondria and possibly increasing NAD + levels in the cytosol, thereby leading to inhibition of NADH-dependent mitochondrial respiration and enhancement of NAD + -dependent glycolysis.
Such important roles of this shuttle have also been demonstrated in vivo. Cardiac-specific deletion of mitochondrial complex I subunit NDUFS4 protein resulted in accumulation of NADH in mitochondria and elevation in NADH/NAD + in rodent heart, which correlated with the accelerated development of heart failure induced by pathological pressure overload or isoproterenol [bib_ref] Mitochondrial complex I deficiency increases protein acetylation and accelerates heart failure, Karamanlidis [/bib_ref] [bib_ref] Normalization of NAD+ redox balance as a therapy for heart failure, Lee [/bib_ref]. Intriguingly, isolated cardiomyocytes from these mice produced lower H 2 O 2 and O 2 than cells from wild-type mice [bib_ref] Mitochondrial complex I deficiency increases protein acetylation and accelerates heart failure, Karamanlidis [/bib_ref] [bib_ref] Normalization of NAD+ redox balance as a therapy for heart failure, Lee [/bib_ref] , indicating that complex I dysfunction leads to reductive stress in the failing heart. Since supraphysiological levels of NADH were reported to inhibit NAD + -dependent sirtuin deacetylase (SIRT1-7) (50), accumulated NADH in mitochondria repressed mitochondrial SIRT3 activity leading to hyperacetylation of mitochondrial The malate/aspartate shuttle exchanges cytosolic NAD(H) with mitochondrial NAD(H) to maintain high cytosolic NAD + levels, which are required for glycolysis, and high mitochondrial NADH levels, which provide electrons for mitochondrial OXPHOS. (B) Inhibition of the malate/aspartate shuttle by silencing GOT2, suppressing SIRT3 activity, or using the chemical inhibitor AOA leads to accumulation of cytosolic NADH and increases in cytosolic NADH/NAD + and reductive ROS generation, indicative of reductive stress. NADH-induced reductive stress shifts cell metabolism from mitochondrial respiration to glycolysis. Enhancement of this shuttle activity by overexpression of AGC1 or recovery of GOT2 activity significantly increases mitochondrial NADH levels and enhances mitochondrial respiration. AGC1, aspartate-glutamate carrier 1; AOA, aminooxyacetic acid; GOT2, glutamate-oxaloacetate transaminase; Mal-Asp shuttle, malate/aspartate shuttle; OXPHOS, oxidative phosphorylation; SIRT3, sirtuin deacetylase family member 3.
proteins [bib_ref] Mitochondrial complex I deficiency increases protein acetylation and accelerates heart failure, Karamanlidis [/bib_ref] [bib_ref] Normalization of NAD+ redox balance as a therapy for heart failure, Lee [/bib_ref] [fig_ref] FIG. 8: Inhibition of the malate/aspartate NADH shuttle induces reductive stress and metabolic reprogramming [/fig_ref]. Specifically, the malate/aspartate shuttle proteins MDH2, a-KG/malate carrier, and GOT2 were found to be hyperacetylated leading to suppression of the transport and oxidation of cytosolic NADH in mitochondria, which correlated with decreases in oxygen consumption and cardiac energetics (manifested by a reduction in phosphocreatine/ATP) [bib_ref] Mitochondrial complex I deficiency increases protein acetylation and accelerates heart failure, Karamanlidis [/bib_ref] [bib_ref] Normalization of NAD+ redox balance as a therapy for heart failure, Lee [/bib_ref]. All of these pathological and biochemical changes were significantly abrogated by feeding the NAD + precursor NAM mononucleotide to mice or overexpressing NAM phosphoribosyltransferase, a rate-limiting enzyme of NAD + biosynthesis, in cardiac tissue [bib_ref] Mitochondrial complex I deficiency increases protein acetylation and accelerates heart failure, Karamanlidis [/bib_ref] [bib_ref] Normalization of NAD+ redox balance as a therapy for heart failure, Lee [/bib_ref]. Therefore, these results support that reductive stress induced by high NADH/NAD + impairs mitochondrial function and energy metabolism in the heart.
By contrast, overexpression of human aspartate-glutamate carrier 1 (AGC1, encoded by SLC25A12 gene) enhanced malate/aspartate shuttle activity on glucose stimulation in rat INS-1E b cells as demonstrated by increases in mitochondrial NAD(P)H levels and cellular glutamate levels (83) [fig_ref] FIG. 8: Inhibition of the malate/aspartate NADH shuttle induces reductive stress and metabolic reprogramming [/fig_ref]. Moreover, AGC1 overexpression also potentiated glucoseinduced increases in glucose oxidation, mitochondrial membrane hyperpolarization, ATP production, and insulin secretion but inhibited lactate secretion compared with empty adenoviral vector-transfected cells [bib_ref] The malate-aspartate NADH shuttle member Aralar1 determines glucose metabolic fate, mitochondrial activity,..., Rubi [/bib_ref] , indicating that elevated NAD(P)H levels in mitochondria promote mitochondrial respiration and energy production. Collectively, these lines of evidence support the views that the malate/aspartate shuttle is essential for carrying electrons from cytosolic NADH to mitochondria for OXPHOS and that disruption of such a shuttle leads to reductive stress and metabolic dysfunction.
## Metabolic coordination under gsh-induced reductive stress
As discussed in the Biosynthesis and Cellular Distribution of GSH section, glutamate is a precursor amino acid of GSH. Glutamate is also a source of a-KG through glutaminolysis, where glutamine is hydrolyzed into glutamate by GLSs (cytosolic GLS1 and mitochondrial GLS2) followed by glutamate conversion into a-KG by GLUDs. Consequently, enhancing glutaminolysis could increase cellular GSH levels and alter cellular redox status and energy metabolism. For example, overexpression of GLS2 markedly elevated cellular glutamate, GSH, and NADH levels as well as GSH/GSSG in three human cancer cell lines, which correlated with diminution in cellular basal ROS levels, implying reductive stress occurs in these cells [bib_ref] Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant..., Hu [/bib_ref]. Interestingly, GLS2-overexpressing cells exhibited increases in cellular a-KG levels, mitochondrial oxygen consumption, and ATP production, suggesting that mitochondrial OXPHOS and the TCA cycle are enhanced under reductive stress. Importantly, reductive stress and metabolic stimulation were significantly normalized by silencing GLS2 (36).
By contrast, GLS1 but not GLS2 was found to mediate HIF-1a-mediated enhancement of glutaminolysis in murine skeletal muscle cells. Activation of HIF-1a signaling by PHD2 deletion under normoxia enhanced glutamine uptake and upregulated GLS1 expression leading to enhanced glutaminolysis and increases in cellular GSH levels and in GSH/GSSG, which correlated with upregulation of antioxidant genes (SOD1, SOD2, catalase, GPx1, and GR) and reduction in cellular and mitochondrial ROS production, indicatives of reductive stress [bib_ref] HIF-1alpha Promotes glutamine-mediated redox homeostasis and glycogendependent bioenergetics to support postimplantation bone..., Stegen [/bib_ref]. Interestingly, this reductive stress was accompanied by decreases in oxygen consumption and palmitate b-oxidation and increases in glycolytic activity and glycogen storage in these cells [bib_ref] HIF-1alpha Promotes glutamine-mediated redox homeostasis and glycogendependent bioenergetics to support postimplantation bone..., Stegen [/bib_ref] , indicating inhibition of mitochondrial function and stimulation of glycolysis and glycogenesis.
Furthermore, a recent study revealed that GSH is required for metabolic integration and reprogramming in murine T cells during inflammation [bib_ref] Glutathione primes T cell metabolism for inflammation, Mak [/bib_ref]. On activation, murine T cells expressed 6-10-fold higher GCLC mRNA than resting T cells, which was accompanied by elevated cellular GSH levels. Activated T cells reprogrammed their metabolism by enhancing fluxes of glutamine into the TCA cycle and of glucose into glycolysis in a c-MYC-dependent manner [bib_ref] Glutathione primes T cell metabolism for inflammation, Mak [/bib_ref]. These metabolic adaptive responses were effectively abolished in T cells with GCLC deletion and recapitulated by exogenous addition of GSH or its precursor NAC [bib_ref] Glutathione primes T cell metabolism for inflammation, Mak [/bib_ref]. Collectively, these findings underscore a close link between GSH-related reductive stress and energy metabolism.
## Concluding remarks
The NAD(H), NADP(H), and GSH/GSSG redox couples are cofactors or/and substrates for many redox and metabolic enzymes. A delicate balance between the reduced and oxidized forms within each redox couple is a prerequisite for support of cellular redox homeostasis and energy metabolism. When that balance is lost, an excess in cellular NAD(P)H and GSH levels leads to reductive stress, metabolic stress, and cell dysfunction.
As an emerging concept, reductive stress complicates, but expands our understanding of the cellular redox environment. Traditionally, a reducing environment is thought to be beneficial for cell functions and biological processes since an oxidizing environment can lead to oxidative damage to proteins, lipids, and nucleic acids. Reductive stress highlights the facts that excess reducing equivalents (NAD(P)H and GSH) are also detrimental to cells via many mechanisms, including disruption of signaling functions of ROS, induction of ROS generation, perturbation of cell metabolism, and inhibition of protein disulfide formation. Thus, mammalian cells must maintain a balanced redox environment to avoid both oxidative and reductive damage.
Current knowledge about the underlying mechanisms of reductive stress and its biological consequences as well as how cells respond to reductive stress remains limited. For example, although it is clear that NADH accumulation under hypoxia induces reductive stress, it is uncertain as to (i) how NADH accumulates under hypoxia beside inhibition of its oxidation by mitochondrial respiration; (ii) whether or not NADH accumulation is compartment-specific; (iii) whether or not the two NADH shuttles are involved in redistribution; (iv) whether or not the NNT enzyme is involved in redistribution; (v) whether and how NADH accumulation affects the metabolic fates of glucose, lipids, and glutamine; and (vi) how cells respond to mitigate this insult. The answers to these questions remain largely open and require future experimental efforts.
In addition, the influences of GSH-induced reductive stress on cellular metabolism and vice versa are also open questions. This work summarizes limited information that is available in the literature on how modulation of GSH biosynthesis affects cellular metabolism. Notably, another important mechanism by which GSH could modulate cellular metabolism is through protein S-glutathionylation. Like phosphorylation, S-glutathionylation is an important posttranslational modification mechanism that regulates biological function, structural protein folding, and subcellular localization of a target protein. Proteins can be glutathionylated through both nonenzymatic and enzymatic reactions.
The former type of S-glutathionylation is primarily processed during oxidative stress and is often nonspecific and irreversible [bib_ref] Redox regulation of mitochondrial function with emphasis on cysteine oxidation reactions, Mailloux [/bib_ref] [bib_ref] Glutathione and mitochondria, Ribas [/bib_ref]. ROS oxidize GSH into GSSG, which reacts with cysteinyl residues (-SH) on proteins to form PSSG. Ample evidence demonstrates that metabolic enzymes are susceptible to glutathionylation under oxidative stress. For example, glycolytic enzymes (e.g., GAPDH, pyruvate kinase, aldolase, phosphoglycerate kinase, triose phosphate isomerase), TCA cycle enzymes (i.e., aconitase, KGDH, IDH3, succinyl-CoA transferase), and mitochondrial OXPHOS protein complexes (Complex I-V) can be glutathionylated, which results in activation or inactivation of their enzymatic activities thus modulating cellular energetics [bib_ref] Site-specific S-glutathiolation of mitochondrial NADH ubiquinone reductase, Chen [/bib_ref] [bib_ref] Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T..., Fratelli [/bib_ref] [bib_ref] Regulation of mitochondrial NADP+-dependent isocitrate dehydrogenase activity by glutathionylation, Is [/bib_ref] [bib_ref] Unearthing the secrets of mitochondrial ROS and glutathione in bioenergetics, Mailloux [/bib_ref] [bib_ref] Reversible inactivation of alpha-ketoglutarate dehydrogenase in response to alterations in the mitochondrial..., Nulton-Persson [/bib_ref].
By contrast, enzyme-driven S-glutathionylation is typically specific and reversible, and is highly controlled by fluctuation in local GSH/GSSG pools [bib_ref] Redox regulation of mitochondrial function with emphasis on cysteine oxidation reactions, Mailloux [/bib_ref] [bib_ref] Glutathione and mitochondria, Ribas [/bib_ref]. The glutathione-Stransferase is the major enzyme mediating S-glutathionylation by adding GSH to cysteinyl residues of a protein producing PSSG [bib_ref] S-glutathionylation: indicator of cell stress and regulator of the unfolded protein response, Townsend [/bib_ref] ; and Grx1 and Grx2 are the primary enzymes catalyzing the reversible reaction [bib_ref] Redox regulation of mitochondrial function with emphasis on cysteine oxidation reactions, Mailloux [/bib_ref]. To date, little is known about whether glutathionylation of proteins, particularly metabolic enzymes, can occur under reductive stress (high GSH/ GSSG) and how this modification impairs cellular metabolic activities. These research topics deserve future investigation.
[fig] FIG. 6: Excess in NADH levels induces reductive stress. [/fig]
[fig] FIG. 8: Inhibition of the malate/aspartate NADH shuttle induces reductive stress and metabolic reprogramming. (A) [/fig]
[table] Table 1: Standard Reduction Potentials (E°¢) of Representative Redox Couples NADH, reduced NAD + ; NADP + , phosphorylated NAD + ; NADPH, reduced NADP + ; O 2 -, superoxide anion; Trx, thioredoxin. [/table]
[table] 20: Freeman H, Shimomura K, Cox RD, and Ashcroft FM. Nicotinamide nucleotide transhydrogenase: a link between insulin secretion, glucose metabolism and oxidative stress. Biochem Soc Trans 34: 806-810, 2006. 21. Freeman HC, Hugill A, Dear NT, Ashcroft FM, and Cox RD. Deletion of nicotinamide nucleotide transhydrogenase: a new quantitive trait locus accounting for glucose intolerance in C57BL/6J mice. Diabetes 55: 2153-2156, 2006. 22. Furchgott RF. Endothelium-derived relaxing factor: discovery, early studies, and identification as nitric oxide. Biosci Rep 19: 235-251, 1999. 23. Gameiro PA, Laviolette LA, Kelleher JK, Iliopoulos O, and Stephanopoulos G. Cofactor balance by nicotinamide nucleotide transhydrogenase (NNT) coordinates reductive carboxylation and glucose catabolism in the tricarboxylic acid (TCA) cycle. J Biol Chem 288: 12967-12977, 2013. 24. Gao Q and Wolin MS. Effects of hypoxia on relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in coronary arterial smooth muscle. Am J Physiol Heart Circ Physiol 295: H978-H989, 2008. 25. Garcia J, Han D, Sancheti H, Yap LP, Kaplowitz N, and Cadenas E. Regulation of mitochondrial glutathione redox status and protein glutathionylation by respiratory substrates. J Biol Chem 285: 39646-39654, 2010. 26. Giorgio M, Trinei M, Migliaccio E, and Pelicci PG. Hydrogen peroxide: a metabolic by-product or a common mediator of ageing signals? Nat Rev Mol Cell Biol 8: 722-728, 2007. 27. Gores GJ, Flarsheim CE, Dawson TL, Nieminen AL, Herman B, and Lemasters JJ. Swelling, reductive stress, and cell death during chemical hypoxia in hepatocytes. Am J Physiol 257: C347-C354, 1989. 28. Gupte SA, Levine RJ, Gupte RS, Young ME, Lionetti V, Labinskyy V, Floyd BC, Ojaimi C, Bellomo M, Wolin MS, and Recchia FA. Glucose-6-phosphate dehydrogenasederived NADPH fuels superoxide production in the failing heart. J Mol Cell Cardiol 41: 340-349, 2006. 29. Han D, Canali R, Garcia J, Aguilera R, Gallaher TK, and Cadenas E. Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione. Biochemistry 44: 11986-11996, 2005. 30. Handy DE and Loscalzo J. Redox Regulation of Mitochondrial Function. Antioxid Redox Signal 16: 1323-1367, 2012. 31. Handy DE and Loscalzo J. Responses to reductive stress in the cardiovascular system. Free Radic Biol Med 109: 114-124, 2017. 32. Handy DE, Lubos E, Yang Y, Galbraith JD, Kelly N, Zhang YY, Leopold JA, and Loscalzo J. Glutathione peroxidase-1 regulates mitochondrial function to modulate redox-dependent cellular responses. J Biol Chem 284: 11913-11921, 2009. 33. Ho HY, Lin YT, Lin G, Wu PR, and Cheng ML. Nicotinamide nucleotide transhydrogenase (NNT) deficiency dysregulates mitochondrial retrograde signaling and impedes proliferation. Redox Biol 12: 916-928, 2017. 34. Holmgren A and Luthman M. Tissue distrubution and subcellular localization of bovine thioredoxin determined by radioimmunoassay. Biochemistry 17: 4071-4077, 1978. 35. Houtkooper RH, Canto C, Wanders RJ, and Auwerx J. The secret life of NAD+: an old metabolite controlling new metabolic signaling pathways. Endocr Rev 31: 194-223, 2010. [/table]
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Heme Oxygenase-1 mRNA Expression in Egyptian Patients With Chronic Liver Disease
The significant relations between the increased HO-1 expression and oxidative stress biomarkers in patients with CLD suggest that HO-1 may play an important role to protect the liver from oxidative stress-dependent damage. Reading this article is recommended to all internists and hepatologists.Background: Chronic liver disease (CLD) is a global medical problem. This disease is associated with increased hepatic oxidative stress. One of the antioxidant enzymes that protect cells against this stress is heme oxygenase-1 (HO-1). Objectives: This study aimed to investigate the mRNA expression of HO-1 in Egyptian patients with CLD and its relation to oxidative stress biomarkers. Patients and Methods: Levels of serum ferritin, carboxyhemoglobin, malondialdehyde (MDA), and erythrocyte-reduced glutathione (GSH) were measured, and HO-1 mRNA expression was detected in 45 CLD patients (15 with nonalcoholic steatohepatitis [NASH], 15 with chronic hepatitis C, and 15 with liver cirrhosis) and 15 healthy controls. Results: HO-1 mRNA expression was increased in patients with NASH, chronic hepatitis C, and liver cirrhosis compared to controls. The expression in cirrhotic patients was significantly higher than that in patients with NASH and chronic hepatitis C. Compared to controls, patients with NASH, chronic hepatitis C, and liver cirrhosis had higher levels of ferritin, carboxyhemoglobin, and MDA and lower levels of GSH. HO-1 mRNA expression was positively correlated with levels of carboxyhemoglobin, serum ferritin, and serum MDA and negatively correlated with levels of erythrocyte GSH in CLD patients. Conclusions: HO-1 mRNA expression was significantly increased in CLD patients, and the increase reflected the severity of the disease. The significant relationship between the increased HO-1 expression and oxidative stress biomarkers in patients with CLD suggests that HO-1 may play an important role in protecting the liver from oxidative stressdependent damage. Therefore, induction of HO-1 could be a novel therapeutic option for CLD.
# Background
Chronic liver disease (CLD) is an important cause of morbidity and mortality and represents a major health problem worldwide. Liver cirrhosis is the final common pathway of many cases of CLD; however, the exact point at which a disease process produces cirrhosis is difficult to define (1). Hepatitis C virus (HCV) infection causes liver disease characterized by inflammation, cell damage, and progressive liver fibrosis leading to cirrhosis [bib_ref] Course and outcome of hepatitis C, Hoofnagle [/bib_ref]. Non-alcoholic steatohepatitis (NASH) is a clinical-pathological condition characterized by a necroinflammatory disorder with fatty infiltration of hepatocytes and may progress to fibrosis or cirrhosis, with a fatal outcome [bib_ref] Nonalcoholic steatohepatitis, Neuschwander-Tetri [/bib_ref]. Although the precise molecular mechanisms by which HCV causes liver injury are not fully understood, recent evidence demonstrates that HCV causes oxidative stress in human liver cells, due to the generation of reactive oxygen species (ROS) [bib_ref] Differential contribution of hepatitis C virus NS5A and core proteins to the..., Garcia-Mediavilla [/bib_ref] [bib_ref] Role of oxidative stress in the pathogenesis of chronic hepatitis C (CHC), Fierbinteanu-Braticevici [/bib_ref]. Moreover, oxidative stress and lipid peroxidation play an important role in the pathogenesis of NASH, as their end products can induce hepatocellular injury and fibrogenesis [bib_ref] Pathogenesis of nonalcoholic steatohepatitis (NASH), Ma [/bib_ref] [bib_ref] Oxidative stress and antioxidants in hepatic pathogenesis, Ha [/bib_ref]. The liver is the major organ that detoxifies excess heme molecules by the action of the heme oxygenase (HO) enzyme that catalyzes the initial and rate-limiting reaction in heme catabolism and cleaves pro-oxidant heme to form iron, carbon monoxide (CO), and biliverdin, which is subsequently converted to bilirubin by biliverdin reductase [bib_ref] The biological significance and physiological role of heme oxygenase, Abraham [/bib_ref]. The liver contains 2 HO isozymes for physiologic degradation of the heme; HO-1 is inducible various stimuli, including cytokines, heavy metals, and reactive oxygen species, while HO-2 is constitutively produced. The antioxidant property of HO is derived from the elimination of pro-oxidant heme and from the biological activities of its reaction products: biliverdin, bilirubin, and iron [bib_ref] Induction of Haem Oxygenase as a Defence Against Oxidative Stress, Stocker [/bib_ref]. Overproduction of biliverdin and bilirubin serves as an antioxidative defense mechanism [bib_ref] Evidence of involvement of bilirubin as physiological protector against oxidative damage, Llesuy [/bib_ref]. Free iron inhibits the de novo synthesis of heme, binds to iron regulatory protein, and stimulates the biosynthesis of ferritin, which exerts an additional antioxidant effect. Another important role of the HO reaction in regulating liver function is to generate CO, which has anti-inflammatory effects and acts as an endogenous regulator to maintain microvascular blood flow [bib_ref] The heme oxygenase-carbon monoxide system: a regulator of hepatobiliary function, Suematsu [/bib_ref]. CO regulates the hepatic vascular tone in a variety of experimental models of conditions such as ischemia-reperfusion, endotoxemia, and heme overloading [bib_ref] Carbon monoxide produced by intrasinusoidally located haem-oxygenase-1 regulates the vascular tone in..., Van Landeghem [/bib_ref] [bib_ref] Heme oxygenase system in hepatic ischemia-reperfusion injury, Richards [/bib_ref]. Recently, much attention has been paid to physiologic and pathophysiologic roles of the HO enzyme in organ homeostasis [bib_ref] Heme oxygenase-1 expression in disease states, Deshane [/bib_ref]. HO-1 has potent cytoprotective effects; it is an anti-oxidant defense enzyme that converts potentially toxic heme into anti-oxidants [bib_ref] Heme oxygenase-1: a novel therapeutic target in oxidative tissue injuries, Takahashi [/bib_ref] and is a stress-responsive protein that is essential for higher eukaryotes to cope with cellular stress and to regulate cellular iron metabolism [bib_ref] The heme oxygenase system: its role in liver inflammation, Wunder [/bib_ref]. Considering these findings, we investigated the relation between HO-1 mRNA expression in Egyptian patients with CLD and oxidative stress biomarkers.
## Objectives
This study investigated the mRNA expression of HO-1 in Egyptian patients with CLD and the relation of HO-1 expression with oxidative stress biomarkers.
# Patients and methods
## Patients
We randomly selected 45 Egyptian patients with CLD from those admitted to the Internal Medicine Department in the Tanta University Hospital between April 2010 and March 2011. The cohort consisted of 15 patients with NASH (6 men and 9 women) with a mean age of 53 ± 8.9 years, 15 patients with chronic hepatitis C (6 men and 9 women) with a mean age of 52 ± 8.2 years, and 15 patients with liver cirrhosis (8 men and 7 women) with a mean age of 54.5 ± 7.6 years. The study protocol was approved by the General Ethics Council of the Tanta University Hospital, Tanta University, Tanta, Egypt, and all patients gave their informed consent to participate in the study. The diagnosis of NASH was established on the basis of the following clinical and histopathological features: (i) abnormal liver biochemistry for more than 3 months; (ii) liver biopsy showing steatosis (10%) in the presence of lobular and/or portal inflammation, with or without Mallory bodies or fibrosis; and (iii) the exclusion of other liver diseases. Diagnosis of chronic hepatitis C was established by serological detection of anti-HCV antibodies and HCV-RNA, and serum transaminase levels greater than 3 times the upper limit of normal for at least 6 months, and confirmed by liver biopsy. The diagnosis of liver cirrhosis was established on the basis of clinical, biochemical, histological, and ultrasound findings. The etiology of cirrhosis was HCV-related in 8 patients, NASH in 4, and cryptogenic in 3 patients. In addition, all cirrhotic patients had schistosomal periportal hepatic fibrosis. Severity of the liver disease was scored according to Child-Pugh classification [bib_ref] Transection of the oesophagus for bleeding oesophageal varices, Pugh [/bib_ref]. Other causes of chronic liver disease, including drugs, alcohol, chronic hepatitis B virus (HBV) infection, autoimmune liver disease, hereditary hemochromatosis, Wilson disease, and α-1 antitrypsin deficiency, were excluded by appropriate serological studies and findings for liver biopsy samples. Hepatocellular carcinoma was ruled out by abdominal ultrasound, spiral abdominal computed tomography, and alpha fetoprotein determination. Patients did not receive antioxidant vitamin or selenium supplementation within the 2 months preceding their inclusion in the study.
## Controls
Fifteen healthy age-and sex-matched volunteers (9 men and 6 women) with a mean age of 50 ± 3.9 years were recruited for participation as controls. They were selected from medical and paramedical staff and from blood donors in the Tanta University Hospital. They gave informed consent to participate in the study. They were seronega- tive for viral hepatitis markers, and for none of them, liver function tests yielded abnormal results.
## Blood collection and biochemical analysis
Fasting blood samples were obtained aseptically from patients and controls and divided into 3 parts; one part was collected in a vacutainer plain tube without anticoagulant, incubated at 37 ºC for half an hour, and centrifuged at 3,000 ×g for 10 min. This serum sample was used for determination of liver function tests, using conventional methods of clinical chemistry, alpha fetoprotein estimation by radioimmunoassay, anti-HCV antibodies detection using commercially available enzyme-linked immunoassay along with serum HCV RNA determination by reverse transcription-polymerase chain reaction (RT-PCR), HBV surface antigen and HBV core antibodies detection by commercial assays, determination of ferritin levels using a kit supplied from Biosystems (21), determination of lipid peroxidation end-product and malondialdehyde (MDA) levels by the thiobarbituric acid method [bib_ref] Determination of aldehyde lipid peroxidation products: Malondialdehyde and 4-hydroxynonenal, Esterbaurer [/bib_ref]. The second fraction was drawn into ethylenediaminetetraacetic acid (EDTA) tubes for estimation of the percent saturation of hemoglobin by carbon monoxide, i.e., carboxyhemoglobin (COHb) concentration, as an indicator of HO-1 activity, using spectrophotometry as previously described [bib_ref] Increased carbon monoxide production in patients with cirrhosis with and without spontaneous..., De Las Heras [/bib_ref]. The erythrocyte pellet was washed 3 times with cold isotonic saline and then diluted with saline to the original blood volume. Hemoglobin concentration was determined using a kit supplied by BioMeriux, France. The erythrocyte-reduced GSH level was measured as previously described [bib_ref] Improved method for the determination of blood glutathione, Beutler [/bib_ref] and expressed as mmol/g Hb. The third part was collected in a heparin sterile vacutainer for determination of HO-1 mRNA expression by RT-PCR. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) using a total RNA isolation system from Promega Corporation, Madison, USA [bib_ref] Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Chomczynski [/bib_ref]. RT-PCR reaction for HO mRNA was performed using QIAGEN's 1-step RT-PCR kit (Hilden, Germany), according to the manufacturer's instructions. The final concentrations of the reaction components (50 µL) were 1× QIAGEN buffer, 400 µM of each dNTP, 0.6 µM of both sense and antisense HO primers, 2 µL of RT-PCR enzyme mix, 5 units of RNase inhibitor, and 2 µg of total RNA. Reverse transcription was achieved by heating the reaction components at 50 ºC for 30 min. The initial PCR activation step was performed by heating at 95 ºC for 15 min. The amplification reaction was carried out by thermal cycler (model 9600, Perkin-Elmer) for 35 cycles of denaturation at 94 ºC for 1 min, annealing at 55 ºC for 1 min, and extension at 72 ºC for 1 min, followed by a final extension step of 72 ºC for 10 min. The HO complementary DNA (cDNA) was amplified using the following primers: sense, 5′-CAGGCAGAGAATGCTGAGTTC-3′ and antisense, 5′-GAT-GTTGAGCAGGAACGCAGT-3′ [bib_ref] Expression of human heme oxygenase-1 in the thick ascending limb attenuates angiotensin..., Quan [/bib_ref]. ß-actin was used as a housekeeping gene with the following primers: sense, 5›-TTCTTTGCAGCTCCTTCGTTGCCG-3› and antisense, 5›-TG-GATGGCTACGTACATGGCTGGG-3›. The amplified product for HO was separated on 2% agarose gel and visualized by ethidium bromide staining under ultraviolet light. Primers for HO were synthesized by Metabion International AG (Martinsried, Germany). The RT-PCR reactions were performed with a Hybaid thermal cycler (Thermo Electron corporation [formerly Hybaid], Waltham, MA, USA). The molecular weight was determined using a DNA ladder purchased from Promega (Madison, WI, USA). The computer program "Quantity one" (version 4.6.3, Bio-Rad, USA) was used to analyze the intensities of the PCR bands.
# Statistical analysis
Results were expressed as mean ± standard deviation (SD). Comparisons between groups were made using Student´s t-test for continuous variables. The correlation between 2 parameters was determined by Pearson's correlation coefficient (r). A P value of less than 0.05 was considered statistically significant.
# Results
The main clinical and biochemical characteristics of patients with CLD are presented in . HO-1 mRNA expression was determined by RT-PCR, as shown in [fig_ref] Figure 1: Representative Agarose Gel Electrophoresis for Amplified PCR Product of Heme Oxygenase-1 Gene... [/fig_ref]. The densities of the bands are expressed in arbitrary units. HO-1 mRNA expression in patients with NASH, chronic hepatitis C, and liver cirrhosis was more than that in controls (P < 0.05, P < 0.001, and P < 0.001, Lane M, DNA molecular marker; Lane 1, blank; Lane 2, Control; Lane 3, nonalcoholic steatohepatitis; Lane 4, chronic hepatitis C; Lane 5, liver cirrhosis (bp = base pair) Bessa SS et al. Heme Oxygenase-1 Expression in Liver Disease respectively). Further, compared to patients with NASH and chronic hepatitis C, patients with cirrhosis had significantly higher HO expression (P < 0.001, for both). HO-1 mRNA expression in chronic hepatitis C patients was significantly more than that in patients with NASH (P < 0.001), as shown in and [fig_ref] Figure 2: Mean + S [/fig_ref]. Serum ferritin, carboxyhemoglobin, and serum MDA levels in patients with NASH, chronic hepatitis C, and liver cirrhosis were significantly higher than the levels in healthy controls. These levels were elevated in cirrhotic patients compared to NASH and chronic hepatitis C patients. Moreover, the levels in patients with chronic hepatitis C were higher than those in patients with NASH (P < 0.01, P < 0.001, and P < 0.001, respectively). On the other hand, erythrocyte GSH levels were decreased in patients with NASH, chronic hepatitis C, and liver cirrhosis compared to controls (P < 0.001, for all) and were significantly lower in cirrhotic patients than in NASH and chronic hepatitis C patients (P < 0.001, for both). Moreover, the erythrocyte GSH level in patients with chronic hepatitis C was significantly lower than those in patients with NASH (P < 0.001) as shown in . [fig_ref] Table 2: Ferritin [/fig_ref] shows that the HO-1 mRNA expression in Child-Pugh class C was more than that in Child-Pugh class A and B (P < 0.001 and P < 0.05, respectively). Moreover, serum ferritin, carboxyhemoglobin, and serum MDA levels in Child-Pugh class C were significantly higher than those in class A and B. In contrast, erythrocyte GSH level in Child-Pugh class C was lesser than that in class A and B (P < 0.001, for both). In the present study, HO-1 mRNA expression was positively correlated with levels of carboxyhemoglobin (r = 0.963, P < 0.001), serum ferritin (r = 0.868, P < 0.001), and serum MDA (r = 0.978, P < 0.001) and negatively correlated with erythrocyte GSH level (r = -0.937, P < 0.001) in CLD patients, as shown in [fig_ref] Figure 3: Correlation Between Heme Oxygenase-1 mRNA Expression and Carboxyhemoglobin Concentration [/fig_ref].
# Discussion
HO-1 is an enzyme that catalyzes the rate-limiting step in heme degradation to result in the formation of iron, carbon monoxide, and biliverdin, which is subsequently converted to bilirubin by biliverdin reductase. The biological effects exerted by the products of this enzymatic reaction have gained much attention. The anti-oxidant, anti-inflammatory, and cytoprotective functions associated with HO-1 are attributable to 1 or more of its degradation products. Induction of HO-1 occurs as an adaptive and beneficial response to various injurious stimuli such as oxidative stress, and this inducible nature of HO-1 signifies its importance in several pathophysiological states such as liver diseases. Thus, HO-1 has emerged as a key target molecule with therapeutic implications [bib_ref] Heme oxygenase-1 as a potential therapeutic target for hepatoprotection, Farombi [/bib_ref]. This study investigated the mRNA expression of HO-1 in Egyptian patients with CLD and its relation to oxidative stress biomarkers, and we showed that HO-1 mRNA expression was increased in patients with NASH, chronic hepatitis C, and liver cirrhosis compared to controls. HO-1 expression in cirrhotic patients was significantly higher than that in patients with NASH and chronic hepatitis C. The HO-1 mRNA expression was increased in Child-Pugh class C compared to Child-Pugh class A and B. Furthermore, HO-1 mRNA expression in CLD patients was positively correlated with levels of carboxyhemoglobin, serum ferritin, and serum MDA and negatively correlated with level of erythrocyte GSH. These results are in agreement with the findings of Malaguarnera et al. [bib_ref] Heme oxygenase-1 levels and oxidative stress-related parameters in non-alcoholic fatty liver disease..., Malaguarnera [/bib_ref] , who showed that HO-1 expression was significantly increased in NASH patients, and the increase reflected the severity of the disease. They observed a significant correlation between the increased levels of HO-1 and ferritin and between the increased levels of HO-1 and lipid peroxidation. Moreover, NASH patients with lower levels of GSH exhibited higher expression of HO-1. Thus, the induction of HO-1 is an adap-
## Characteristic controls (n = 15) nash n (n = 15) hcv n (n = 15) liver cirrhosis (n = 15)
Age, y 50 ± 3.9 53 ± 8.9 52 ± 8. Bessa SS et al. Heme Oxygenase-1 Expression in Liver Disease tive response against oxidative damage elicited by lipid peroxidation, and this may be critical in the progression of the disease. This was supported by Nan et al. [bib_ref] Heme oxygenase-1 prevents non-alcoholic steatohepatitis through suppressing hepatocyte apoptosis in mice, Nan [/bib_ref] , who reported that HO-1 plays an important role in NASH. Regarding alcoholic steatohepatitis, Yao et al. [bib_ref] Heme oxygenase-1 upregulated by Ginkgo biloba extract: potential protection against ethanol-induced oxidative..., Yao [/bib_ref] have shown that the induction of HO-1 by Ginkgo biloba is associated with a decrease in liver damage caused by ethanol feeding for 90 d in rats. This is probably due to the enhanced anti-oxidative capacity against ethanol-induced oxidative stress and maintenance of cellular redox balance [bib_ref] The protective role of HO-1 and its generated products (CO, bilirubin, and..., Yao [/bib_ref] [bib_ref] Protective role of HO-1 for alcohol-dependent liver damage, Nussler [/bib_ref]. Conflicting data are available on HO-1 in hepatitis C. demonstrated that HCVexpressing human hepatoma cells have increased levels of HO-1 and decreased Bach 1 expression. Abdalla et al. [bib_ref] Down-regulation of heme oxygenase-1 by hepatitis C virus infection in vivo and..., Abdalla [/bib_ref] , on the contrary, observed a decrease in HO-1 and HO-1 mRNA in liver biopsies from HCV-infected patients. The expression of HO-1 was also reduced in cell lines that stably express the HCV core protein, which suggests that core gene products are capable of regulating HO-1 expression. Clearly, further studies are necessary to reconcile these conflicting results. Concerning the effects of HO-1 induction on hepatitis C, Shan et al. [bib_ref] Reciprocal effects of micro-RNA-122 on expression of heme oxygenase-1 and hepatitis C..., Shan [/bib_ref] have shown a decrease in HCV replication, an effect similar to that described by , in HBV infection. Moreover, Zhu et al. [bib_ref] Heme oxygenase-1 suppresses hepatitis C virus replication and increases resistance of hepatocytes..., Zhu [/bib_ref] showed that targeted overexpression of HO-1 led to a significant inhibition of HCV replication without affecting other parameters of cell viability in human hepatoma cells. These studies have recently been extended by others, who have shown that the HO-1 product biliverdin interfered with HCV replication via direct modulation of the antiviral interferon-α response in 2 human hepatoma replicon cell lines [bib_ref] The heme oxygenase 1 product biliverdin interferes with hepatitis C virus replication..., Lehmann [/bib_ref]. Thus, HO-1 might serve as a specific therapeutic target for the treatment of chronic HCV infection, although the results are somewhat contradictory. With respect to liver cirrhosis, have shown that HO-1 mRNA and protein expression is increased in hepatocytes and some Kupffer cells in the early phase of the disease, while HO-2 expression in these cells is unchanged. In cirrhotic livers, mainly those with biliary cirrhosis, both HO-1 and HO-2 are increased, as shown by Goh et al. [bib_ref] Nitric oxide synthase and heme oxygenase expressions in human liver cirrhosis, Goh [/bib_ref]. HO-1 was localized mainly in Kupffer cells, while HO-2 was localized in the cytoplasm of the hepatocytes. Similar results were obtained by in patients with post-hepatic cirrhosis. They showed that HO-1 expression increased in the liver, being mainly distributed in Kupffer cells and hepatocytes. Moreover, a study on cirrhotic patients undergoing liver transplantation has shown that HO-1 is upregulated through heme-independent stimuli, according to the development of portal hypertension, and that induced HO-1 plays a pathophysiological role in portal hypertension through CO production [bib_ref] Increased heme oxygenase-1 gene expression in the livers of patients with portal..., Matsumi [/bib_ref]. In the current study, carboxyhemoglobin concentration in patients with NASH, chronic hepatitis C, and liver cirrhosis was significantly higher than that in healthy controls. The concentration was elevated in cirrhotic patients compared to NASH and chronic hepatitis C patients. These results are consistent with the findings of previous studies [bib_ref] Carboxyhemoglobin and its correlation to disease severity in cirrhotics, Tran [/bib_ref] [bib_ref] Increased plasma carbon monoxide in patients with viral cirrhosis and hyperdynamic circulation, Tarquini [/bib_ref]. In cirrhotic patients with spontaneous bacterial peritonitis, CO production, evaluated as the CO concentration in the exhaled air and blood CO-Hb level, is further increased and may participate in circulatory alterations [bib_ref] Increased carbon monoxide production in patients with cirrhosis with and without spontaneous..., De Las Heras [/bib_ref]. In our study, levels of serum ferritin in patients with NASH, chronic hepatitis C, and liver cirrhosis were significantly higher than those in healthy controls. High ferritin levels have previously been reported in NASH [bib_ref] Heme oxygenase-1 levels and oxidative stress-related parameters in non-alcoholic fatty liver disease..., Malaguarnera [/bib_ref] and chronic HCV-related hepatitis [bib_ref] Iron storage, lipid peroxidation and glutathione turnover in chronic anti-HCV positive hepatitis, Farinati [/bib_ref] [bib_ref] Serum ferritin and hepatic glutathione concentrations in chronic hepatitis C patients related..., Barbaro [/bib_ref] , and our study clearly confirms this finding. An imbalance in the pro-oxidant/antioxidant equilibrium in favor of pro-oxidants constitutes the oxidative stress phenomenon, a condition that may induce a number of pathophysiological events in the liver [bib_ref] Oxidative stress and antioxidants in hepatic pathogenesis, Ha [/bib_ref]. In this study, serum MDA level was significantly higher and erythrocyte GSH level was significantly lower in patients with NASH than in healthy controls. Similar findings were reported by previous studies [bib_ref] Heme oxygenase-1 levels and oxidative stress-related parameters in non-alcoholic fatty liver disease..., Malaguarnera [/bib_ref] [bib_ref] Oxidative stress and enzymatic antioxidant status in patients with nonalcoholic steatohepatitis, Koruk [/bib_ref]. Meanwhile, we also observed that compared to healthy controls, patients with chronic hepatitis C had higher serum MDA and lower erythrocyte GSH levels. These results are in accordance with the findings of other studies [bib_ref] Role of oxidative stress in the pathogenesis of chronic hepatitis C (CHC), Fierbinteanu-Braticevici [/bib_ref] [bib_ref] glutathione and glutathione peroxidases in blood of patients with chronic liver diseases, Czuczejko [/bib_ref]. The present study provided evidence for the protective role of HO-1 in the body's response to oxidative stress. Due to its beneficial effects, the targeted induction of HO-1 is considered to have major therapeutic potential for the treatment of inflammatory liver diseases to prevent progression of early stages of liver injury [bib_ref] Heme oxygenase-1 as a therapeutic target in inflammatory disorders of the gastrointestinal..., Vijayan [/bib_ref]. In this context, it is interesting to note that induction of HO-1 expression by natural compounds contributes to protection against liver damage in various experimental models [bib_ref] Natural heme oxygenase-1 inducers in hepatobiliary function, Volti [/bib_ref]. Current knowledge on the applications of HO-1 as a therapeutic target still seems precocious and critical questions remain to be answered before clinical interventions become available. Because induction of HO-1 expression may hold therapeutic promises, studies designed to identify clinically relevant methods of delivering such treatment, such as gene therapy to increase the induction of HO-1 or methods to deliver individual by-products of heme degradation by HO (e.g., CO and bilirubin), would be warranted. From this study, we conclude that HO-1 mRNA expression is significantly increased in CLD patients, and the increase reflects the severity of the disease. The significant relationship between the increased HO-1 expression and oxidative stress biomarkers in patients with CLD suggest that HO-1 may play an important role in protecting the liver from oxidative stress-induced damage. Therefore, the induction of HO-1 might be a novel therapeutic option for CLD. However, large-scale clinical studies are needed to better clarify the exact role of the heme-HO system in CLD and the potential clinical applications of inducing the HO-1 system.
Heme Oxygenase-1 Expression in Liver Disease Bessa SS et al.
[fig] Figure 1: Representative Agarose Gel Electrophoresis for Amplified PCR Product of Heme Oxygenase-1 Gene at 550 bp and ß-actin at 457 bp as a Housekeeping Gene. [/fig]
[fig] Figure 2: Mean + S. D. of Heme Oxygenase-1 mRNA Expression in Patients With Chronic Liver Disease and Controls [/fig]
[fig] Figure 3: Correlation Between Heme Oxygenase-1 mRNA Expression and Carboxyhemoglobin Concentration (A), Serum Ferritin Level (B), Serum Malondialdehyde Level (C) and Reduced Glutathione Level (D) in Patients With Chronic Liver Disease Heme Oxygenase-1 Expression in Liver Disease Bessa SS et al. [/fig]
[table] Table 2: Ferritin [/table]
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10.1186/s12955-020-01581-z
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CCBY
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7552532
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33046091
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s2orc_pubmed_articles
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A systematic review of the association between dietary patterns and health-related quality of life
Background: Health related quality of life (HRQOL) is a potent indicator of individual's happiness and life satisfaction.The way in which the HRQOL is affected by the diet is a topic of constant interest and debate among researchers. Evaluating the association between single nutrients or foods and HRQOL fails to take into consideration the complex interactions between nutrients. Also, the findings from previous investigations on the relationship between dietary patterns and HRQOL have been inconsistent. Therefore, our aim was to assess the existing evidence regarding the relationship between the dietary patterns and HRQOL by conducting a systematic review.Methods:A literature search was conducted in PubMed, Scopus, Web of Sciences and Google scholar databases from inception to March 2020, to identify studies that investigated associations between the dietary patterns (regardless of methods used to define dietary patterns) and HRQOL domains. Two researchers independently checked titles and abstracts, evaluated full-text studies, extracted data, and appraised their quality using the Newcastle-Ottawa Scale (NOS).Results:Thirteen studies (four longitudinal, and nine cross-sectional studies), with a total of 43,445 subjects, were included. Of the studies included in this review, eight studies evaluated the association between "Mediterranean" dietary patterns (MDP) and HRQOL, while five studies examined the association between different dietary patterns ("Healthy", "Unhealthy", "Western", "Fruit and vegetable", "Bread and butter" and etc.) and HRQOL. Excluding three studies which showed no significant association, healthy dietary patterns such as MDP, "Healthy" and "Fruit and vegetable" dietary patterns were associated with better HRQOL in physical and mental components scores. The quality assessment of included studies according to NOS criteria were ranged between medium to high quality.Conclusion:According to the current evidence, "Healthy" dietary patterns and "Mediterranean" dietary patterns are associated with better dimension scores of HRQOL in both physical and mental summaries. While, unhealthy dietary patterns and "Western" dietary patterns are associated with lower scores of HRQOL. Further longitudinal studies are required to clarify the association between dietary patterns and HRQOL
# Introduction
In recent years, life expectancy has increased in most countries, resulting in an increased prevalence of persons living with disabilities and chronic diseases [bib_ref] Global, regional, and national age-sex-specific mortality for 282 causes of death in..., Roth [/bib_ref] [bib_ref] Global, regional, and national incidence, prevalence, and years lived with disability for..., James [/bib_ref]. The quality of life is a very complex concept and contains different psychological, physical, social, and cultural aspects of well-being and health-related Page 2 of 15 [bib_ref] 18:337 • fast, convenient online submission • thorough peer review by experienced..., Vajdi [/bib_ref] quality of life (HRQOL) improvement is one of the most important aims of healthcare systems. HRQOL is a multidimensional concept, which subjectively measures an individual's social, emotional, functional and physical well-being [bib_ref] Health-related quality of life in French adolescents and adults: norms for the..., Baumann [/bib_ref]. HRQOL represents an individual's perception of how health affects a person's life quality and overall well-being and is measured with either specific questionnaires (e.g., Hospital Anxiety and Depression scale (HADS)) or generic one's (e.g., the 36-item Short Form (SF-36), the 12-item Short Form (SF-12)) [bib_ref] Dietary patterns and quality of life in older adults: a systematic review, Govindaraju [/bib_ref].
Various factors, such as economic dependence [bib_ref] Determinants of health-related quality of life in elderly in Tehran, Tajvar [/bib_ref] , living situations [bib_ref] Who should measure quality of life?, Addington-Hall [/bib_ref] , and lifestyle factors such as physical activity [bib_ref] Physical activity level and health-related quality of life in the general adult..., Bize [/bib_ref] , and dietary habits [bib_ref] Nutrition and quality of life in older adults, Amarantos [/bib_ref] [bib_ref] Mental health problems in relation to eating behavior patterns, nutrient intakes and..., Farhangi [/bib_ref] can affect HRQOL. Among them, healthy dietary habits play an important role in our state of mental and physical health and prevention and treatment of non-communicable diseases [bib_ref] Self-reported physical activity behavior of a multi-ethnic adult population within the urban..., Baldew [/bib_ref] [bib_ref] Assessing the impact of dietary habits on health-related quality of life requires..., Ruano-Rodríguez [/bib_ref]. It is well established that an unhealthy diet can cause a reduction in physiological function and increasing the risk of disease development [bib_ref] Muscle strength and body mass index as long-term predictors of mortality in..., Rantanen [/bib_ref] [bib_ref] Decreased cytokine expression in peripheral blood leukocytes of patients with severe sepsis, Cabioglu [/bib_ref] , that there is a significant association between diet and alterations in immune and cognitive functions [bib_ref] Life style characteristics associated with nutritional risk in elderly subjects aged 80-85..., Pearson [/bib_ref] and consequently that an improvement in diet is an important factor in the improvement of physiological function [bib_ref] Nutritional assessment of demented patients: a descriptive study, Magri [/bib_ref]. For instance, previous studies have shown that greater adherence to the "Mediterranean" diet (MED) is associated with a significant improvement in general psychological and physical health [bib_ref] Dietary patterns and quality of life in older adults: a systematic review, Govindaraju [/bib_ref] [bib_ref] Assessing the impact of dietary habits on health-related quality of life requires..., Ruano-Rodríguez [/bib_ref]. In another study by Amarantos et al. [bib_ref] Nutrition and quality of life in older adults, Amarantos [/bib_ref] , it has been highlighted that "Good nutrition improves HRQOL by promoting health, preventing dietary deficiency disease, and ameliorating or averting secondary malnutrition that is caused by or associated with other disease" [bib_ref] Nutrition and quality of life in older adults, Amarantos [/bib_ref] , p.1).
Beyond single foods or nutrients, the assessment of whole dietary patterns is likely to provide a better explanation of diet-health relations. It is well established that people do not eat isolated nutrients and instead consume meals containing of a diversity of foods with complex combinations of nutrients that are likely to be interactive [bib_ref] Dietary pattern analysis: a new direction in nutritional epidemiology, Hu [/bib_ref]. Whole-of-diet analysis represent a wider picture of a combination of foods and nutrients, such as the synergetic, additive, and antagonist effect of the foods [bib_ref] Dietary pattern and health-related quality of life among breast cancer survivors, Kim [/bib_ref] and provide researchers the opportunity to account for the interactions between different nutrients [bib_ref] Dietary patterns and diet quality: approaches to assessing complex exposures in nutrition, Mcnaughton [/bib_ref] [bib_ref] Dietary patterns, approaches, and multicultural perspective, Tucker [/bib_ref]. Thus, dietary patterns may be more predictive of HRQOL and disease risk than foods or nutrients in isolation. Dietary patterns are derived based on empirical approach using statistical methods including principal component analysis (PCA) or cluster analysis [bib_ref] Dietary pattern analysis: a new direction in nutritional epidemiology, Hu [/bib_ref]. PCA create groups by intercorrelated dietary variables, while cluster analysis groups individuals into categories according to their reported mean consumptions of foods [bib_ref] Empirically derived eating patterns using factor or cluster analysis: a review, Newby [/bib_ref].
Few studies have examined the relationship between dietary patterns and HRQOL and their results are inconsistent and the majority of the studies in literature have been limited by cross-sectional study design. For example, studies by Mozzillo et al. [bib_ref] Unhealthy lifestyle habits and diabetes-specific health-related quality of life in youths with..., Mozzillo [/bib_ref] , Holmes et al.and Perez-Tasigchana et al. (UAM-cohort) [bib_ref] Mediterranean diet and healthrelated quality of life in two cohorts of community-dwelling..., Perez-Tasigchana [/bib_ref] have not found any significant relationship between dietary patterns and HRQOL. However, some studies have reported the association of namely "Western" or "Unhealthy" dietary patterns with physical and mental chronic disease [bib_ref] Dietary patterns and metabolic syndrome-a review of epidemiologic evidence, Baxter [/bib_ref] [bib_ref] Dietary patterns and depression risk: a meta-analysis, Li [/bib_ref] [bib_ref] Dietary patterns and cardiovascular disease, Williams [/bib_ref] and poor HRQOL [bib_ref] Dietary pattern and health-related quality of life among breast cancer survivors, Kim [/bib_ref] [bib_ref] Associations between dietary patterns and longitudinal quality of life changes in colorectal..., Gigic [/bib_ref] [bib_ref] Empirically derived dietary patterns and health-related quality of life in the SUN..., Ruano [/bib_ref]. Also, several studies reported that "Western" dietary pattern (increased intake of saturated fat and refined foods along with low intake of vegetable and fruits) is inversely associated with healthy factors such as immunity [bib_ref] Fast food fever: reviewing the impacts of the Western diet on immunity, Myles [/bib_ref] and chronic diseases [bib_ref] The global cardiovascular risk transition: associations of four metabolic risk factors with..., Danaei [/bib_ref] [bib_ref] Western diet, family history of colorectal cancer, NAT2, GSTM-1 and risk of..., Slattery [/bib_ref]. While dietary patterns recognized in each study may be different from each other, some important characteristics of the healthy dietary pattern such as high intake of fruits, vegetables, legumes, seafood, whole grains, and low intake of refined grains, processed meat and sweetened foods have been suggested to be related to positive health benefits [bib_ref] Healthy dietary pattern is inversely associated with non-alcoholic fatty liver disease in..., Adriano [/bib_ref] [bib_ref] Dietary approaches to stop hypertension (DASH) dietary pattern is associated with reduced..., Asghari [/bib_ref]. Moreover, "Mediterranean" style dietary pattern (MEDP) is also associated with decreased risk of chronic disease and improved HRQOL [bib_ref] Relationship of the adherence to the Mediterranean diet with health-related quality of..., Alcubierre [/bib_ref] [bib_ref] Adherence to a Mediterranean diet is associated with a better health-related quality..., Bonaccio [/bib_ref] [bib_ref] Mediterranean diet and quality of life: Baseline cross-sectional analysis of the PREDIMED-PLUS..., Galilea-Zabalza [/bib_ref]. This pattern particularly consists of the intake of nonrefined cereals and products, vegetables, fruits, olive oil and non-fat or low-fat dairy products is a known primary preventive tool against chronic cardiovascular events [bib_ref] Dietary patterns: a Mediterranean diet score and its relation to clinical and..., Panagiotakos [/bib_ref] [bib_ref] Mediterranean' dietary pattern for the primary prevention of cardiovascular disease, Rees [/bib_ref] [bib_ref] Jahangiry L. Gender differences in major dietary patterns and their relationship with..., Farhangi [/bib_ref]. Numerous evidences have demonstrated that "Mediterranean" dietary pattern (MDP) reduces cardiovascular risk, improves survival from coronary heart disease (CHD), improved glycemic control and decreased risk of type 2 diabetes [bib_ref] Mediterranean eating pattern, Boucher [/bib_ref].Given the conflicting results and lack of systematically reviewed publication of earlier studies, the aim of this study was to systematically review published data to evaluate the relationship between dietary patterns and HRQOL among general population without age or disease restrictions.
# Methods
## Search strategy
A systematic search was conducted using Web of Sciences, Scopus, PubMed and Google scholar databases to the studies evaluated the relationship between dietary patterns and HRQOL from.
inception to March 2020. No language restriction was used. In search strategy, we used a combination of the MeSH (Medical Subject Headings) terms including the following: (Diet OR dietary OR patterns OR factor analysis OR cluster analysis OR principal component analysis OR diet patterns OR diet pattern OR
## Inclusion criteria
Studies were evaluated for eligibility using the inclusion and exclusion criteria in [fig_ref] Table 2: Inclusion and exclusion criteria for studies [/fig_ref]. The search results were uploaded into EndNote software (version X8, for Windows, Thomson Reuters, Philadelphia, PA, USA) and duplicates were removed. Therefore retrieved articles were merged and the review process has been facilitated. Two reviewers (MAF and MV) independently assessed the titles and abstracts of all studies identified in the search. Studies not meeting the eligibility criteria were eliminated. Furthermore, the reference lists of relevant reviews and of included articles were also checked for further studies. Full-texts of relevant articles were retrieved if meeting the eligibility criteria and findings Web of science (("dietary patterns" OR "dietary patterns" OR "Diet" OR "diet pattern" OR "diet patterns" OR "eating patterns" OR "eating patterns" OR "food pattern" OR "food pattern" OR "principal component analysis" OR " factor analysis ") AND ( "Life Quality" OR " life qualities " OR "Quality of Life" OR "health related quality of life" OR "HRQOL" OR " QOL " OR " EQ-5D " OR " EuroQol 5 Dimensions " OR " SF 12" OR " SF 36")) 478 were re-screened. Any discrepancies were discussed between the two authors. The patient/Population; Intervention; Comparator; Outcome (PICO) question was as follows: in human models (P), does healthy dietary pattern (I) compared to unhealthy dietary pattern (C), affect HRQOL (O)?
## Data extraction
Data extraction was conducted by two independent reviewers (MAF and MV), and any disagreements were resolved by consensus. The following data were extracted using a standard form: first author's name, study location, publication year, study design, sample size, age and gender of subjects, type of study population, dietary pattern assessment method, total number of participants and the number of case and controls, the HRQOL assessment tool and information about adjustments for possible confounders the main results.
## Quality assessment
The methodological quality of included studies was evaluated using the Newcastle-Ottawa scale (NOS) adopted for cross-sectional and cohort studies. The 9-point NOS scale has scoring ranges from 0 to nine.
The tool assesses the studies based on three dimensionsselection, compatibility, exposure or outcome. Both authors rated the article independently and discussed the ratings.
# Results
The current study follows the Preferred Reporting Items of Systematic Reviews and Meta-Analysis (PRISMA) guidelines for reporting the systematic reviews [bib_ref] Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement, Moher [/bib_ref]. The flowchart of the study selection process is described in screened for title and abstract review and 58 articles were eligible for full text review. Finally, excluding 45 papers because of not meeting the inclusion criteria -not assessing the dietary pattern with either factor analysis or MDP method, not assessing the statistical associations between dietary patterns and HRQOL, design of intervention-a total of thirteen articles were included in the current systematic review. The characteristics of included papers are presented in [fig_ref] Table 3: Characteristics of studies included in the systematic review owing to reporting the... [/fig_ref]. Two longitudinal study [bib_ref] Associations between dietary patterns and longitudinal quality of life changes in colorectal..., Gigic [/bib_ref] [bib_ref] Diet and physical activity in people with intermediate cardiovascular risk and their..., Sanchez-Aguadero [/bib_ref] , two cohort studies analyzing the baseline characteristics [bib_ref] Mediterranean diet and healthrelated quality of life in two cohorts of community-dwelling..., Perez-Tasigchana [/bib_ref] [bib_ref] Empirically derived dietary patterns and health-related quality of life in the SUN..., Ruano [/bib_ref] and nine cross-sectional studies [bib_ref] Dietary pattern and health-related quality of life among breast cancer survivors, Kim [/bib_ref] [bib_ref] Unhealthy lifestyle habits and diabetes-specific health-related quality of life in youths with..., Mozzillo [/bib_ref] [bib_ref] Relationship of the adherence to the Mediterranean diet with health-related quality of..., Alcubierre [/bib_ref] [bib_ref] Adherence to a Mediterranean diet is associated with a better health-related quality..., Bonaccio [/bib_ref] [bib_ref] Mediterranean diet and quality of life: Baseline cross-sectional analysis of the PREDIMED-PLUS..., Galilea-Zabalza [/bib_ref] [bib_ref] Relationship between adherence to the mediterranean diet and health-related quality of life..., Zaragoza-Marti [/bib_ref] [bib_ref] Associations of diet quality with health-related quality of life in older Australian..., Milte [/bib_ref] [bib_ref] Association of dietary patterns with systemic inflammation, quality of life, disease severity,..., Moravejolahkami [/bib_ref] were included. Studies were published between 2016 and 2019 and seven of these studies were carried out in Spain [bib_ref] Dietary pattern and health-related quality of life among breast cancer survivors, Kim [/bib_ref] [bib_ref] Mediterranean diet and healthrelated quality of life in two cohorts of community-dwelling..., Perez-Tasigchana [/bib_ref] [bib_ref] Empirically derived dietary patterns and health-related quality of life in the SUN..., Ruano [/bib_ref] [bib_ref] Relationship of the adherence to the Mediterranean diet with health-related quality of..., Alcubierre [/bib_ref] [bib_ref] Mediterranean diet and quality of life: Baseline cross-sectional analysis of the PREDIMED-PLUS..., Galilea-Zabalza [/bib_ref] [bib_ref] Diet and physical activity in people with intermediate cardiovascular risk and their..., Sanchez-Aguadero [/bib_ref] [bib_ref] Relationship between adherence to the mediterranean diet and health-related quality of life..., Zaragoza-Marti [/bib_ref] , two in Italy [bib_ref] Unhealthy lifestyle habits and diabetes-specific health-related quality of life in youths with..., Mozzillo [/bib_ref] [bib_ref] Adherence to a Mediterranean diet is associated with a better health-related quality..., Bonaccio [/bib_ref] , one in France, one in USA [bib_ref] Associations between dietary patterns and longitudinal quality of life changes in colorectal..., Gigic [/bib_ref] , one in Australia [bib_ref] Associations of diet quality with health-related quality of life in older Australian..., Milte [/bib_ref] and one in Iran [bib_ref] Association of dietary patterns with systemic inflammation, quality of life, disease severity,..., Moravejolahkami [/bib_ref]. Studies where cross-sectional data were derived from longitudinal, cohort or even randomized trials were reported as cross-sectional. The largest sample size belonged to the study of Bonaccio et al. [bib_ref] Adherence to a Mediterranean diet is associated with a better health-related quality..., Bonaccio [/bib_ref] considering 16,936 participants and the lowest samples size was belonged to the study of Gigic et al. [bib_ref] Associations between dietary patterns and longitudinal quality of life changes in colorectal..., Gigic [/bib_ref] with 192 participants. The studies in the present review evaluated the relationship between dietary patterns evaluated with PCA or MDP score with HRQOL. We did not exclude disease status; accordingly five studies evaluated the dietary patterns with factor or cluster analysis [bib_ref] Dietary pattern and health-related quality of life among breast cancer survivors, Kim [/bib_ref] [bib_ref] Associations between dietary patterns and longitudinal quality of life changes in colorectal..., Gigic [/bib_ref] [bib_ref] Empirically derived dietary patterns and health-related quality of life in the SUN..., Ruano [/bib_ref] [bib_ref] Association of dietary patterns with systemic inflammation, quality of life, disease severity,..., Moravejolahkami [/bib_ref] , while eight studies evaluate the MDP [bib_ref] Unhealthy lifestyle habits and diabetes-specific health-related quality of life in youths with..., Mozzillo [/bib_ref] [bib_ref] Mediterranean diet and healthrelated quality of life in two cohorts of community-dwelling..., Perez-Tasigchana [/bib_ref] [bib_ref] Relationship of the adherence to the Mediterranean diet with health-related quality of..., Alcubierre [/bib_ref] [bib_ref] Adherence to a Mediterranean diet is associated with a better health-related quality..., Bonaccio [/bib_ref] [bib_ref] Mediterranean diet and quality of life: Baseline cross-sectional analysis of the PREDIMED-PLUS..., Galilea-Zabalza [/bib_ref] [bib_ref] Diet and physical activity in people with intermediate cardiovascular risk and their..., Sanchez-Aguadero [/bib_ref] [bib_ref] Relationship between adherence to the mediterranean diet and health-related quality of life..., Zaragoza-Marti [/bib_ref] [bib_ref] Associations of diet quality with health-related quality of life in older Australian..., Milte [/bib_ref]. Moreover, among all of the studies, one study was carried out among breast cancer survivals [bib_ref] Dietary pattern and health-related quality of life among breast cancer survivors, Kim [/bib_ref] , one among subjects with intermediate cardiovascular risk [bib_ref] Diet and physical activity in people with intermediate cardiovascular risk and their..., Sanchez-Aguadero [/bib_ref] , one in colorectal cancer [bib_ref] Associations between dietary patterns and longitudinal quality of life changes in colorectal..., Gigic [/bib_ref] , one in multiple sclerosis [bib_ref] Association of dietary patterns with systemic inflammation, quality of life, disease severity,..., Moravejolahkami [/bib_ref] , one among patients with type 1 diabetes [bib_ref] Unhealthy lifestyle habits and diabetes-specific health-related quality of life in youths with..., Mozzillo [/bib_ref] , one among patients with type 2 diabetes [bib_ref] Relationship of the adherence to the Mediterranean diet with health-related quality of..., Alcubierre [/bib_ref] , one in patients with minor digestive symptomsand others among general apparently healthy individuals. From the perspective of age, two studies were among older individual [bib_ref] Mediterranean diet and healthrelated quality of life in two cohorts of community-dwelling..., Perez-Tasigchana [/bib_ref] [bib_ref] Relationship between adherence to the mediterranean diet and health-related quality of life..., Zaragoza-Marti [/bib_ref] while others were performed among general population .
Major variability was observed between the HRQOL assessment tools. Five studies [bib_ref] Mediterranean diet and healthrelated quality of life in two cohorts of community-dwelling..., Perez-Tasigchana [/bib_ref] [bib_ref] Empirically derived dietary patterns and health-related quality of life in the SUN..., Ruano [/bib_ref] [bib_ref] Adherence to a Mediterranean diet is associated with a better health-related quality..., Bonaccio [/bib_ref] [bib_ref] Diet and physical activity in people with intermediate cardiovascular risk and their..., Sanchez-Aguadero [/bib_ref] [bib_ref] Relationship between adherence to the mediterranean diet and health-related quality of life..., Zaragoza-Marti [/bib_ref] [bib_ref] Associations of diet quality with health-related quality of life in older Australian..., Milte [/bib_ref] measured HRQOL with standard questionnaires of SF-36, SF-12 reporting physical components scores (PCS) and mental components scores (MCS) along with scores obtained for eight domains. Studies which carried out among cancer patients used the European Organization for Research and Treatment of Cancer Quality-of-life Questionnaire (EORTC QLQ) for measuring HRQOL [bib_ref] Dietary pattern and health-related quality of life among breast cancer survivors, Kim [/bib_ref] [bib_ref] Associations between dietary patterns and longitudinal quality of life changes in colorectal..., Gigic [/bib_ref] , one study used Food and Benefit Assessment (FBA) questionnaire, One study carried out among multiple sclerosis patients used Multiple Sclerosis Quality Of Life-54 questionnaire (MSQOL-54) [bib_ref] Association of dietary patterns with systemic inflammation, quality of life, disease severity,..., Moravejolahkami [/bib_ref] , one study used Pediatric Quality of Life Inventory 3.0 Diabetes Module (PedsQL 3.0 DM) [bib_ref] Unhealthy lifestyle habits and diabetes-specific health-related quality of life in youths with..., Mozzillo [/bib_ref] , and one used Audit of Diabetes-Dependent Quality of Life (ADDQoL-19) [bib_ref] Relationship of the adherence to the Mediterranean diet with health-related quality of..., Alcubierre [/bib_ref]. Dietary assessment was also measured with variable tools. Most of the studies used food frequency questionnaires (FFQ) [bib_ref] Mediterranean diet and healthrelated quality of life in two cohorts of community-dwelling..., Perez-Tasigchana [/bib_ref] [bib_ref] Associations between dietary patterns and longitudinal quality of life changes in colorectal..., Gigic [/bib_ref] [bib_ref] Empirically derived dietary patterns and health-related quality of life in the SUN..., Ruano [/bib_ref] [bib_ref] Relationship of the adherence to the Mediterranean diet with health-related quality of..., Alcubierre [/bib_ref] [bib_ref] Adherence to a Mediterranean diet is associated with a better health-related quality..., Bonaccio [/bib_ref] [bib_ref] Diet and physical activity in people with intermediate cardiovascular risk and their..., Sanchez-Aguadero [/bib_ref] [bib_ref] Relationship between adherence to the mediterranean diet and health-related quality of life..., Zaragoza-Marti [/bib_ref] [bib_ref] Associations of diet quality with health-related quality of life in older Australian..., Milte [/bib_ref] [bib_ref] Association of dietary patterns with systemic inflammation, quality of life, disease severity,..., Moravejolahkami [/bib_ref] , while one study evaluated dietary intake using three day non-consecutive food record [bib_ref] Dietary pattern and health-related quality of life among breast cancer survivors, Kim [/bib_ref] , one study with non-consecutives 24 h recall methodand one with Traditional MDP score by 17-point questionnaire to assess adherence [bib_ref] Mediterranean diet and healthrelated quality of life in two cohorts of community-dwelling..., Perez-Tasigchana [/bib_ref]. Among the studies evaluated MDP, one study evaluated traditional MDP score by 17-point questionnaire [bib_ref] Mediterranean diet and quality of life: Baseline cross-sectional analysis of the PREDIMED-PLUS..., Galilea-Zabalza [/bib_ref] , four studies [bib_ref] Adherence to a Mediterranean diet is associated with a better health-related quality..., Bonaccio [/bib_ref] [bib_ref] Diet and physical activity in people with intermediate cardiovascular risk and their..., Sanchez-Aguadero [/bib_ref] [bib_ref] Relationship between adherence to the mediterranean diet and health-related quality of life..., Zaragoza-Marti [/bib_ref] [bib_ref] Associations of diet quality with health-related quality of life in older Australian..., Milte [/bib_ref] measured MED score by Trichopoulou et al. [bib_ref] Diet and overall survival in elderly people, Trichopoulou [/bib_ref] , one study used the MED Quality Index (KIDMED) score [bib_ref] Unhealthy lifestyle habits and diabetes-specific health-related quality of life in youths with..., Mozzillo [/bib_ref] , one study with three approaches of MDP index, prevention with MED (PREDIMED) score and Trichopoulou's MED score [bib_ref] Mediterranean diet and healthrelated quality of life in two cohorts of community-dwelling..., Perez-Tasigchana [/bib_ref] and one with relative "Mediterranean" diet score (rMED) method [bib_ref] Relationship of the adherence to the Mediterranean diet with health-related quality of..., Alcubierre [/bib_ref].
The quality assessment of the included studies is presented in . The quality total score of studies ranged between 6 and 9 while most of them had medium and strong qualities scored by NOS scaling method. Based on our search of the literature, no prior study has assessed the relationship between other dietary patterns such as the Asian dietary patterns (traditional "Japanese" and "Chinese" diets and etc.), "Nordic", or "French" diets and HRQOL. In summary among thirteen included studies, ten studies found significant relations between dietary patterns (dietary patterns derived with factor or cluster analysis or MDP) and HRQOL while three studies did not observe any significant relations between dietary patterns and HRQOL [bib_ref] Unhealthy lifestyle habits and diabetes-specific health-related quality of life in youths with..., Mozzillo [/bib_ref] [bib_ref] Mediterranean diet and healthrelated quality of life in two cohorts of community-dwelling..., Perez-Tasigchana [/bib_ref]. Because of the great heterogeneity between the methodological approaches, study designs and report of results, the data synthesis and meta-analysis was not possible.
# Discussion
## Principal findings
In the current systematic review, the studies reporting the relationship between dietary patterns and HRQOL were reviewed. Most of the included studies showed the relationship between dietary patterns and HRQOL with only three exceptions showing no association in subjects with minor digestive problems and type1 diabetes [bib_ref] Unhealthy lifestyle habits and diabetes-specific health-related quality of life in youths with..., Mozzillo [/bib_ref] [bib_ref] Mediterranean diet and healthrelated quality of life in two cohorts of community-dwelling..., Perez-Tasigchana [/bib_ref]. Of the studies evaluating the dietary patterns reported higher adherence to healthy dietary patterns are associated with better scores of HRQOL in one or more dimensions [bib_ref] Dietary pattern and health-related quality of life among breast cancer survivors, Kim [/bib_ref] [bib_ref] Associations between dietary patterns and longitudinal quality of life changes in colorectal..., Gigic [/bib_ref] [bib_ref] Empirically derived dietary patterns and health-related quality of life in the SUN..., Ruano [/bib_ref] [bib_ref] Associations of diet quality with health-related quality of life in older Australian..., Milte [/bib_ref] [bib_ref] Association of dietary patterns with systemic inflammation, quality of life, disease severity,..., Moravejolahkami [/bib_ref]. Also, higher adherence to MDP was associated with better scores of HRQOL . To our knowledge, this is the first systematic review to examine the effect of dietary patterns on HRQOL among general population without age or disease restrictions.
## Details from previous research/studies
To date, only one systematic review has been carried out to investigate the relationship of dietary patterns and quality of life in older people [bib_ref] Dietary patterns and quality of life in older adults: a systematic review, Govindaraju [/bib_ref]. Like our results, they revealed that healthy dietary patterns were related to better quality of life in one or more domains and adherence to MED were significantly related to improvement in at least one of the quality of life domains. In another study by Ruano et al. [bib_ref] Empirically derived dietary patterns and health-related quality of life in the SUN..., Ruano [/bib_ref] evaluating the baseline data of the SUN cohort, two major dietary patterns of the "Western" dietary pattern (rich in processed pastries and red meats) and the MDP (high in olive oil, vegetables and fruits) was identified among 11,128 participants. The "Western" dietary pattern was inversely associated with all domains of HRQOL. The magnitude of these differences between the participants in the highest versus lowest quintile of adherence to the "Western" dietary pattern ranged from 20.8 (for mental health) to 23.5 (for vitality). In opposite, the MDP was associated with better HRQOL domains while scores ranged from + 1.3 (for physical functioning) to + 3.4 (for vitality). In the study by Kim et al. [bib_ref] Dietary pattern and health-related quality of life among breast cancer survivors, Kim [/bib_ref] and Gigic et al. [bib_ref] Associations between dietary patterns and longitudinal quality of life changes in colorectal..., Gigic [/bib_ref] among patients with colorectal and breast cancer, patients with higher adherence to "Western" diet had lower chances to improve in physical functioning, diarrhea and constipation. While, patients following a "Fruit and vegetable" and "Healthy" diet indicated improving diarrhea and dyspnea scores. The deleterious effects of "Western" dietary patterns have been widely mentioned in numerous studies; its association with metabolic syndrome, obesity, insulin resistance [bib_ref] Associations of dietary patterns with metabolic syndrome and insulin resistance: a cross-sectional..., Arisawa [/bib_ref] , and risk of cardiovascular disease [bib_ref] Dietary patterns in relation to cardiovascular disease incidence and risk markers in..., Mertens [/bib_ref] [bib_ref] Major dietary patterns are related to plasma concentrations of markers of inflammation..., Lopez-Garcia [/bib_ref]. Moreover, considering the relationship between
## Table 4 newcastle-ottawa quality assessment scale (nos) for studies included in the systematic review
One star represents a score of 1, and a study can be awarded a maximum score of 9 in total. The items were scored "*"if the answer was "YES, " and "−" if the answer was "NO" or "UNCLEAR. " The final quality scores were as follows: low quality = 0-3; moderate quality = 4-6; high quality ≥ 7 Cross-sectional * -* * * ** * 8
Bonaccio et al. [bib_ref] Adherence to a Mediterranean diet is associated with a better health-related quality..., Bonaccio [/bib_ref] Cross-sectional * * * * ** ** * 9
Milte et al. [bib_ref] Associations of diet quality with health-related quality of life in older Australian..., Milte [/bib_ref] Cross-sectional * * * * ** ** * 9
Alcubierre et al. [bib_ref] Relationship of the adherence to the Mediterranean diet with health-related quality of..., Alcubierre [/bib_ref] Page 12 of 15 [bib_ref] 18:337 • fast, convenient online submission • thorough peer review by experienced..., Vajdi [/bib_ref] "Western" dietary pattern and mental component summary of HRQOL, some studies reported an inverse relationship between "Western" dietary pattern and depression, anxiety [bib_ref] The association of major dietary patterns with depression, anxiety and stress in..., Nasir [/bib_ref] , mental [bib_ref] Association between dietary patterns and mental disorders in pregnant women in Southern..., Paskulin [/bib_ref] and cognitive disorders [bib_ref] Dietary patterns and cognitive function in Korean older adults, Kim [/bib_ref]. One possible explanation is that high in saturated and trans fats, refined sugars, red and processed meat could reduce the brain derived neurotropic factor (BDNF) concentrations and to inhibit its expression via several pro-inflammatory cytokine production [bib_ref] Regulation of brainderived neurotrophic factor mRNA levels in hippocampus by neuronal activity, Castren [/bib_ref] [bib_ref] A high-fat, refined sugar diet reduces hippocampal brain-derived neurotrophic factor, neuronal plasticity,..., Molteni [/bib_ref].
In the study of Moravejolahkami et al. [bib_ref] Association of dietary patterns with systemic inflammation, quality of life, disease severity,..., Moravejolahkami [/bib_ref] , examining the association between dietary patterns and HRQOL among 261 multiple sclerosis patients, results showed that ''Fruits, vegetables, low fat dairy-based'' pattern and ''Mediterranean-Like'' dietary pattern were associated with higher physical and mental health composite scores (P adjusted < 0.001). In another study by Sanchez-Aguadero et al. [bib_ref] Diet and physical activity in people with intermediate cardiovascular risk and their..., Sanchez-Aguadero [/bib_ref] , greater adherence to the MED was related to higher scores on the SF-12 social functioning and mental component. After adjustment for confounders, for each point of increase in the MED adherence score, there was an increase of 1.17 points in the mental component value (p < 0.01). It is well documented that MDP is potentially able to protect against numerous chronic disease including cardiovascular events [bib_ref] Mediterranean diet and prevention of coronary heart disease in the elderly, Dontas [/bib_ref] [bib_ref] Dietary Patterns and Mediterranean Diet Score and Hazard of Recurrent Coronary Heart..., Shikany [/bib_ref] metabolic syndrome [bib_ref] The effect of Mediterranean diet on metabolic syndrome and its components: a..., Kastorini [/bib_ref] and diabetes [bib_ref] Reduction in the incidence of type 2 diabetes with the Mediterranean diet:..., Salas-Salvado [/bib_ref]. Also, from mental component summary point of view, MED could potentially decrease the risk of depression [bib_ref] Mediterranean diet and depression, Sanchez-Villegas [/bib_ref] cognitive decline and dementia [bib_ref] Mediterranean diet, cognitive function, and dementia: a systematic review, Lourida [/bib_ref] [bib_ref] Mediterranean diet, stroke, cognitive impairment, and depression: a meta-analysis, Psaltopoulou [/bib_ref]. Higher consumption of vegetables and fruits as a characteristic of MDP is associated with improved HRQOL [bib_ref] Physical exercise, vegetable and fruit intake and health-related quality of life in..., Gong [/bib_ref]. Therefore, it is not out of expected that MED is related to improve in both mental and physical domain of HRQOL.
In addition, less than 25% of the studies (3 out of 13 studies) included in this review reported no significant association between dietary patterns and HRQOL. Mozzillo et al. [bib_ref] Unhealthy lifestyle habits and diabetes-specific health-related quality of life in youths with..., Mozzillo [/bib_ref] fails to show any significant relationship between adherence to the MED and HRQOL, maybe due to the low number of patients with poor diet quality in their study. Perez-Tasigchana et al. [bib_ref] Mediterranean diet and healthrelated quality of life in two cohorts of community-dwelling..., Perez-Tasigchana [/bib_ref] found that PREDIMED scores were related to a marginally better PCS in the Seniors-ENRICA cohort and also reported no association between MED score and any of the HRQOL domains in the UAM-cohort. The studies were performed 10 years apart and both the studies used different methods to measure dietary pattern and HRQOL, and found consistent findings. In a cross sectional study by Holmes et al.; it was noted that no significant differences in HRQOL were found between dietary patterns. They used cluster analysis to derive dietary patterns and FBA questionnaire for assessment of HRQOL.
## Summary
There is an obvious fact that existing studies on dietary patterns focus mainly on MED or "Western" dietary patterns, and very little attention has been given to the effect of the other dietary patterns such as the "Japanese", "Nordic", "French", or "Chinese" traditional diet on HRQOL and more studies are required on these neglected dietary patterns. Accordingly, our study focuses more on MDP and "Western" dietary patterns. The "Nordic" diet is characterized by high intake of root vegetables, grains, berries, nuts, and seafood. The "Nordic" diet is similar to the MED but the "Nordic" diet emphasizes canola oil more than olive oil [bib_ref] The association between adherence to the New Nordic Diet and diet quality, Bjornara [/bib_ref]. A recent cross-sectional study showed that high adherence to the "Nordic" diet was associated with a healthier lifestyle [bib_ref] Adherence to the Healthy Nordic Food Index in the Norwegian Women and..., Jensen [/bib_ref]. Side dishes in "Japanese" traditional diet include several species of fish that are a rich source of high quality protein as well as omega-3 acids, which are believed to be beneficial for human health [bib_ref] Very long-chain n-3 fatty acids and human health: Fact, fiction and the..., Calder [/bib_ref].
As reported in the current review, most of the studies regarding the associations between dietary patterns and HRQOL were considered the healthy benefits of MDP in improvement of quality of life. Although there was a great heterogeneity in estimating the MED score, however, almost all of the studies had consistent results reporting the positive effects of the MED on HRQOL. In spite of observed associations between HRQOL and dietary patterns, the possible underlying mechanisms are unclear. In fact, all of the life domains including personal satisfaction, social interactions or economical characteristics could have direct relationships with food behaviors and eating ways and, together with the physical and mental health, could explain what we consider to be a good or poor quality of life [bib_ref] Dietary patterns and health outcomes, Kant [/bib_ref].
# Strengths and limitations
The current broad systematic review was performed according to PRISMA guidelines and was included all of the studies up to March 2020 in its literature search. We combined the studies in numerous disease and age groups because of the limited number of studies. Moreover, the included studies were large observational studies with cross-sectional design and therefore the causal inference could not be relied. Additionally, the dietary assessments and quality of life were both assessed by self-reported tools and might be a source of bias. Lastly, it will be better to develop a nutrition-specific quality of life assessment tool to better interpretation of the results of the effects of diet on the quality of life. The dietary assessment, quality of life measurements and data visualization varied from one study to another and the great heterogeneity among included studies regarding the design, dietary assessment, quality of life assessment tools and statistics made us unable to run a meta-analysis.
# Conclusion
In conclusion, according to our findings, MDP and "Healthy" dietary patterns are associated with better dimension scores of HRQOL in both physical and mental summaries. While, "Unhealthy" dietary patterns and "Western" dietary patterns are associated with lower scores of HRQOL. Adjusting for the potential confounders, the results might be identifiable for final causal inference. Because of the great heterogeneity between the methodological approaches, designs and report of results, the meta-analysis was not possible. Further longitudinal studies are required to clarify the association between dietary patterns and HRQOL.
[fig] FFQ: Food Frequency Questionnaire, PedsQL 3.0 DM Pediatric Quality of Life Inventory 3.0 Diabetes Module, MSQOL-54 Multiple Sclerosis Quality Of Life-54 questionnaire, EORTC QLQ-C30 European Organization for Research and Treatment of Cancer Quality-of-life Questionnaire Core 30, EORTC QLQ-BR23 The EORTC Breast Cancer-Specific Quality of Life Questionnaire, ADDQoL-19 Audit of Diabetes-Dependent Quality of Life, IMI Italian Mediterranean diet index, CHD coronary heart disease, PREDIMED score prevention with Mediterranean diet score rMED Relative Mediterranean diet score, CVD cardiovascular disease, PCS Physical component score, MCS Mental component score, MEDP Mediterranean style dietary pattern, MDP Mediterranean dietary pattern MED Mediterranean diet, HRQOL Health-related quality of life, SF-12, The 12-item Short Form, SF-36 The 36-item Short Form, EQ-5D The European Quality of Life-5 Dimensions, QOL Quality of Life, [/fig]
[fig] Abbreviations: ADDQoL-19: Audit of Diabetes-Dependent Quality of Life; EQ-5D: The European Quality of Life-5 Dimensions; FFQ: Food Frequency Questionnaire; HRQOL: Health-related quality of life; KDQOL-36: Kidney Disease Quality of Life 36-item survey; MANSA: The Manchester Short Appraisal was used to assess self-rated quality of life; MCS: Mental component score; MDP: Mediterranean dietary pattern; MED: Mediterranean diet; MeSH: Medical subject headings; MSQOL-54: Multiple Sclerosis Quality of Life-54 questionnaire; NOS: The Newcastle-Ottawa Scale; PCS: Physical component score; PedsQL 3.0 DM: Pediatric Quality of Life Inventory 3.0 Diabetes Module; PREDIMED: Score prevention with Mediterranean diet score; rMED: Relative Mediterranean diet score; SF-12: The 12-item Short Form; SF-36: The 36-item Short Form. [/fig]
[table] Table 1: Search strategy and number of publications in each electronic database [/table]
[table] Table 2: Inclusion and exclusion criteria for studies [/table]
[table] Table 3: Characteristics of studies included in the systematic review owing to reporting the association between dietary patterns and health-related quality of life [/table]
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Correction to: Mutations in the nucleotide binding and hydrolysis domains of helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function
[fig] Figure 6: Effect of nucleotide cofactors on nuclease activity of HpMutS2 and mutants. Plots showing time dependent depletion of substrate DNA (a, b, c) Single-stranded DNA and d, e, f Holliday junction. DNA substrates (1 nM) were incubated with HpMutS2 and mutants (150 nM) at 37°C. [/fig]
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Ruptured caesarean scar ectopic pregnancy: a diagnostic dilemma in a resource-limited setting
Background: Caesarean scar pregnancy (CSP) remains a very rare form of ectopic pregnancy associated with serious life threatening obstetric complications and even death in case of late diagnosis and treatment.Case presentation:We report a case of a ruptured caesarean scar pregnancy in a 29 year-old gravida 5, para 3 with a past obstetric history of two consecutive caesarean sections done 9 and 5 years ago respectively. The patient presented with intermittent lower abdominal pains on a 20 weeks gestation associated with mild epigastralgia and 2 previous episodes of mild pervaginal bleeding (2 and 1 months ago respectively before consultation) managed with injectable progesterone. Her evolution 4 h later was marked by an increase in the intensity of the abdominal pain, an unmeasurable blood pressure and a feeble pulse. Immediate paracentesis revealed 10 cc of fresh non coagulating blood. The diagnosis of ruptured ectopic pregnancy with abundant hemoperitoneum was considered and an emergency laparotomy with fluid and blood resuscitation was carried out. A midline laparotomy revealed a ruptured caesarean scar ectopic pregnancy with an abundant hemoperitoneum. Careful resection of the placenta and repair of the ruptured isthmic region of the uterus was carried out. Recovery after surgery was without complications and the patient was discharged on the 6th day following surgery.Conclusion:Caesarean scar pregnancy remains a very rare obstetric condition. Late diagnosis of this condition can be associated with serious life threatening obstetric complications. The rarity of the condition warrants a high index of suspicion among clinicians.
# Background
A caesarean scar pregnancy (CSP) is a very rare form of pregnancy that occurs when the developing blastocyst implants on a previous caesarean scar [bib_ref] Caesarean scar pregnancy, Ash [/bib_ref] [bib_ref] Ectopic pregnancy in previous caesarean section scar, Hong [/bib_ref]. Like all ectopic pregnancies, it can be potentially life threatening given the risk of heavy haemorrhage and uterine rupture [bib_ref] Pregnancy located below the internal Os-cervical and caesarean scar ectopics, Bari [/bib_ref] [bib_ref] Ectopic Pregnancy in caesarean section scar: a case report, Aich [/bib_ref]. Although the number of reported cases seems to increase with the increasing rate of caesarean deliveries [bib_ref] Caesarean scar pregnancy, Ash [/bib_ref] , the number of cases remain very few with a reported incidence ranging from 1 in 2216 to 1 in 1800 pregnancies [bib_ref] Caesarean scar ectopic pregnancy, Devarajan [/bib_ref].
Given its very rare nature, it constitutes a diagnostic dilemma among clinicians, especially in cases of late presentation with severe obstetric haemorrhage and in resource-limited settings where first trimester ultrasound scans are not routinely performed [bib_ref] Low-lying-implantation ectopic pregnancy: a cluster of cesarean scar, cervico-isthmus, and cervical ectopic, Tsai [/bib_ref]. Failure to identify this condition on time could contribute to increase maternal morbi-mortality. A few cases have been reported in literature but none to our knowledge has been reported in Cameroon. We present a case of ruptured ectopic pregnancy at 20 weeks gestation on a successfully tried double caesarean section scar.
## Open access
## Bmc research notes
*Correspondence: [email protected] 1 Kekem District Hospital, Kekem, West Region, Cameroon Full list of author information is available at the end of the article
## Case presentation
A 29-year-old black Cameroonian of Bamileke ethnicity, gravida 5, para 3 with a past obstetric history of two consecutive caesarean sections done 9 and 5 years ago. Also noted was a successful trial of scar 2 years after the second caesarean section and a uterine evacuation following a miscarriage at 8 weeks of gestation.
The patient before consultation in our facility had been hospitalised twice in an integrated health centre for mild pelvic discomfort and two episodes of bleeding per vaginum, for which she was managed with injectable progesterone and discharged with favourable evolution.
The client was then received in our emergency department at 20 weeks gestation with mild to moderate intermittent lower abdominal pains associated to mild epigastralgia. The client however had a history of fever 24 h before consultation but no urgency, frequency nor mictalgia were reported. The parameters at entry had as temperature 37 °C, blood pressure of 110/60 mmHg, Pulse rate of 94 beats per minute. Other physical examination findings included mild generalised abdominal tenderness on superficial and deep palpation which was worse at the pelvic region. The provisional diagnosis of threatened abortion (due to malaria or asymptomatic bacteriuria was considered) with acute appendicitis in pregnancy as differential. A rapid diagnostic test for malaria was done which was negative. The team on duty directly went into management with Omeprazole 20 mg tablets (1 tablet 12 hourly), phloroglucinol 80 mg injectable (1 ampoule 8 hourly) and Ampicilline injectable (1 g 8 hourly) while thick blood smears, urinalysis, obstetric ultrasound, and full blood count were requested for the next morning.
Her evolution 4 h later was marked by an increase in the intensity of the abdominal pain which became generalised with altered general condition. The blood pressure was unmeasurable and the pulse feeble. Immediate paracentesis revealed 10 cc of fresh non coagulating blood. The diagnosis of ruptured ectopic pregnancy with abundant hemoperitoneum was considered and because of hemodynamic instability, the patient was immediately prepared for an emergency laparotomy with fluid and blood resuscitation.
A midline subumbilical laparotomy [fig_ref] Figure 1: Image showing midline laparotomy and hemoperitoneum [/fig_ref] was carried under general anaesthesia. The perioperative findings included: an abundant hemoperitoneum [fig_ref] Figure 1: Image showing midline laparotomy and hemoperitoneum [/fig_ref] estimated to two litres; intact membranes containing the foetus in the abdominal cavity; rupture line along the old caesarean scar at the isthmic uterine region, detached placental tissue most of which was still inserted and covering the internal cervical os (see [fig_ref] Figure 2: Image showing the uterus and the intact membranes Fig [/fig_ref]. The uterus and the intact membranes were carefully exteriorised. The hemoperitoneum was reduced with the help of sterile abdominal towels. Careful detachment of the placenta and repair of the opening with vicryl 1 suture was done. The patient was placed on antibioprophylaxis with Ampicilline injectable (1 g 8 hourly for 2 days) and analgesics (1000 mg of injectable paracetamol 8 hourly for 3 days). The postoperative recovery was uneventful and the patient was discharged 7 days following surgery. Follow-up of the patient to 1 year after surgery was uneventful.
# Discussions and conclusions
Caesarean scar pregnancy (CSP) is the rarest form of ectopic pregnancy and can be associated to serious and life threatening obstetric complications. The incidence of CSP varies from 1 in 1800 to 1 in 2226 pregnancies [bib_ref] Caesarean scar pregnancy, Ash [/bib_ref] and accounts for 6% of ectopic pregnancies in women with prior cesarean deliveries [bib_ref] • fast, convenient online submission • thorough peer review by experienced researchers..., Petrides [/bib_ref]. Given its location and possibility of growth with associated normally increasing titres of beta-hCG, a high suspicion index remains the only efficient way to identify and manage this life threatening obstetric condition on time. Even though the main cause of CSP is not known, in literature, it has been associated to history of uterine trauma, caesarean section [bib_ref] Ectopic pregnancies caesarean section scars: the 8 year experience of one medical..., Maymon [/bib_ref] , invitro fertilization, manual placental removal, adenomyosis and myomectomy [bib_ref] Caesarean scar pregnancy, Ash [/bib_ref] [bib_ref] Caesarean scar ectopic pregnancy, Devarajan [/bib_ref] [bib_ref] Caesarean scar ectopic pregnancy: a single centre case series: original article, Michener [/bib_ref]. In most cases of CSP, the gestation sac is usually completely surrounded by myometrium and the fibrous tissue of the scar, quite separate from the endometrial cavity [bib_ref] Caesarean scar pregnancy, Ash [/bib_ref]. These forms are generally termed "intramural pregnancies" (that is, completely confined to the myometrium) as opposed to the other forms which develop without total confinement to the myometrium [bib_ref] Firsttrimester diagnosis and management of pregnancies implanted into the lower uterine segment..., Jurkovic [/bib_ref]. The most efficient and readily available tool for early diagnosis is transvaginal ultrasound. Suggested sonographic criteria to be considered when making diagnosis of CSP in literature include: empty uterine cavity; location of trophoblast mainly between bladder and anterior uterine wall; thin or absent layer of myometrium between the gestational sac and the bladder; identification of a discontinuity in the anterior wall of the uterus on a sagittal view running through the amniotic sac and an empty endo-cervical canal [bib_ref] Firsttrimester diagnosis and management of pregnancies implanted into the lower uterine segment..., Jurkovic [/bib_ref] [bib_ref] Clinical dilemmas and risks of misdiagnosis and mismanagement associated with endogenous caesarean..., Paterson [/bib_ref]. No classical treatment methods have been described in literature but treatment methods range from close monitoring to viability and term to conservative and radical surgery in case of uterine rupture [bib_ref] Ectopic pregnancy in previous caesarean section scar, Hong [/bib_ref] [bib_ref] Evidence-based management of non-tubal ectopic pregnancies, Alalade [/bib_ref].
In our case, the patient had undergone two caesarean sections with a successful trial of the two scars and a dilatation and curettage for an incomplete abortion. The trial of the double scar and the subsequent dilatation and curettage might have compromised the integrity of the caesarean scar thereby exposing her to CSP. In CSP, it is thought that implantation villi find their way into the myometrium through a microtubular tract between the caesarean section scar and the endometrial canal [bib_ref] Caesarean scar pregnancy, Ash [/bib_ref]. The placenta and the conceptus were totally expulsed from the uterine opening with an intact posterior uterine wall.
Most cases of CSP are usually diagnosed in the first trimester. Patients are generally amenorrheic women with painless vaginal bleeding early in pregnancy (39%), mild abdominal pain or discomfort (16%) [bib_ref] Caesarean scar pregnancy, Ash [/bib_ref] [bib_ref] Caesarean scar ectopic pregnancy, Devarajan [/bib_ref]. In case of rupture, patients will present with severe acute pain of sudden onset and profuse bleeding and hypovolemic shock. However, 9% of patients with non-ruptured forms might present just with abdominal pains and may be asymptomatic with incidental diagnosis in 37% of cases [bib_ref] Caesarean scar pregnancy, Ash [/bib_ref]. This constitutes the diagnostic dilemma as none of these signs directly point to caesarean scar pregnancy as the closest possible diagnosis. This therefore calls on a high index of suspicion of CSP especially in women presenting with any of the above symptoms and any of the risk factors. On suspicion of CSP radiological evaluation is mandatory [bib_ref] Ectopic Pregnancy in caesarean section scar: a case report, Aich [/bib_ref]. In most cases without rupture or onset of rupture, clinical examination is usually unremarkable. The uterus and the abdomen are usually tender in case of rupture [bib_ref] Caesarean scar ectopic pregnancy: a single centre case series: original article, Michener [/bib_ref]. Failure of diagnosis and extension into the second trimester is usually associated with a higher risk of rupture and haemorrhage. It was the case with the client as the diagnosis was only made when the life of the patient was seriously threatened.
In the above presented case, our patient had presented twice with mild pervaginal bleeding in the first trimester which was managed with injectable progesterone. No first trimester ultrasound was done by the patient because of financial barriers. The influence of the low income setting here on the health of the patient is clear. This is a cause for concern as most pregnant women in our setting start antenatal consultations only in the second trimester and the ultrasounds are done only in the late second trimester. The very rare nature of ectopic pregnancies in the developed world has also been associated to missed and mismanaged cases of CSP even when first trimester ultrasound scans are readily available [bib_ref] Clinical dilemmas and risks of misdiagnosis and mismanagement associated with endogenous caesarean..., Paterson [/bib_ref]. Early diagnosis and management of this life threatening condition is therefore even more impeded in a resource-limited setting given that the access of pregnant women to ultrasound scans is still limited.
The working diagnosis (threatened abortion due to malaria or asymptomatic bacteriuria with acute appendicitis in pregnancy as differential) was considered given the very low suspicion index of CSP within the team. This low suspicion index and the absence of the ultrasonographer allowed for progression of the condition to its severe and life threatening stage. In case of rupture, medical treatment with methotrexate or curettage is not a suitable option. Emergency laparotomy with resection of the ectopic and repair of the uterus or hysterectomy are suitable options. In our case, given the future fertility desire expressed by the couple and peroperative findings, we completed resection of the ectopic and repaired the uterine opening.
Caesarean scar pregnancy remains a very rare obstetric condition. Late diagnosis of this condition can be associated with serious life threatening obstetric complications. Given the rare nature ectopic pregnancies and more specifically CSP, clinicians should have a high index of suspicion especially in amenorrheic women with risk factors. First trimester ultrasonography remains indispensable in the appropriate screening for such conditions especially in clients presenting with risk factors. Improving access to prenatal services (while emphasizing on the need to take up these services early in pregnancy and systematically meeting competent ultrasonographers in the first trimester especially in patients with risk factors for CSP) could go a long way to improve the condition and the prognosis of patients with CSP.
Abbreviation CSP: caesarean scar pregnancy.
Authors' contributions ABA, BK, and VNA contributed equally in the diagnosis, management and follow up of the patient, ABA and PNN drafted the manuscript. All authors read and approved the final manuscript.
[fig] Figure 1: Image showing midline laparotomy and hemoperitoneum [/fig]
[fig] Figure 2: Image showing the uterus and the intact membranes Fig. 3 Image showing the rupture line along the old caesarean scar [/fig]
[fig] Author details 1: Kekem District Hospital, Kekem, West Region, Cameroon. 2 Department of Biomedical Sciences, University of Dschang, Dschang, Cameroon. 3 Ibal Sub-Divisional Hospital, Oku, North-west Region, Cameroon. 4 Department of Obstetrics and Gynaecology, Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, Yaoundé, Cameroon. 5 Obstetrics and Gynaecology Unit, Yaoundé Central Hospital, Yaoundé, Cameroon. [/fig]
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The synergistic effect of high temperature and ozone on the number of deaths from circulatory system diseases in Shijiazhuang, China
Introduction: Urban ozone pollution in China is becoming increasingly serious.Climate warming, high temperatures, and ozone pollution all have significant impacts on human health.However, the synergistic effects of high temperatures and ozone pollution in summer on human health are rarely studied.China is at a critical stage of environmental pollution control.Assessing the health impact of high temperatures and ozone exposure on the number of deaths from circulatory diseases is of great significance for formulating ozone-related prevention and control policies.Methods: This study uses daily data on deaths from circulatory system diseases in Shijiazhuang from June to August during the summer of 2013-2016, as well as concurrent meteorological data and concentration of O 3 and PM 2.5 pollution data.The generalized additive model (GAM) with Poisson distribution, smooth curve threshold effect, and saturation effect method is used to control for confounding effects.Results: The study evaluates the impact of short-term exposure to temperature and ozone on deaths from circulatory system diseases and the synergistic effect after controlling for confounding factors.The results show that the impact of temperature and ozone on deaths from circulatory system diseases in Shijiazhuang is nonlinear, with a temperature threshold of 27.5°C and an ozone concentration threshold of 100 μg/m 3 .With an increase of temperature by 1°C, the risk of deaths for total population, men and women are 6.8%, 4.6% and 9.3%, respectively.The increase in temperature and ozone concentration has a greater impact on women; in men, the increase has a lag effect of 2 to 3 days, but the lag did not affect women.Discussion: In conclusion, high temperatures and high ozone concentration have synergistic enhancement effects on circulatory system diseases.Prevention and scientific management strategies of circulatory system diseases in high temperatures and high ozone environments should be strengthened.KEYWORDShigh temperature and ozone concentration, circulatory diseases, the number of deaths, synergistic effect, health effect
# Introduction
China used to suffer from serious air pollution.With the Chinese government's efforts to reduce emissions, PM 2.5 and other fine particulate matter pollution has been significantly improved.However, ozone (O 3 ) pollution has become increasingly serious in recent years, and has become a serious problem for China's urban environment [bib_ref] Anthropogenic drivers of 2013-2017 trends in summer surface ozone in China, Li [/bib_ref].Especially against the background of global warming, the frequency and intensity of heat wave events has increased.The dual effects of high temperatures and ozone pollution may coexist for a long time, and will seriously threaten people's health and become a new focus of attention [bib_ref] Heat waves in the United States: mortality risk during heat waves and..., Anderson [/bib_ref].
According to the Lancet Countdown China report, the number of heatwave exposure days per capita in China increased by 4.51 days in 2020 compared to the 1986-2005 average, resulting in an increase of about 92% in heatwave related deaths, and the impact on human health is increasing.High ozone pollution can lead to an increase in related diseases and deaths [bib_ref] Tropospheric ozone distributions over Europe during the heat wave in July 2007..., Eremenko [/bib_ref] [bib_ref] The relation between temperature, ozone, and mortality in nine French cities during..., Filleul [/bib_ref].Previous studies focused more on the impact of one atmospheric environmental condition on health, such as high temperatures or ozone.However, there are few studies on whether high temperatures and ozone exposure have synergistic effects on human health.
The Beijing-Tianjin-Hebei region is an area with serious air pollution, and the ozone concentration has been on the rise in recent years [bib_ref] Characteristics of ozone and its relationship with meteorological factors in Beijing-Tianjin-Hebei region, Wang [/bib_ref].Shijiazhuang, the capital city of Hebei Province, is also a representative city in northern China.In this paper, ozone concentration data published on the website of the Ministry of Environmental Protection of China during 2013-2016 and circulatory system disease deaths in Shijiazhuang during the same period are used.Based on epidemiological analysis, a generalized additive model and nonparametric binary response model are used to evaluate the effects of air temperature and short-term exposure to ozone on the number of deaths from circulatory system diseases in Shijiazhuang.The objective is to further explore the impact risks of high temperatures and O 3 pollution on human health in an environment with multiple exposures, so as to strengthen the proactive prevention awareness of highly sensitive people and provide a basis for the government to formulate scientific prevention and control policies.
# Data and methods
## Data sources
This study is conducted in Shijiazhuang (114° 48'e, 38° 03'n), the capital of Hebei Province in northern China.The number of daily circulatory disease deaths in Shijiazhuang during the summer (from 1 June to 31 August) from 2013 to 2016 is obtained from the Chinese Center for Disease Control and Prevention.According to the 10th edition of the International Classification of Diseases (IDC-10, coded as I00-99), deaths from coronary heart disease, ischemic heart disease, ischemic stroke, cerebral hemorrhage, and cerebral infarction are included.
The data of daily mean temperature, relative humidity, and air pressure in Shijiazhuang during the summer (from 1 June to 31 August) from 2013 to 2016 are provided by Hebei Meteorological Information Center.Data of O 3 (O 3 -8h) and PM 2.5 average daily concentration of atmospheric pollutants during the same period are obtained from the website of the Ministry of Environmental Protection of China.All of this data are quality-controlled before being released by professional organizations.
# Research methods
The daily death number of circulatory system diseases is calculated according to time series, and the influence of temperature and O 3 short-term exposure on death number of circulatory system diseases is evaluated using a generalized additive model (GAM) of Poisson distribution [bib_ref] Synergistic effect of temperature and O 3 on the numbers of COPD..., Fu G Q，tian [/bib_ref] [bib_ref] Effects of PM 2.5 exposure in different air quality grades on daily..., Fu [/bib_ref].Before assessing the synergistic effect of air temperature and O 3 on the daily death number of circulatory system diseases, the influences of daily mean air temperature and O 3 concentration on daily death number of circulatory system diseases are studied, respectively, to determine whether there is a curve relationship.In this model, the daily death number of circulatory system diseases is taken as the dependent variable, the air temperature (O 3 ) as the independent variable, and the regression spline function is used to control the confounding effects of time trend (Time), annual change (Year), Holiday effect (Holiday), relative humidity (RH), and PM i iday
In the formula, Y t is the daily death number of circulatory diseases on day t, E (Yt|X) is the expected daily number of deaths from circulatory system diseases on day t, α is the intercept, β is the regression coefficient, Χ is the temperature T (ozone O 3 ), s() is a nonlinear spline function, and df is the degree of freedom.
Secondly, stratified thresholds of daily mean temperature and O 3 concentration are analyzed according to threshold effect and saturation effect of smooth curves, and tested by logarithmic likelihood ratio.Third, the synergistic effect of air temperature and O 3 on the daily death number of circulatory diseases is analyzed.
All results are expressed as relative risk (RR) and 95% confidence interval (95% CI), with p < 0.05 as the test of statistical significance.In addition, the impact on different genders is assessed.
# Results
## Background analysis of air pollution in shijiazhuang
According to GB 9137-88 and HJ 633-2012, the concentrations of major air pollutants O 3 and PM 2.5 in Shijiazhuang are calculated from June to August in the summers of 2013-2019, and their
## Statistical characteristics of circulatory system disease deaths, pollutant concentration, and meteorological elements in shijiazhuang
Table [fig_ref] TABLE 1: Statistical characteristics of circulatory system disease deaths and meteorological elements and air... [/fig_ref] shows the statistical characteristics of circulation system deaths and meteorological environment elements in Shijiazhuang.In the summer (1 June to 31 August) from 2013 to 2016, 4,420 people died from circulatory diseases in Shijiazhuang city, of which 54.3% were men and 45.7% were women.The average daily death number from circulatory system diseases in Shijiazhuang city was 12.0, and the maximum daily death number was 40.0.During the corresponding period, the average daily temperature was 26.8°C, the relative humidity was 64.7%, O 3 concentration was 123.6 μg/m 3 , and PM 2.5 concentration was 71 μg/m 3 .
## Exposure-response relationship between daily mean temperature, ozone, and deaths from circulatory diseases in shijiazhuang
Figure [fig_ref] FIGURE 2: Variation curves of average temperature, O 3, and circulatory system disease deaths... [/fig_ref] shows the exposure-response relationship between daily mean temperature, ozone concentration, and the number of deaths from circulatory diseases in Shijiazhuang.The model controls the confounding effects of time trend, annual change, holiday effect, relative humidity, and PM 2.5 concentration.The daily mean temperature, ozone concentration, and the number of deaths from circulatory diseases show nonlinear changes.The increased risk of daily deaths from circulatory diseases is 1.6% (95%CI: 1.001, 1.032) for every 1°C increase in daily mean temperature.The increased risk of daily death from circulatory disease is 2.1% for every 10 μg/m 3 increase in O 3 concentration (95%CI: 1.007, 1.034).It can be seen that rising temperatures and increasing ozone concentration are associated with an increased risk of death from circulatory diseases.
## Effects of air temperature and ozone on circulatory disease deaths under different thresholds
Based on the previous analysis, the smoothing curve threshold effect and saturation effect method are used to analyze the threshold effect of temperature and ozone on the death number of circulatory system diseases.Table [fig_ref] TABLE 2: Relative risk and 95% confidence interval [/fig_ref] shows the relative risk and 95% confidence interval (95%CI) of the influence of daily mean temperature on the number of deaths from circulatory diseases under the stratification of temperature and ozone threshold.When the daily mean temperature is higher than 27.5°C, the daily death number of circulatory diseases changes steadily with temperature.The relative risk (RR) of increased daily deaths from circulatory diseases is 0.997 (95%CI:0.976,1.019) for every 1°C increase in mean temperature.If the RR is less than 1, no risk relationship is found.The RR of daily deaths from circulatory diseases increases by 1.049 (95%CI: 1.017, 1.083) for every 1°C increase in mean temperature above 27.5°C.RR greater than 1 is associated with an increased risk of 4.9% and is tested for significance at p < 0.05.The log-likelihood ratio of air temperature between the two layers is 0.023, and the threshold stratification is statistically significant.
Similarly, when the O 3 concentration is lower than 100 μg/m 3 , the RR for increasing the number of daily deaths from circulatory diseases is 0.994 (95%CI: 0.965, 1.025) with an increase of 10 μg/m 3 , and no risk relationship is found.When O 3 concentration is higher than 100 μg/m 3 , the RR of daily deaths from circulatory diseases increases by 1.037 (95%CI: 1.015, 1.059) and the risk increases by 3.7% for every 10 μg/m 3 increase in O 3 concentration (p < 0.0001).The risk relationship passes the significance test.
## Synergistic effects of air temperature and ozone on circulatory disease deaths
According to the stratification of ozone concentration and daily mean temperature threshold, the influence of every 1°C increase in daily mean temperature on the number of deaths from circulatory system diseases and the relationship between men and women are calculated, respectively, under different ozone concentrations and different temperature threshold intervals (Table [fig_ref] TABLE 3: Relative risk and 95% confidence interval [/fig_ref].When the O 3 concentration is less than 100 μg/m 3 , the temperature effects of different stratifications are different.When the temperature is less than 27.5°C, no significant risk relationship is found among the total population or among men.
When the temperature is higher than 27.5°C, with an increase of temperature by 1°C, the risk of the total number of deaths from circulatory diseases is 6.8% (95% CI:0.929, 1.228), but there are only 33 samples, and p < 0.05 is not statistically significant.When the O 3 concentration is greater than 100 μg/m 3 and the temperature is lower than 27.5°C, no significant risk is found in the total population or among men.However, when the temperature is greater than or equal to 27.5°C, every 1°C increase in daily mean temperature has an increased risk for the total number of deaths from circulatory diseases, for men and women, of 6.8% (95%CI: 1.025, 1.114), 4.6% (95%CI: 0.988, 1.109), and 9.3% (95%CI: 1.029, 1.162).In conclusion, when the O 3 concentration is greater than 100 μg/m3 and the air temperature is greater than 27.5°C, both the total number and the number of male and female deaths show the greatest risk effect value, indicating that higher air temperature and high ozone pollution have a synergistic enhancement effect on the number of circulatory deaths, especially for women.
## Lagged effects of temperature and ozone on deaths from circulatory diseases
Based on the analysis of the synergistic effect of temperature and ozone on the death number of circulatory system diseases, the lag effect is further analyzed.Table shows the relative risk and 95% confidence interval (95%CI) of the total number of deaths in the circulatory system in Shijiazhuang for every 1°C increase in temperature when O 3 ≥ 100 μg/m 3 and T ≥ 27.5°C, with 0 to 9 days lag.It can be seen that the lag effect of the synergistic effect of temperature and ozone is more complex.The risk of 3 days lag (T.3) is 6.9% (95%CI:1.029,1.111), and the risk of 9 days lag (T.9) increases to 7.7% (95%CI: 1.034, 1.121), p < 0.001, where the statistical significance is increased.For men, the risk increases to 6.4% at 2 to 3 days lag, and reaches the maximum of 7.7% at 7 days lag (95%CI: 1.021, 1.136), which passes the significance test of p < 0.05.While for women, it is still the same day that has the greatest impact risk and no lagged effect is found.In conclusion, the synergistic effect of air temperature and ozone has the highest risk for women and no lag effect is found, while for men there is a lag effect of 2 to 3 days and 7 days.The total number shows three high risk values on the same day, 3 days, and 9 days, respectively.
# Discussion
It is found that the effects of daily mean temperature and ozone pollution on the number of deaths from circulatory diseases in summer are nonlinear.The threshold of daily mean temperature is 27.5°C, and the threshold of ozone pollution is 100 μg/m 3 .High temperatures in summer increase the risk of death from circulatory diseases (14, 15).In terms of the temperature threshold index, the local comfortable temperature is used as a reference in many Chinese cities [bib_ref] Interaction effects between ambient temperature and PM2.5 and O3 on mortality in..., Zhang [/bib_ref] [bib_ref] Cardiovascular mortality risk attributable to ambient temperature in China, Yang [/bib_ref] to obtain the temperature threshold that has an impact on the number of deaths from cardiovascular and cerebrovascular diseases.For example, the temperature in Chengdu, Harbin, Changsha, and Guangzhou is 22.2°C, 20.6°C, 25.1°C, and 26.5°C, respectively.In Shanghai [bib_ref] Impact of average daily temperature on stroke mortality in community: a time-series..., Chen [/bib_ref] , the median daily mean temperature of 18.2°C is taken as the reference, and the risk effect of high temperatures of 30.1°C (95th percentile of temperature) on stroke can reach 26%.In Hong Kong [bib_ref] Effects of temperature on mortality in Hong Kong: a time series analysis, Yi [/bib_ref] , the 75th percentile of 27.8°C is used as the control, and the mortality risk of cardiovascular and cerebrovascular accidents is 9% (95%CI: 1.006, 1.125) when the temperature is higher than 31.5°C(99th percentile).
Giang PN et al.showed that when the average temperature in Vietnam is higher than 26°C, the risk of admission for cardiovascular and cerebrovascular diseases increase with the increase of temperature.In this paper, aiming at the influence of summer temperature on circulatory diseases, the threshold of 27.5°C is higher than the annual comfortable temperature, but it is equivalent to the 55th percentile for summer.The death risk of circulatory system diseases caused by high temperatures is mainly related to heat stimulation of the nervous regulation of the circulatory system, increased sweating, blood viscosity, blood vessel dilation, accelerated blood circulation, tachycardia, blood pressure changes, and internal blood insufficiency [bib_ref] High ambient temperature and mortality: a review of epidemiologic studies from, Basu [/bib_ref].When O 3 concentration is higher than 100 μg/m 3 , the risk of daily deaths from circulatory diseases in Shijiazhuang increased by 3.7% with every increase of 10 μg/m 3 in O 3 concentration, and the risk is statistically significant (p < 0.05).Gu et al. [bib_ref] Seasonal variation in the acute effect of ozone on emergency ambulance dispatches..., Gu [/bib_ref] studied the exposureresponse relationship between ozone and the number of emergency patients with cardiovascular and cerebrovascular diseases in Ningbo, and showed that when ozone concentration increased by 10 μg/m 3 in the warm season, the number of emergency patients with cardiovascular and cerebrovascular diseases increased by 1.17%.Tao et al. [bib_ref] Estimated acute effects of ambient ozone and nitrogen dioxide on mortality in..., Tao [/bib_ref] studied the acute effects of ozone pollution in the Pearl River Delta, and the total mortality rate increased by 0.81% when ozone concentration increased by 10 μg/m 3 , which is consistent with the results of this study.Dong et al.
(24) conducted a meta-analysis of short-term ozone exposure and mortality risk in a Chinese population, and demonstrated that the rise of atmospheric ozone concentration would lead to an increase in non-accidental total mortality, cardiovascular system disease mortality, and respiratory system disease mortality.However, Hu et al. [bib_ref] Time-series analysis of association between ozone concentration and daily emergency ambulance dispatches..., Hu [/bib_ref] studied the relationship between atmospheric ozone concentration and residents' first aid in Shijiazhuang city from 2013 to 2015 and found that, when ozone concentration increased by 10 μg/m 3 , the number of residents requiring first aid for respiratory diseases increased by 1.21%, but there was no significant change in the number of residents requiring first aid for circulatory diseases.This may be related to seasonal differences and whether to adjust the confounding effect of PM 2.5 pollutant [bib_ref] Seasonal variation in the acute effect of ozone on emergency ambulance dispatches..., Gu [/bib_ref] [bib_ref] Effects of season and temperature on gastrointestinal bleeding in patients with ischemic..., Ma [/bib_ref].This study mainly focuses on summer and adjusts the confounding effect of PM 2.5 .Ozone has a strong oxidizing ability, and short-term exposure to ozone causes the increase of systemic oxidative stress, which is related to human platelet activation and blood pressure increase, thus affecting cardiovascular health [bib_ref] Autonomic effects of controlled fine particulate exposure in young healthy adults: effect..., Day [/bib_ref].
The synergistic effect of temperature and ozone on population health is less studied.The North China Plain is a region with high temperatures and ozone concentrations in the summer.It is found that, when O 3 ≥ 100 μg/m 3 and T ≥ 27.5°C, the risk of death in the circulatory system is the highest 6.8% (95%CI:1.025,1.114), with it growing with every 1°C increase in temperature.When O 3 < 100 μg/m 3 and T ≥ 27.5°C, the risk of circulatory death is still 6.8% (95%CI:0.929,1.228), but is not statistically significant.When T < 27.5°C, no risk relationship is found whether ozone concentration is greater than 100 μg/ m 3 or not.It shows that high temperatures mainly affect the death number of circulatory system diseases in summer, and high temperatures and high ozone pollution have a synergistic enhancement effect.The study by Zhang et al. [bib_ref] Interaction effects between ambient temperature and PM2.5 and O3 on mortality in..., Zhang [/bib_ref] showed that the interaction between air temperature and pollutants had a very complex relationship on the number of deaths from diseases.When high temperatures and high ozone concentration co-existed, there was a synergistic strengthening effect on the number of deaths from respiratory and cardiovascular diseases, which was consistent with the results of this study.Zhang [bib_ref] Interaction effects between ambient temperature and black carbon and PM2.5 on mortality..., Zhang [/bib_ref] studied the interaction effect between air temperature and PM 2.5 pollutant in Beijing and showed that when the air temperature was higher than 24°C, the risk of death from cardiovascular and cerebrovascular diseases caused by air temperature and PM 2.5 together reached 3.97%, which increased the risk of death from circulatory diseases in a high temperature and high pollution environment.Ren C (30) studied the short-term effects of air temperature and ozone on the total mortality in 60 communities in the eastern United States and pointed out that high temperatures could regulate the risk of ozone death, with certain regional differences.
In the study of the lag effect and gender difference, this study finds that under a high temperature and high ozone concentration environment, there is a 3-day lag effect on the total number of deaths on men from circulatory system diseases, but there is a lag effect on women.Zhang et al. ( 16) performed a single ozone lag analysis and found that the risk of death from cardiovascular and cerebrovascular diseases increased by 0.66% (95%CI: 0.42, 0.90) TABLE The relative risk and 95% confidence interval (95%CI) of the effect of temperature increases of 1°C with different lag days on the total number of circulatory system deaths and gender in Shijiazhuang.
## T (°c)
Total number Men Women 22) analyzed the influence of ozone concentration on cardiovascular and cerebrovascular diseases by using the reception data of emergency vehicles in Ningbo and found that when ozone concentration increased by 10 μg/m 3 , the excess risk of the number of emergency patients for cardiovascular and cerebrovascular diseases was greater for men than women, and there was no lag effect.There are some similarities and differences between the above studies and the results of this study.In many studies, there is a lag analysis for high temperature, and there is also a lag analysis for the impact of pollutants, but the analysis of the synergistic lag of temperature and ozone is rare.Theoretically, there is a lag effect on circulatory disease from high temperatures, and there is also a lag effect from ozone.The high temperature and high ozone environment in summer enhances the risk of circulatory death, and the lag results obtained in this study are reliable.In this study, Shijiazhuang, a representative city in northern China with frequent high temperatures in summer and serious ozone pollution, is selected.Disease data are circulatory system disease death data, and the selected cities and health conditions are more representative than outpatient case data.This study analyzes the single factor influence relationship, threshold index, synergistic effect, and lag effect of temperature and ozone, which is more comprehensive than previous analysis.It reflects the relationship based on the impact of temperature and ozone pollution on the number of deaths from circulatory system diseases, which is a common issue, but there may be certain limitations in individual exposure.
# Conclusion
In this paper, daily circulatory system disease death data, meteorological data, and O 3 and PM 2.5 concentration pollution data in Shijiazhuang city from June to August in the summers of 2013 to 2016 were used to evaluate the impact of high temperatures and short-term exposure to O 3 on the number of deaths from circulatory system diseases after controlling the confounding effect.It was found that high temperatures and O 3 pollution had a synergistic effect on circulatory system diseases.It provides evidence for strengthening the prevention and scientific management of circulatory system diseases under high temperatures and in high ozone environments.
[fig] FIGURE 2: Variation curves of average temperature, O 3, and circulatory system disease deaths in Shijiazhuang from June to August, 2013 to 2016. [/fig]
[table] TABLE 1: Statistical characteristics of circulatory system disease deaths and meteorological elements and air pollutants in Shijiazhuang from June to August, 2013 to 2016. [/table]
[table] TABLE 2: Relative risk and 95% confidence interval (95%CI) for the effect of temperature and O3 on the number of circulatory system deaths in Shijiazhuang from June to August, 2013 to 2016. [/table]
[table] TABLE 3: Relative risk and 95% confidence interval (95%CI) of circulatory system deaths with every 1°C increase between different concentrations of temperature and O 3 interval. [/table]
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10.3390/insects13090766
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CCBY
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9504246
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36135467
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s2orc_pubmed_articles
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Functions of Egg-Coating Substances Secreted by Female Accessory Glands in Alderflies, Fishflies and Dobsonflies (Megaloptera)
# Introduction
Insect eggs are protected physically and chemically, to allow them to survive harsh environmental conditions and biological attacks. The chorion differs interspecifically in structure, reflecting different physiological needs and environmental hazards [bib_ref] Novel morphological and physiological aspects of insect eggs, Trougakos [/bib_ref]. Deposited eggs are highly vulnerable to oophagous predators and parasitoids, and eggs are therefore also protected directly by the parents or indirectly by maternal secretory chemicals that cover the eggs [bib_ref] Chemical protection of insect eggs, Blum [/bib_ref] [bib_ref] Insect accessory reproductive glands: Key players in production and protection, Gillott [/bib_ref]. Further, maternal secretion of chemical substances onto the laid eggs and the cleaning of each egg contributes to antimicrobial activity [bib_ref] Presence of antibacterial peptides on the laid egg chorion of the medfly..., Marchini [/bib_ref] [bib_ref] Maternal care provides antifungal protection to eggs in the European earwig, Boos [/bib_ref] [bib_ref] Eggshell spheres protect brown widow spider (Latrodectus geometricus) eggs from bacterial infection, Makover [/bib_ref]. Most insects coat
# Materials and methods
## Examined species
We observed the egg masses laid by females of two species of alderflies (Sialidae: Sialis), nine species of fishflies (Corydalidae: Chauliodinae; Anachauliodes, Nigronia, Parachauliodes, Neochauliodes), and 23 species of dobsonflies (Corydalidae: Corydalinae; Protohermes, Nevromus, Neoneuromus, Acanthacorydalis) . Light-trapped females in the field and those emerging from laboratory-reared larvae were placed in individual glass vessels (65 mm in diameter, 90 mm in height) at 25 ± 1 - C (14:10 h L:D cycle) after measuring their head width (between the outer margin of the right and left eyes) and forewing length (distance from the tip to the base) using slide calipers. A wet filter paper was placed on the bottom of the vessel to prevent desiccation, and the top was covered with nylon mesh and a glass lid. Adults were given 10% sucrose solution every day, which was dropped onto the adult mouthpart until they stopped drinking.
Field-collected larvae were placed in individual glass vessels (65 mm in diameter, 90 mm in height) with small stones on the bottom acting as refuges. Well-aerated tap water, not exceeding 5 mm in depth, was provided and replaced daily. The larvae were fed one or two living last-instar larvae of a chironomid midge every day. The rearing vessels were stored in an incubator at a constant temperature of 15 ± 1 - C, 20 ± 1 - C, or 30 ± 1 - C (14:10 h L:D cycle) according to the water temperature of the larval habitat of each species. When the larvae stopped feeding for one week, they were individually relocated to moist peat moss, in which they made holes for pupation, and maintained at 25 ± 1 - C (14:10 h L:D cycle). Adults usually emerged after about a 14-day prepupal period and about a 10-day pupal period.
The two egg masses parasitized by the egg parasitoids were found in the field and preserved in 80% ethanol. The normal and parasitized eggs were counted separately under a binocular microscope (×4) to calculate % parasitism. . Information on species examined in this study with the mean and standard errors (SEs) of the head width, forewing length, and weight of accessory glands (AGs) in the field-collected females and/or those reared from the field-collected larvae. -: not measured.
## Female accessory gland substances
The accessory gland of the reproductive organs was dissected out of the female body under low-temperature anesthesia (insects were kept at −20 - C until they stopped moving) and weighed. The gland was immediately broken with fine forceps in an 8 mL glass vial with a polyethylene lid. After removing the gland wall, the liquid substance was preserved at −30 - C prior to the experiments, to examine its ability to protect eggs from desiccation, prevent feeding by ants, and avoid predation by oophagous ladybird beetles. A portion of the substance in the vials was dried for long-term preservation (usually over one month). In this case, a drop of distilled water was added to the dried substance and mixed with fine forceps before the experiments. Therefore, all the experiments did not accurately reproduce the actual substance concentration present in the female glands, but as in the laboratory operation, the substances were dried once secreted to cover the eggs in the field.
Desiccation protection was assessed based on the water loss of 1% agarose gel kept at 25 ± 1 - C for 24 h in small glass tubes (7 mm in diameter, 25 mm in height), the openings of which were covered with 0.02 mm nylon mesh (N = 3) or nylon mesh + accessory gland substances (N = 3). The glass tubes covered with nylon mesh + distilled water (N = 3) were also used, because a drop of distilled water was added to the partly dried accessory gland substances in the vials for uniform coverage of the nylon mesh with the substances dissolved in water. The experiments were performed once in 10 species and in triplicate (#1-3) in six species. The glass tubes containing the agarose gel were weighed before (W 0 ) and after (W 1 ) 24 h at 25 - C, and finally after drying in an oven at 80 - C over the next 24 h (W 2 ). The water-loss percentage was calculated as (W 0 − W 1 )/(W 0 − W 2 ) × 100.
Ants are major predators of insects [bib_ref] Role of ants in pest management, Way [/bib_ref] and insect eggs [bib_ref] Chemical protection of insect eggs, Blum [/bib_ref] and can usually recognize the presence of repellent compounds. They are ideal for use in bioassays of potential noxious substances [bib_ref] Aposematism in the burying beetle? Dual function of anal fluid in parental..., Lindstedt [/bib_ref]. In this study, the predation avoidance function was assessed using the large ant species Camponotus japonicus Mayr (Hymenoptera: Formicidae) and small ant species Formica japonica Motschoulsky (Hymenoptera: Formicidae) as potential egg predators. These two ant species have highly variable food habits [bib_ref] Food habits of some ant species inhabiting the Taisho lava of the..., Yoshimoto [/bib_ref]. Workers of these ants were collected from Minamiosawa, Hachioji, Tokyo, central Japan. They were placed in individual glass vials (30 mm in diameter, 65 mm in height) with a piece of wet cotton for water supply and maintained at 25 ± 1 - C (14:10 h L:D cycle). Following a 24 h starvation period, a pair of glass capillaries (1 mm in inner diameter) was placed in each vial through a cotton plug. These capillaries contained 10% sucrose solution + distilled water (5 µL/mL) and 10% sucrose solution + accessory gland substances (5 mg/mL), respectively. Ten vials with ants and three vials without ants (control) were prepared for each ant species. Before all of the sucrose solution was consumed (after 0.5-6 h for large C. japonicus and 11-25 h for small F. japonica), the reduction (L mm) from the initial liquid level was measured using slide calipers. The amount of sucrose solution consumed was calculated as follows: π (0.5 mm) 2 (L ant -L control ). Following the experiments, the ant head width was measured at the widest part.
Adults of predatory ladybird beetles are also general predators of insect eggs (e.g., [bib_ref] Feeding preference of three lady beetle predators of the hemlock woolly adelgid..., Butin [/bib_ref] [bib_ref] Cannibalism in two subtropical lady beetles (Coleoptera: Coccinellidae) as a function of..., Bayoumy [/bib_ref]. In this study, adult ladybird beetles, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), were collected in Tokyo, Saitama, Yamanashi, and Nagano Prefectures in central Japan. These field-collected individuals were starved for one day and then used for egg feeding experiments. The ovulated mature eggs were dissected out of the female abdomen and placed on a wet filter paper in a small Petri dish (26 mm in inner diameter, 14 mm in height), arranged in an alternating pattern of five uncoated eggs and five coated eggs by dipping them into the thawed female accessory gland substances. After 24 h, at 25 ± 1 - C (14:10 h L:D cycle), from putting each beetle in the Petri dish, we counted eggs by dividing into three categories, intact, broken, and lost (eaten), as determined under a binocular microscope (×4). If all eggs were intact, the data were excluded from the analysis. Ants are major predators of insects [bib_ref] Role of ants in pest management, Way [/bib_ref] and insect eggs [bib_ref] Chemical protection of insect eggs, Blum [/bib_ref] and can usually recognize the presence of repellent compounds. They are ideal for use in bioassays of potential noxious substances [bib_ref] Aposematism in the burying beetle? Dual function of anal fluid in parental..., Lindstedt [/bib_ref]. In this study, the predation avoidance function was assessed using the large ant species Camponotus japonicus Mayr (Hymenoptera: Formicidae) and small ant species Formica japonica Motschoulsky (Hymenoptera: Formicidae) as potential egg predators. These two ant species have highly variable food habits [bib_ref] Food habits of some ant species inhabiting the Taisho lava of the..., Yoshimoto [/bib_ref]. Workers of these ants were collected from Minamiosawa, Hachioji, Tokyo, central Japan. They were placed in individual glass vials (30 mm in diameter, 65 mm in height) with a piece of wet cotton for water supply and maintained at 25 ± 1 °C (14:10 h L:D cycle). Following a 24 h starvation period, a pair of glass capillaries (1 mm in inner diameter) was placed in each vial through a cotton plug. These capillaries contained 10% sucrose solution + distilled water (5 μL/mL) and 10% sucrose solution + accessory gland substances (5 mg/mL), respectively. Ten vials with ants and three vials without ants (control) were prepared for each ant species. Before all of the sucrose solution was consumed (after 0.5-6 h for large C. japonicus and 11-25 h for small F. japonica), the reduction (L mm) from the initial liquid level was measured using slide calipers. The amount of sucrose solution consumed was calculated as follows: (0.5 mm) 2 (Lant-Lcontrol). Following the experiments, the ant head width was measured at the widest part.
## Statistics
Values are shown as mean ± standard error (SE). Pearson's correlation analysis was used to examine the log-log relationship between the mean female body size (head width) and the mean accessory gland weight among 29 species , and the residuals from the estimated regression equation were used to analyze the relative accessory gland size on their phylogenetic relationships of Megaloptera. In the analysis of the ability of accessory gland substances to prevent the desiccation of the eggs, differences in the mean water loss (%) among the three treatment groups were tested by the analysis of variance (ANOVA). The paired t-test was used to detect difference in the mean amount of sucrose solution consumed by each ant when the solutions with and without female accessory gland substances were presented simultaneously. In the experiments of the ability of the accessory gland substances to prevent egg predation by the ladybird beetles, differences in frequencies of intact, broken, and lost (eaten) eggs were examined by the chi-square test.
# Results
## Egg masses and accessory gland substances
Female alderflies and fishflies, excluding Nigronia, laid single-layered egg masses [fig_ref] Figure 2: Typical egg masses of Megaloptera [/fig_ref]. In contrast, all species of dobsonflies laid multi-layered egg masses with a hemispherical shape [fig_ref] Figure 2: Typical egg masses of Megaloptera [/fig_ref]. The female accessory gland was a single pouch in alderflies [fig_ref] Figure 3: Megaloptera female accessory glands [/fig_ref] , a single elongated tube in fishflies [fig_ref] Figure 3: Megaloptera female accessory glands [/fig_ref] , and a paired pouch in dobsonflies [fig_ref] Figure 3: Megaloptera female accessory glands [/fig_ref]. Liquid substances present in the female accessory gland were usually pale to dark brown liquid in alderflies and fishflies [fig_ref] Figure 3: Megaloptera female accessory glands [/fig_ref]. The gland substances were yellow, green, orange, and brown in species of dobsonflies and showed more varied properties: sticky in Protohermes species [fig_ref] Figure 3: Megaloptera female accessory glands [/fig_ref] and powdered after drying in Nevromus, Neoneuromus, and Acanthacorydalis [fig_ref] Figure 3: Megaloptera female accessory glands [/fig_ref]. In the latter three genera, the egg mass was wholly covered with a hard material [fig_ref] Figure 2: Typical egg masses of Megaloptera [/fig_ref]. (a) Parachauliodes continentalis, in situ, and accessory gland substance one day after secretion; (d,e) Protohermes grandis, in situ, and accessory gland substance one day after secretion; (f-h) Neoneuromus ignobilis: (f) dissected accessory gland, (g) pale green color of the accessory gland substance just after excretion, (h) accessory gland substance dried to a hardened white powder.
[formula] (b) (c) (d) (h) (f) (g) (i) (j) (k) (l) (e) [/formula]
The female body size of Megaloptera varied greatly in head width and forewing length among genera and species Two egg masses of Protohermes grandis were parasitized by the hymenopteran parasitoid Oooencyrtus yoshidai in the field; one in Niigata Prefecture and the other on Sado Island, central Japan; 16.2% and 28.1% of eggs were parasitized, respectively.
## Functions of accessory gland substances
Female accessory gland substances prevented water loss from the 1% agarose gel, excluding Parachauliodes continentalis and Neoneuromus coomani [fig_ref] Figure 4: Megaloptera female accessory gland substance effects against dehydration [/fig_ref]. In this experiment, alderflies (Sialis) and some fishflies (e.g., Neochauliodes) could not be examined Two egg masses of Protohermes grandis were parasitized by the hymenopteran parasitoid Oooencyrtus yoshidai in the field; one in Niigata Prefecture and the other on Sado Island, central Japan; 16.2% and 28.1% of eggs were parasitized, respectively.
## Functions of accessory gland substances
Female accessory gland substances prevented water loss from the 1% agarose gel, excluding Parachauliodes continentalis and Neoneuromus coomani [fig_ref] Figure 4: Megaloptera female accessory gland substance effects against dehydration [/fig_ref]. In this experiment, alderflies (Sialis) and some fishflies (e.g., Neochauliodes) could not be examined because the gland substances sufficient to cover the whole nylon mesh of the tube opening could not be obtained from their small accessory glands.
Insects 2022, 13, x FOR PEER REVIEW 10 of 2 because the gland substances sufficient to cover the whole nylon mesh of the tube openin could not be obtained from their small accessory glands. The head widths of the large and small ant species used in the feeding experimen were 2.24 mm (N = 90, SE = 0.06) and 1.16 mm (N = 79, SE = 0.01), respectively. The additio of the accessory gland substances to 10% sucrose solution decreased the feeding rate o water loss (%) estimated using agarose gel kept at 25 - C for 24 h (for explanations of Cont., Water, and AGS, see. This experiment was performed in triplicate (#1-3) in Parachauliodes continentalis, Protohermes similis, P. grandis, P. immaculatus, Neoneuromus ignobilis, and Acanthacorydalis asiatica, while there was no replication for other species. The results of ANOVA are also shown (NS: p > 0.05).
The head widths of the large and small ant species used in the feeding experiments were 2.24 mm (N = 90, SE = 0.06) and 1.16 mm (N = 79, SE = 0.01), respectively. The addition of the accessory gland substances to 10% sucrose solution decreased the feeding rate of large ants in two of 11 species tested in the experiment [fig_ref] Figure 5: Effect of female accessory gland substances from several species of Megaloptera on... [/fig_ref]. In the small ant species, the rate of feeding on the sucrose solution was decreased by adding the accessory gland substances in five of 11 species tested [fig_ref] Figure 5: Effect of female accessory gland substances from several species of Megaloptera on... [/fig_ref]. large ants in two of 11 species tested in the experiment [fig_ref] Figure 5: Effect of female accessory gland substances from several species of Megaloptera on... [/fig_ref]. In the small ant species, the rate of feeding on the sucrose solution was decreased by adding the accessory gland substances in five of 11 species tested [fig_ref] Figure 5: Effect of female accessory gland substances from several species of Megaloptera on... [/fig_ref]. In the egg choice experiment, the ladybird beetles ate more eggs without accessory gland substances than those with the substances when given the both types of eggs . The higher survival of eggs coated with the accessory gland substances was statistically significant in 13 of 15 species, excluding Sialis tohokuensis and P. continentalis. . Predation of eggs of 15 Megaloptera species coated and uncoated with female accessory gland substances (AGS) after 24 h exposure to ladybird beetles, Harmonia axyridis (see. p-values were determined by the chi-square test.
# Discussion
## Functions of egg-coating substances
All species of Megaloptera examined laid their eggs in a mass [fig_ref] Figure 2: Typical egg masses of Megaloptera [/fig_ref]. Mature eggs in the ovarioles were milky white, whereas the laid egg masses showed a variety of colors [fig_ref] Figure 2: Typical egg masses of Megaloptera [/fig_ref] due to coating with female accessory gland substances of different colors [fig_ref] Figure 3: Megaloptera female accessory glands [/fig_ref]. Egg or egg mass color may be one of the adapted traits for survival until hatching, particularly for eggs without direct parental care [bib_ref] Evolutionary ecology of insect egg coloration: A review, Guerra-Grenier [/bib_ref]. Crypsis, warning signals, and photoprotection are most important functions of insect egg adaptive coloration [bib_ref] Evolutionary ecology of insect egg coloration: A review, Guerra-Grenier [/bib_ref]. The egg masses of Megaloptera were found on the underside of leaves, tree trunks and branches, and on the surface of rocks near the streams and ponds [bib_ref] The egg masses, eggs, and first-instar larvae of Eastern North American Corydalidae, Baker [/bib_ref] [bib_ref] Life history and habits of Sialis rotunda and S. californica in western..., Azam [/bib_ref] [bib_ref] Oviposition of the dobsonfly (Corydalus cornutus, Megaloptera) on a large river, Mangan [/bib_ref] [bib_ref] Egg-laying sites of the alderfly Sialis yamatoensis (Megaloptera: Sialidae) around ponds in..., Takeuchi [/bib_ref] [bib_ref] The life history of the alderfly Sialis japonica (Megaloptera: Sialidae) in Ayukaeri..., Takeuchi [/bib_ref]. Yellow and green colors may be used as background-matching camouflage for living leaves, while brown matches tree trunks and branches, and white pale-colored tree trunks or rock surfaces. This type of crypsis may function to prevent predation by diurnal predators visually searching for food, such as birds, lizards, and mammals. The warning coloration of unpalatable or toxic eggs is still unclear in insects [bib_ref] Evolutionary ecology of insect egg coloration: A review, Guerra-Grenier [/bib_ref]. In the present study, we did not examine whether or not the eggs of Megaloptera are unpalatable or toxic for the predators that have the ability to learn by sight. Some pigments have an UV-protecting function, but the egg masses of Megaloptera are usually laid on the underside of the leaves, tree branches, and rocks, because the hatched larvae must fall into the water. This type of egg coloration may be rare in insects [bib_ref] Evolutionary ecology of insect egg coloration: A review, Guerra-Grenier [/bib_ref].
During egg development, the egg coating may help maintain the humidity of eggs. Our assessment of desiccation protection by the accessory gland substances suggested that most species can prevent water loss from the eggs by using these accessory gland substances as coatings [fig_ref] Figure 4: Megaloptera female accessory gland substance effects against dehydration [/fig_ref]. The sticky substances secreted by the female Protohermes species seemed to have a strong effect against desiccation. In addition, the hardened white powders produced by Neoneuromus and Acanthacoridalis were more protective against desiccation compared with the substances produced by Parachauliodes [fig_ref] Figure 4: Megaloptera female accessory gland substance effects against dehydration [/fig_ref]. The desiccation tolerance of insect eggs may also arise from physiological processes, such as dormancy and osmoregulation, during embryonic development [bib_ref] Insects with survival kits for desiccation tolerance under extreme water deficits, Thorat [/bib_ref]. However, egg dormancy has not been reported in any species of Megaloptera to date. Egg clustering in a mass is another factor preventing the desiccation of eggs. Single eggs and single-layered egg masses may be disadvantageous with regard to desiccation. In general, egg clustering may reduce the amount of egg surface exposed to ambient conditions, thereby reducing desiccation [bib_ref] Egg deposition patterns in butterflies: Why do some species cluster their eggs..., Stamp [/bib_ref]. In fact, under conditions of low humidity, the hatching rate was lowest in butterfly eggs experimentally arranged in a single loose layer and highest in those arranged in three tightly packed layers [bib_ref] The evolution of egg clustering in butterflies: A test of the egg..., Clark [/bib_ref]. Thus, the multi-layered egg mass in dobsonflies may also promote the retention of humidity.
Most insects coat their eggs with substances produced in the female accessory glands, which is assumed to provide eggs with protection against predators, parasitoids, and microorganisms [bib_ref] Chemical protection of insect eggs, Blum [/bib_ref] [bib_ref] Insect accessory reproductive glands: Key players in production and protection, Gillott [/bib_ref]. There have been no clear reports of predators eating megalopteran eggs in the field. In our first feeding experiment, two species of ants were used as potential predators, because ants are prominent predators of insect bodies and eggs [bib_ref] Chemical protection of insect eggs, Blum [/bib_ref] [bib_ref] Role of ants in pest management, Way [/bib_ref]. The accessory gland substances extracted from several species effectively decreased the consumption of sucrose solution by the ants [fig_ref] Figure 5: Effect of female accessory gland substances from several species of Megaloptera on... [/fig_ref]. This repellent effect against ant feeding may be more advantageous for insects laying eggs in a mass, because all of the eggs in a cluster may be consumed if discovered by foraging worker ants as a result of recruitment [bib_ref] Chemical protection of insect eggs, Blum [/bib_ref]. Ladybird beetles are also the general predators of insect eggs (e.g., [bib_ref] Feeding preference of three lady beetle predators of the hemlock woolly adelgid..., Butin [/bib_ref] [bib_ref] Cannibalism in two subtropical lady beetles (Coleoptera: Coccinellidae) as a function of..., Bayoumy [/bib_ref]. Our second feeding experiment using adult ladybird beetles showed that the accessory gland substances of most species effectively decreased the risk of predation (Table 2, [fig_ref] Figure 6: Characteristics of the egg mass and female accessory gland substances [/fig_ref].
Although five hymenopteran parasitoids (Trichogramma tajimaense, T. semblidis, Oooencyrtus longicauda, O. protohermesis, and O. yoshidai) and one dipteran parasitoid (Pseudogaurax idiogenes) have been reported to parasitize the eggs of Megaloptera [fig_ref] Table 3: Parasitoids known from Megaloptera egg masses [/fig_ref] [bib_ref] Life history and habits of Sialis rotunda and S. californica in western..., Azam [/bib_ref] [bib_ref] A new species of Pseudogaurax Malloch (Diptera: Chloropidae) reared from dobsonfly egg-masses..., Melo [/bib_ref] [bib_ref] The life history and feeding habits of Sialis cornuta Ross in a..., Pritchard [/bib_ref] [bib_ref] A new species of Trichogramma (Hymenoptera: Trichogrammatidae) parasitic on eggs of the..., Yashiro [/bib_ref] [bib_ref] The egg-parasite of Sialis lutaria: A study of the influence of the..., Salt [/bib_ref] [bib_ref] Biological studies of two hymenopterous parasites of aquatic insect eggs, Martin [/bib_ref] [bib_ref] Description of three new species of Ooencyrtus (Hymenoptera: Encyrtidae) from China, Zhang [/bib_ref] , it is difficult to determine the effectiveness of accessory gland substances for avoidance of egg parasitism. We could not prepare the adult parasitoids and host egg masses simultaneously, but did obtain two egg masses of P. grandis parasitized by O. yoshidai in the field [fig_ref] Table 3: Parasitoids known from Megaloptera egg masses [/fig_ref]. The parasitism is usually higher in single-layered egg masses of Sialis alderflies than multi-layered egg masses of Protohermes dobsonflies (with a sticky cover) and Corydalus dobsonflies (with a hardened white cover). Interestingly, there have been no reports of parasitism on fishfly egg masses despite being laid in a single layer, as in alderflies. The egg masses are much larger than parasitoid ovipositors [fig_ref] Figure 7: Three females of the egg parasitoid Ooencyrtus yoshidai on the multi-layered egg... [/fig_ref] , and the eggs located on the surface of the egg mass are expected to be parasitized. However, it is likely that these parasitoids attack the egg masses during oviposition [bib_ref] A new species of Pseudogaurax Malloch (Diptera: Chloropidae) reared from dobsonfly egg-masses..., Melo [/bib_ref]. If so, some multi-layered and covered egg masses may be subject to a high degree of parasitism. [fig_ref] Figure 6: Characteristics of the egg mass and female accessory gland substances [/fig_ref]. Characteristics of the egg mass and female accessory gland substances (AGS) mapped on the phylogeny of Megaloptera [bib_ref] Similar pattern, different paths: Tracing the biogeographical history of Megaloptera (Insecta: Neuropterida)..., Jiang [/bib_ref]. Different background colors show different genera. * Not statistically significant; -Not examined in this study. For explanation of the log-scale residual of accessory gland (AG) weight, see the statistics section in the main text. With the exception of Nigronia, the multi-layered egg masses were all semispherical in shape (see [fig_ref] Figure 2: Typical egg masses of Megaloptera [/fig_ref].
The chemical characteristics of the accessory gland substances seem to be species/generaspecific in Megaloptera. However, the chemicals comprising the accessory gland substances are still unknown. In particular, there is interest in the chemical characteristics and synthetic processes of the sticky accessory gland substances secreted by female Protohermes and the liquids forming the hardened white covers secreted by female Neoneuromus, Acanthacorydalis, and their lineages [fig_ref] Figure 3: Megaloptera female accessory glands [/fig_ref]. Chemical information is required to better understand the structure and function of the diverse female accessory gland substances in Megaloptera. The antimicrobial activity of egg-coating chemicals will be examined in future studies. [fig_ref] Figure 7: Three females of the egg parasitoid Ooencyrtus yoshidai on the multi-layered egg... [/fig_ref]. Three females of the egg parasitoid Ooencyrtus yoshidai on the a sticky cover secreted from the female accessory glands of Protoherme
## Peer review
The chemical characteristics of the accessory gland su cies/genera-specific in Megaloptera. However, the chemicals gland substances are still unknown. In particular, there is inter teristics and synthetic processes of the sticky accessory gland male Protohermes and the liquids forming the hardened white Neoneuromus, Acanthacorydalis, and their lineages (Figures 3 and is required to better understand the structure and function of th gland substances in Megaloptera. The antimicrobial activity of Pseudogaurax idiogenes Wheeler c Brazil (Sao Paulo) not so high [bib_ref] A new species of Pseudogaurax Malloch (Diptera: Chloropidae) reared from dobsonfly egg-masses..., Melo [/bib_ref] a Egg masses laid on natural substrates. b Egg masses on artificial boards. c This dipteran parasitid in an egg mass is sometimes parasitized by the unknown small wasp parasitoid.
## Evolutionary patterns of egg mass characteristics
The evolutionary patterns of the egg mass structure and function in Megaloptera are shown by the molecular phylogenetic tree [bib_ref] Similar pattern, different paths: Tracing the biogeographical history of Megaloptera (Insecta: Neuropterida)..., Jiang [/bib_ref]. The data from the present and previous studies are summarized in [fig_ref] Figure 6: Characteristics of the egg mass and female accessory gland substances [/fig_ref] , although no information on eggs is available for alderflies of Austrosialis, Caribesialis, Haplosyalis, Indosialis, Leptosialis, and Stenosialis; fishflies of Apochauliodes, Archichauliodes, Ctenochauliodes, Dysmicohermes, Madachauliodes, Nothochauliodes, Orohermes, Platychauliodes, Protochauliodes, Puri, and Taeniochauliodes; and dobsonflies of Chloroniella. Single-layered egg masses may be plesiomorphic because alderflies and fishflies, excluding Nigronia, lay eggs in one layer. Egg masses of Nigronia are single layered in some masses and multi-layered in others, but not a hemispherical shape, even in the latter cases [bib_ref] The egg masses, eggs, and first-instar larvae of Eastern North American Corydalidae, Baker [/bib_ref] [bib_ref] A preliminary survey of the Megaloptera of Oklahoma, Arnold [/bib_ref]. In contrast, all dobsonflies lay a multi-layered hemispherical egg mass. However, a published photograph of the multi-layered egg mass laid by Chloronia hieroglyphica is slightly unclearand should be confirmed in future.
The female accessory gland was a single pouch in alderflies (also see [bib_ref] Rapid evacuation of spermatophore contents and male post-mating behavior in alderflies (Megaloptera:..., Hayashi [/bib_ref] , single elongated tube in fishflies, and paired pouch in dobsonflies. The female accessory gland substances were brown in alderflies and brown to dark brown in fishflies [bib_ref] The egg masses, eggs, and first-instar larvae of Eastern North American Corydalidae, Baker [/bib_ref] [bib_ref] A preliminary survey of the Megaloptera of Oklahoma, Arnold [/bib_ref]. In contrast, the accessory gland substances of dobsonflies varied in color among genera and species. The accessory gland weight relative to the body (residual in log scale) differed among genera. Protohermes have a larger accessory gland, which may be related to the sticky substances covering the egg mass, while all fishfly genera examined (Anachauliodes, Parachauliodes, Neochauliodes) have a smaller accessory gland. Although based on a small sample size, the accessory gland of Acanthacorydalis is relatively small. We must study the reasons why the relative size of female accessory glands varies among genera and species.
# Conclusions
The female accessory gland substances of Megaloptera are used for egg coating and show high diversity in color and other properties. The coloration of egg masses may play a role in crypsis on background substrates such as leaves, tree trunks and branches, and stones. We experimentally demonstrated that the accessory gland substances prevent severe desiccation of eggs during development and avoid predation by oophagous predators such as ladybird beetles and ants. Unfortunately, no information is available on the chemical compounds and synthesis of these egg-coating substances in Megaloptera. Further studies are required for chemical analysis of these substances and to examine other functions such as egg-parasitoid avoidance and antimicrobial activity to understand the evolution of insect egg structure and properties.
## Institutional review board statement: not applicable.
Informed Consent Statement: Not applicable.
# Data availability statement:
The data presented in this study are available on request from the authors.
[fig] Figure 1: (a) The glass tubes containing 1% agarose gel used to examine the effects of female accessory gland substances (AGS) on desiccation rates. The entire opening of the tube was covered with nylon mesh only (control), nylon mesh + water (control for AGS dissolved in distilled water if partly dried), and nylon mesh + AGS. (b) Glass vessels used to assess the preference for 10% sucrose solution, with and without AGS in the glass capillaries, of the large and small ant species Camponotus japonicus and Formica japonica. (c) Egg choice of five coated (pale brown) and five uncoated (whitish) eggs with AGS of the egg-eating ladybird beetle Harmonia axyridis. The beetle was placed in a Petri dish (26 mm inner diameter) for 24 h. [/fig]
[fig] Figure 2: Typical egg masses of Megaloptera. (a) Sialis melania, in dorsal view; (b) Anachauliodes laboissierei, in dorsal view; (c) Nigronia serricornis, in dorsal view; (d) Parachauliodes japonicus, in dorsal view; (e) Parachauliodes continentalis, in dorsal view; (f) Protohermes grandis, in lateral-dorsal view; (g) Protohermes immaculatus, in dorsal view; (h) Nevromus sp., in dorsal view; (i) Neoneuromus ignobilis, in dorsal view; (j) ditto, in ventral view; (k) Acanthacorydalis fruhstorferi, in laterodorsal view; (l) ditto, in ventral view. [/fig]
[fig] Figure 3: Megaloptera female accessory glands (ag) and ovaries (ov). (a) Sialis melania, in situ; (b,c) [/fig]
[fig] Figure 4: Megaloptera female accessory gland substance effects against dehydration. Mean (± SE, = 3) water loss (%) estimated using agarose gel kept at 25 °C for 24 h (for explanations of Con Water, and AGS, seeFigure 1a). This experiment was performed in triplicate (#1-3) in Parachauliod continentalis, Protohermes similis, P. grandis, P. immaculatus, Neoneuromus ignobilis, and Acanthacory alis asiatica, while there was no replication for other species. The results of ANOVA are also show (NS: p > 0.05). [/fig]
[fig] Figure 5: Effect of female accessory gland substances from several species of Megaloptera on avoidance of predation by the large ant species Camponotus japonicus (top) and the small ant species Formica japoninca (bottom). Two capillaries, one with sucrose solution only (S) and another with sucrose solution plus accessory gland substance (S + AGS), were offered simultaneously to each [/fig]
[fig] Figure 6: Characteristics of the egg mass and female accessory gland substances (AGS) mapped on the phylogeny of Megaloptera[37]. Different background colors show different genera. * Not statistically significant; -Not examined in this study. For explanation of the log-scale residual of accessory gland (AG) weight, see the statistics section in the main text. With the exception of Nigronia, the multi-layered egg masses were all semispherical in shape (seeFigure 2). [/fig]
[fig] Figure 7: Three females of the egg parasitoid Ooencyrtus yoshidai on the multi-layered egg mass with a sticky cover secreted from the female accessory glands of Protohermes grandis. Scale bar: 1 mm. [/fig]
[fig] Author: Contributions: Conceptualization, P.Y., X.L. and F.H.; methodology, P.Y. and F.H.; investigation and analysis, all authors; writing-original draft preparation, P.Y. and F.H.; writing-review and editing, X.L.; visualization, P.Y., X.L. and F.H. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This research was funded by JST SPRING grant number JPMJSP2156 (P.Y.) and National Natural Science Foundation of China (No. 32170448) (X.L.). [/fig]
[table] Table 3: Parasitoids known from Megaloptera egg masses: Trichogramma (Hymenoptera: Trichogrammatidae), Ooencyrtus (Hymenoptera: Encyrtidae) and Pseudogaurax (Diptera: Chloropidae). The percentages of parasitized eggs per parasitized egg mass are also shown (N is the number of parasitized egg masses examined). Protohermes grandis Thunberg Ooencyrtus yoshidai Noyes & Hirose Japan (Nagano) [36] Protohermes grandis Thunberg Ooencyrtus yoshidai Noyes & Hirose [/table]
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10.1111/1759-7714.13191
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CCBY
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6825913
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31529678
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s2orc_pubmed_articles
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Anaplastic thyroid carcinoma mimicking a malignant pleural mesothelioma: Clues for diagnosis
Pleural metastasis of thyroid carcinoma is very rarely encountered in the evaluation of pleural effusion and diagnosis may be challenging. However, an anaplastic transformation of papillary thyroid carcinoma (PTC), although a rare condition, should be considered even after a prolonged period of patient follow-up. Here we report a case of anaplastic thyroid carcinoma mimicking malignant pleural mesothelioma diagnosed nine years after the initial diagnosis of PTC and detail the clues used to orient and confirm the diagnosis.
# Introduction
Metastasis of thyroid carcinoma to the pleura is a rare condition, although anaplastic transformation of previous papillary thyroid carcinoma (PTC) is one of the most aggressive cancers. In this situation, the diagnostic exploration of a prevailing pleural effusion can be a challenge for pulmonary physicians. Indeed, in usual cases of anaplastic thyroid carcinoma (ATC), metastases occur in the thyroid and regional lymph nodes rather than at a distant site. Characterization of the pleural fluid may sometimes contain subtle clues which may assist clinicians in making the correct diagnosis.
Here, we briefly report and discuss a difficult diagnostic case of anaplastic thyroid carcinoma which developed at the pleural site mimicking a malignant pleural mesothelioma.
## Case report
A 77-year-old patient presented with a history of progressive dyspnea of one week due to a pleural effusion on the left side. He had never smoked and retired after working as a warehouseman including a few years in shipyards where he had been exposed to asbestos. He was mainly treated for an obesity-hypoventilation syndrome with noninvasive ventilation. He had a past medical history of PTC treated by total thyroidectomy nine years previously and additional radioiodine therapy for mediastinal relapse one year later with a prolonged survey showing he maintained a stable condition.
On examination, the patient required 8 L/min oxygen support and chest radiograph showed a massive pleural effusion on the left side. Thoracic computed tomography (CT) detected atelectasis of the left and right lower lobes and pleural effusion on the left side. The 18F-fluoro-2-deoxy-d-glucose positron emission tomography/computed tomography (PET/CT) fused imaging found important 18-FDG uptake in the left pleura (SUVmax 12 g/mL,) and moderate hypermetabolism of two mediastinal (SUVmax 7.6 g/mL) and one subdiaphragmatic pillar (SUVmax 8 g/mL) nodes. Because of his severe respiratory condition and despite a strong suspicion of malignant mesothelioma, a pleuroscopy for diagnosis was counterindicated. A pigtail pleural catheter was placed and percutaneous pleural biopsies performed. Analysis of the pleural fluid showed an exudate and cytology revealed predominantly lymphocytes (82%) without any malignant cells. Blood thyroglobulin was at its lowest level for the last five years (2.8 ng/mL) and thyroglobulin in the pleural fluid was at the lower detection limit (1.1 ng/mL). Echography of the neck did not detect any local relapse.
Pathology of the pleural biopsies showed a malignant poorly differentiated proliferation of fusiform cells . Immunohistochemical staining was negative for both thyroid transcriptase factor-1 (TTF1) and thyroglobulin, but showed strong positivity to paired-box gene 8 (PAX-8). Moreover, mesothelial markers such as calretinin, cytokeratin 5/6 and WT-1 were negative. Diagnosis of anaplastic thyroid carcinoma at the pleural site was confirmed and the patient was referred to a cancer endocrinologist.
# Discussion
Thyroid cancers, the most common malignant tumors of endocrine organs, are frequently found in the papillary subtypes but rarely metastasize to the pleura. The incidence is reported to be <0.1% of malignant pleural effusions. 1 Anaplastic transformation is a rare evolution of this disease. As a result, diagnosis of malignant pleural effusion due to anaplastic thyroid carcinoma is extremely rare and there have only been two previously published clinical cases. For Abe and colleagues a diagnosis was made following autopsy of a patient who presented with a massive pleural effusion with slowly progressive lung nodules. The lung nodules contained typical papillary thyroid carcinoma associated with undifferentiated thyroglobulin-negative component suggestive of anaplastic transformation. 2 Kim et al. reviewed fewer than 20 reported cases of anaplastic transformation at different metastatic sites and found only one case of pleural thickening with pleural effusion in which diagnosis was made by pleural biopsies with immunohistochemical staining suggestive of both PTC and anaplastic components.In our case, the clinical work-up was led by the diagnosis of a pleural exudate in an asbestos-exposed patient with a PET-CT evaluation mimicking a malignant pleural mesothelioma. It highlights the diagnostic difficulties in such situations of malignant pleural effusion in a patient with a previous history of PTC, in particular when a thoracoscopic assessment of the pleural cavity is not feasible.
First, a simple dosage of thyroglobulin in the pleural effusion may be helpful. This protein is known as a marker of PTC relapse after total thyroidectomy when the serum level is high, reflecting the presence of thyroid papillary cells. Following this suggestion, it has been reported that elevated pleural fluid thyroglobulin could be a potential marker of pleural metastasis from PTC.However, ATC is known for losing thyroglobulin or TTF1 expression.Unlike the two previously reported cases, our case did not show mixed cellular proliferation, only ATC histology. This is in accordance with the low level of pleural fluid thyroglobulin found in our patient.
Radioiodine-based imaging is not helpful in the case of ATC as undifferentiated cells do not absorb the iodine, but PET-CT provided important metabolic information.
A final diagnosis was obtained from tissue morphology and immunohistologic profile in all the cases discussed here. ATC is typically negative for thyglobulin and TTF1, and the PAX-8 transcription factor appears to have a useful diagnostic value (expressed in 79% of ATC cells). 1,6 This marker, a nephric-lineage transcriptor factor for organogenesis of the thyroid gland, kidney, and mullerian system, is mainly shared by renal carcinoma. BRAF mutation that was found in our patient may also help since it is commonly found in thyroid carcinoma, but not in primary pleural processes such as mesothelioma. Similar diseasespecific markers may also be useful to differentiate other rare tumors or diseases developing in the pleura, such as multiple myeloma.In conclusion, we report the first case of singlecomponent anaplastic thyroid carcinoma at the unique pleural metastatic site mimicking a malignant pleural mesothelioma. Owing to its scarcity and poor prognosis, this disease may be underdiagnosed. It could be evoked typically in patients with a past history of PTC, even after long-term patient follow-up. Pleural thyroglobulin, PAX-8 immunostaining and molecular analysis may also be useful to orient and confirm the diagnosis.
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10.1186/1754-1611-7-7
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CCBY
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3621683
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23517522
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s2orc_pubmed_articles
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Effect of substrate stiffness on early human embryonic stem cell differentiation
Background:The pluripotency and self renewing properties of human embryonic stem cells (hESC) make them a valuable tool in the fields of developmental biology, pharmacology and regenerative medicine. Therefore, there exists immense interest in devising strategies for hESC propagation and differentiation. Methods involving simulation of the native stem cell microenvironment, both chemical and physical, have received a lot of attention in recent years. Equally important is evidence that cells can also sense the mechanical properties of their microenvironment. In this study, we test the hypothesis that hESCs accept mechanical cues for differentiation from the substrate by culturing them on flexible polydimethylsiloxane (PDMS) of varying stiffness. Results: PDMS substrates were prepared using available commercial formulations and characterized for stiffness, surface properties and efficiency of cell attachment and proliferation. Across different substrate stiffness, cell numbers, cell attachment and cell surface area were found to be similar. Expression of pluripotency markers decreased with increased time in culture across all PDMS substrates of varying stiffness. Analysis of gene expression of differentiation markers indicates that the differentiation process becomes less stochastic with longer culture times. Conclusions: We evaluated the utility of PDMS substrates for stem cell propagation and substrate mediated differentiation. The stiffness affected gene expression of pluripotent and differentiation markers with results indicating that these substrate systems could potentially be used to direct hESC fate towards early mesodermal lineages. This study suggests that coupled with soluble factors, PDMS substrates could potentially be useful in generating defined populations of differentiated cells.
# Background
Human embryonic stem cells (hESCs) are characterized by their ability to self renew and to differentiate into any diploid human cell type. This property makes them a valuable tool for studying the basic biology of lineage specification, and for applications in fields such as pharmacology and tissue engineering [bib_ref] Embryonic stem cells as a model to study cardiac, skeletal muscle, and..., Wobus [/bib_ref]. The control of stem cell fate has chiefly been attributed to genetic specifications and cellular response to signals from the surrounding niche in the form of chemical, mechanical and matrix factors [bib_ref] Niche-mediated control of human embryonic stem cell selfrenewal and differentiation, Peerani [/bib_ref]. Multiple studies have shown that soluble factors such as fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs) and Wnts can regulate stem cell behaviour [bib_ref] Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human..., Xu [/bib_ref] [bib_ref] Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells, Chadwick [/bib_ref] [bib_ref] Differentiation of Human Embryonic Stem Cells to Neural Lineages in Adherent Culture..., Gerrard [/bib_ref] [bib_ref] Defining the Role of Wnt/?-Catenin Signaling in the Survival, Proliferation, and Self-Renewal..., Dravid [/bib_ref] and have been used as chemical cues in methodologies to generate clinically relevant cell populations. Mammalian cells also generate and are exposed to forces in vivo and in vitro; and these forces can influence stem cell fates by modulating cell shape, cytoskeletal structure and interaction with the extra cellular matrix (ECM) [bib_ref] Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment, Mcbeath [/bib_ref] [bib_ref] Matrix elasticity directs stem cell lineage specification, Engler [/bib_ref]. Simulating in-vivo mechanical deformations by imposing substrate strains has been shown to influence stem cell differentiation and such responses to mechanical loading depend not only on the type of stem cell but also on the state of differentiation and the type of strain applied [bib_ref] Cyclic strain induces mouse embryonic stem cell differentiation into vascular smooth muscle..., Shimizu [/bib_ref] [bib_ref] Anisotropic mechanosensing by mesenchymal stem cells, Kurpinski [/bib_ref] [bib_ref] Inhibition of human embryonic stem cell differentiation by mechanical strain, Saha [/bib_ref]. Such studies indicate that even the mechanical properties of the culture system may be modified and tailored to generate a desired cell population. Despite these numerous efforts, the creation of efficient, reliable, and scalable differentiation protocols has remained largely elusive.
Recent data suggesting that the extracellular matrix influences stem cell fate has led to interest in research involving control of stem cell fate by directing ECM geometry/ topography, mechanical properties, transmission of mechanical and biophysical factors to the cell, and the control of cell geometry [bib_ref] Exploring and Engineering the Cell Surface Interface, Stevens [/bib_ref] [bib_ref] Conversion of ES cells to columnar epithelia by hensin and to squamous..., Takito [/bib_ref]. In most cases, individual stem cells do not survive in suspension and their adhesion to a matrix is therefore essential for viability. The ECM as a major niche element provides not only a scaffold for cellular support, migration and proliferation, but also acts as the surrounding microenvironment that influences the cellular fate decision by presenting physical and chemical cues as well as binding soluble factors [bib_ref] Exploring and Engineering the Cell Surface Interface, Stevens [/bib_ref]. In vitro, the substrate acts as the primary ECM component, and while a feeder layer of inactivated mouse embryonic fibroblasts (MEF) has been the traditional gold standard, polymeric materials have also been investigated for their ability to support hESC propagation. We have previously reviewed the potential of bio-inspired polymers in determining human stem cell fate, which not only possess the advantage of being a xeno-free culture system but can also be tailored to very specific needs [bib_ref] Role of bioinspired polymers in determination of pluripotent stem cell fate, Abraham [/bib_ref].
Early proof of the influence of ECM stiffness on stem cell differentiation was provided by qualitative studies involving mouse mammary epithelial cells that showed increased differentiation when grown on soft gel collagen substrates as opposed to Tissue Culture Plastic [bib_ref] Substrate properties influencing ultrastructural differentiation of mammary epithelial cells in culture, Emerman [/bib_ref]. ECM control of stem cell fate by regulating growth factor diffusion has been demonstrated by artificially tethering a growth factor to a substrate, which increased survival of human mesenchymal stem cells (MSCs) [bib_ref] Tethered epidermal growth factor provides a survival advantage to mesenchymal stem cells, Fan [/bib_ref]. In additional studies, the ECM was also found to be a more potent differentiation cue for MSCs than standard induction cocktails [bib_ref] Growth factors, matrices, and forces combine and control stem cells, Discher [/bib_ref]. Tissue-level elasticity has been shown to be able to determine lineage and phenotype commitment in naïve MSCs. Later studies showed that human MSCs could be kept quiescent by growing them on polyacrylamide substrates that mimicked the properties of marrow while preserving their multilineage potential [bib_ref] Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but..., Winer [/bib_ref]. When NIH/3 T3 cells were cultured on polydimethylsiloxane (PDMS) substrates patterned with varying stiffness, the cells accumulated preferentially on the stiffer regions of the substrates with differential remodelling of ECM on stiff vs. compliant areas, which led to the suggestion that migration, and not proliferation, was responsible [bib_ref] Repositioning of cells by mechanotaxis on surfaces with micropatterned Young's modulus, Gray [/bib_ref]. In seminal studies, Engler and colleagues showed that matrices whose elasticities were comparable to brain tissue ("soft matrices") were neurogenic and stiffer and rigid matrices (with elasticities comparable to muscle and bone tissue, respectively) were respectively myogenic and osteogenic [bib_ref] Matrix elasticity directs stem cell lineage specification, Engler [/bib_ref]. Substrate compliance was also demonstrated to positively influence survival and functionality of mouse ESC-derived hepatocyte like cells [bib_ref] Functional modulation of ES-derived hepatocyte lineage cells via substrate compliance alteration, Li [/bib_ref].
In this study, we aimed to understand the role of substrate stiffness in two dimensional hESC culture and hoped to devise a PDMS based culture system for directing hESC differentiation. Specifically, we chose to focus on how modulating the mechanical properties of the substrate affected cell density and cell shape, along with maintaining pluripotency and the possibility of lineage specification. hESC (BGO1v) were cultured on commercial Polydimethylsiloxane of varying stiffness both in the presence and absence of basic FGF. Presence of pluripotent cells in culture was determined using Alkaline Phosphatase Assay assay and qPCR was performed at various time points to assess pluripotency and differentiation.
# Results
## Synthesis and characterization of pdms substrates
Cell culture substrates with varying stiffness were prepared from PDMS by varying the base to cross linking agent ratio from 10:1, 20:1, 40:1. In the following discussion, the various substrates will be referred to by their base: crosslinker ratio e.g. PDMS 10:1. Tangent moduli were calculated from the tensile testing data [fig_ref] Figure 1: Testing mechanical properties of the three PDMS formulations used in cell culture... [/fig_ref] and ranged between 0.078 MPa to 1.167 MPa [fig_ref] Table 1: Tangent moduli of PDMS substrates [/fig_ref]. The data for 10:1 PDMS show that the polymer has at least two distinct tangent moduli: lower stiffness was observed at low strain levels than at higher strains. The transition was not observed in 20:1 and 40:1 PDMS, although it is likely that this was because the samples failed before they reached the strain level at which the transition occurs. Surface roughness of the prepared cell culture substrates as determined by tapping mode in AFM was shown to lie between 0.8 nm and 1.0 nm over a 20 μm 2 area [fig_ref] Figure 2: Surface roughness measured by AFM [/fig_ref] with no major surface features present. Contact angle measurements [fig_ref] Table 2: Contact angles prior to surface treatment [/fig_ref] indicated that surface hydrophilicity prior to fibronectin treatment decreased with decreasing stiffness of the substrate.
## Cellular attachment, proliferation and morphology
Cells grown on PDMS substrates initially proliferated at a rate similar to cells grown on mouse embryonic fibroblasts and fibronectin coated Tissue culture polystyrene (TCPS), which were used as controls. However, after seven days, there were more cells on the various PDMS substrates compared to the TCPS controls, indicating that PDMS could be used as a platform for hESC propagation and differentiation (data not shown). In order to determine that this difference in proliferation was a result of the effect of the substrates on the cells and was not merely a consequence of viable cell attachment, cell densities were measured after 12 hours. The results indicate that in the early stages, even in the absence of cell-cell contact, cellular attachment was comparable across substrates. Cell shape (also independent of cell-cell contact) and cell surface area was also comparable across substrates at this early time point, with no statistically significant differences with increase in substrate stiffness [fig_ref] Figure 4: A variant hESC line [/fig_ref].
## Self renewal and lineage specification
Cells collected from substrates after 4 and 7 days in culture were re-plated onto inactivated MEF layers and cultured in complete hESC medium for 4 days and stained for AP activity. We found that a subpopulation of pluripotent cells remained among the cells cultured on all substrates at Day 4 and at later points (cells collected from substrates on Day 7), while some differentiated cells could be found within most cells stained for AP [fig_ref] Figure 5: Alkaline Phosphatase Activity for cells collected from various substrates [/fig_ref]. These results suggest that these differentiated cells could be early progenitors capable of taking on normal hESC morphology and interacting with cells in a way conducive to proper colony formation.
Gene expression analysis across all substrates at various time points in culture showed that expression of pluripotency markers tended to decrease over time in the absence of basic FGF, as was anticipated. Differential gene expression Expression Index (EI) analysis [fig_ref] Table 3: Expression indices across substrates [/fig_ref] indicates that across all time points, cells on the various PDMS substrates were more differentiated than those on TCPS. Among the various PDMS substrates, early differentiation appears to follow a stochastic process, with EI values falling sharply between Day 4 and Day 7.
Across the various PDMS substrates, pluripotency markers decrease over time, with the exception of Nanog in cells cultured on PDMS 20:1 [fig_ref] Figure 6: Comparison of individual gene expression across substrates [/fig_ref] , B, C). Nanog expression peaked at Day 10 on PDMS 20:1 and was significantly higher in cells grown on this substrate than the other two at this time point. Expression of the three representative differentiation markers tested (NeuroD, IGF2, AFP)increased on almost all PDMS substrates from Day 4 to Day 7, with change in NeuroD expression being the most, followed by that of AFP. Few exceptions included IGF2 expression on PDMS 40:1 and AFP expression on PDMS 40:1. On all PDMS substrates, NeuroD expression peaks at Day 7 but by Day 10 falls to slightly lower levels, while still remaining higher than Day 4 [fig_ref] Figure 6: Comparison of individual gene expression across substrates [/fig_ref]. NeuroD expression on the softest substrate was significantly higher than the other two at this early time point. IGF2 and AFP expression show a consistent increase with time, but were the highest on PDMS 20:1 [fig_ref] Figure 6: Comparison of individual gene expression across substrates [/fig_ref] , F).
# Discussion
We have shown here that substrate stiffness affects cellular spreading, proliferation and gene expression of hESCs. PDMS was chosen as the substrate because it is easy to handle, inexpensive, does not swell in contact with water and can be micropatterned using techniques such as soft lithography. Surface treatments with UV radiation or ethanol, such as those used here; also do not affect material properties. Other properties of PDMS, such as thermal stability, transparency and chemical inertness, make it particularly useful in bioengineering, in spite of potential batch to batch variabilities. We were able to fabricate substrates with stiffness varying from 0.078 to 1.167 MPa, a range similar to that reported by others [bib_ref] The motility of normal and cancer cells in response to the combined..., Chevolleau [/bib_ref] [bib_ref] Focal adhesion size controls tension-dependent recruitment of α-smooth muscle actin to stress..., Goffin [/bib_ref]. We generated even stiffer substrates by using a base: crosslinker ratio of 5:1 (PDMS 5:1) and performed initial characterization studies but decided to focus on softer substrates for cell culture and gene expression analysis. However, even within the given range of stiffness tested, substrate mediated biological effects were observed. Although proliferation increased on substrates, cell spreading did not increase with increased stiffness. These results are in direct contrast with previously reported studies conducted with terminally differentiated cells [bib_ref] Cell locomotion and focal adhesions are regulated by substrate flexibility, Pelham [/bib_ref] [bib_ref] Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion, Yeung [/bib_ref]. Our results tend to indicate that hESCs might react differently to substrate stiffness when compared with terminally differentiated cells like fibroblasts or endothelial cells [bib_ref] Cell locomotion and focal adhesions are regulated by substrate flexibility, Pelham [/bib_ref] [bib_ref] Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion, Yeung [/bib_ref]. These differences in cellular behaviour merits further studies in determining how cells in their undifferentiated state respond to substrate stimuli. Cells on stiffer substrates tend to exert larger traction forces [bib_ref] Tissue cells feel and respond to the stiffness of their substrate, Discher [/bib_ref] and it may be hypothesised that substrates affect cell development by affecting cellular migration and movement via the dynamics and size of adhesion sites. Substrate compliance are a major factor in cell culture studies and the probable molecular responses to these substrates have been discussed in detail in literature [bib_ref] Tissue cells feel and respond to the stiffness of their substrate, Discher [/bib_ref] [bib_ref] Cell type-specific response to growth on soft materials, Georges [/bib_ref].
Our data indicate that at early time points, stiffness mediated differentiation follows a rather stochastic process, but certain trends begin to appear with longer duration in culture [fig_ref] Table 3: Expression indices across substrates [/fig_ref]. By Day 10, the mesodermal marker IGF2 was found to be the most highly expressed gene, with its expression being the highest on PDMS 20:1, one of the stiffer substrates. The dynamics of differentiation across different substrates reveals interesting trends. Specifically, the PDMS 20:1 demonstrates stronger dynamics in AFP and IGF2 expression [fig_ref] Figure 6: Comparison of individual gene expression across substrates [/fig_ref] , F), while other conditions do not show such an obvious trend in differentiation. One possible hypothesis for the mechanism of this increased AFP and IGF2 expressions correlated with stiffness is that stiffer substrates provide an environment that more closely mimics those experienced by migrating mesodermal and endodermal cells in the early embryo. Our data also indicates that there is greater increase in IGF2 (mesoderm) expression [fig_ref] Figure 6: Comparison of individual gene expression across substrates [/fig_ref] , when compared with AFP (endoderm) expression [fig_ref] Figure 6: Comparison of individual gene expression across substrates [/fig_ref] , indicating a greater propensity for increased mesodermal differentiation on PDMS 20:1 substrate. At this juncture, it is also interesting to note that MSCs -which originate from the mesoderm -respond highly to substrate stiffness based cues for lineage specification. Recent studies involving hESCs cultured in three dimensional polymeric substrates with a broad range of elasticities also indicated that as stiffness increased, mesodermal differentiation was favoured over endodermal lineages [bib_ref] The influence of scaffold elasticity on germ layer specification of human embryonic..., Zoldan [/bib_ref].
While the molecular mechanisms linking substrate stiffness and hESC differentiation still remain to be explored, we can speculate this to be either a direct effect of mechanical properties of the substrate on cellular differentiation events, or an indirect effect related to the changes in cell spreading and migration. Thus the stiffness mediated increase in cell attachment might be mimicking the environment of migrating mesoderm cells, thereby supporting the growth and differentiation of more adhesive cells. We also need to take into account that altering substrate rigidity also affects the overall chemical composition of the substrate which could affect differentiation. However, in previous studies on polyacrylamide gels of different formulations but similar stiffness, cell morphology remained similar [bib_ref] Cell locomotion and focal adhesions are regulated by substrate flexibility, Pelham [/bib_ref]. One caveat to keep in mind is that not all mechanosensitive cell types respond similarly to changes in substrate stiffness [bib_ref] Cell type-specific response to growth on soft materials, Georges [/bib_ref]. Multiple tissues may have similar elasticities, and cell types respond differently to mechanical signals, in a manner somewhat similar to what they experience in their native tissue [bib_ref] Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion, Yeung [/bib_ref].
# Conclusions
In this study, we have demonstrated that fibronectincoated PDMS substrates are capable of supporting hESC attachment and proliferation. We have shown here that substrate stiffness affects proliferation, while cell spreading and cell attachment remains comparable across the range of stiffness tested. With increased time in culture, differentiation increased and gene expression associated with mesodermal differentiation was upregulated as stiffness increased from soft to stiff, suggesting that the substrate is an important variable that needs to be carefully considered in developing protocols for stem cell propagation and differentiation. Future studies will focus on whether such a system can be used to bring about terminal differentiation of hESCs towards defined cell types of the mesodermal lineage.
# Materials and methods
## Esc culture
## Substrate synthesis and characterization
The PDMS substrate was prepared from the commercially available Sylgard 184 silicone elastomer kit (Dow Corning, Midland, MI) by mixing the base and the curing agent in varying ratios. Specifically, PDMS with base: crosslinker w/w ratio 10:1, 20:1, and 40:1 were prepared. The pre-polymer mixtures were mixed thoroughly for at least 5 minutes, degassed, and poured into 35 mm polystyrene tissue culture Petri dishes. PDMS was then cured for at least 60 hours at 22-33°C. Samples were stored at room temperature in a vacuum desiccator.
Tensile testing was done to characterize the bulk mechanical properties of the substrate. Specifically, 1 mm dog-bone shaped strips were subjected to a tensile load at a strain rate of 10 mm/min and the test was conducted to failure. The elastic modulus was determined manually by calculating the slope of the stress strain curve within linear limits.
Topographical and phase images were taken on an MFP-3D AFM (Asylum Research, Santa Barbara, CA). Images were obtained in non-contact (AC/tapping) mode and height, amplitude and phase images were taken using a silicon cantilever (AC-240 TS, Olympus Instruments) at a scan speed 1 Hz at 512 pixels/line. The scan size was 20 μm × 20 μm.
Surface wetting properties of the various substrates were evaluated by measuring the static water contact angles via the sessile drop method using a Ramé-Hart Goniometer/ Tensiometer (Model 500) equipped with a special optical system and a CCD camera and the image was analyzed using DROPImage Advanced for contact angle determination.
## Cell culture on pdms substrates
Before conducting cell culture experiments, PDMS substrates were sterilized by treating them with ethanol under ultraviolet light for 1 hour, followed by a second round of UV exposure for another 30 minutes. To promote cell attachment to the various substrates studied, plates coated with various formulations of PDMS and the polystyrene plates were treated with 2.5 mL of 10 μg/mL of human plasma fibronectin (Chemicon, Cat. No. FC010) overnight at 37°C. Substrates were then washed twice with PBS and cells were seeded at 50,000 cells per 35 mm plate. To promote differentiation, cells were grown in differentiation medium (hESC medium without bFGF). Cells grown on inactivated MEF feeders in hESC medium were used as controls. Medium was changed on the second day and on every following day. On the 4 th , 7 th , and 10 th day cells were collected from the plates by treating them with collagenase and trypsin as described above. Cells that remained attached following enzymatic passaging were collected using a rubber cell scraper. Cell counts were performed using a hemocytometer. The collected cells were snap frozen in liquid nitrogen and stored at −80°C for subsequent gene expression analysis. All experiments were performed with three replicates per condition.
## Alkaline phosphatase activity
Some of the collected cells were also seeded onto plates with inactivated MEF feeders and propagated in hESC medium for 4 days after which an Alkaline Phosphatase Substrate Kit (Vector Labs, Cat. No. VC-SK-5100-KI01) was used to assay for alkaline phosphatase (AP) activity.
## Cell surface area calculations
To study how substrates affect morphology independent of cell-cell contact, cells were plated at a density of 10,000 cells per 35 mm plate and grown in hESC growth medium. Cells seeded at the same density on acellularized MEF layers were used as controls. After 12 hours, images of cells from different experimental conditions were captured using a Nikon Eclipse TS100 inverted microscope and a Nikon Coolpix 5000 digital camera. Area was calculated using ImageJ software by manually outlining the cell perimeter with each area measurement performed twice. Cell density was calculated by manually counting the number of cells in the field of vision and extrapolating to the cell density in each of the 35 mm dishes. Each condition was performed with three replicate plates, and images of multiple cells were captured from each plate. All cells that were in contact with other cells were excluded from the analysis.
# Gene expression analysis
For gene expression analysis, samples were prepared by isolating total RNA using TRIZOL (Invitrogen, Cat. No. 10296-010, Carlsbad, CA) according to manufacturer's instructions. Briefly, cell pellets were treated with TRIZOL and chloroform, RNA from the aqueous phase was precipitated in isopropyl alcohol, washed with 75% ethanol, and dissolved in water. RNA was quantified using a UV-vis Spectrophotometer (Biomate 3, Thermo Scientific, Waltham, MA). cDNA was reverse-transcribed from 1 μg of total RNA according to manufacturer's protocols using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat. No. 4368814 Foster City, CA). The reactions were incubated for 10 minutes at 25°C and for 120 minutes at 37°C. Expression of pluripotent and differentiation genes was analyzed using quantitative real time RT-PCR using Taqman primers (Applied Biosystems, Foster City, CA); performed in an ABI HT7900 system (Applied Biosystems, Foster City, CA) and the data were acquired using sequence detection system software (SDS v2.2.1, Applied Biosystems, Foster City, CA). The original replicates (n = 3) for each condition were tested in duplicate, and all failed reactions (termed "undetermined" by the software) were excluded from the analysis. ΔCt values were obtained by normalizing the Ct values against the endogenous 18S ribosomal RNA. Data analysis for differential expression between the different samples was conducted in triplicate and Student's t-test was conducted to ascertain the significance of differential expression.
Expression Index was used to detect the relative differentiation state of cells and was based on average Ct values from triplicate measurements and used the mathematical model described in Noaksson et al. [bib_ref] Monitoring differentiation of human embryonic stem cells using real-time PCR, Noaksson [/bib_ref]
[fig] Figure 1: Testing mechanical properties of the three PDMS formulations used in cell culture studies. Stress Strain profiles of A) 10:1 PDMS; B) 20:1 PDMS and C) 40:1 PDMS substrates. [/fig]
[fig] Figure 2: Surface roughness measured by AFM. Height (Topography) of the three PDMS samples. A: PDMS 5: 1 RMS roughness: 1.0 nm over 20 micron area B: PDMS 10: 1 RMS roughness: 0.8 nm over 20 micron area C: PDMS 20: 1 RMS roughness: 0.8 nm over 20 micron area. [/fig]
[fig] C ells/m m 2, Figure 3: 10: 1 PDMS 20: 1 PDMS 40: 1 TCPS Cell Density across substrates, independent of cell-cell contact. Cell density after 12 hours was used as an indicator for degree of cell attachment across various substrates. Error bars represent standard deviation. [/fig]
[fig] Figure 4: A variant hESC line (BG01v) was cultured on MEF feeders that have been inactivated with mitomycin-C. Cells were cultured in hESC medium, which consisted of Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Gibco, Cat. No. 11320-033) supplemented with 20% knockout serum replacement (KSR; Gibco, Cat No. 10828-028), 1 mM L-glutamine (Gibco, Cat. No. 25030-081), 0.1 mM minimal essential medium Cell Morphology across substrates, independent of cell-cell contact. Cell surface area after 12 hours on various substrates, with 20 cells measured per condition from triplicate samples. Area of cells grown on PDMS 40:1 was significantly lower than that of the other substrates. Error bars represent standard deviation. [/fig]
[fig] Figure 5: Alkaline Phosphatase Activity for cells collected from various substrates. Cells collected from different substrates after 4 and 7 days in culture were re-plated on feeders in the presence of complete hESC medium for four days, following which they were stained for AP activity. A: Cells from 10: 1 PDMS, B: Cells from 20: 1 PDMS, C: Cells from TCPS, D: Cells from MEF. [/fig]
[fig] Figure 6: Comparison of individual gene expression across substrates. Genes analyzed included A: Oct 4 (Pluripotent) B: Nanog (Pluripotent) C: Sox 2 (Pluripotent) D: Neuro D (Ectoderm) E: IGF2 (Mesoderm) F: AFP (Endoderm). (* indicates p < 0.01 and ** indicates p < 0.05). [/fig]
[table] Table 1: Tangent moduli of PDMS substrates [/table]
[table] Table 2: Contact angles prior to surface treatment [/table]
[table] Table 3: Expression indices across substrates [/table]
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10.1121/1.4950820
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CCBY
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27250188
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s2orc_pubmed_articles
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Eliciting the most prominent perceived differences between microphones.
The attributes contributing to the differences perceived between microphones (when auditioning recordings made with those microphones) are not clear from previous research.Consideration of technical specifications and expert opinions indicated that recording five programme items with eight studio and two microelectromechanical system microphones could allow determination of the attributes related to the most prominent inter-microphone differences.Pairwise listening comparisons between the resulting 50 recordings, followed by multi-dimensional scaling analysis, revealed up to 5 salient dimensions per programme item; 17 corresponding pairs of recordings were selected exemplifying the differences across those dimensions.Direct elicitation and panel discussions on the 17 pairs identified a hierarchy of 40 perceptual attributes.An attribute contribution experiment on the 31 lowest-level attributes in the hierarchy allowed them to be ordered by degree of contribution and showed brightness, harshness, and clarity to always contribute highly to perceived intermicrophone differences.This work enables the future development of objective models to predict these important attributes.
# I. introduction
To describe the sonic characteristics of a microphone, manufacturers can supply several standardised measurements that describe its objective performance .It has been noted by [bib_ref] The evaluation of microphones, part 1: Measurements, Olive [/bib_ref] ,, and others that these measurements do not always correlate well with perceived characteristics, and they have suggested other objective measures [bib_ref] Test method for the evaluation of ringing in microphones, Green [/bib_ref] [bib_ref] Methods for the subjective assessment of small impairments in audio systems including..., Hebrock [/bib_ref] [bib_ref] The evaluation of microphones, part 1: Measurements, Olive [/bib_ref].However, even these suggested measures do not directly correlate with specific subjective attributes.Identifying the perceptual attributes that vary between microphones, and the extent to which these attributes vary, would be a step toward more perceptually meaningful microphone comparisons.
Relevant perceptual descriptors and attributes can be identified by using elicitation experiments, and these have been widely conducted for loudspeakers, musical acoustics, concert hall acoustics, and multi-channel audio systems, e.g., [bib_ref] Timbral description of musical instruments, Disley [/bib_ref] ; [bib_ref] Elicitation of attributes for the evaluation of audio-on-audio interference, Francombe [/bib_ref] ; [bib_ref] Dimension analyses of perceived sound quality of sound-reproducing systems, Gabrielsson [/bib_ref] ; [bib_ref] Perceived sound quality of soundreproducing systems, Gabrielsson [/bib_ref] ; [bib_ref] Unravelling the perception of spatial sound reproduction: Language development, verbal protocol analysis..., Koivuniemi [/bib_ref] ; [bib_ref] Identification of some perceptual dimensions underlying loudspeaker dissimilarities, Lavandier [/bib_ref] ; [bib_ref] Concert hall acoustics assessment with individually elicited attributes, Lokki [/bib_ref]. [bib_ref] Perceived sound quality of soundreproducing systems, Gabrielsson [/bib_ref] elicited 55 descriptors of the differences between loudspeakers, and analysed these descriptors to find the 8 most prominent corresponding attributes: clearness/distinctness, sharpness/hard-softness, brightness/darkness, fullness-thinness; feeling of space, nearness; disturbing sounds, and loudness.More recent work by [bib_ref] Identification of some perceptual dimensions underlying loudspeaker dissimilarities, Lavandier [/bib_ref] found two salient dimensions relating to the perceived differences between loudspeakers from a multi-dimensional scaling (MDS) analysis.These were labeled: bass/treble balance and medium emergence.This research was followed by a study by [bib_ref] Objective characterization of perceptual dimensions underlying the sound reproduction of 37 single..., Michaud [/bib_ref] on a larger set of loudspeakers, finding three salient dimensions: bass/treble balance, medium emergence, and feeling of space.In the work of [bib_ref] Unravelling the perception of spatial sound reproduction: Language development, verbal protocol analysis..., Koivuniemi [/bib_ref] , which was focused on spatial sound systems, four timbral attributes were found: richness, hardness, emphasis, and tone colour.
In the research of musical acoustics, [bib_ref] Timbral description of musical instruments, Disley [/bib_ref] performed experiments into musical timbre using 15 attributes: bright, clear, warm, thin, harsh, dull, nasal, metallic, wooden, rich, gentle, ringing, pure, percussive, and evolving. [bib_ref] A subjective ratings scale for timbre, Pratt [/bib_ref] identified three scales to describe the timbre of musical instruments: dull/brilliant, cold/warm, and pure/rich.
Although there may be some overlap between the dimensions, descriptors, scale labels, and attributes identified in these previous studies and those that would be pertinent to the assessment of microphones, it cannot be concluded that they will all be relevant, nor that they will be sufficient.There has, however, been a relatively small amount of research specifically into the perceived differences between microphones.
Research by De Man and Reiss (2013) was focused on the methods used to obtain subjective data, rather than on identifying specific perceptual attributes, and found both multi-stimulus and pairwise-comparison approaches to be suitable.asked listeners to describe the characteristics of three dynamic microphones and reduced the responses to the descriptors harsh, edgy, warm, (not enough, or no) low-end and (not enough, or extended) highend.As part of a subsequent study into the perceptual effects of ringing in microphones, [bib_ref] Methods for the subjective assessment of small impairments in audio systems including..., Hebrock [/bib_ref] found that, of 13 suggested attributes, the 9 most frequently used by listeners were detailed, dull, muffled, open, thin, warm, harsh, nasal, and smooth.In the first study, only three microphones were considered, and all had similar specifications.In the second study, listeners characterised stimuli only in terms of attributes specified by the experimenters.It is therefore possible that other attributes might have been found to be important had a wider range of microphones been evaluated in the first study and had listeners not been limited in their responses in the second.
Research by aimed to identify some of the perceptual attributes that differ between perceptually very similar microphones, but the results of the listening experiments indicated the responses were no better than chance.This may have been due to the magnitude of the differences between the stimuli being too small.
It is apparent, therefore, that although multiple attributes have been found that contribute to, or that might contribute to, the perceived differences between microphones, the relative degrees of their contributions are untested and there might be other attributes that contribute equally or even more.Hence, this study aims to determine the full set of attributes in terms of which microphones differ, to label these attributes appropriately, and to find the relative contributions of these attributes to perceived inter-microphone differences.The challenge of modeling these attributes in terms of objective physical metrics can then be addressed in future studies.In order that the study should not be limited to experimenter-proposed attributes, the approach taken employs elicitation experiments whereby listeners report freely on perceived differences between recordings made using alternative microphones.To avoid the problems encountered by , a wide range of microphones is selected, programme items are chosen that exhibit a wide range of differences, and a hybrid elicitation method is employed.
The experiment methods are further explained in Sec.II, and broken down into five distinct phases.The specific procedures and results for the five phases are presented in Secs.III-VII, and the results are discussed in Secs.VIII and IX.
# Ii. experiment methods
There are two approaches to elicitation experiments: direct and indirect.Direct elicitation methods involve asking participants to verbally describe the perceptual sensations evoked by stimuli, whereas indirect elicitation experiments require subjects to rate these sensations without explicit description.
A common indirect elicitation method is MDS in which participants rate the similarity of every pairwise combination of a set of stimuli, and an analysis is then conducted which attempts to position each stimulus in a multidimensional group space so that the pairwise distances match the pairwise similarity ratings.This has been used in several studies on audio codecs, tools, and products to find the number of salient dimensions across which a stimulus set varies [bib_ref] Dimension analyses of perceived sound quality of sound-reproducing systems, Gabrielsson [/bib_ref] [bib_ref] Application of multidimensional scaling to subjective evaluation of coded speech, Hall [/bib_ref] [bib_ref] A hybrid technique for validating unidimensionality of perceived variation in a spatial..., Neher [/bib_ref].However, MDS analysis is only a data reduction tool, reducing the potentially large number of perceptual differences between stimuli into a smaller number of orthogonal dimensions.Each dimension does not necessarily correlate with a single perceptual attribute and dimensions are not identified as relating to particular attributes but are, instead, simply numbered.
One of the most common direct elicitation methods is free choice profiling (FCP), in which each participant develops his/her own set of words or phrases to describe the attributes that they can perceive to differ between stimuli.This is often followed by a panel discussion stage where common and similar terms are grouped, across all participants, to arrive at a single list of agreed descriptors that might each correspond to a particular attribute [bib_ref] Elicitation of attributes for the evaluation of audio-on-audio interference, Francombe [/bib_ref] [bib_ref] Audio descriptive analysis and mapping of spatial sound displays, Zacharov [/bib_ref].In the FCP stage, to ensure that all differences within a stimulus set are elicited, each stimulus must be compared directly with every other stimulus.This can be a difficult and time-consuming task for subjects and can lead to listener fatigue, potentially resulting in noisy data and/or missed attributes.
The hybrid method used in this study combined both approaches to make the elicitation task simpler and thereby increase the likely quality of the results.First, a similarity rating experiment and MDS analysis was conducted to identify stimulus pairs exhibiting large differences.This analysis was conducted similarly to the work of [bib_ref] A hybrid technique for validating unidimensionality of perceived variation in a spatial..., Neher [/bib_ref] , [bib_ref] Application of multidimensional scaling to subjective evaluation of coded speech, Hall [/bib_ref].These stimulus pairs (rather than the full stimulus set) were then used in an FCP experiment, similar to that conducted by [bib_ref] Elicitation of attributes for the evaluation of audio-on-audio interference, Francombe [/bib_ref].This was followed by a panel discussion to group the elicited terms and to agree on an attribute label to represent the terms in each group.
Previous studies have used the frequency with which a term has been elicited as an indicator of the importance of the attributes that it describes [bib_ref] Elicitation of attributes for the evaluation of audio-on-audio interference, Francombe [/bib_ref].However, this approach has the potential to underestimate the importance of attributes which listeners hear clearly but find difficult to describe.In the current study, therefore, once attribute labels had been agreed, a novel attribute contribution experiment was conducted, asking subjects to explicitly rate the degree to which each of the attributes contribute to the overall difference between each stimulus pair.
The full study was conducted in five phases.
- Phase 1: Determined suitable microphones and programme items based on objective factors that are known to differ between microphones, in order to make recordings likely to be able to reveal the attributes comprising the most prominent inter-microphone differences.This is described in Sec.III.- Phase 2: Employed pairwise similarity ratings and MDS to reveal the number of salient dimensions and to identify exemplary stimulus pairs.This is described in Sec.IV.
- Phase 3: Used an FCP approach to elicit terms from listeners that describe the differences between the exemplary stimuli.This is described in Sec.V. - Phase 4: Used panel discussions to group the elicited terms to reduce redundancy and to identify and label the underlying perceptual attributes.This is described in Sec.VI. - Phase 5: Determined the degree to which each agreed attribute contributes to perceived inter-microphone differences, by way of an attribute contribution experiment.This is described in Sec.VII.
## Iii. phase 1: microphones and sources
There are over 1500 studio microphones in existence, with varying degrees of similarity and difference .For this study, microphones were selected that were expected to exemplify the full range of potential inter-microphone differences.
## A. microphone selection
The selection of microphones was conducted in two parts.First, in order to select microphones based on objective parameters, examples were listed that exemplified the main differences in microphone design.Second, since some perceptual differences might not correlate with standard objective measures, recording engineers were asked to suggest additional microphones that they felt sounded significantly different from those listed. describes manufacturer guidelines for the measurement and documentation of the transduction type, sensitivity, frequency response, directivity pattern, and self-noise of a microphone.In addition to these five factors, other research has suggested that the diaphragm size and transient response of a microphone may affect perceived sound quality [bib_ref] Choosing the right microphone by understanding design tradeoffs, Bartlett [/bib_ref] [bib_ref] Methods for the subjective assessment of small impairments in audio systems including..., Hebrock [/bib_ref].It has also been suggested that the head-basket and, for a condenser microphone, the capsule termination type may be relevant.
## Objective selection
## Bs
From a list of commonly used studio microphones, microphones were selected that represented the extremes for each of these objective parameters.Thus, for each continuously-variable parameter, one microphone in the selected set was chosen due to it having a particularly high value of that parameter; one was chosen due to it having a particularly low value; and the other microphones will have intermediate values of that parameter (but each will represent an extreme for another parameter).For categorical parameters (e.g., transduction type) at least one microphone was chosen in each category.
For the parameters diaphragm size and frequency response, categories were selected.For diaphragm size, microphones were categorised as either large diaphragm (diameter >16 mm) or small diaphragm (diameter <16 mm).For frequency response, microphones were categorised as either flat or tailored, where tailored refers to any microphone whose on-axis frequency response includes a region exhibiting gain more than 3 dB greater than that at 1 kHz .Since measurements of transient response are not included in , transient response is estimated from transduction type and diaphragm size: a smalldiaphragm condenser microphone is likely to have a fast transient response; a large-diaphragm dynamic microphone is likely to have a slow transient response.
Application of these criteria resulted in the selection of the eight studio microphones shown in Table [fig_ref] TABLE I: Selected microphones and their objective characteristics [/fig_ref].
## Expert suggestions
A list of the eight selected microphones was presented to five experienced audio engineers.The engineers were asked if they felt that any perceptual characteristics that differ between microphones were not accounted for by the microphones on the list and, if they did, to suggest additional microphones to illustrate these characteristics.All five engineers responded that the list exemplified all relevant perceptual differences.To avoid any bias arising from knowledge of the microphones chosen, none of these engineers took part in subsequent phases of the study.
## Additional non-studio microphones
It is unlikely that the audio engineers would have considered non-studio microphones.MEMS (microelectromechanical systems) microphones are used in a wide range of low-power devices, such as tablets and mobile telephones.They can be designed to have similar frequency response and self-noise to that of a typical studio microphone [bib_ref] Evaluation for smartphone sound measurement applications, Kardous [/bib_ref] [bib_ref] Acoustic sensors, Sessler [/bib_ref] , but the perceived quality of the recorded signal can be very different.
A pilot experiment was conducted with 12 commercially available MEMS microphones from 4 manufacturers.Ten subjects were asked to rate the basic audio quality, as defined in ITU-R BS.1116-1 (1994) and ITU-R BS. , of recordings made with the 12 microphones using a multiple stimulus comparison test interface.Recordings were made of pop music, classical music, and speech.The two MEMS microphones reported as having the highest and lowest quality over all three programme items were added to the list for the current study:
- Wolfson WM7131 (Edinburgh, Scotland): MEMS microphone reported as having the highest quality in a pilot study.
- Knowles SPU0410HR5H (Itasca, IL): MEMS microphone reported as having the lowest quality in a pilot study.
## B. programme item selection
In previous work, it was found that musical sources were good at revealing the perceptual differences between microphones (better than vocal sources) [bib_ref] Validation of experimental methods to record stimuli for microphone comparisons, Pearce [/bib_ref].Therefore only musical sources were considered for this study.
The sources were selected to have characteristics likely to reveal the objective inter-microphone differences listed in Sec.III A 1: double bass, drums, acoustic guitar, string quartet, and trumpet, each played unaccompanied.The double bass was plucked, playing a jazz turn-around (duration 8 s) resulting in a stimulus with little high-frequency content and low sound pressure level (SPL).Drums consisted of a snare, hi-hats, and cymbals playing a simple rhythm (duration 7 s) across all pieces of the kit; this produced a nonharmonic frequency spectrum and high level of highfrequency energy.The acoustic guitar played continuous sixstring strummed chords (duration 9 s) with a pick; this produced fast transients, a high level of high-frequency content, and a harmonic frequency spectrum.The string quartet played the first four bars (duration 9 s) of Vivaldi's Summer (mvt.1), producing a large dynamic range, broadband harmonic frequency spectrum, and slow transients.Finally the trumpet played a loud fanfare (duration 12 s), generating a high SPL at the microphone, and was intended to excite as many distortions in the microphones as possible.
## C. recording of the stimuli
Previous experiments [bib_ref] Validation of experimental methods to record stimuli for microphone comparisons, Pearce [/bib_ref] have identified a suitable method for recording stimuli for intermicrophone perceptual comparisons: a multi-microphone array with a maximum inter-microphone spacing of no more than 150 mm, in an ITU-R BS 1116 compliant listening room (ITU-R BS.1116-1, 1994).
The five selected programme items were therefore recorded in this way, using the microphone arrangement shown in Fig. , to provide 50 stimuli for Phase 2. All microphones were recorded with a Presonus Digimax FS (Baton Rouge, LA) microphone preamplifier feeding an RME Fireface 800 (Haimhausen, Germany) audio interface.The MEMS microphones were supplied with 2.7 V power and recorded through the instrument inputs of the preamplifier due to their high output impedances.The input gain on the preamplifier was adjusted for each microphone to produce the same digital input level when excited with pink noise replayed through a Genelec 1032 (Iisalmi, Finland)
[formula] C ¼ Cardioid, B ¼ Bidirectional, O ¼ Omnidirectional) (C ¼ Condenser, D ¼ Dynamic, R ¼ Ribbon) Sensitivity (mV/Pa) Self-noise (dBA) Diaphragm size (S ¼ Small, L ¼ Large) [/formula]
Frequency response loudspeaker 1.5 m from the array, at a measured level of 74 dB SPL at the array.Each source was positioned with its acoustic centre 1.5-2 m directly in front of the array.
[formula] (F ¼ Flat, T ¼ Tailored) Headbasket (C ¼ Cylindrical, N ¼ Non-Cylindrical) Capsule termination (E ¼ Edge-Terminated, C ¼ Centre-Terminated) AKG C12 (Vienna, [/formula]
It is acknowledged that placing a microphone in an array in close proximity to other microphones may alter its off-axis response; however, this study does not seek to determine the off-axis characteristics of a particular microphone but, rather, to compare on-axis characteristics across microphones.
## Iv. phase 2: similarity ratings
Using the recordings made in Phase 1, pairwisecomparison tests were conducted in order to find programme items and microphone pairs that exhibited differences across each salient dimension, for use in an FCP experiment.
Stimuli were presented diotically over a pair of Sennheiser HD650 headphones with a Focusrite VRM Box (High Wycombe, England) interface, with the VRM feature disabled.All stimuli were loudness-matched by a panel of five listeners, using a method-of-adjustment test, to a listening level judged to be comfortable.
## A. similarity ratings
To reduce the potential for listener fatigue, each of the five programme items was presented in a separate listening test.Prior to each test, subjects were presented with all ten stimuli, which could be auditioned at will, for the programme item under assessment in order to allow them to familiarise themselves with these stimuli.They were then presented with a smaller version of the test interface in order to allow them to familiarise themselves with the rating task.Nine listeners were asked to rate the similarity of each pair of stimuli on a 100 point scale with endpoints labeled as "most similar" and "least similar" taking into consideration only the range of similarities within the ten-stimulus set.All listeners were undergraduate students on the Music and Sound Recording course at the University of Surrey; all had participated in multiple listening tests previously, and all had passed a taught module in technical listening.
Each of the five tests used a graphical user interface which comprised one page of recordings of a single programme item.Each test contained all 45 pair-wise comparisons, and the listener moved a slider to indicate the perceived similarity between the two stimuli in each pair.To ensure that each pair was considered and rated, each slider had to be moved from its original position, least similar, before the test software would show the test as completed.If a listener wished to rate a pair as least similar then they were required to move the slider away from this point and back again.No restrictions were placed on the order in which listeners rated stimulus pairs nor on the number of times each pair could be auditioned.Ordering of the tests, stimulus pair ordering within each page, and stimulus ordering within each pair, were all randomised for each listener.
## B. multi-dimensional scaling analysis
MDS analysis of the similarity ratings was conducted for each programme item independently to find: (i) for each programme item, the number of salient dimensions across which the ten recordings differed; and (ii) for each dimension within each programme item, a pair of stimuli exhibiting a large difference, for use in Phase 3. MDS analysis was conducted in SPSS version 21 using the PROXSCAL algorithm.
# Dimension analysis
After the work of [bib_ref] Multidimensional scaling, Kruskal [/bib_ref] , [bib_ref] Multidimensional perceptual unfolding of spatially processed speech I: Deriving the stimulus space..., Martens [/bib_ref] , [bib_ref] A hybrid technique for validating unidimensionality of perceived variation in a spatial..., Neher [/bib_ref] , and [bib_ref] Perceptually-motivated audio morphing: Warmth, Brookes [/bib_ref] , the number of salient dimensions in a dataset was deemed to be the dimensionality of the simplest MDS solution having a normalised raw stress of <0.1 where adding a further dimension would increase the squared correlation (r 2 ) by no more than 0.05.
Using these criteria, the number of salient dimensions used by the listeners to differentiate between the tested stimuli was found individually for the bass (three), drums (two), guitar (three), strings (four), and trumpet (five) programme items.Plots of the normalised raw stress for each programme item are included in the supplementary material. 1The maximum number of dimensions for an MDS solution for a set of N stimuli is N À 1; however, as the number of dimensions increases above ðN À 1Þ=4 the risk of a degenerate solution increases [bib_ref] Multidimensional scaling, Kruskal [/bib_ref].
One indication of a degenerate solution is that the distances between the data are equal, or nearly equal, and thus the data lie on a circle [bib_ref] Applications of multidimensional scaling in psychometrics, Takane [/bib_ref].Visual inspection of each of the MDS solutions confirmed that this was not the case.
Although the shapes of the group spaces appear sensible, it is still possible that a solution might be degenerate.If this is the case then the range of microphones taken forward to subsequent phases might be sub-optimal, increasing the statistical noise in their results.If results of subsequent phases appear overly noisy, or if listeners report significant difficulty with their tasks, then the question of degeneracy will be revisited.
## Selection of stimulus pairs
The MDS solution with the identified dimensionality for each programme item was plotted as a set of twodimensional group space projections, and these plots are included in the supplementary material. 1For each programme item, a pair of microphones spaced most widely across each orthogonal dimension was identified.Across all 5 programme items, a total of 17 microphone pairs were selected.These are shown in Table [fig_ref] TABLE II: Mean dissimilarity scores from Phase 2 experiment for the 17 selected stimulus... [/fig_ref] along with the mean dissimilarity scores which will be used in Section VII.
## V. phase 3: elicitation of descriptors
With stimulus pairs selected that exhibit differences across each of the main perceptual dimensions of inter-microphone difference, a direct elicitation experiment was conducted to find terms describing these differences.The FCP method was used, where the response format is not limited and subjects are free to use as many terms as required to describe the differences between stimuli.
Fifteen final-year undergraduate students on the Music and Sound recording course at the University of Surrey participated in this experiment.All had participated in multiple listening tests previously, and all had passed a taught module in technical listening, but none had previously participated in this study.For the avoidance of potential bias, subjects were given no information about the nature of the stimuli.
The test interface comprised 17 pages; 1 stimulus pair per page.On each page, subjects were asked to type in a text box as many terms or short phrases as required to describe the differences between the stimuli.Presentation order of the pages was randomised between subjects.
Listeners had commented previously that in performing some comparisons they focused primarily on particular portions of the stimuli.To facilitate this practice, ease the task, and potentially reduce statistical noise in the results, subjects were provided with the facility to define and loop sections of the audio.Stimuli were reproduced with the same set up as Phase 2.
A total of 768 descriptive terms were elicited.
## Vi. phase 4: descriptor grouping & attribute labelling
To remove redundancy from the elicited terms panel discussions were held, similar to those in the work of [bib_ref] Audio descriptive analysis and mapping of spatial sound displays, Zacharov [/bib_ref] and [bib_ref] Elicitation of attributes for the evaluation of audio-on-audio interference, Francombe [/bib_ref].All 15 subjects who participated in the FCP experiment participated in these panel discussions.
Each of the 768 elicited terms was printed onto an individual card, with the associated stimulus pair on the rear of the card.The cards were then presented to the subjects one at a time, asking the subjects to group together any elicited terms referring to the same perceptual attribute.During the discussions, two sets of the original reproduction setup were available for auditioning each stimulus pair upon request.
The discussions reduced the 768 elicited terms into 38 groups.Subjects were then asked, for each group, to produce a label and a description for the corresponding perceptual attribute.Whilst conducting this stage of the discussion, subjects decided to arrange the groups into a hierarchy, and two additional, mid-level, empty groups were added to help structure the hierarchy.This resulted in a total of 40 groups and associated attributes, shown in Fig. . The attributes corresponding to the two additional groups are denoted with an asterisk.
From Fig. , it can be seen that each higher-level attribute, such as spectral content, can be considered as a combination of the lower level attributes: low-frequency content (LF content), mid-frequency content (MF content), and brightness in this case.
It is interesting to note that the number of attributes identified is greater than the total number of dimensions revealed by the MDS analysis in Phase 2. Although this could suggest that there is a degree of remaining redundancy, it is likely to indicate that at least some of those dimensions correspond to multiple attributes varying in parallel within the tested stimulus set.It is also interesting that several of the agreed perceptual attributes might more commonly be considered to be acoustic parameters (e.g., noise level, dynamic range, LF content).However, the panel felt that these acoustic parameters were directly perceptible and identifiable and therefore could also be considered to be perceptual attributes.
## Vii. phase 5: attribute contribution
Phase 4 generated a list of 40 attributes in a hierarchy (with 31 attributes at the lowest level) that exemplify the differences between the recorded stimuli.However, it is not clear the extent to which each of these contributes to the overall difference between stimuli.
In order to determine which attributes contribute the most, an attribute contribution experiment was conducted.In this experiment, subjects were presented with each of the 17 selected pairs of stimuli in turn and asked to rate the contribution of each attribute to the overall perceived difference between the 2 stimuli in the pair.The test interface comprised a separate page for each stimulus pair (Fig..Thirtyone sliders were presented to the subject, each pertaining to one of the lowest-level attributes and assigned a unique colour.The order of these was randomised for each subject, but maintained across different stimulus pairs.When hovering the mouse cursor over a slider, the definition of the attribute, agreed upon in Phase 4, would appear.
The attribute contribution chart, shown in Fig., updated in real time to display a pie chart that represents the contribution of each of the sliders to the overall differences.Subjects were asked to make this pie chart representative of the overall difference.As in Phase 3, listeners were able to define and loop corresponding sections of the two stimuli to facilitate their preferred mode of listening.
The tests were split over three individual sessions containing six, six, and five pages, respectively.The pages were randomly ordered for each subject, and each subject rated each stimulus pair only once.Stimuli were again reproduced as in Phases 2 and 3.
The attribute contribution test was completed by the same 15 subjects as in Phases 3 and 4. Four additional listeners, with the same level of listening experience and training, completed the experiment in order to check for bias in the original subjects due to their involvement with the panel discussions.
## A. attribute contribution results
Since the subjects were asked to make the attribute contribution pie chart representative of the overall difference between the stimuli, the absolute position of each slider has no meaning (e.g., all sliders set to 20 will produce the same pie chart as all sliders set to 100).For the analysis, it was the percentage contribution of each attribute to the overall difference that was analysed.
## Subject comparison
A univariate analysis of variance (ANOVA) was performed with the percentage contribution as the dependent variable, and the independent variables of stimulus pair, attribute, and the subject group (original 15 subjects or the additional 4 subjects).The subject group did not have a statistically significant effect (p ¼ 1.000): subject group did not affect the overall result.
However, the interaction between the attribute and the subject group had a statistically significant effect (p ¼ 0.001).Although this implied that the two groups of subjects were responding differently for some attributes, the F statistic was low (F ¼ 2.010) compared to that for other significant variables, such as attribute (p < 0.001, F ¼ 36.138).Partial eta squared for this interaction was also low (g 2 p ¼ 0:07).It was therefore concluded that this interaction effect was very small compared to other factors.The full ANOVA table is shown in Table [fig_ref] TABLE III: Full factorial ANOVA table for the attribute contribution experiment [/fig_ref].
From this analysis, it was concluded that the original subjects were largely unbiassed in their judgements.Therefore, all 19 subjects were considered as a single group in all subsequent analysis.
# Overall attribute contributions
The relative contribution of each attribute to the perceived differences between the tested microphones was sought.This contribution can be determined from the product of two factors for each tested stimulus pair: (i) the attribute's contribution to the overall difference between the two stimuli (i.e., the percentage contribution in the Phase 5 results), and (ii) the relative magnitude of the overall difference between those two stimuli (i.e., the mean dissimilarity score shown in Table [fig_ref] TABLE II: Mean dissimilarity scores from Phase 2 experiment for the 17 selected stimulus... [/fig_ref] , divided by the mean of all mean dissimilarity scores).
The mean contributions (across stimulus pairs) and 95% confidence intervals for each attribute are shown in Fig. .
From this it can be seen that brightness contributed the most overall to the differences between the microphones.The second-highest-contributing factor was noise level.However, it can be seen from Fig. that the 95% confidence intervals are larger for noise level than for any other attribute.This suggests that the ratings of noise level contribution were not consistent across programme items.The full rank ordering of attributes, and mean contributions, are shown in Table [fig_ref] TABLE IV: Attributes ordered by overall contribution to inter-microphone differences [/fig_ref].
## Attribute contributions by microphone type
There was a concern that brightness was contributing highly to the overall difference due primarily to the MEMS microphones having a high-frequency resonance.Additionally, the large 95% confidence intervals for noise level implied that the ratings for this attribute differed greatly across stimulus pairs and it was felt that the noise performances of the MEMS microphones might have been largely responsible for this.To investigate further, analyses were conducted by microphone type, considering separately: (i) studio-vs-studio microphone pairs and (ii) MEMS-vs-studio microphone pairs.
Figure [fig_ref] FIG. 5: FIG [/fig_ref] shows the mean contribution of each attribute for the studio-studio and MEMS-studio comparisons separately.An ANOVA indicated that the effect of comparison type (studio-or MEMS-studio) was statistically significant (p ¼ 0.001, F ¼ 10.93).
A one-way ANOVA, performed for each attribute individually, with comparison type as the factor, showed that the contributions of brightness, honky, nasal, tinny-ness, harshness, noise level, noise spectrum, recording noise, and instrument noise differed significantly according to comparison type.These attributes are shaded grey in Fig. [fig_ref] FIG. 5: FIG [/fig_ref].
Even though brightness was rated differently in the studio-and MEMS-studio comparisons, this factor is rated the highest in both comparison groups.However, the second most prominent attribute overall, the noise level, contributes very little to the difference in the studio-studio comparisons.
## Attribute contribution by programme item
To analyse the effect of programme item on the contribution of each attribute, the results broken down by programme item are shown in Fig. .The range covered by the y axis on Fig. is much larger than that on Figs. [fig_ref] FIG. 5: FIG [/fig_ref].This is because noise level (the second-highest contributor overall) contributes a large percentage to the overall difference for the bass programme item, but contributes very little for the other programme items.This might be due to the low SPL produced by the bass and/or to the absence of high-frequency programme content to mask the microphone's self-noise.
This explains the large confidence intervals for noise level in the overall analysis, Fig. . Brightness, harshness, and clarity (the highest, third-highest, and fourth-highest overall contributors, respectively) contribute relatively large percentages to the inter-microphone differences for the majority of the programme items.
# Viii. discussion
Comparing the microphone descriptors and attributes highlighted by [bib_ref] Methods for the subjective assessment of small impairments in audio systems including..., Hebrock [/bib_ref] against the attributes identified in the current study, it can be seen that there is little commonality; harshness, warmth, and LF content being the only shared attributes.It is notable, however, that this list includes the third most highly contributing attribute identified in the current study (harshness), as well as the higher-level attribute warmth, which is split into subattributes in the current study.
It is also worth noting that some other attributes of Hebrock et al. might in fact be similar (or even equivalent) to some in the current study.A further panel discussion such as that employed in Phase 4 might lead to, for example, extended high-end being grouped with brightness, or detailed being grouped with clarity.It might even group dull and muffled and identify them each as being equivalent to a lack of brightness.These possibilities underline the importance of Phase 4 to the current study.
Even allowing for these potential similarities or equivalences, however, the current study still identifies several additional attributes as being important.This confirms the value of the use of a wide range of microphones and programme items and of the adopted free elicitation approach.
The situation is similar when considering the findings of the loudspeaker-based studies reviewed in Sec.I. Clarity and brightness are the only attributes shared with the lowest level of the current study's hierarchy, but both are in the top four according to degree of contribution.The higher-level attribute tone is also shared but the current study splits this into six component attributes.Again, there is also a possibility of similarity or equivalence between seemingly different attributes; for example, bass/treble balance, could be considered as a combination of LF content and brightness.
When considering the musical acoustics based studies reviewed in Sec.I, the attributes brightness, clarity, harshness, and nasal are shared with the lowest level of the current study's hierarchy, and the first three of these are in the top four highest contributing (top three if noise level is discounted).The higher-level attributes warmth and ringing are also shared but are split into multiple sub-attributes in the current study.Finally, again, there is a possibility of similarity/ equivalence; for example, the dull/brilliant scale may refer to the same perceptual attribute as brightness from this study.It is interesting to note that the attribute noise level, found to be the second largest contributing factor in this study, was not revealed in any of the previous studies.It was only revealed in the current study due to the deliberate selection of a very wide range of microphones (including MEMS microphones) and of programme items with a very wide range of characteristics (including the bass, which produced very little high frequency sound).
Thus, it seems that: (i) three of the four attributes found by the current study to contribute the most to perceived inter-microphone differences were also identified in previous studies; (ii) additional attributes were revealed by the current study, as a result of it focusing specifically on microphones, evaluating a wide range of microphones and programme items and employing free elicitation rather than allowing listeners to choose only from a limited prescribed attribute list; (iii) higher level attributes identified in previous studies can be broken down into multiple subattributes, each making a specific contribution; and (iv) panel discussions have the potential to identify equivalences between elicited descriptors and thereby reduce redundancy in attribute sets.The attributes contributing to the differences perceived between microphones (when auditioning recordings made with those microphones) are not clear from previous research, and perceived microphone characteristics do not always correlate well with manufacturers' standard measurements.As a step toward developing a perceptually relevant set of measures of microphone quality, a five-phase study was conducted to determine the perceptual dimensions across which microphones differ and to find the relative contributions of the corresponding attributes to perceived intermicrophone differences.
In Phase 1, consideration of microphone technical specifications and expert opinions from audio engineers indicated that recording five programme items (double bass, drums, acoustic guitar, string quartet, and trumpet) with eight studio and two MEMS microphones (listed in Sec.III A) would provide suitable stimuli to reveal the attributes comprising the most prominent inter-microphone differences.Such recordings were therefore made for use as stimuli in a listening test.
In Phase 2, pairwise listening comparisons between the resulting 50 stimuli, followed by multi-dimensional scaling analysis, revealed 17 salient dimensions and 17 corresponding pairs of stimuli exemplifying the differences across those dimensions.
In the FCP elicitation, in Phase 3, a total of 768 terms described the differences that listeners heard between the stimuli in each exemplary pair.Phase 4 then employed panel discussions to group the elicited terms and reduce redundancy, and identified a hierarchy of 40 perceptual attributes (Fig. .
Finally, in Phase 5, an attribute contribution experiment determined, for the 31 descriptors at the lowest level of the hierarchy, the degree to which each of them contributed to perceived inter-microphone differences.The results of this experiment allowed the attributes to be ordered by degree of contribution, and this ordering is shown in Table [fig_ref] TABLE IV: Attributes ordered by overall contribution to inter-microphone differences [/fig_ref].
Further analysis revealed that, overall, brightness is the attribute contributing the most to inter-microphone differences (this was the case for all programme items and for the majority of microphone pairs).Noise level, although ranked second overall, only contributes highly when microphones differing greatly in self-noise are used to record a source that lacks high frequency content.Brightness, harshness, and clarity were shown to contribute highly for all programme items and for all microphone pairs.Future work will develop models of the attributes contributing the most to perceived inter-microphone differences, in terms of objectively-measurable parameters.Such models could facilitate microphone development and testing by manufacturers, and microphone selection by users.
[fig] FIG.: Hierarchy the attributes corresponding to the groups generated by the panel discussions of Phase 4. Attributes marked with an asterisk correspond to groups containing no elicited terms from Phase 2 and were created by the panel to assist in structuring the hierarchy. [/fig]
[table] TABLE I: Selected microphones and their objective characteristics. [/table]
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10.4056/sigs.1344279
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CCBY
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3111990
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21677865
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s2orc_pubmed_articles
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Data shopping in an open marketplace: Introducing the Ontogrator web application for marking up data using ontologies and browsing using facets
In the future, we hope to see an open and thriving data market in which users can find and select data from a wide range of data providers. In such an open access market, data are products that must be packaged accordingly. Increasingly, eCommerce sellers present heterogeneous product lines to buyers using faceted browsing. Using this approach we have developed the Ontogrator platform, which allows for rapid retrieval of data in a way that would be familiar to any online shopper. Using Knowledge Organization Systems (KOS), especially ontologies, Ontogrator uses text mining to mark up data and faceted browsing to help users navigate, query and retrieve data. Ontogrator offers the potential to impact scientific research in two major ways: 1) by significantly improving the retrieval of relevant information; and 2) by significantly reducing the time required to compose standard database queries and assemble information for further research. Here we present a pilot implementation developed in collaboration with the Genomic Standards Consortium (GSC) that includes content from the StrainInfo, GOLD, CAMERA, Silva and Pubmed databases. This implementation demonstrates the power of ontogration and highlights that the usefulness of this approach is fully dependent on both the quality of data and the KOS (ontologies) used. Ideally, the use and further expansion of this collaborative system will help to surface issues associated with the underlying quality of annotation and could lead to a systematic means for accessing integrated data resources.
# Introduction
The field of molecular biology is now a dataintensive discipline, which can largely be attributed to recent advancements in 'omics technologies. Due to the increasing affordability of these technologies, there is now an everexpanding, increasingly democratized and complex array of distributed data resources for the scientific researcher to contend with. The most recent Nucleic Acids Research (NAR) database special issue states that there are now over 1200 published biological databases in the accompanying online Database Collection [bib_ref] Nucleic Acids Research Database Issue and online Database Collection: a community of..., Cochrane [/bib_ref]. Now, more than ever before, we require better tools to enable researchers to exploit this growing body of information, including the ability to work at the intersection of data held in different resources [bib_ref] Working together to put molecules on the map, Field [/bib_ref].
## Faceted browsing
Here we explore an approach -that of faceted browsing -for pulling together and viewing biological data resources in a new way. This approach has been successfully used in eCommerce for managing the exploration of large and complex search spaces. Faceted browsing is the use of information of different types (presented in facets, frames or windows) to quickly compare and select criteria about products. In other words, individual products are placed under multiple classification hierarchies and can therefore be viewed by users in a multitude of ways. This method is particularly prevalent in Web sites that have extensive product catalogues, such as iTunes and Amazon, where items are described by their key attributes like price, manufacturer/publisher or genre. Consider, for example, visiting Amazon to buy a television. Rather than being presented with a list of thousands of different items, users are asked to narrow down their choice by picking elements from different facets i.e. dimensions that describe the product they are looking for. For televisions, facets might include: screen-size, brand, pricerange, refresh-rate and so on. As the user selects particular characteristics, the number of matching candidates in the product catalogue is fed back on the screen and the list becomes more manageable. The major benefit of faceted browsing, compared to traditional keyword searches, is that users need not have any prior knowledge of either the content or structure of the underlying resources. Research within the information retrieval and human-computer interaction communities has shown faceted browsing to be both popular and effective in data exploration. The field of biology has arguably one of the largest and best product back catalogues of any science discipline. In order to utilize these data more effectively, we suggest here that biological data can be packaged-up and described as data products. However, unlike a multinational company that can control the descriptions of its product lines, biological data is highly distributed and heterogeneously described. In order to achieve a truly open biological linked-data marketplace, we need standardized and robust descriptions of our data products.
## Annotating data products to support faceted browsing
Many types of biological data still lack informative descriptions -even those that are minimal [bib_ref] Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI..., Taylor [/bib_ref] [bib_ref] The minimum information about a genome sequence (MIGS) specification, Field [/bib_ref] and while the use of ontologies for annotation is growing, there are still large resources that remain unannotated in any meaningful way. In such cases, it is still possible to extract information directly from a variety of data sources (or literature) to create useful annotations that support navigation and discovery. This can be done by matching content to relevant lists of terms of special interest to users. Consensus lists of such terms from different knowledge domains are collectively known as Knowledge Organization Systems (KOS) and can range from simple glossaries and dictionaries (or controlled vocabularies) through to more complex classification schemes, taxonomies, thesauri, gazetteers and ontologies.
Here we present the Ontogrator web application, where we have used a set of KOS to demonstrate how data can be marked-up to create informative facets for search and discovery. We are in particular interested in the use of ontologies as facets. Ontologies can be loosely defined as sets of concepts or terms that also contain explicit relationships between them. Perhaps the best-known example of an applied ontology in the field of Molecular Biology is the Gene Ontology (GO) [bib_ref] Gene ontology: tool for the unification of biology. The Gene Ontology Consortium, Ashburner [/bib_ref] , but there are now a wide range of available ontologies [bib_ref] The OBO Foundry: coordinated evolution of ontologies to support biomedical data integration, Smith [/bib_ref] opening up a range of options for future aggregation and Ontogration of data.
# Material and methods
The Ontogrator Web application provides a Java-Script GUI (graphical user interface) running within a web browser. This Web application fetches data on demand from a back-end comprising a set of REST (representational state transfer) web services supported by a LAMP (Linux, Apache, MySQL, PHP) software stack. A MySQL database is constructed and indexed specifically to support the functions of the browser GUI. The back-end service (see [fig_ref] Figure 1: The Ontogrator platform [/fig_ref] performs the following key functions: A -Data acquisition: ingestion of raw data from primary sources; B -Semantic indexing: detecting concepts in the data using text mining; C -Browsing services: providing the client with an efficient conceptbased retrieval service; D -Data and facet updates: periodic refreshing of the underlying resources. Standards in Genomic Sciences
## A -data acquisition
Data to be imported is converted to tabular format and pre-processed using a PHP script which is customized for each data source. This identifies which columns should be scanned for terms as well as constructing a unique identifier for each record. For example, a data resource with a habitat column would be marked for matching against the Environment Ontology. Once the input processors have been constructed, the remainder of the processing is fully automated. The import scripts create appropriate tables in the back-end database to hold both the data and any hits found during semantic indexing.
## B -semantic indexing
Concept annotation is performed by Terminizer, an external Web service that detects mentions of ontological concepts from a given ontology in a given textual passage. Word stemming and phrase rearrangement are employed to spot approximate matches, as well as blacklisting to remove common false positives. In addition to this generic ontology matching service, Ontogrator can call upon other external Web services. For example, in the genomic Ontogrator instance, the uBioservice is used for detecting species identifiers and GAZis used for identification of geo-locations.
## C -browsing services
A collection of PHP scripts are installed on the server to be called by the Web application. They provide the following functions over a RESTful interface:
- List the available data sources - Return meta-data about the columns available in a given data source - List the items from a data source which match any given set of concepts or identifiers - Give a detailed breakdown of exactly how an item matches against a set of concepts or identifiers. In our demonstration system, the term lookup is sufficiently fast to provide the user with an interface that is updated in a timely manner. As the speed of the queries depends on straightforward indexing of the underlying database, we anticipate that this would continue to work as more data was added. As all the operations are read-only, server-side replication of the database and/or Web server threads could be employed to maintain performance in the light of increased demand. It may also be useful for people to access these REST services directly, but at this point they are provided solely to feed the web application and we do not advertise them as a public API.
## D -data and facet updates
As the underlying data sources and ontologies change, these updates need to be reflected in the Ontogrator. For example, scripts in the back-end can be set to update the content of each resource (i.e. add new rows or update entries that have been changed) whenever a new version is published. Only the affected rows need to be reprocessed. On the other hand, an update to ontology resources (used for semantic indexing) would necessitate a full re-processing of all data sources as a background batch process.
## Ontogrator web interface
The Ontogrator interface, as viewed in a browser, is divided into two principal sections [fig_ref] Figure 2: The Ontogrator Interface [/fig_ref]. The top half of the page contains facet browsers, one for each of the available ontologies or taxonomies. The bottom half contains a tabular viewer for displaying results. In between these sections is a toolbar which displays the current search query (i.e. restrictions applied in the faceted windows). All sections can be interactively resized by the user. Individual items in the results table contain a hyper-link back to their entry in the source database. Typically, these entries contain significantly more information than is displayed in the view window of Ontogrator. Additionally, items can be doubleclicked to display a pop-up information window which gives details about the exact set of ontological concepts which were associated with this item. This view can be helpful when trying to discover exactly why a particular item has been considered to be a match for the current filters. The user interface is built using the Ext JS toolkit. This provides for a highly interactive interface composed of familiar widgets such as tree browsers, tables and combo-boxes. The Ext JS li-brary enables the interface to function correctly on all contemporary browsers. The user interface has been engineered to ensure that some results are always visible on screen in order to avoid the common problem of users failing to scroll long pages and thus failing to see the searches in action. The number of visible facets is currently limited to four, as this allows side-by-side display of them without consuming too much page real estate. As discussed previously, the architecture is expected to scale well, so there is no fundamental limit preventing the use of more facets, or, for example, allowing four to be selected from a larger pool.
# Results
## An instance of ontogrator for the genomic standards consortium (gsc)
To demonstrate the utility of facetted browsing as applied to biological data, we have worked within the Genomic Standards Consortium (GSC) [bib_ref] The minimum information about a genome sequence (MIGS) specification, Field [/bib_ref] to produce an instance of the Ontogrator system populated with content from genomic, metagenomic, marker gene sequences and culture collection databases. The data resources chosen included: 1) the Genomes OnLine Database [bib_ref] The Genomes On Line Database (GOLD) in 2009: status of genomic and..., Liolios [/bib_ref] (GOLD), 2) the Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis Database [bib_ref] CAMERA: a community resource for metagenomics, Seshadri [/bib_ref] (CAMERA), 3) the SILVA comprehensive ribosomal RNA database [bib_ref] SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA..., Pruesse [/bib_ref] and 4) the StrainInfo.net database [bib_ref] Knowledge accumulation and resolution of data inconsistencies during the integration of microbial..., Dawyndt [/bib_ref]. Furthermore, we also including text from a selection of 5) PubMed abstracts to illustrate the value of integrating information about molecules, organisms and the literature at the same time. In this instance of Ontogrator, facets were chosen that organize the meta-data content by taxonomy, anatomy, environment and location, the latter two axes of data organization being of special interest to the GSC [bib_ref] Working together to put molecules on the map, Field [/bib_ref]. We have created these four facets using the following KOS: 1) a species taxonomy (represented by the NCBI taxonomy, 2) anatomy (modeled by MIAA, environmental factors (represented by EnvOand geographical locations (represented by GAZ.
## Using ontogrator to explore marked up data
In the screenshot of the Ontogrator online interface as shown in [fig_ref] Figure 2: The Ontogrator Interface [/fig_ref] , the initial view of the ontograted resources shows a default data source and the root terms of the different ontologies. Each time the user adjusts the query by picking one or more terms in a facet, the results table is updated showing items and hit counts from the selected data resource. It is this continual display of visual feedback about the distribution of results that gives users the navigational awareness that enables the successful exploration of unfamiliar data. In [fig_ref] Figure 2: The Ontogrator Interface [/fig_ref] , the query retrieves data products from the five data sources that are related to Scandinavian Peninsula and Dairy product. Once a resource is selected as active (e.g., StrainInfo in [fig_ref] Figure 2: The Ontogrator Interface [/fig_ref] , its instances appear in a conventional tabular form with each row being a direct hyperlink back to the primary resource. The number of hits in each of the non-active resources is also shown, allowing the user to quickly ascertain which of them might also be worth browsing (e.g., there are fourteen hits relating to scholarly publications for the same query in the PubMed database that may be of interest). Additionally, the distribution of hits across the facet is also given, so that the user is guided in their exploration. For instance, if dairy product is showing 4 hits, expanding that node might reveal that cheese accounts for 1, milk for 2 and fermented dairy product for 1 of those results. Drilling down through the facet in this way allows the user to adjust the granularity of the query to a level with which they are comfortable.
# Discussion
Here we discuss the main strengths and challenges of the proposed approach, as well as future developments that could be integrated into Ontogrator.
## Searching using the power of ontologies
A major benefit of ontology-driven searching is the automatic broadening of retrieval results, e.g., look for bone but find tibia. Such searching is far more powerful than traditional keyword searching in which query terms must match the results explicitly. Using the current content of the Ontogrator genomic instance, a search for bacterial cultures in StrainInfo isolated from dairy products in Scandinavian Peninsula (see [fig_ref] Figure 2: The Ontogrator Interface [/fig_ref] returns, among others, an entry from a culture of Leuconostoc isolated from kefir in Stockholm. Even though this result contained none of the chosen query terms, the underlying subsumption hierarchy -of the species taxonomy, the Environment ontology and a geographical location Gazetteer -enabled the system to infer that Leuconostoc was a kind of bacteria, kefir was a kind of (fermented) dairy product and Stockholm was located in the Scandinavian Peninsula. This example clearly shows the potential of semanticallyenhanced facets for broadening the search space over conventional, keyword-based information retrieval systems.
Ontogrator facilitates the exploration and discovery of unexpected patterns in concept co-occurrences across facets, which might lead to the generation of novel research hypotheses. It supports both data drill-down to focus the results, and roll-up to generalize the queries. The top levels of the faceted trees give a useful overview of the query distribu-tion over the data set; e.g., which species or countries are overrepresented, and how many hits are available. For example, using the facets provided here, a user could select habitat=soil from the Environment dimension and then see how soil samples are distributed across countries by referring to the occurrence of entries now shown in the location facet.
No prior knowledge of either the content or structure of these resources is needed by the users, as the faceted browsing interface provides both the query vocabulary and navigational feedback. In contrast to existing interfaces based on keyword searching, by using ontologies Ontogrator overcomes the guess the keyword problem and provides the user with a new yet familiar way to explore distributed data sets in a unified environment.
## Future features
An obvious future direction for the Ontogrator platform to take without any further modification is to increase the number of data resources ontograted, either by increasing the number of resources in the GSC portal, or by building other community portals (e.g., model organisms, clinical trials, or environmental data). In addition to facets based on existing ontologies and taxonomies (which are represented as trees), new facet types could be imagined that use other ways (i.e. KOS) in which to organize data. For example, some facets might be better represented graphically, for example a schematic representation of the human digestive tract for exploring human microbiome project data; or a geopolitical map facet for exploring samples marked up with geolocations. Furthermore, a phylogenetic tree facet can be used to display entries according to their evolutionary relatedness, or a semantic network of concepts can be used to represent dimensions that have not yet been formally represented. We can also envisage more relaxed matching of resource entries in cases when there are few hits using the standard ontological matching or when different resources have been semantically indexed by different, yet related ontologies. Matches in these cases could be based on semantic distances between pre-computed database annotations and/or user queries. We could use the semantic layer (i.e. ontological annotations) to enable cross-database retrievals through the auto-mated discovery of mappings based on semantic distances between conceptual tags. This approach should provide retrieval of data instances that are, for example, similar to dairy products even though the dataset has not been indexed by such tags. For example, a future interface could support functions like users who searched for this have also searched for this and this.
## Capturing the user experience
As a third party data aggregator, the quality and accuracy of data annotation is of paramount importance when retrieving data via Ontogrator. The ultimate test for the impact of such systems is the end users. In order to continually improve the application and the data that underpins it, we aim to provide a feedback mechanism where users of the Ontogrator application can rate the appropriateness of data sets based on their search criteria and alert data suppliers of problems with their data.
## Driving ontology development
The Ontogrator could be used to help mature and improve the ontologies it relies upon. More precisely, it could implement a mechanism to provide feedback on terms that have either been overrepresented in data (and may need further specialization) or do not exist in the current hierarchy (e.g., a term clearing house can be provided for the submission of new terms to existing ontologies). Similarly, Ontogrator could be open to user-driven updates of annotations/mappings of the Ontograted resources (e.g., a user can indicate that a returned entry is not relevant to a particular query, so the software could have the ability to learn e.g., by removing the annotations and/or by re-training the mapping tools.
# Conclusions
We argue that the combined approach of faceted browsing and resource aggregation is an effective solution for aligning and mining information across a collection of related databases. Furthermore, by combining the power of searching over information resources with ontologies, complex distributed data sets can be searched over whilst leveraging the combined knowledge of expert communities.
[fig] Figure 1: The Ontogrator platform. Ontologies, or other KOS, and selected content are processed for use in Ontogrator. After data acquisition and annotation (semantic indexing), browsing services enable exploration and discovery through the web application. [/fig]
[fig] •: List the concepts which match a given substring (for autocomplete when searching) [/fig]
[fig] Figure 2: The Ontogrator Interface. 1) Users pick facet terms by hierarchical browsing or through keyword search. 2) Continuous feedback about the distribution of hits across the facet helps to guide exploration.3) The number of hits in all available resources is displayed in real-time. 4) Hits include hyperlinks back to the primary source. [/fig]
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10.1155/2020/9456350
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CCBY
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7528019
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33029181
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s2orc_pubmed_articles
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Comparatively Evaluating the Role of Herb Pairs Containing Angelicae Sinensis Radix in Xin-Sheng-Hua Granule by Withdrawal Analysis
e present study aims to investigate the roles of herb pairs containing Angelicae Sinensis Radix (Danggui) in Xin-Sheng-Hua Granule (XSHG) on hemolytic and aplastic anemia (HAA) mice. HAA model mice were induced by acetyl phenylhydrazine and cyclophosphamide; then the samples of XSHG and its decomposed recipes (DY, DC, DT, DH, DJ, and DZ) were orally administrated to these mice. Indicators of peripheral blood routine, organ index, and ATPase activities were tested. Moreover, the main effective components in these samples were also analyzed by UHPLC-TQ-MS/MS. Clear separation between the control and model groups from score plot of principal component analysis (PCA) was easily seen, indicating that HAA model was successfully conducted. Afterwards, relative distance calculation method between dose groups and control group from PCA score plot was adopted to evaluate the integrated effects of hematinic function of different samples. And the orders of hematinic effects were as follows: XHSG > DJ > DT > DZ > DH > DC > DY. Further analysis of these samples by UHPLC-TQ-MS/MS revealed that XSHG underwent complicated changes when herb pairs containing Danggui were excluded from XSHG, respectively. Compared with XSHG, the vast majority of active compounds in sample DY (formula minus herb pair Danggui-Yimucao) decreased significantly, which could partly explain why herb pair Danggui-Yimucao made great contribution to XSHG. ese findings showed that withdrawal analysis method is a valuable tool to analyze the impacts of herb pairs containing Danggui on XSHG, which could lay foundation to reveal the compatibility rules of this formula.HindawiEvidence-Based Complementary and Alternative Medicine Volume 2020, Article ID 9456350, 13 pages https://doi.
# Introduction
Traditional Chinese medicines (TCMs) have made tremendous contributions to the health of the ancient Chinese people for a long time. To achieve maximum therapeutic effect of certain diseases, TCMs are usually used in combination with several specific herbs instead of single herbs [bib_ref] Convergence: where west meets east, Tian [/bib_ref]. Xin-Sheng-Hua Granule (XSHG), originating from the classic and typical formulae Sheng-Hua Decoction, has function of promoting blood circulation, nourishing blood, and relieving pains [bib_ref] Shenghua decoction reduces uterine bleeding and regulates T-cell paradigm in human deciduas..., Li [/bib_ref]. And the formula is the boiled water extract of seven medical herbs, Angelicae Sinensis Radix (Danggui, DG), Leonuri Herba (Yimucao, YMC), Chuanxiong Rhizoma (Chuanxiong, CX), Persicae Semen (Taoren, TR), Carthami Flos (Honghua, HH), Cirsii Zingiberis Rhizoma Carbonisatum (Jiangtan, JT), and Glycyrrhizae Radix Et Rhizoma Praeparata Cum Mell (Zhigancao, ZGC), at a proportion of 80 : 90 : 30 : 8 : 5 : 5 : 5 [bib_ref] Simultaneous determination of hydroxysafflor yellow A, ferulic acid and ammonium glycyrrhizinate in..., Zhang [/bib_ref]. According to the basic theory of TCM, XSHG is usually clinically applied to treat postpartum diseases including uterine hemorrhage, postpartum abdominal pain, and retention of the lochia after delivery [bib_ref] Comparative analysis of the main bioactive components of Xin-Sheng-Hua granule and its..., Pang [/bib_ref]. And it is well known that postpartum diseases are usually associated with blood deficiency. erefore, studying the effects of nourishing blood of XSHG is significant for researchers to discover its action mechanism.
In ancient China, two certain herbs are frequently used together in many formulae for the treatment of several diseases, which are also called herb pairs, Yaodui or Duiyao. ey usually could achieve the effects of assistance, restraint, suppression, mutual antagonism, or mutual enhancement. As the fundamental composition units of TCM formulae, herb pairs are significant for researchers to study the compatibility rules of complicated formulae [bib_ref] Identification and comparative analysis of the major chemical constituents in the extracts..., Yang [/bib_ref]. Interestingly, a series of herb pairs containing Danggui could be formed when Danggui was combined with other six single herbs from XSHG, respectively, including herb pairs Danggui-Yimucao, Danggui-Chuanxiong, Danggui-Taoren, Danggui-Honghua, Danggui-Jiangtan, and Danggui-Zhigancao. Ever since a long time ago, herb pairs including Danggui have usually been applied clinically for the treatment of women's diseases [bib_ref] Herb pairs containing Angelicae Sinensis Radix (Danggui): a review of bio-active constituents..., Jin [/bib_ref]. Based on our previous studies, the synergistic and complementary effects of promoting blood circulation and nourishing blood could be discovered in herb pairs Danggui-Chuanxiong and Danggui-Honghua [bib_ref] Comparative metabolomics analysis on hematopoietic functions of herb pair Gui-Xiong by ultra-high-performance..., Li [/bib_ref] [bib_ref] Comparative metabolomics analysis on invigorating blood circulation for herb pair Gui-Hong by..., Li [/bib_ref]. Undoubtedly, herb pairs containing Danggui made extremely significant contribution to this formula. In order to disclose the compatibility rules of XSHG from the aspects of herb pairs, it has become particularly important for researchers to investigate the contribution of these herb pairs to XSHG. As one of the decomposed analysis methods, the withdraw analysis has been applied to explore the compatibility rules of multiherb formulae due to the advantages of simplifying the complexity [bib_ref] Integrated metabolomics and network pharmacology approach to explain possible action mechanisms of..., Pang [/bib_ref] , suggesting that this method could be effectively applied to study the compatibility rules of XSHG.
To investigate the roles of herb pairs containing Danggui in the formulae on hemolytic and aplastic anemia (HAA) mice, the sample XSHG and six different samples of decomposed recipes were prepared separately, including sample DY (the formulae minus herb pair Danggui-Yimucao), DC (minus Danggui-Chuanxiong), DT (minus Danggui-Taoren), DH (minus Danggui-Honghua), DJ (minus Danggui-Jiangtan), and DZ (minus Danggui-Zhigancao). en principal component analysis (PCA) model was applied to evaluate integrated effects of hematinic function of XSHG and these decomposed recipes [bib_ref] Strategy of integrated evaluation on treatment of traditional Chinese medicine as "interaction..., Duan [/bib_ref]. Additionally, twenty-one bioactive constituents in these samples [bib_ref] 1H-qNMR for direct quantification of stachydrine in Leonurus japonicus and L. cardiaca, Kuchta [/bib_ref] [bib_ref] Carthami flos suppresses neutrophilic lung inflammation in mice, for which nuclear factor-erythroid..., Kim [/bib_ref] [bib_ref] RP-thin layer chromatographic method for the quantification of three gingerol homologs of..., Khan [/bib_ref] [bib_ref] Separation and characterization of phenolic compounds and triterpenoid saponins in licorice (Glycyrrhiza..., Qiao [/bib_ref] , including five phthalides, four aromatic acids, three alkaloids, four flavonoids, three gingerols, and two other components, were also determined simultaneously by ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry (UHPLC-TQ-MS/MS). And the content variations of active components were further compared between the formulae and the decomposed recipes in order to discover potential reasons of their effects.
In this study, the withdrawal analysis approach was originally applied to study the compatibility rules of XSHG from the aspects of herb pairs. Besides, a sensitive, reliable, and powerful technique capable of quantifying 21 marker components in XSHG was firstly proposed and validated by UHPLC-TQ-MS/MS method within 20 min. According to the above analysis, the contribution of these herb pairs to the formula XSHG was systematically investigated to discover their action characteristics in XSHG. Furthermore, our findings could lay foundation to further reveal the compatibility rules of XSHG.
# Materials and methods
## Chemicals and reagents.
e herbs were generously provided by Revolence Pharmaceutical Co., Ltd. (Jiangsu, China), in February 2016, including Angelica sinensis (Oliv.) Diels, Leonurus japonicus Houtt., Ligusticum chuanxiong Hort., Prunus persica (L.) Batsch, Carthamus tinctorius L., roasted rhizome of Zingiber officinale Rosc, and Radix Glycyrrhizae Preparata; detailed information is summarized in [fig_ref] Table 1: e results of peripheral blood routine between control group and treatment groups [/fig_ref]. e seven herbs were kindly identified by Dr. Qinan Wu, doctor of pharmacognosy (College of pharmacy, Nanjing University of Chinese Medicine, Nanjing, China), and the voucher specimens were deposited at the Herbarium of Nanjing University of Chinese Medicine.
Acetyl phenylhydrazine (APH) was obtained from Tianjin Institute of Fine Chemicals retrocession (batch number: 20130816, Tianjin, China). Cyclophosphamide (CP) was purchased from Jiangsu Hengrui Medicine Co., Ltd. (batch number: 15031218). Reference compounds of trigonelline (1), protocatechuic acid (3), hydroxysafflor yellow A (4), amygdalin (6), chaffier acid (7), liquiritin (9), ferulic acid (10), zingerone, senkyunolide I (13), senkyunolide H [bib_ref] RP-thin layer chromatographic method for the quantification of three gingerol homologs of..., Khan [/bib_ref] , glycyrrhizic acid (16), 6-gingerol (17), ligustilide [bib_ref] Integrative drug efficacy assessment of Danggui and European Danggui using NMR-based metabolomics, Zhang [/bib_ref] , and butylidenephthalide [bib_ref] Metabolomics-based mechanisms exploration of Huang-Lian Jie-Du decoction on cerebral ischemia via UPLC-Q-TOF/MS..., Zhu [/bib_ref] were purchased from Sichuan Shinning Biotech Institute (Chengdu, China). Stachydrine hydrochloride (2), chlorogenic acid (5), leonurine hydrochloride (8), isoliquiritoside [bib_ref] 1H-qNMR for direct quantification of stachydrine in Leonurus japonicus and L. cardiaca, Kuchta [/bib_ref] , liquiritigenin [bib_ref] Separation and characterization of phenolic compounds and triterpenoid saponins in licorice (Glycyrrhiza..., Qiao [/bib_ref] , senkyunolide A (18), 6-shogaol (21) were obtained from National Institute for the Control of Pharmaceutical and Biological Product (Beijing, China). e chemical structures of these chemical standards were given as shown in . By using the method of peak area normalization, the purity of all standards was over 98%. Acetonitrile with HPLC grade was obtained from Merck (Darmstadt, Germany); deionized water was purified using a Milli-Q water purification system (Bedford, MA, USA); the remaining reagents were analytical grade.
## Herbal extraction.
e mixture (699 g) of DG-YMC-CX-TR-HH-JT-ZGC was mixed (80 : 100 : 30 : 8 : 5 : 5 : 5).
en the mixture was extracted with boiling water (1 : 8, w/v) for three times (2 h, 1.5 h, and 1.5 h). e extract was filtered through gauze, and the filtrate was subsequently evaporated with rotary evaporation under vacuum at 60°C, and the extract of XSHG was finally obtained. To prepare the DY, DC, DT, DH, DJ, and DZ extracts, the mixtures of decomposed recipes were extracted using the same preparation methods. Taking the case of the preparation of sample DY, a total of 159 g mixed powder of CX-TR-HH-JT-ZGC (30 : 8 : 5 : 5 : 5) was decocted by refluxing with water (1 : 8, w/ v) for 2 h, 1.5 h, and 1.5 h, respectively. en the extract was filtered and concentrated to obtain the DY sample. e specific compositions for the different preparations were listed in [fig_ref] Table 2: Amounts [/fig_ref] Chinese Medicine. All efforts were made to relief the suffering of animals. After 10 days of acclimatization, 100 mice were randomly divided into 10 groups with 10 mice in each: the control (K), model (M), positive drug control group (Fu-Fang-A-Jiao-Jiang, AJ), and the groups XSHG, DY, DC, DT, DH, DJ, and DZ. Except the control group, the mice in other groups were hypodermically injected with 2% APH saline solution at dose of 20 mg·kg −1 and 10 mg·kg −1 body weight on days 1 and 4 accordingly; 2 h after the hypodermic injection with APH saline solution on day 4, the mice were injected with CP saline solution intraperitoneally at a dose of 20 mg·kg −1 on days 4, 5, 6, and 7 [bib_ref] Urine and plasma metabonomics coupled with UHPLC-QTOF/MS and multivariate data analysis on..., Li [/bib_ref]. As a result, HAA mice were induced, and the experimental period was 7 days. e mice in positive drug control group were orally given Fu-Fang-A-Jiao-Jiang at a dose of 0.1 g·kg −1 , and the mice in XHSG group were intragastrically administrated with XHSG extracts at a dose of 4.86 g·kg −1 . By using the body surface area normalization method, the animal dose of XSHG, DY, DC, DT, DH, DJ, and DZ groups was inferred based on the human daily dose. And the formula was described as follows: human dose of crude herbs in clinic × 0.018/200 × 1000 × the multiple of clinical equivalency dose [bib_ref] Comparative metabolomics analysis on invigorating blood circulation for herb pair Gui-Hong by..., Li [/bib_ref]. e dose of XSHG was equivalent to one time of the adult daily dose of the formula XSHG crude drugs (36 g, from XSHG in which DG-YMC-CX-TR-HH-JT-ZGC were 12.36 g, 15.45 g, 4.64 g, 1.24 g, 0.77 g, 0.77 g, and 0.77 g, respectively) according to clinical prescription. And the doses of DY, DC, DT, DH, DJ, and DZ extracts were 1.11, 2.57, 3.02, 3.09, 3.09, and 3.09 g·kg −1 , respectively. Control and model groups were orally given the same volume of saline solution. All animals were intragastrically administrated one time each day for continuous 7 days since the beginning of reproducing HAA model.
## Sample collection.
Before the gavage administration on day 7, the mice of all groups were weighted by electronic balance. Half hour after the last administration of the extracts, blood samples (0.5 mL) were collected by orbital venous plexus method into 1.5 mL centrifuge tube with 1.5% EDTA-2Na (EDTA-2Na/blood: 1/9, v/v). en blood samples were used to measure peripheral blood routine and ATPase activities of red cell membrane. Moreover, the whole liver, spleen, and thymus of mice in every group were isolated and then weighed by electronic balance. All experiments were finished within 3 h after sample collection.
## Effect indicators of nourishing blood.
A total of 300 μL blood was applied to measure the peripheral blood routine by Sysmex XS 800i (Shanghai, China) type fully automatic blood analyzer. And the peripheral blood routine in this experiment includes white blood cell count (WBC/10 9 L −1 ), red blood cell count (RBC/10 12 L −1 ), hemoglobin (HGB/ g·L −1 ), neutrophils ratio (NE/%), lymphocyte ratio (LY/%), monocyte ratio (MO/%), haematocrit (HCT), and platelet count (PLT/10 9 L −1 ). Also, thymus index, spleen index, and liver index of mice in every group were obtained according to the following equation: thymus index � the weight of thymus/the body weight of mice; spleen index � the weight of spleen/the body weight of mice; liver index � the weight of liver/the body weight of mice, respectively.
Additionally, the ATPase activities of red cell membrane, including Na + -K + and Ca 2+ -Mg 2+ ATPase, were carried out in a 96-well microplate reader as reported before. By using commercial Na + , K + and Ca 2+ , Mg 2+ ATPase kits (Nanjing Jiancheng Bioengineering Institute, China), the ATPase activities were assayed by measuring the release of inorganic phosphate (Pi) from the hydrolysis of ATPase according to the manufacturer's instructions. Briefly, the activities of Na + -K + and Ca 2+ -Mg 2+ ATPase were determined by measuring the amount of inorganic phosphate with malachite green dye method and then expressed as micromoles per milliliter blood. By using a malachite-based Biomol Green reagent, released inorganic phosphate (Pi) was assayed [bib_ref] Chromatographic isolation of the functionally active MutS protein covalently linked to deoxyribonucleic..., Monakhova [/bib_ref].
## Quantitative analysis of marker components
## Preparation of standard solutions.
A mixed stock solution containing the reference standards 1-21 was prepared by dissolving them in 90% methanol and then this mixed standard stock solution was diluted with 90% methanol to a series of appropriate concentrations for the construction of calibration curves. All the samples were stored at 4°C until use and filtered through a 0.22 μm cellulose membrane prior to injection.
## Preparation of sample solutions.
e concentrated extracts of XSHG, DY, DC, DT, DH, DJ, and DZ for the treatment of mice were diluted to 100 times with Millipore water, respectively. Subsequently, the extracts of different samples were centrifuged at 13000 rpm for 10 min, and the supernatant was stored at 4°C. Before the UHPLC-TQ-MS/ MS analysis, the sample solutions were filtered through 0.22 μm membrane.
## Uhplc-tq-ms/ms instrumentation and conditions.
A Waters ACQUITY UHPLC system (Waters, Milford, MA, USA) was performed comprised of a binary pump solvent delivery system, an online degasser, and an autosampler. Chromatographic separations were carried out on a ermo Scientific Hypersil GOLD (100 mm × 3 mm, 1.9 μm) column. e mobile phase was composed of A (acetonitrile) and B (water with 0.1% formic acid) using a gradient program as follows: 0-2 min, isocratic 5% A; 2-12 min, linear gradient 5%-40% A; 12-18 min, linear gradient 40%-95% A; 18-19 min, linear gradient 95%-5% A; and 19-20 min, isocratic 5%-5% A. e flow rate of the mobile phase was set at 0.40 mL·min −1 , and the injection volume was 2 μL. e column temperature and the autosampler were conditioned at 35°C and 4°C, respectively.
Mass spectrometry analysis was operated using a Xevo Triple Quadrupole tandem quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) equipped with an electrospray ionization source (ESI). e mass data was collected from m/z 100 to 1000, and the TQ mass spectrometer was performed in both positive and negative modes. Additionally, multiple reaction monitoring (MRM) was applied for the MS spectra. e parameters of MS analysis were as follows: drying gas, N 2 ; cone gas flow, 50 L·h −1 ; desolvation gas flow, 1000 L·h −1 ; capillary voltage, 3000 V; desolvation temperature, 550°C; source temperature, 150°C. Dwell time was automatically designed by software. e collision energy and cone voltage were optimized for each compound, and the selected values are shown in .
# Uhplc-tq-ms/ms method validation.
e UHPLC method was validated for linearity, limits of quantification and detection (LOQs and LODs), interday and intraday precision, stability, accuracy, and matrix effect according to some reports on quantification analysis.
A series of standard solutions containing the 21 analytes were injected in duplicate, and the calibration curves could be constructed by the peak areas versus the corresponding concentrations of the analytes. e values of LOD and LOQ were acquired using diluted standard solution when the signal-to-noise ratios (S/N) were about 3 and 10, respectively.
To evaluate the precision of the developed method, six replicates of the standard solution were examined for intraday precision, and then the sample was further analyzed during consecutive three days for interday precision. To estimate the repeatability, six independent sample solutions (XSHG sample) were prepared by the above method, and the variations of these samples were determined by UHPLC-TQ-MS/MS. One of XSHG sample solutions was kept at room temperature and analyzed at 0, 2, 4, 8, 12, 18, and 24 h, respectively, to evaluate the stability of these samples. e accuracy of this method was confirmed with the spike recovery test. e recovery was performed by spiking the marker analytes with low (50%), middle (100%), and high (150%) levels to 1.0 g of the XSHG sample, which had been analyzed in the previous study. e triplicate spiked samples were then extracted and processed according to the sample preparation methods mentioned above. To compare the spiked samples, a blank sample without standards was also similarly prepared and analyzed.
Because the ion enhancement or suppression could have the negative impact on the analysis of these compounds, the slope comparison method was applied to study the matrix effect [bib_ref] Evaluation of zirconium dioxide-based sorbents to decrease the matrix effect in avocado..., Lozano [/bib_ref]. For the construction of standard addition calibration curves, the sample extracts were spiked with appropriate amounts of standards to perform the recovery measurement. Subsequently, the slopes of calibration curves from standard addition experiments were compared with the slopes of calibration curves from 90% methanol standards under the same concentration levels; thus the ratio (slope matrix/slope solvent) was used to evaluate the matrix effect.
## Statistical analyses.
All of the results were presented as mean ± standard deviation (SD) and the statistical program SPSS 19.0 was applied to analyze their variations.
Additionally, one-way analysis of variance (ANOVA) with Dunnett's test was used to calculate the statistical comparison. In all cases, P < 0.05 was regarded to be significant.
# Results
## Evaluation of hematinic function
[formula] 3.1.1. Peripheral Blood Routine Analysis. [/formula]
e peripheral blood routine data are shown in [fig_ref] Table 1: e results of peripheral blood routine between control group and treatment groups [/fig_ref]. Compared with control group, all the indicators of peripheral blood routine of model group had statistical differences (P < 0.05). Among them, HGB, RBC, LY%, and HCT in model group were obviously decreased in comparison with control group (P < 0.01), while the indicators of WBC, NE%, MO%, and PLT increased significantly (P < 0.01). All of the results above demonstrated that the blood deficiency mice were successfully induced. Compared with model group, WBC, MO%, and HCT% in XHSG group and the groups of decomposed recipes were significantly reduced (P < 0.05); except for the DY and DC group, RBC, HGB, and LY (%) in other groups were obviously increased (P < 0.05); NE (%) and PLT in the DT group decreased significantly (P < 0.05). And in comparison with the XSHG group, except WBC, NE (%), and MO (%), other indicators in DY group had remarkable difference (P < 0.01); RBC, HGB, LY (%), and PLT in DC group showed statistical difference (P < 0.05); WBC and LY (%) in DH group changed significantly (P < 0.05); LY (%) in the DT, DJ, and DZ groups were remarkably reduced (P < 0.05).
## Effects on immune organ index.
e effects on immune organ index for each group are shown in [fig_ref] Figure 2: e effects of immune organ index and ATPase activities for administration groups... [/fig_ref]. As shown in [fig_ref] Figure 2: e effects of immune organ index and ATPase activities for administration groups... [/fig_ref] -2(c), in the model group, thymus index was decreased, while liver index and spleen index increased conspicuously (P < 0.01). In the XSHG and DJ groups, the liver index and spleen index were significantly reduced and the thymus index was increased obviously (P < 0.05). ymus index in DY and DC groups had statistical difference (P < 0.01), and the DT group could significantly affect the liver index and spleen index (P < 0.01) compared to the model group. Moreover, the DH and DZ groups had significant effects on the spleen index (P < 0.01). Compared with the XSHG group, except DJ group, liver index in other groups significantly increased (P < 0.05); thymus index in DC, DT, DH, and DZ groups obviously reduced (P < 0.05); and spleen index in DY and DC groups was conspicuously increased (P < 0.01). [fig_ref] Figure 2: e effects of immune organ index and ATPase activities for administration groups... [/fig_ref] and 2(e), the Na + -K + and Ca 2+ -Mg 2+ ATPase activities of red cell membrane for the model group were significantly reduced compared to the control group (P < 0.01), indicating that the blood deficiency mice could affect the ATPase activities. After administration, all groups could obviously improve the Na + -K + ATPase activity (P < 0.01); except the DY group, the Ca 2+ -Mg 2+ ATPase activities in other groups could be significantly increased (P < 0.05). Compared to the Evidence-Based Complementary and Alternative Medicine XSHG group, the Na + -K + and Ca 2+ -Mg 2+ ATPase activities of red cell membrane for DY and DC groups were reduced conspicuously (P < 0.05); and the Na + -K + ATPase activity in DT group and the Ca 2+ -Mg 2+ ATPase activity for DH group significantly decreased (P < 0.05).
## Effects on atpase activity. as shown in
## Integrated effects of hematinic function.
To investigate the integrated effects of hematinic function, the raw data of effect indicators in different groups were imported into EZinfo 2.0 software for PCA analysis. In the PCA scores [fig_ref] Figure 3: PCA score plot between control group and dose groups [/fig_ref] , each point represented an individual sample and the similar effects of the administration groups would be clustered together. Clear separation between control and model group from the PCA scores could be easily seen, and other treatment groups scattered across these two groups.
ese results suggested that the model and control group had different effect indicators, indicating that the blood deficiency mice were successfully copied.
Furthermore, the relative distances between administration groups (the model, XSHG, DY, DC, DT, DH, DJ, and DZ groups) and the control group from PCA score plot of effect indicators were calculated for quantization [bib_ref] Strategy of integrated evaluation on treatment of traditional Chinese medicine as "interaction..., Duan [/bib_ref] [bib_ref] Integrative drug efficacy assessment of Danggui and European Danggui using NMR-based metabolomics, Zhang [/bib_ref]. e jumping-off point of the integrated effects for treatment groups was defined as the mean integration effects for normal rats from PCA; then the relative distances of different drug groups were obtained. From , it was found that the relative distances of the treatment groups were decreased compared to model group, suggesting that the samples of XHSG and its decomposed recipes could regulate the HAA mice to the normal state. Remarkably, the distances of DY and DC groups were relatively long, while the relative distances of DJ and DT groups were shorter than the others, which indicated that the DY and DC groups had poor curative effects on the treatment of anemia mice. And the orders of hematinic effects of dose groups were as follows:
[formula] XHSG > DJ > DT > DZ > DH > DC > DY. [/formula]
ese results demonstrated that the herb pairs containing Danggui played different roles in this formulae, and herb pairs Danggui-Yimucao and Danggui-Chuanxiong made great contribution to hematinic effects of XSHG.
## Quantitative analysis of samples using uhplc-tq-ms/ms
## Optimization of the chromatographic conditions and
Mass Spectrometric Conditions. Due to various structural types of active components in the sample XSHG, the UHPLC conditions were studied on a ermo Scientific Hypersil GOLD column (100 mm × 3 mm, 1.9 μm). Compared with methanol, better separation efficiency could be achieved for the low polarity components (gingerols) when using acetonitrile. And the additives of formic acid could reduce the peak tailing and increase the retention time of aromatic acids. Finally, the mobile phase of 0.1% formic acid solution, acetonitrile, was selected. For the good separation of high polarity constituents (alkaloids), the initial mobile phase was adjusted at 95% aqueous phase-5% organic phase. Although stachydrine hydrochloride and trigonelline had similar retention time on this chromatographic condition, the two components could be distinguished by their characteristic fragmentation. e flow rate and column temperature were set at 0.4 mL·min −1 and 40°C. Typical chromatograms of multiple components with MRM mode are shown in. e target compounds were examined separately under the ESI + or ESI − mode according to the intensity and sensitivity of their signals. To obtain the characteristic transition for MRM determination of each compound, at least two precursor/product ion pairs of the compound were chosen in this investigation. By comparing the response of the obtained ion pairs, the highest sensitive and specific ion pairs were finally applied for MS/MS detection. Afterwards, the values of collision energy and cone voltage were optimized by the Intellistart program. In this study, protonated molecules [M + H] + were determined with abundant ions in the analysis of phthalides, alkaloids, and gingerols, while organic acids, flavonoids, and amygdalin exhibited deprotonated molecules [M -H] − with the most abundant intensities. For the gingerols (6-gingerol, 6-shogaol, and zingerone), m/z 137 as the daughter ions could be observed in these analytes. Additionally, m/z 137 could also be detected in the MS fragment ions of senkyunolide A and liquiritigenin. As for senkyunolide I and senkyunolide H, Evidence-Based Complementary and Alternative Medicine they could produce the same precursor/product ion pairs of 225 ⟶ 91, thus leading to the mutual interference. Fortunately, the two analytes could be baseline separated under the optimized UHPLC conditions. All the MRM transitions and parameters optimized in the investigation are summarized in . , the correlation coefficient values greater than 0.9937 were obtained for all the standards within the test ranges. eir values of LOD and LOQ for MS/MS determination were in the ranges from 0.62 to 9.89 ng/mL and 1.96 to 30.58 ng/mL, respectively. Other results about method validation are listed in . e overall intra-and interday variations (RSDs) of the 21 standards ranged from 1.06% to 3.26% and 1.43% to 4.38%, respectively. e repeatability and stability expressed as RSDs were in the ranges of 1.85%-4.86% and 1.67%-4.59%, respectively. e recoveries of 21 analytes ranged from 94.79% to 102.76% with RSD between 1.31% and 4.78%. Additionally, the slop ratio values of matrix curve to the neat standard solution curve ranged from 0.91 to 1.07, suggesting that the matrix effect was not significant under the optimized conditions. Consequently, these results revealed that the proposed UHPLC-TQ-MS/MS method was accurate, sensitive, and repeatable for the quantitation of these target constituents. [fig_ref] Table 2: Amounts [/fig_ref] , respectively. In order to study the roles of herb pairs containing Danggui on the formulae, the theorized amounts of the analytes in disassembled prescription were originally introduced. Apart from aromatic acids and phthalides, other analytes were the marker compounds and each compound could only be found in one certain plant; thus, the theorized amounts of these analytes in discomposed recipes were their observed amounts in XSHG. For example, stachydrine hydrochloride could only be found in herb Yimucao, and the amount of stachydrine hydrochloride in XSHG was 2148.92 mg, and then the theorized amount of stachydrine hydrochloride in the decomposed recipes was 2148.92 mg.
## Method validation. as shown in
# Quantitative analysis of
Annoyingly, aromatic acids and phthalides could be detected in Danggui and Chuanxiong, while these constituents could just be found from Chuanxiong in the disassembled samples. To solve the problem, aromatic acids and phthalides were also determined in the single herbs of Danggui (240 g) and Chuanxiong (90 g), and the preparation of these samples all adopted the above extraction methods. e results showed that the amounts of these target compounds were different in Danggui and Chuanxiong. en the proportionality coefficient of aromatic acids or phthalides in Chuanxiong could be obtained according to the following equation: (Amount in CX (90 g))/(Amount in CX (90 g) + Amount in DG (240 g)). For example, the amount of ligustilide in the single herbs of Chuanxiong (90 g) and Danggui (240 g) was 242.28 mg and 453.36 mg, respectively; thus the proportionality coefficient of ligustilide in Chuanxiong could be calculated: 242.28 mg/ (242.28 mg + 453.36 mg) � 0.348. Afterwards, the theorized amounts of these constituents in XSHG (only from Chuanxiong) could be further deduced: Amount e. � Proportionality coefficient of CX × Amount of the analyte in XSHG. Using ligustilide as a case, the proportionality coefficient of ligustilide in CX was 0.348 according to the above analysis result, and the amount of ligustilide in XSHG 572.22 mg; then the theorized amount of ligustilide could be calculated: Amount e. � 0.348 × 572.22 mg � 199.26 mg. e theorized amounts of the compounds in XSHG are also presented in [fig_ref] Table 2: Amounts [/fig_ref]. Although there were some deficiencies in the calculation of theorized amounts for aromatic acids and phthalides, the proportionality coefficient of these constituents in Danggui and Chuanxiong might be an effective way to calculate the theorized amounts of Chuanxiong in the disassembled formula.
## E concentration variations of target compounds and chemical interaction analysis.
To discover the potential reasons of the contribution of herb pairs containing Danggui to XSHG, the source data was firstly standardized by the statistical program SPSS 19.0; then standardized data was imported into MassLynx software [bib_ref] Integrative drug efficacy assessment of Danggui and European Danggui using NMR-based metabolomics, Zhang [/bib_ref] [bib_ref] Metabolomics-based mechanisms exploration of Huang-Lian Jie-Du decoction on cerebral ischemia via UPLC-Q-TOF/MS..., Zhu [/bib_ref]. And the PCA score plot (two principal components) of seven different groups is given in [fig_ref] Figure 3: PCA score plot between control group and dose groups [/fig_ref]. Compared to other samples, the sample DJ and DZ were closer to XSHG, while the sample DY and DC were farther away from this formula. ese results indicated that each herb pair was the indispensable part of the formulae, and herb pairs Danggui-Yimucao, Danggui-Chuanxiong might be particularly important for this formula.
Moreover, the increased rate � (Amount Obs. − Amount e. )/Amount e . was applied to evaluate the amount variations of different analytes in decomposed recipes. Based on the above equation, the increased rates of active constituents were calculated accordingly. And the heat map of the increased rates of target constituents in different separated prescriptions is given in. In the heat map, the increased rate of 0 indicates that the observed amount was equal to its theorized amount, otherwise denoting amount increases (>0) or amount decreases (<0). Altogether, the increased rates of active constituents in the sample DJ and DT were higher than those in other samples, while the increased rates of target compounds in the sample DC and DY were lower than those in other samples. e heat map illustrated that herb pairs containing Danggui played significant roles in XSHG, and various herb pairs might make different contribution to the formula.
ese results above suggested that vast majority of active compounds decreased to some extent when herb pairs Danggui-Yimucao and Danggui-Chuanxiong were eliminated from XSHG. us, this fact could be the importantly potential reason of different contributions of herb pairs containing Danggui to XSHG on HAA mice.
# Discussion
TCM formulae were the indispensable part of human life, which play significant roles for disease treatment in the developing countries. However, the obscure composition of TCM medicines limited their clinical application [bib_ref] Comprehensive chemical profiling of Yindan Xinnaotong soft capsule and its neuroprotective activity..., Pang [/bib_ref]. Lots of efforts have been devoted to clarifying the effective material basis of TCM medicines, such as isolating and identifying single effective ingredients [bib_ref] Bioactive equivalence of combinatorial components identified in screening of an herbal medicine, Liu [/bib_ref] [bib_ref] Screening of minor bioactive compounds from herbal medicines by in silico docking..., Wu [/bib_ref] [bib_ref] A strategy for screening of high-quality enzyme inhibitors from herbal medicines based..., Song [/bib_ref]. However, these studies disregarded the integrative therapeutic effects of multiple effective compounds in TCM medicines. Herb pairs are the basis units of TCM formulae, which are formed by two herbs. e study of herb pairs could help to discover the effective material basis of the relevant TCM formulae [bib_ref] A network pharmacology approach to investigate the blood enriching mechanism of Danggui..., Shi [/bib_ref]. XSHG prescription was composed of seven herbs, including Danggui, Yimucao, Chuangxiong, Taoren, Honghua, Jiangtan, and Zhigancao. In the clinic, XSHG could exhibit good therapeutic effects on anemia via increasing hematopoietic stem cell differentiation and proliferation [bib_ref] Integrated metabolomics and network pharmacology approach to explain possible action mechanisms of..., Pang [/bib_ref]. Strikingly, different herb pairs could be formed when the herb Danggui was combined with other six herbs [bib_ref] Herb pairs containing Angelicae Sinensis Radix (Danggui): a review of bio-active constituents..., Jin [/bib_ref]. erefore, the herb pairs containing Danggui could play significant roles for its treatment of anemia. To investigate the potential effective material basis, the roles of herb pairs containing Danggui in XSHG were comparatively evaluated using withdraw analysis. Fu-Fang-A-Jiao-Jiang is a famous TCM formula descending from "Jing Yue Quan Shu" (Ming Dynasty). It has been approved by National Medical and Products Administration (NMPA) to replenish qi and nourish blood. Modern research revealed that Fu-Fang-A-Jiao-Jiang could effectively promote hematopoietic effects through improving bone marrow hematopoietic microenvironment; thus, it was selected as positive drug in this study [bib_ref] Hematopoietic effects and mechanisms of Fufang E׳jiao Jiang on radiotherapy and chemotherapy-induced..., Liu [/bib_ref].
Combining the chemical analysis and bioactive evaluation, the results discovered that herb pairs Danggui-Yimucao and Danggui-Chuanxiong made great contributions to the treatment effects of XSHG. Danggui, which is also called "female ginseng," has good effects for the treatment of complicated gynecological disorders through nourishing and tonifying blood [bib_ref] Herb pair Danggui-Honghua: mechanisms underlying blood stasis syndrome by system pharmacology approach, Yue [/bib_ref]. Yimucao has the effects on promoting blood flow for regulating menstruation, and it has been extensively used to treat postpartum hemorrhage [bib_ref] Comparative analysis of the main active constituents from different parts of Leonurus..., Tan [/bib_ref]. Chuanxiong could exert key roles in regulating menstruation and relieving pain and activating blood circulation [bib_ref] A systematic review on the rhizome of Ligusticum chuanxiong Hort, Chen [/bib_ref]. Modern research has demonstrated that Danggui-Chuanxiong showed obvious synergistic effects on regulating peripheral blood routine, energy metabolic enzymes, and immune organs [bib_ref] Enriching blood effect comparison in three kinds of blood deficiency model after..., Li [/bib_ref] [bib_ref] Study on antioxidant interaction of different preparations and proportions of Danggui-Chuanxiong drug..., Huang [/bib_ref]. In our study, when the herb pair Danggui-Chuanxiong was excluded from XSHG, the hematinic function decreased obviously, which was consistent with the previous studies [bib_ref] Enriching blood effect comparison in three kinds of blood deficiency model after..., Li [/bib_ref] [bib_ref] Study on antioxidant interaction of different preparations and proportions of Danggui-Chuanxiong drug..., Huang [/bib_ref].
# Conclusions
e withdrawal analysis method was applied to evaluate the contribution of herb pairs containing Danggui to XSHG on HAA mice. With PCA analysis, a clear separation of healthy control group and deficiency group was obtained, and the administration groups were located between model group and control group. Among these dose groups, the group XSHG was the closest to the control group, while the group DY was the farthest away from the control group. In order to reveal the potential reasons of different contributions of six herb pairs to XSHG, the content variations of these samples were also analyzed by UHPLC-TQ-MS/MS method. And the contents of target constituents in XSHG underwent different changes when herb pairs containing Danggui were excluded from this formula, respectively. In particular, the contents variations of DY and DC groups mostly decreased to some extent compared with that of XSHG group. e data of pharmacodynamics and the contents changes of the analytes in the decomposed recipes suggested similar results that a series of herb pairs containing Danggui were indispensable parts of the formulae, and herb pairs Danggui-Yimucao might be particularly important. Our results in the present study could lay foundation to further reveal the compatibility mechanism of XSHG. Furthermore, this original method might provide a good reference for interpreting the drug interactions from the aspects of herb pairs.
## Data availability
e data used to support the findings of this study are available from the corresponding author upon request.
## Conflicts of interest
e authors declare no conflicts of interest.
[fig] Figure 2: e effects of immune organ index and ATPase activities for administration groups in HAA mice (x ± s, n � 10). (a) Liver index; (b) thymus index; (c) spleen index; (d) Na + -K + -ATPase; (e) Ca 2+ -Mg 2+ -ATPase). # P < 0.05 and ## P < 0.01 versus control group; * P < 0.05 and * * P < 0.01 versus model group; Δ P < 0.05 and Δ P < 0.01 versus XSHG group. K, control group; M, model group; XSHG, Xin-Sheng-Hua granules; DY, XSHG minus Danggui-Yimucao; DC, minus Danggui-Chuanxiong; DT, minus Danggui-Taoren; DH, minus Danggui-Honghua; DJ, minus Danggui-Jiangtan; DZ, minus Danggui-Zhigancao. [/fig]
[fig] Figure 3: PCA score plot between control group and dose groups (Model, XSHG, DY, DC, DT, DH, DJ, and DZ). (a) 2D plot of the integrated hematinic effects; (b) 2D plot of the amounts of the 21 analytes in different samples. [/fig]
[fig] Figure 4: : e UHPLC-TQ-MS/MS analysis MRM chromatogram of 21 analytes in the samples of XSHG and its decomposed recipes. [/fig]
[fig] Figure 5: : e heat map of increased rates of target constituents in six different separated prescriptions (DY, DC, DT, DH, DJ, and DZ). e increased rate of 0 indicates that the observed amount was equal to its theorized amount, otherwise denoting amount increases (>0) or amount decreases (<0) in the separated prescriptions. [/fig]
[table] Table 1: e results of peripheral blood routine between control group and treatment groups (x + s, n � 8). * * 18.61 ± 1.32 * * 38.01 ± 2.13 * * 447.25 ± 28.97 * * XSHG 5.73 ± 0.96 * * 7.66 ± 0.32 * * 140.75 ± 5.18 * * 28.89 ± 1.54 66.45 ± 3.06 * * 19.49 ± 1.11 * * 37.03 ± 2.24 * * [/table]
[table] Table 2: Amounts (mg) of the 21 compounds in XSHG and six samples of decomposed recipes (n � 91.00 ± 2.79 ## 81.04 ± 2.68 ## 76.15 ± 2.19 n.d. 10 267.52 ± 20.76 82.98 ± 5.75 74.16 ± 3.52 n.d. 99.20 ± 8.67 ## 99.87 ± 7.65 ## 91.91 ± 7.95 ## 85.35 ± 6.04 11 21.93 ± 1.43 21.93 ± 1.43 15.27 ± 1.31 * * 16.51 ± 1.58 * * 19.44 ± 1.24 * 18.68 ± 1.40 * * n.d. 16.45 ± 1.07 * * 12 2.17 ± 0.17 2.17 ± 0.17 1.62 ± 0.11 * * 1.82 ± 0.12 * * 3.48 ± 0.22 ## 2.77 ± 0.12 ## 2.20 ± 0.16 n.d. 13 134.92 ± 7.07 60.39 ± 2.26 74.46 ± 2.47 ## n.d. 70.98 ± 3.34 ## 59.66 ± 1.82 63.81 ± 1.57 65.80 ± 1.81 # 14 73.81 ± 6.36 26.46 ± 1.79 36.44 ± 2.55 ## n.d. 34.83 ± 1.05 ## 30.17 ± 1.67 ## 30.87 ± 0.97 ## 32.57 ± 1.72 ## 15 2.51 ± 0.24 2.51 ± 0.24 2.62 ± 0.22 3.18 ± 0.14 ## 3.30 ± 0.24 ## 2.75 ± 0.22 2.41 ± 0.16 n.d. 16 430.17 ± 20.84 430.17 ± 20.84 423.49 ± 22.98 460.62 ± 21.44 ## 474.48 ± 28.63 ## 395.46 ± 15.28 * * 397.73 ± 19.17 * n.d. 17 47.87 ± 2.69 47.87 ± 2.69 28.97 ± 2.00 * * 39.33 ± 2.60 * * 49.91 ± 3.02 33.66 ± 2.62 * * n.d. 39.09 ± 2.24 * * 18 437.86 ± 32.07 173.97 ± 10.72 187.92 ± 17.68 n.d. 206.68 ± 12.24 ## 172.80 ± 7.15 172.44 ± 11.35 162.31 ± 11.21 * 19 572.17 ± 33.48 199.26 ± 9.15 152.60 ± 6.28 * * n.d. 228.98 ± 14.30 ## 195.75 ± 13.48 175.37 ± 6.65 * 166.62 ± 7.41 * * 20 36.96 ± 3.61 20.07 ± 0.56 11.25 ± 0.62 * * n.d. 17.47 ± 0.88 * * 11.79 ± 0.63 * * 12.77 ± 0.75 * * 10.68 ± 0.79 * * 21 1.43 ± 0.10 1.43 ± 0.10 0.84 ± 0.059 * * 1.26 ± 0.09 1.78 ± 0.12 ## 1.52 ± 0.12 n.d. 1.31 ± 0.08 * * n.d. � not detectable; * P < 0.05 and * *P < 0.01 (the observed amount decreased in comparison with its theory amount); # P < 0.05 and ## P < 0.01 (the observed amount increased in comparison with its theory amount). a Obs., the observed amount of each compound in different samples. b e theorized amount of each compound in the samples of decomposed recipes. Except for aromatic acids and phthalides, the theorized amounts of other compounds in the decomposed recipes were the observed amounts of the analytes in XSHG accordingly; for aromatic acids and phthalides, the theorized content could be calculated: Amount e. � Proportionality coefficient of CX × Amount of the analyte in XSHG.1, trigonelline; 2, stachydrine hydrochloride; 3, protocatechuic acid; 4, hydroxysafflor yellow A; 5, chlorogenic acid; 6, amygdalin; 7, caffeic acid; 8, leonurine hydrochloride; 9, liquiritin; 10, ferulic acid; 11, zingerone; 12, isoliquiritoside; 13, senkyunolide I; 14, senkyunolide H; 15, liquiritigenin; 16, glycyrrhizic acid; 17, 6-gingerol; 18, senkyunolide A; 19, ligustilide; 20, butylidenephthalide; 21, 6-shogaol. [/table]
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10.1016/j.onehlt.2019.100095
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CCBY
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6538949
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31193679
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s2orc_pubmed_articles
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Examination of unintended consequences of antibiotic use restrictions in food-producing animals: Sub-analysis of a systematic review
## Context
With increasing attention paid to the rapid rise in antimicrobial resistance and its resulting health and economic consequences, there is mounting pressure to develop strategies to promote prudent use of antibiotics in humans and in agriculture. Though the World Health Organization (WHO) has made recommendations on prudent use of antimicrobials in food-producing animals as early as 1997, they recently undertook a rigorous process, following international standards, to develop and publish formal guidelines on this topic. These WHO Guidelines recommended both a reduction and restriction of antibiotics in food-producing animals, and were informed by our recent systematic review and meta-analysis showing that such measures likely reduce antibiotic resistance in animals and also in certain human populations (particularly those having direct contact with animals) [bib_ref] Restricting the use of antibiotics in food-producing animals and its associations with..., Tang [/bib_ref]. Evidence though of potential unintended consequences is less clear. There are concerns that restrictions of antibiotic use in food-producing animals may negatively impact animal health and welfare, resulting in increased rates of infection and a paradoxical increase in antibiotic use for therapy [bib_ref] Temporal relationship between decrease in antimicrobial prescription for Danish pigs and the..., Jensen [/bib_ref] [bib_ref] Raised without antibiotics: impact on animal welfare and implications for food policy, Karavolias [/bib_ref]. Furthermore, antibiotic growth promoters have been used to maximize growth, production, and feed efficiency, resulting in some hesitation in response to complete bans of these products. [bib_ref] Benefits and risks of antimicrobial use in food-producing animals, Hao [/bib_ref] Increasing evidence suggests though, that the benefit of antibiotics for productivity is likely minimal in industrialized production, [bib_ref] The livestock reservoir for antimicrobial resistance: a personal view on changing patterns..., Aarestrup [/bib_ref] [bib_ref] The routine use of antibiotics to promote animal growth does little to..., Collignon [/bib_ref] [bib_ref] Growth promoting antibiotics in food animal production: an economic analysis, Graham [/bib_ref] with no significant long-term negative impacts seen when antibiotic growth promoters are eliminated. [bib_ref] Growth promoting antibiotics in food animal production: an economic analysis, Graham [/bib_ref] [bib_ref] Changes in the use of antimicrobials and the effects on productivity of..., Aarestrup [/bib_ref] [bib_ref] The effect of discontinuing the use of antimicrobial growth promoters on the..., Emborg [/bib_ref] [bib_ref] Commentary: benefits and risks of antimicrobial use in food-producing animals, Schlundt [/bib_ref]. McEwen et al. conducted a narrative review of 14 studies that examined unintended consequences of national-level restrictions of antibiotic use in food-producing animals. Five studies reported no adverse consequences, while the others reported increases in certain diseases in the animals, increased antibiotic use for therapeutic purposes, and decreased feed efficiency. These effects tended to be small, temporary and likely to be mitigated by improved biosecurity, hygiene, and animal housing and husbandry practices. The authors concluded that the implementation of strategies to restrict antibiotic use in foodproducing animals should not be delayed.
To add to this evidence base, we present here a sub-analysis of our previously published systematic review [bib_ref] Restricting the use of antibiotics in food-producing animals and its associations with..., Tang [/bib_ref]. The methods have been described in detail in that publication. [bib_ref] Restricting the use of antibiotics in food-producing animals and its associations with..., Tang [/bib_ref] In summary, we searched electronic databases Agricola Inclusion criteria were original studies describing interventions to reduce antibiotic use in food-producing animals, and that compared proportions of antibioticresistant bacterial isolates in animals or humans between intervention and comparator groups. Any interventions that reduced or restricted one or more antibiotics, to any extent, were considered; these included mandatory or voluntary bans, antibiotic-free or organic production systems, national reduction targets, or requiring veterinary consultation or culture and sensitivity testing prior to antibiotic use. For this subanalysis, we specifically identified the subset of studies that report unintended consequences of interventions that restrict antibiotic use in food-producing animals; the key findings from this sub-analysis are summarized below.
## Findings
Of the 181 studies included in the original systematic review, 47 were included in this sub-analysis, on the basis of the studies explicitly reporting information on potential unintended consequences associated with antibiotic restriction strategies. Detailed characteristics and quality assessments of the individual studies can be found in our prior publication [bib_ref] Restricting the use of antibiotics in food-producing animals and its associations with..., Tang [/bib_ref]. The unintended consequence that was most frequently examined in this subset of studies was bacterial contamination and/or food safety. None explored adverse effects on human health or decrease in food availability for human consumption.
## Antibiotic use (n = 5)
One study found an increase in the use of non-restricted antibiotic growth promoters (AGPs) after the ban of one specific AGP [bib_ref] Effect of abolishment of the use of antimicrobial agents for growth promotion..., Aarestrup [/bib_ref]. Four studies reported that though there was an increase in the use of therapeutic antibiotics to treat individual animals, there remained a reduction in the total amount of antibiotics used [bib_ref] Effects of reducing antimicrobial use and applying a cleaning and disinfection program..., Dorado-Garcia [/bib_ref] [bib_ref] Impact of Denmark's ban on antimicrobials for growth promotion, Jensen [/bib_ref] [bib_ref] Effects of restricted antimicrobial exposure on antimicrobial resistance in fecal Escherichia coli..., Morley [/bib_ref].
## Food safety (n = 34)
Fifteen studies found an increased rate of bacterial contamination in retail meats when antibiotic restrictions were applied [bib_ref] Comparison of types and antimicrobial susceptibility of staphylococcus from conventional and organic..., Bombyk [/bib_ref] [bib_ref] Prevalence and antimicrobial resistance of campylobacter spp. and salmonella serovars in organic..., Cui [/bib_ref] [bib_ref] Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles..., El-Shibiny [/bib_ref] [bib_ref] Comparison of prevalence, antimicrobial resistance, and occurrence of multidrug-resistant salmonella in antimicrobial-free..., Gebreyes [/bib_ref] [bib_ref] Prevalence and antimicrobial susceptibility of thermophilic campylobacter in organic and conventional broiler..., Heuer [/bib_ref] [bib_ref] Longitudinal study of distributions of similar antimicrobial-resistant salmonella serovars in pigs and..., Keelara [/bib_ref] [bib_ref] Prevalence, characterization, and antimicrobial susceptibility of salmonella Gallinarum isolated from eggs produced..., Lee [/bib_ref] [bib_ref] Antimicrobial resistance in enterococcus spp. strains isolated from organic chicken, conventional chicken,..., Miranda [/bib_ref] [bib_ref] Antimicrobial resistance in Enterobacteriaceae strains isolated from organic chicken, conventional chicken and..., Miranda [/bib_ref] [bib_ref] Antimicrobial resistance in Escherichia coli strains isolated from organic and conventional pork..., Miranda [/bib_ref] [bib_ref] Comparison of antimicrobial resistance in Escherichia coli, Staphylococcus aureus, and listeria monocytogenes..., Miranda [/bib_ref] [bib_ref] Prevalence and antibiotic resistance pattern of salmonella serovars in integrated crop-livestock farms..., Peng [/bib_ref] [bib_ref] Relationship between antimicrobial drug usage and antimicrobial susceptibility of gram-positive mastitis pathogens, Pol [/bib_ref] [bib_ref] Ecological dynamics of campylobacter in integrated mixed crop-livestock farms and its prevalence..., Salaheen [/bib_ref]. Eighteen studies reported either no difference in contamination rates or less contamination in the intervention group when the use of antibiotics was restricted [bib_ref] Isolation, virulence, and antimicrobial resistance of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin..., Abdalrahman [/bib_ref] [bib_ref] Prevalence and distribution of salmonella in organic and conventional broiler poultry farms, Alali [/bib_ref] [bib_ref] Antimicrobial resistance in E. coli isolates from conventionally and organically reared poultry:..., Alvarez-Fernandez [/bib_ref] [bib_ref] Influence of housing systems on microbial load and antimicrobial resistance patterns of..., Alvarez-Fernandez [/bib_ref] [bib_ref] Prevalence and antimicrobial resistance among campylobacter spp. in Louisiana retail chickens after..., Han [/bib_ref] [bib_ref] Prevalence and antimicrobial resistance of salmonella serovars in conventional and organic chickens..., Lestari [/bib_ref] [bib_ref] Effect of conventional and organic production practices on the prevalence and antimicrobial..., Luangtongkum [/bib_ref] [bib_ref] Prevalence, concentrations, and antibiotic sensitivities of salmonella Serovars in poultry from retail..., Mazengia [/bib_ref] [bib_ref] Influence of farming methods on microbiological contamination and prevalence of resistance to..., Miranda [/bib_ref] [bib_ref] Microbiological quality and antimicrobial resistance of Escherichia coli and Staphylococcus aureus isolated..., Miranda [/bib_ref] [bib_ref] Organic or antibiotic-free labeling does not impact the recovery of enteric pathogens..., Mollenkopf [/bib_ref] [bib_ref] Fluoroquinolone-resistant campylobacter isolates from conventional and antibiotic-free chicken products, Price [/bib_ref] [bib_ref] The persistence of fluoroquinoloneresistant campylobacter in poultry production, Price [/bib_ref] [bib_ref] Contamination of post-harvest poultry products with multidrug resistant Staphylococcus aureus in Maryland-Washington..., Teramoto [/bib_ref] [bib_ref] Prevalence and antimicrobial resistance of campylobacter in antimicrobial-free and conventional pig production..., Thakur [/bib_ref] [bib_ref] Contamination rates and antimicrobial resistance in bacteria isolated from "grass-fed" labeled beef..., Zhang [/bib_ref]. One study showed variable results depending on the bacteria in question [bib_ref] Contamination rates and antimicrobial resistance in enterococcus spp, Zhang [/bib_ref].
## Animal health (n = 4)
Two studies in dairy herds reported increased prevalence of intramammary infections and mastitis pathogens with restriction of antibiotic use (due to organic production) [bib_ref] Relationship between antimicrobial drug usage and antimicrobial susceptibility of gram-positive mastitis pathogens, Pol [/bib_ref] [bib_ref] Prevalence and antibiotic resistance of mastitis pathogens isolated from dairy herds transitioning..., Park [/bib_ref] , while a third study showed no difference in mastitis between groups [bib_ref] Comparison of antibiotic resistance of udder pathogens in dairy cows kept on..., Roesch [/bib_ref]. The single study that examined mortality reported no difference in either mortality rate or mean age at mortality in intervention versus comparator groups [bib_ref] Effects of reducing antimicrobial use and applying a cleaning and disinfection program..., Dorado-Garcia [/bib_ref].
## Animal production (n = 3)
Two studies reported adverse effects on animal production with increased feeding time to achieve target weight and increased production cycle duration [bib_ref] Effects of reducing antimicrobial use and applying a cleaning and disinfection program..., Dorado-Garcia [/bib_ref] [bib_ref] Effects of restricted antimicrobial exposure on antimicrobial resistance in fecal Escherichia coli..., Morley [/bib_ref]. One study showed variable results, with increased parity but lower milk yield in dairy cows [bib_ref] Reproductive performance, udder health, and antibiotic resistance in mastitis bacteria isolated from..., Garmo [/bib_ref]. The effects of antibiotic restrictions on animal production vary likely as they depend upon concurrent management changes implemented to promote animal health. For example, when Denmark banned antibiotic growth promoters, productivity improved likely due to a multimodal strategy that included increased veterinary oversight and changes to feed composition to include whole wheat and feeding enzymes. [bib_ref] Commentary: benefits and risks of antimicrobial use in food-producing animals, Schlundt [/bib_ref] [bib_ref] Evidence-based policy for controlling antimicrobial resistance in the food chain in Denmark, Wielinga [/bib_ref].
## Costs and economics (n = 2)
One study estimated increased costs in animal production due to increased feeding time to reach target weight, when antibiotic use is restricted [bib_ref] Effects of restricted antimicrobial exposure on antimicrobial resistance in fecal Escherichia coli..., Morley [/bib_ref]. Another study reported decreased veterinary costs with antibiotic restriction; the specific cost inputs and drivers of this cost difference were not reported [bib_ref] Effects of reducing antimicrobial use and applying a cleaning and disinfection program..., Dorado-Garcia [/bib_ref].
## Interpretation of findings
This sub-analysis of our comprehensive systematic review suggests that unintended consequences are uncommonly reported in studies that are designed to examine the effect of antibiotic restrictions in foodproducing animals on antibiotic resistance. Of the 181 studies included in our original systematic review, only 47 reported any unintended consequences. Of these, nearly one-third reported unintended consequences in the discussion section of the publication, without specifying these in a research question or objective.
Despite theoretical concerns that restrictions in antibiotic use in food-producing animals may result in numerous harms to both animal and human health, these are not borne out in our sub-analysis. The associations between unintended consequences and antibiotic restrictions are mixed across all outcome domains, with no clear or consistent trend. Half of the studies reporting on safety of retail food products suggest increased contamination when antibiotic restriction measures are in place. Because no study examined human health outcomes, the clinical significance of this is unclear.
We recognize that unintended consequences were not specifically the focus of our systematic review. As a result, this sub-analysis does not comprehensively capture all studies on this topic. Furthermore, all but two of the studies were undertaken in the United States of America or in Europe. Generalizability of our findings may therefore be limited, especially to low and lower-middle-income countries where management and hygiene practices may be less developed. However, our study complements the previously-mentioned paper on this topic by McEwen et al., by virtue of our identification of a number of additional studies not covered by their recent review. Together, our two reviews provide value in summarizing an informative, though small, body of literature examining potential harms of interventions that restrict antibiotic use in food-producing animals. We demonstrate that future research on antibiotic restrictions in agriculture should more specifically consider their impact on unintended consequences. The increasing global efforts to reduce and restrict antibiotic use in food-producing animals present the perfect opportunity to conduct rigorous evaluations of potential harms and to provide insight regarding the role of local context in the relationship between antibiotic restriction andUnintended consequences of interventions restricting antibiotic use in food-producing animals.
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## Contributors
Each of the 12 authors meets the authorship requirements as established by the International Committee of Medical Journal Editors in the Uniform Requirements for Manuscripts Submitted to Biomedical Journals. All authors were involved in the design and development of the study. HG created the search strategy and conducted the literature search in electronic databases. DN conducted the grey literature search. KT and NC screened all studies for inclusion into the original systematic review and performed all study quality assessments. SC, PR, and HB provided input on studies where consensus could not be reached. KT, NC, DN, AP, and NS, performed data extraction. All authors contributed to data interpretation and data analysis. KT drafted the manuscript and all authors revised it critically for content. All authors have full access to all data and can take responsibility for the integrity of the data and accuracy of the data analysis. All authors have read and approved the manuscript.
## Role of the funding source
The WHO was involved in both the original systematic review and meta-analysis, as well as this sub-study. They were involved in developing the research question, the study design and the study protocol. They had no involvement in data extraction or interpretation of findings. The authors have been given permission by the WHO to publish this article. All had full access to all of the data and can take responsibility for the integrity of the data and the accuracy of the data analysis.
## Acknowledgements
This study was commissioned and paid for by the World Health Organization (WHO). They were involved in developing the research question, the study design and the study protocol. They had no involvement in data extraction or interpretation of findings. The authors have been given permission by the WHO to publish this article. The corresponding author had full data and had final responsibility for the decision to submit for publication. Copyright in the original work on which this article is based belongs to the WHO. The authors have been given permission to publish this article. The authors alone are responsible for the views expressed in this publication and do not necessarily represent the views, decisions, or policies of the World Health Organization.
## Conflict of interest
JK is the principal investigator on an unrestricted grant in aid to conduct an epidemiological study of invasive pneumococcal disease in humans, including impact of pneumococcal vaccines (Pfizer Canada). He is also the local co-investigator on contract of a clinical trial of a maternal pertussis vaccine (GSK Canada). All other authors declare no conflicts of interest other than the WHO funding of this study. Abbreviations: AGP -Antibiotic growth promoters; ↑ = increased in the intervention compared to the comparator group; ↓ = decreased in the intervention compared to the comparator group; and ↔ = no difference between the intervention and comparator groups. Where Red = favors comparator group; Green = favors intervention group; Yellow = no difference between intervention and comparator group.
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Sex-specific strategies of resource allocation in response to competition for light in a dioecious plant
[fig] Figure S2: Results on reproductive traits in males as a function of replicates. Data are given as means per replicate population ± SE. Abbreviation: n = number. Red and black dots represent respectively low and high densities. [/fig]
[fig] Figure S3: Results on reproductive traits in females as a function of replicates. Data are given as means per replicate population ± SE. Red and black dots represent respectively low and high densities. [/fig]
[table] Table S1: Plant height Mean lenght of the first ramification Plant diameter Plant biomass Total seed weight [/table]
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Xanthohumol and Gossypol Are Promising Inhibitors against Babesia microti by In Vitro Culture via High-Throughput Screening of 133 Natural Products
Human babesiosis caused by Babesia microti is an emerging threat for severe illness and even death, with an increasing impact worldwide. Currently, the regimen of atovaquone and azithromycin is considered as the standard therapy for treating human babesiosis, which, however, may result in drug resistance and relapse, suggesting the necessity of developing new drugs to control B. microti. In this regard, natural products are promising candidates for drug design against B. microti due to their active therapeutic efficacy, lower toxicity, and fewer adverse reactions to host. Here, the potential inhibitors against B. microti were preliminarily screened from 133 natural products, and 47 of them were selected for further screening. Gossypol (Gp) and xanthohumol (Xn) were finally shown to effectively inhibit the growth of B. microti with IC 50 values of 8.47 µm and 21.40 µm, respectively. The cytotoxicity results showed that Gp and Xn were non-toxic to erythrocytes at a concentration below 100 µm. Furthermore, both of them were confirmed to be non-toxic to different types of cells in previous studies. Our findings suggest the potential of Gp and Xn as effective drugs against B. microti infection.
# Introduction
Babesiosis is an emerging zoonosis induced by the intraerythrocytic protozoa of the genus Babesia, which may cause enormous harm, threat, and loss to the livestock industry and even public health [bib_ref] Babesiosis vaccines: Lessons learned, challenges ahead, and future glimpses, Rathinasamy [/bib_ref] [bib_ref] Diagnosis, Treatment and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis, Sanchez [/bib_ref]. Facing these challenges, the Center for Disease Control and Prevention regarded it as a nationally notifiable disease and has been monitoring it in the United States since January 2011 [bib_ref] The centers for disease control and prevention and state health departments should..., Jajosky [/bib_ref] [bib_ref] Rise in babesiosis cases, Ingram [/bib_ref]. To date, more than 100 Babesia species have been reported, but only a few of them can infect humans and cause human babesiosis, including Babesia microti, Babesia divergens, Babesia duncani, and Babesia venatorum. B. microti is the primary agent for human babesiosis and can be transmitted by tick Ixodes scapularis, blood transfusion, and placenta [bib_ref] From mice to ticks to an increasing number of highly susceptible humans, Westblade [/bib_ref]. Babesiosis cases are distributed throughout the world, especially in the United States, Asia, and Europe, and show an upward trend worldwide [bib_ref] Human babesiosis in china: A systematic review, Chen [/bib_ref] [bib_ref] Human babesiosis in europe: What clinicians need to know, Hildebrandt [/bib_ref]. The clinical manifestations are similar to those of malaria, including fever, chills, hemolysis, hemoglobinuria, splenomegaly, and jaundice [bib_ref] The centers for disease control and prevention and state health departments should..., Jajosky [/bib_ref]. The symptoms for the patients infected with B. microti depend on their age and physical health status. The young and those in good health are always asymptomatic,
## Preparation of infected erythrocytes
The parasitemia of B. microti-infected KM mice was microscopically determined on blood smears. When the parasitemia reached 15-20%, 100 µL B. microti-infected blood was collected from each mouse for further analysis. Briefly, mouse blood was collected into sterile vacuum tubes containing anticoagulant with EDTA (100 µL/mL blood), followed by centrifugation at 3000 rpm for 5 min at 4 - C. Next, the supernatant, especially the layer of white blood cells, was removed and the pellet was resuspended with cold Puck's saline glucose buffer (PSG) to the original volume and then gently mixed. The above step was repeated three times or more until the supernatant was clear. Finally, the resulting mixture was resuspended 1:1 with Puck's saline glucose plus extra glucose (PSG + G) and stored at 4 - C for further analysis. [bib_ref] Surface Antigen 1 Is a crucial secreted protein that mediates babesia microti..., Li [/bib_ref]. The culture plate was incubated at 37 - C with a gas mixture of 2% O 2 , 5% CO 2 , and 93% N 2 at a constant pressure in a modular incubator chamber (Billups-Rothenberg, Del Mar, CA, USA). The culture was split every three days and the medium was replaced with HL-20 every 24 h as a routine management until 72 h as follows: 110 µL supernatant was discarded and 0.3 µL of the culture was drawn from the bottom for parasitemia determination, followed by adding 110 µL HL-20 medium into the well and gently mixing. The thin smears were fixed with ice cold 50% ethanol and 50% methanol (v:v) and stained with Giemsa (Thermo Fisher Scientific, Cleveland, OH, USA). Finally, the percentage of parasitized erythrocytes was determined by microscopy and flow cytometry.
## In vitro culture of b. microti
## Preliminary screening and rescreening of natural products
A total of 133 natural products (different herbal extracts) obtained from the small molecule natural product library (Selleck Chemicals, Houston, TX, USA) were used for screening potential drugs against B. microti by in vitro culture. The general information of the 133 natural products is shown in [fig_ref] Table 1: The summary of cytotoxicity of gossypol and xanthohumol on different cell lines [/fig_ref]. The assays were performed in triplicate and in 96-well flat-bottom plates. For initial screening, 133 drugs were diluted separately by HL-20 medium to the final concentration of 10 µm (50 µm for rescreening). Cells treated with DMSO (0.01%) alone were used as negative controls. DA is a recommended drug reported to be effective in inhibiting B. microti and it was used as a positive control in this study. During the experiment, B. microti was cultured under the drug-free condition for 24 h and the parasitemia was determined as the initial parasitemia. Next, 110 µL supernatant was replaced with the HL-20 medium that contained drugs. The parasitemia was determined separately at 24 h, 48 h, and 72 h by microscopy and flow cytometry. The 1 mg/mL Hoechest-33258 (Beyotime Biotechnology, Shanghai, China) was diluted by phosphate buffer saline (PBS) that had passed through 0.22 µm filters to 100 µg/mL. Next, 500 µL PBS, 10 µL Hoechest-33258 (100 µg/mL), and 30 µL erythrocyte pellet collected from 96-well plates were mixed gently in 1.5 mL tubes and were incubated in the dark at 37 - C for 30 min. Finally, the parasitemia was determined by flow cytometry and calculated for every sample based on 100,000 cells. The data were analyzed by GraphPad Prism (version 6.0, La Jolla, CA, USA) (with significant differences indicated as **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05). The parasitemia at 72 h was compared with the parasitemia of negative and positive controls. The drugs with significant differences from DMSO and no significant differences from DA in efficacy were selected for rescreening.
## Combination of gp and xn on in vitro culture
Gp and Xn were combined to determine whether their effects are additive, synergistic, or antagonistic. The combination assay was performed using the Different concentration combinations were diluted by HL-20 medium in 96-well flat-bottom plates in triplicate. DA at 1 µm was used as the positive control. The culture plate was incubated with a mixture gas of 2% O 2 , 5% CO 2 , and 93% N 2 at 37 - C at a constant pressure. After 72 h of incubation, 0.6 µL of the culture was collected from the bottom to make smears for parasitemia determination. The parasitemia data for the combination assay were analyzed using the CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA) based on the Chou-Talalay method [bib_ref] Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in..., Chou [/bib_ref].
## Cell toxicity assay
Gp and Xn were added into the HL-20 medium at the final concentrations of 1 µm, 10 µm, 50 µm, and 100 µm, respectively. The culture conditions for the cytotoxicity assay consisted of 110 µL HL-20 medium that contained different concentrations of drugs and 40 µL infected mouse RBCs, which were mixed in the 96-well plates in triplicate. The culture medium was changed every 24 h to 72 h, from which 10 µL was collected for blood count. Further significance tests were performed by GraphPad Prism 6 (Graphpad Software, La Jolla, CA, USA).
## In vivo inhibition assay
KM mice (six weeks) were given intraperitoneal injection of B. microti that had been preserved in liquid nitrogen. A drop of blood was collected from the tail vein for Giemsa staining to determine the parasitemia after 24 h of injection. On the fifth day, the KM mice were injected intramuscularly and separately with DMSO (3 mg/kg), DA (3 mg/kg), Gp, and Xn in triplicate. The injection concentration gradient was 1 mg/kg, 3 mg/kg, and 5 mg/kg for Gp and 1 mg/kg, 5 mg/kg, and 10 mg/kg for Xn. Parasitemia was determined by microscopy.
# Results
## Forty-seven natural products showed significant inhibitory effects on b. microti in preliminary screening
The inhibitory effects of 133 natural products (10 µm) on B. microti were explored by in vitro culture, and 47 of them were selected for rescreening through comparison with DMSO and DA [fig_ref] Figure 1: The preliminary screening of 133 natural products against B [/fig_ref] and. For comparison with DMSO, the inhibitory effects were calculated using the formula: ((parasitemia for DMSO minus parasitemia for natural products)/parasitemia for DMSO) × 100% [fig_ref] Figure 2: The calculated inhibitory effects of 133 natural products on B [/fig_ref] and. Significance analysis was carried out and the natural products with no significant difference from DA were selected for rescreening. A comparison with DA and DMSO further selected 47 natural products with the code numbers of A7, A8, B4, B5, B8, B9, C10, E5, E9, E10, E11, G3, G4, G5, G8, H2, H4, H7, H8, 2A1, 2A4, 2A5, 2B1, 2B4, 2B6, 2C3, 2D1, 2D2, 2D3, 2D4, 2D5, 2D6, 2E2, 2E3, 2E5, 2E6, 2F1, 2F2, 2F3, 2F5, 2G2, 2G4, 2G5, 2H1, 2H2, 2H4, and 2H5. The full names of the 47 natural products are detailed in [fig_ref] Table 1: The summary of cytotoxicity of gossypol and xanthohumol on different cell lines [/fig_ref]. [fig_ref] Table 1: The summary of cytotoxicity of gossypol and xanthohumol on different cell lines [/fig_ref]. The inhibitory effects of 47 natural products on B. microti were rescreened under 50 μm [fig_ref] Figure 3: The rescreening of 47 natural products against B [/fig_ref]. Among them, 2F1 (dioscin) was discarded due to the dissolution of RBCs. After 72 h in vitro culture, the data were subjected to significance analysis, and the parasitemia for five of the natural products was shown to be not significantly different from that of DA: 2E6 (Xn), G3 (cryptotanshinone), G5 (honokiol), 2F3 (palmatine chloride), and B5 (Gp) with inhibition rates of 66.97%, 47.58%, 51.50%, 48.64%, and 63.05%, respectively [fig_ref] Figure 4: The calculated inhibitory effects of 47 natural products on B [/fig_ref]. The chemical structure and formula of the five natural products obtained from the small molecule natural product library (Selleck Chemicals, Houston, TX, USA) are shown in [fig_ref] Figure 1: The preliminary screening of 133 natural products against B [/fig_ref]. The inhibitory effects of 47 natural products on B. microti were rescreened under 50 µm [fig_ref] Figure 3: The rescreening of 47 natural products against B [/fig_ref]. Among them, 2F1 (dioscin) was discarded due to the dissolution of RBCs. After 72 h in vitro culture, the data were subjected to significance analysis, and the parasitemia for five of the natural products was shown to be not significantly different from that of DA: 2E6 (Xn), G3 (cryptotanshinone), G5 (honokiol), 2F3 (palmatine chloride), and B5 (Gp) with inhibition rates of 66.97%, 47.58%, 51.50%, 48.64%, and 63.05%, respectively [fig_ref] Figure 4: The calculated inhibitory effects of 47 natural products on B [/fig_ref]. The chemical structure and formula of the five natural products obtained from the small molecule natural product library (Selleck Chemicals, Houston, TX, USA) are shown in [fig_ref] Figure 1: The preliminary screening of 133 natural products against B [/fig_ref]. To confirm the results of parasitemia by microscopy, parasitemia under the treatment of 47 natural products for 72 h was also determined by flow cytometry. Hoechst was used as a marker for B. microti detection and the parasitemia was calculated by the percent of infected RBCs in 100,000 RBCs. A comparison with DMSO showed that B. microti was significantly inhibited by 2E6 (Xn) and B5 (Gp) with inhibition rates of 64.53% and 53.20%, respectively [fig_ref] Figure 5: The inhibitory effects of 47 natural products on B [/fig_ref]. Meanwhile, the inhibition rates for 2E6 and B5 showed no significant differences from that of DA (63.99%). The To confirm the results of parasitemia by microscopy, parasitemia under the treatment of 47 natural products for 72 h was also determined by flow cytometry. Hoechst was used as a marker for B. microti detection and the parasitemia was calculated by the percent of infected RBCs in 100,000 RBCs. A comparison with DMSO showed that B. microti was significantly inhibited by 2E6 (Xn) and B5 (Gp) with inhibition rates of 64.53% and 53.20%, respectively [fig_ref] Figure 5: The inhibitory effects of 47 natural products on B [/fig_ref]. Meanwhile, the inhibition rates for 2E6 and B5 showed no significant differences from that of DA (63.99%). The To confirm the results of parasitemia by microscopy, parasitemia under the treatment of 47 natural products for 72 h was also determined by flow cytometry. Hoechst was used as a marker for B. microti detection and the parasitemia was calculated by the percent of infected RBCs in 100,000 RBCs. A comparison with DMSO showed that B. microti was significantly inhibited by 2E6 (Xn) and B5 (Gp) with inhibition rates of 64.53% and 53.20%, respectively [fig_ref] Figure 5: The inhibitory effects of 47 natural products on B [/fig_ref]. Meanwhile, the inhibition rates for 2E6 and B5 showed no significant differences from that of DA (63.99%). The other three of the five natural products, 2F3, G3, and G5 (palmatine chloride, cryptotanshinone, and honokiol), were excluded due to no significant difference between their inhibitory effect and that of DMSO by flow cytometry determination. Finally, only Xn and Gp were identified to have significant inhibitory effects on B. microti and thus selected for further analysis. other three of the five natural products, 2F3, G3, and G5 (palmatine chloride, cryptotanshinone, and honokiol), were excluded due to no significant difference between their inhibitory effect and that of DMSO by flow cytometry determination. Finally, only Xn and Gp were identified to have significant inhibitory effects on B. microti and thus selected for further analysis.
## Cytotoxicity of gp and xn
After treatment with 1 μm, 10 μm, 50 μm, and 100 μm Gp for 72 h, the values of RBC counts were 1.44 × 10 9 /mL, 1.46 × 10 9 /mL, 1.22 × 10 9 /mL, and 1.04 × 10 9 /mL, respectively. For 1 μm, 10 μm, 50 μm, and 100 μm Xn, the values of RBC counts were 1.38 × 10 9 /mL, 1.41 × 10 9 /mL, 1.40 × 10 9 /mL, and 1.34 × 10 9 /mL, respectively. All the values of RBC counts showed no significant difference from the negative control for all the concentration gradients of Xn [fig_ref] Figure 6: The cytotoxicity assay of xanthohumol [/fig_ref] and Gp [fig_ref] Figure 6: The cytotoxicity assay of xanthohumol [/fig_ref] according to the statistical analysis. These results indicated that Gp and Xn were non-toxic to RBCs below 100 μm and their inhibitory effects on B. microti could be further evaluated. The cytotoxicity of Gp and Xn on other cells and organisms have been extensively investigated in previous studies [bib_ref] Synthesis of xanthohumol analogues and discovery of potent thioredoxin reductase inhibitor as..., Zhang [/bib_ref] [bib_ref] Antiproliferative and cytotoxic activity of xanthohumol and its non-estrogenic derivatives in colon..., Logan [/bib_ref] [bib_ref] Chiral gossypol derivatives: Evaluation of their anticancer activity and molecular modeling, Zhang [/bib_ref] [bib_ref] A safety study of oral xanthohumol administration and its influence on fertility..., Hussong [/bib_ref]. Based on the results of cytotoxicity of Xn and Gp on erythrocytes and the reports of their cytotoxicity on other cells, Xn and Gp are non-toxic at IC50 values of 21.40 μm and 8.47 μm, respectively, and they can be considered as effective inhibitor candidates against B. microti [fig_ref] Table 1: The summary of cytotoxicity of gossypol and xanthohumol on different cell lines [/fig_ref].
## Cytotoxicity of gp and xn
After treatment with 1 µm, 10 µm, 50 µm, and 100 µm Gp for 72 h, the values of RBC counts were 1.44 × 10 9 /mL, 1.46 × 10 9 /mL, 1.22 × 10 9 /mL, and 1.04 × 10 9 /mL, respectively. For 1 µm, 10 µm, 50 µm, and 100 µm Xn, the values of RBC counts were 1.38 × 10 9 /mL, 1.41 × 10 9 /mL, 1.40 × 10 9 /mL, and 1.34 × 10 9 /mL, respectively. All the values of RBC counts showed no significant difference from the negative control for all the concentration gradients of Xn [fig_ref] Figure 6: The cytotoxicity assay of xanthohumol [/fig_ref] and Gp [fig_ref] Figure 6: The cytotoxicity assay of xanthohumol [/fig_ref] according to the statistical analysis. These results indicated that Gp and Xn were non-toxic to RBCs below 100 µm and their inhibitory effects on B. microti could be further evaluated. The cytotoxicity of Gp and Xn on other cells and organisms have been extensively investigated in previous studies [bib_ref] Synthesis of xanthohumol analogues and discovery of potent thioredoxin reductase inhibitor as..., Zhang [/bib_ref] [bib_ref] Antiproliferative and cytotoxic activity of xanthohumol and its non-estrogenic derivatives in colon..., Logan [/bib_ref] [bib_ref] Chiral gossypol derivatives: Evaluation of their anticancer activity and molecular modeling, Zhang [/bib_ref] [bib_ref] A safety study of oral xanthohumol administration and its influence on fertility..., Hussong [/bib_ref]. Based on the results of cytotoxicity of Xn and Gp on erythrocytes and the reports of their cytotoxicity on other cells, Xn and Gp are non-toxic at IC 50 values of 21.40 µm and 8.47 µm, respectively, and they can be considered as effective inhibitor candidates against B. microti [fig_ref] Table 1: The summary of cytotoxicity of gossypol and xanthohumol on different cell lines [/fig_ref]. other three of the five natural products, 2F3, G3, and G5 (palmatine chloride, cryptotanshinone, and honokiol), were excluded due to no significant difference between their inhibitory effect and that of DMSO by flow cytometry determination. Finally, only Xn and Gp were identified to have significant inhibitory effects on B. microti and thus selected for further analysis.
## Cytotoxicity of gp and xn
After treatment with 1 μm, 10 μm, 50 μm, and 100 μm Gp for 72 h, the values of RBC counts were 1.44 × 10 9 /mL, 1.46 × 10 9 /mL, 1.22 × 10 9 /mL, and 1.04 × 10 9 /mL, respectively. For 1 μm, 10 μm, 50 μm, and 100 μm Xn, the values of RBC counts were 1.38 × 10 9 /mL, 1.41 × 10 9 /mL, 1.40 × 10 9 /mL, and 1.34 × 10 9 /mL, respectively. All the values of RBC counts showed no significant difference from the negative control for all the concentration gradients of Xn [fig_ref] Figure 6: The cytotoxicity assay of xanthohumol [/fig_ref] and Gp [fig_ref] Figure 6: The cytotoxicity assay of xanthohumol [/fig_ref] according to the statistical analysis. These results indicated that Gp and Xn were non-toxic to RBCs below 100 μm and their inhibitory effects on B. microti could be further evaluated. The cytotoxicity of Gp and Xn on other cells and organisms have been extensively investigated in previous studies [bib_ref] Synthesis of xanthohumol analogues and discovery of potent thioredoxin reductase inhibitor as..., Zhang [/bib_ref] [bib_ref] Antiproliferative and cytotoxic activity of xanthohumol and its non-estrogenic derivatives in colon..., Logan [/bib_ref] [bib_ref] Chiral gossypol derivatives: Evaluation of their anticancer activity and molecular modeling, Zhang [/bib_ref] [bib_ref] A safety study of oral xanthohumol administration and its influence on fertility..., Hussong [/bib_ref]. Based on the results of cytotoxicity of Xn and Gp on erythrocytes and the reports of their cytotoxicity on other cells, Xn and Gp are non-toxic at IC50 values of 21.40 μm and 8.47 μm, respectively, and they can be considered as effective inhibitor candidates against B. microti [fig_ref] Table 1: The summary of cytotoxicity of gossypol and xanthohumol on different cell lines [/fig_ref].
## The inhibitory effect of xn and gp on the in vitro culture of b. microti
After treating B. microti for 72 h with 1 µm, 10 µm, and 50 µm of Gp and Xn, the parasitemia was 4.20%, 2.07%, and 1.93% for Gp and 3.87%, 3.93%, and 2.07% for Xn, respectively. The results of Gp and Xn showed significant difference from that of DMSO (p < 0.0001), but no significant difference from that of DA (p > 0.05). Meanwhile, 10 µm Gp and 50 µm Xn were shown to extensively inhibit the reproduction of B. microti. Based on the parasitemia at 72 h, the inhibitory effects of Gp [fig_ref] Figure 7: The inhibitory effect of gossypol and xanthohumol on the in vitro culture... [/fig_ref]
## The inhibitory effect of xn and gp on the in vitro culture of b. microti
After treating B. microti for 72 h with 1 μm, 10 μm, and 50 μm of Gp and Xn, the parasitemia was 4.20%, 2.07%, and 1.93% for Gp and 3.87%, 3.93%, and 2.07% for Xn, respectively. The results of Gp and Xn showed significant difference from that of DMSO (p < 0.0001), but no significant difference from that of DA (p > 0.05). Meanwhile, 10 μm Gp and 50 μm Xn were shown to extensively inhibit the reproduction of B. microti. Based on the parasitemia at 72 h, the inhibitory effects of Gp [fig_ref] Figure 7: The inhibitory effect of gossypol and xanthohumol on the in vitro culture... [/fig_ref]
## The inhibitory effect of xn and gp combination on the in vitro culture of b. microti
Six dilutions (0.25 × IC 50 , 0.5 × IC 50 , IC 50 , 2 × IC 50 , and 4 × IC 50 ) of Gp and Xn were combined, respectively. The parasitemia data were analyzed by CompuSyn software with the combination index (CI) values for synergism (less than 0.90), additive effect (from 0.90 to 1.10), or antagonism (more than 1.10). The CI values for the combination of drugs at IC 50 , IC 75 , IC 90 , and IC 95 were 1.81893, 1.59855, 1.40495, and 1.28689, respectively, indicating the antagonism of the Gp and Xn combination on B. microti.
## Inhibitory effect of xn and gp on the in vivo culture of b. microti
At 24 h post administration of Xn and Gp, the parasitemia for the treatment with 1mg/kg, 3 mg/kg, and 5 mg/kg Gp showed slight increases from 20.57% to 24.97%, 19.33% to 21.93%, and 17.77% to 22.17% with growth rates of 21.39%, 13.45%, and 24.76%, respectively [fig_ref] Figure 8: The inhibitory effect of Gp [/fig_ref]. Consistent with Gp, after treatment of 1 mg/kg Xn, the parasitemia was slightly increased, in contrast to a significant decrease from 24.77% to 19.87% and 23.23% to 18.87% in the parasitemia of groups treated with 5 mg/kg and 10 mg/kg Xn, respectively [fig_ref] Figure 8: The inhibitory effect of Gp [/fig_ref]. The results indicated 5 mg/kg and 10 mg/kg Xn had better inhibitory effects on the in vivo culture of B. microti relative to that of 1mg/kg, 3 mg/kg, and 5 mg/kg Gp. The growth rate was much higher in the DMSO treatment than it was in the treatment with Gp or Xn.
## The inhibitory effect of xn and gp combination on the in vitro culture of b. microti
Six dilutions (0.25 × IC50, 0.5 × IC50, IC50, 2 × IC50, and 4 × IC50) of Gp and Xn were combined, respectively. The parasitemia data were analyzed by CompuSyn software with the combination index (CI) values for synergism (less than 0.90), additive effect (from 0.90 to 1.10), or antagonism (more than 1.10). The CI values for the combination of drugs at IC50, IC75, IC90, and IC95 were 1.81893, 1.59855, 1.40495, and 1.28689, respectively, indicating the antagonism of the Gp and Xn combination on B. microti.
## Inhibitory effect of xn and gp on the in vivo culture of b. microti
At 24 h post administration of Xn and Gp, the parasitemia for the treatment with 1mg/kg, 3 mg/kg, and 5 mg/kg Gp showed slight increases from 20.57% to 24.97%, 19.33% to 21.93%, and 17.77% to 22.17% with growth rates of 21.39%, 13.45%, and 24.76%, respectively [fig_ref] Figure 8: The inhibitory effect of Gp [/fig_ref]. Consistent with Gp, after treatment of 1 mg/kg Xn, the parasitemia was slightly increased, in contrast to a significant decrease from 24.77% to 19.87% and 23.23% to 18.87% in the parasitemia of groups treated with 5 mg/kg and 10 mg/kg Xn, respectively [fig_ref] Figure 8: The inhibitory effect of Gp [/fig_ref]. The results indicated 5 mg/kg and 10 mg/kg Xn had better inhibitory effects on the in vivo culture of B. microti relative to that of 1mg/kg, 3 mg/kg, and 5 mg/kg Gp. The growth rate was much higher in the DMSO treatment than it was in the treatment with Gp or Xn. . microti-caused human babesiosis has predominated and presented a gradual upward trend throughout the world [bib_ref] The centers for disease control and prevention and state health departments should..., Jajosky [/bib_ref]. To date, a series of drugs are available, and among them, ID and DA are two effective drugs widely used in the treatment of animal babesiosis. For human babesiosis, the regimen of clindamycin and quinine was initially used as the standard treatment [bib_ref] Atovaquone and azithromycin for the treatment of babesiosis, Krause [/bib_ref]. However, increasing problems emerged, particularly the adverse effects for patients, such as tinnitus, diarrhea, gastroenteritis, and decreased hearing [bib_ref] Atovaquone and azithromycin for the treatment of babesiosis, Krause [/bib_ref]. The combination of atovaquone and azithromycin was regarded as an alternative therapy in curing human babesiosis caused by B. microti, due to fewer adverse effects and higher therapeutic efficacy [bib_ref] Management strategies for human babesiosis, Smith [/bib_ref]. However, under the regimen of atovaquone and azithromycin, increasing evidence indicated higher dosage, longer duration, and even treatment failure in some immunocompromised patients [bib_ref] Clinical and molecular evidence of atovaquone and azithromycin resistance in relapsed babesia..., Simon [/bib_ref]. A possible explanation is associated with the mutations in the binding regions of the target proteins. A previous article reported that the treatment of an immunocompromised patient with atovaquone and azithromycin for six weeks showed the emergence of resistant haplotypes, leading to relapse, due to the mutation of tyrosine to cysteine amino acid substitution in CYTb (atovaquone binding region) and arginine to cysteine amino acid substitution in RPL4 (azithromycin binding region) [bib_ref] Clinical and molecular evidence of atovaquone and azithromycin resistance in relapsed babesia..., Simon [/bib_ref]. While the combination of atovaquone and azithromycin is the most effective therapy against B. microti infection, drug resistance, relapse, and treatment failure are all pressing concerns, suggesting that the development of new therapies, especially new drugs, may be the most effective method to control B. microti infection.
# Discussion
## Compared with
# Discussion
Compared with B. divergens, B. duncani, and B. venatorum, B. microti-caused human babesiosis has predominated and presented a gradual upward trend throughout the world [bib_ref] The centers for disease control and prevention and state health departments should..., Jajosky [/bib_ref]. To date, a series of drugs are available, and among them, ID and DA are two effective drugs widely used in the treatment of animal babesiosis. For human babesiosis, the regimen of clindamycin and quinine was initially used as the standard treatment [bib_ref] Atovaquone and azithromycin for the treatment of babesiosis, Krause [/bib_ref]. However, increasing problems emerged, particularly the adverse effects for patients, such as tinnitus, diarrhea, gastroenteritis, and decreased hearing [bib_ref] Atovaquone and azithromycin for the treatment of babesiosis, Krause [/bib_ref]. The combination of atovaquone and azithromycin was regarded as an alternative therapy in curing human babesiosis caused by B. microti, due to fewer adverse effects and higher therapeutic efficacy [bib_ref] Management strategies for human babesiosis, Smith [/bib_ref]. However, under the regimen of atovaquone and azithromycin, increasing evidence indicated higher dosage, longer duration, and even treatment failure in some immunocompromised patients [bib_ref] Clinical and molecular evidence of atovaquone and azithromycin resistance in relapsed babesia..., Simon [/bib_ref]. A possible explanation is associated with the mutations in the binding regions of the target proteins. A previous article reported that the treatment of an immunocompromised patient with atovaquone and azithromycin for six weeks showed the emergence of resistant haplotypes, leading to relapse, due to the mutation of tyrosine to cysteine amino acid substitution in CYTb (atovaquone binding region) and arginine to cysteine amino acid substitution in RPL4 (azithromycin binding region) [bib_ref] Clinical and molecular evidence of atovaquone and azithromycin resistance in relapsed babesia..., Simon [/bib_ref]. While the combination of atovaquone and azithromycin is the most effective therapy against B. microti infection, drug resistance, relapse, and treatment failure are all pressing concerns, suggesting that the development of new therapies, especially new drugs, may be the most effective method to control B. microti infection.
Natural products, due to their broad range of activity, have been extensively studied in several diseases, especially in anti-parasite and anti-cancer therapies. In this study, natural products were considered as promising candidates for drug design against B. microti infection. The 133 natural products were obtained from the small molecule natural product library (Selleck Chemicals, Houston, TX, USA) and were preliminarily screened at 10 µm. Among them, 47 natural products were determined to be significantly different from DMSO, suggesting they could inhibit the replication of B. microti. After rescreening the 47 natural products at 50 µm, five of them were shown to have strong inhibitory effects on B. microti under microscopic examination, those products were 2F3 (palmatine chloride), B5 (Gp), 2E6 (Xn), G3 (cryptotanshinone), and G5 (honokiol). Palmatine chloride as a hydrochloride salt of palmatine has strong anti-malarial activity with low cytotoxicity and is also valuable as an Alzheimer's disease modifying strategy [bib_ref] Screening of a library of traditional chinese medicines to identify anti-malarial compounds..., Nonaka [/bib_ref] [bib_ref] integrating in vitro and in silico approaches to evaluate the "dual functionality"..., Haj [/bib_ref]. Cryptotanshinone, a quinoid diterpene isolated from Salvia miltiorrhizabunge, has shown various anti-cancer activities [bib_ref] Cryptotanshinone induces cell cycle arrest and apoptosis of nsclc cells through the..., Kim [/bib_ref]. Honokiol originally obtained from magnolia extract is reported to have the activity of inhibiting multiple malignancies [bib_ref] Honokiol Is a foxm1 antagonist, Halasi [/bib_ref]. However, palmatine chloride, cryptotanshinone, and honokiol were excluded through parasitemia determination by flow cytometry. Gp and Xn were finally identified to significantly inhibit the growth of B. microti by the combined determination of microscopy and flow cytometry. While microscopy is widely used in parasitemia calculation, this method has many disadvantages [bib_ref] Diagnosis, Treatment and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis, Sanchez [/bib_ref] [bib_ref] development of a real-time polymerase chain reaction assay for sensitive detection and..., Bloch [/bib_ref] [bib_ref] Evaluation of a fluorescence-based method for antibabesial drug screening, Guswanto [/bib_ref]. For instance, this method is not applicable or may consume vast resources of manpower in large-scale screening of potential drugs. Moreover, the errors caused by factitious factors are inevitable and may influence the results of screening to a great extent. The fluorescence-based method where SYBR green I (SG I) binds to the double-stranded DNA of the parasites has been extensively applied in anti-malaria drug screening [bib_ref] Evaluation of a fluorescence-based method for antibabesial drug screening, Guswanto [/bib_ref] [bib_ref] A New Real-Time PCR assay for improved detection of the parasite babesia..., Teal [/bib_ref]. Due to the absence of a nucleus in erythrocytes, the cells that were labeled by fluorescence were all the parasites-infected erythrocytes. The flow cytometry used in this study possesses the feature of quick analysis and is the most advanced cell analysis technology available to date [bib_ref] Evaluation of a fluorescence-based method for antibabesial drug screening, Guswanto [/bib_ref]. Therefore, the flow cytometry screening eliminated three of the five drugs that were selected by microscopy. However, flow cytometry also has the disadvantage of high cost, and may cost too much in large-scale preliminary screening [bib_ref] Evaluation of a fluorescence-based method for antibabesial drug screening, Guswanto [/bib_ref]. Therefore, these two methods were combined in the present study: microscopy examination in a preliminary large-scale screening and flow cytometry in rescreening confirmation. Collectively, microscopy combined with flow cytometry will facilitate resource utilization and parasitemia calculation.
Lactate dehydrogenase (LDH), an indispensable active enzyme, was reported to be involved in glycolysis, mediating the reversible metabolism of pyruvate to lactate [bib_ref] Lactate dehydrogenase 5: An old friend and a new hope in the..., Augoff [/bib_ref]. The inhibition of LDH will lead to the death of parasites, which has been reported in Plasmodium falciparum, Toxoplasma gondii, and Babesia bovis [bib_ref] The kinetic properties and sensitivities to inhibitors of lactate dehydrogenases (ldh1 and..., Dando [/bib_ref] [bib_ref] Identification of babesia bovis l-lactate dehydrogenase as a potential chemotherapeutical target against..., Bork [/bib_ref]. Gp, extracted from cotton seeds, is a competitive inhibitor against the binding of reduced nicotinamide adenine dinucleotide to LDH [bib_ref] Identification of babesia bovis l-lactate dehydrogenase as a potential chemotherapeutical target against..., Bork [/bib_ref]. Therefore, Gp was regarded as a promising drug candidate in anti-tumor, anti-parasite, and anti-HIV research [bib_ref] The kinetic properties and sensitivities to inhibitors of lactate dehydrogenases (ldh1 and..., Dando [/bib_ref]. However, at a concentration over 500 µm, Gp was reported to be cytotoxic and accompanied with hemolysis by in vitro culture [bib_ref] Antimalarial activity of new gossypol derivatives, Razakantoanina [/bib_ref]. In this study, cytotoxicity assays were performed at 1 µm, 10 µm, 50 µm, and 100 µm of Gp and Xn, respectively. Both of them exhibited no toxicity to erythrocytes and thus can be used as candidates for drug design. The cytotoxicity to other cells and organisms has been extensively investigated in previous studies. Gp was shown to cause 50% death of chronic lymphocytic leukemia cells at 30 µM [bib_ref] A BH3 mimetic, induces apoptosis in chronic lymphocytic leukemia cells, Balakrishnan [/bib_ref]. For epithelial cervix cancer cells (HeLa), brain malignant glioma cells (U87), and gastric cancer cells (M85), the IC 50 values were 31.3 µm, 59.6 µm, and 39.7 µm, respectively [bib_ref] Chiral gossypol derivatives: Evaluation of their anticancer activity and molecular modeling, Zhang [/bib_ref]. The toxicity assay in vivo was also conducted, and the median lethal doses (LD 50 ) were tested as 2315 mg/kg, 550 mg/kg, and 35 mg/kg for rat, pig, and mouse, respectively [bib_ref] Reproductive toxicologic effects of gossypol on male rabbits: Biochemical, enzymatic, and electrolytic..., Shaaban [/bib_ref] [bib_ref] biochemical changes and histological alteration induced by gossypol in testicular and hepatic..., El-Sharaky [/bib_ref] [bib_ref] Reversible Antispermatogenic effect of gossypol in langur monkeys (presbytis entellus entellus), Sharma [/bib_ref] [bib_ref] Postweaning exposure to gossypol results in epididymis-specific effects throughout puberty and adulthood..., Romualdo [/bib_ref] [bib_ref] A polyphenolic aldehyde from cotton plant, interferes with swine granulosa cell function, Basini [/bib_ref]. Xn is a prenylated chalcone derived from a hop plant, Humulus lupulus L. [bib_ref] The antileishmanial activity of xanthohumol is mediated by mitochondrial inhibition, Monzote [/bib_ref] [bib_ref] Broad spectrum anti-infective potential of xanthohumol from hop (humulus lupulus l.) in..., Gerhauser [/bib_ref] , which possesses a broad spectrum against virus, bacteria, fungi, and plasmodium [bib_ref] Broad spectrum anti-infective potential of xanthohumol from hop (humulus lupulus l.) in..., Gerhauser [/bib_ref]. Several studies explored the anticarcinogenic properties of Xn as a cancer chemo-preventive agent by modulating the enzymes involved in carcinogen metabolism and detoxification [bib_ref] Cancer chemopreventive activity of xanthohumol, a natural product derived from hop, Gerhäuser [/bib_ref]. Xn can also eliminate reactive oxygen species, thereby inhibiting the production of nitric oxide and superoxide anion radicals. The biological properties of Xn are extensive and have attracted the attention of an increasing number of researchers; however, few reports are available about the inhibitory effects of Xn on parasites. Thus far, the anti-leishmanial activity of Xn was only shown to be mediated by inhibiting mitochondrial activities [bib_ref] The antileishmanial activity of xanthohumol is mediated by mitochondrial inhibition, Monzote [/bib_ref]. To test the cytotoxicity, an MTT assay was performed by incubating HeLa, A549 (hypotriploid alveolar basal epithelial cells), and HepG2 (human liver hepatocellular carcinoma cell line) with Xn, and the IC 50 values were determined as 40.4 µm, 30.5 µm, and 35.0 µm, respectively [bib_ref] Synthesis of xanthohumol analogues and discovery of potent thioredoxin reductase inhibitor as..., Zhang [/bib_ref]. Furthermore, the toxicity of Xn to the HCT116 and HT29 as well as the hepatocellular carcinoma cell lines HepG2 and Huh7 was experimentally determined in previous studies, and the IC 50 values were 40.8 µm, 50.2 µm, 25.4 µm, and 37.2 µm, respectively [bib_ref] Antiproliferative and cytotoxic activity of xanthohumol and its non-estrogenic derivatives in colon..., Logan [/bib_ref]. Additionally, female BALB/c mice were fed with 1000 mg Xn/kg body weight, and no signs of Xn-toxicity or adverse effects were observed in normal organs [bib_ref] Xanthohumol Feeding does not impair organ function and homoeostasis in Mice, Dorn [/bib_ref]. The treatment of female Sprague Dawley rats with 1000 mg Xn/kg body weight only showed weak hepatotoxicity and did not affect the development of the rats [bib_ref] A safety study of oral xanthohumol administration and its influence on fertility..., Hussong [/bib_ref]. Xn was also used at an escalating dose in menopausal women, leading to no sex hormones, blood clotting, or acute toxicity [bib_ref] Pharmacokinetics of Prenylated Hop Phenols in Women Following Oral Administration of a..., Van Breemen [/bib_ref]. All these results indicate that Gp and Xn with IC 50 values of 8.47 µm and 21.40 µm, respectively, were non-toxic by in vitro and in vivo assays. In this article, the combination of Gp and Xn showed an antagonistic effect on B. microti in an in vitro test, indicating that monotherapy is better than combination therapy. For the combination therapy strategy, the drugs with a similar target will be more suitable, such as mycophenolate mofetil, AGI-5198, disulfiram, olutasidenib, and brequinar, which are potential dehydrogenase inhibitors and whose combination with Gp against B. microti needs to be further studied. Previous studies have shown that Xn could inhibit the cyclooxygenase 1 (COX-1) and COX-2 activities that are involved in mucosal protection. Diclofenac sodium, lornoxicam, and ketorolac tromethamine salt are selective COX inhibitors, whose combination with Xn against B. microti could also be further investigated in the future.
Based on the preliminary screening and rescreening of 133 natural products, Gp and Xn were identified as two potential drugs against B. microti. On one hand, Gp (IC 50 8.47 µm) and Xn (IC 50 21.40 µm) can extensively inhibit the growth of B. microti at a low concentration. On the other hand, both of them were shown to be non-toxic to erythrocytes and other types of cells or organisms at the IC 50 8.47 µm of Gp or IC 50 21.40 µm of Xn. These results indicate their potential use for drug design against babesiosis caused by B. microti.
# Conclusions
In this study, the inhibitory effects of 133 natural products on B. microti were preliminarily screened by microscopy, and 47 of them were further screened by flow cytometry. The combined screening and in vitro culture results revealed Gp and Xn (with IC 50 values of 8.47 µm and 21.40 µm, respectively) as promising chemotherapeutic agents against B. microti. Moreover, the in vivo results also indicated their effective inhibition on B. microti. This study provides new insights for the design and development of novel drugs against B. microti, which may effectively inhibit B. microti infection.
# Supplementary materials:
The following are available online at http://www.mdpi.com/2076-393X/8/4/613/ s1, [fig_ref] Figure 1: The preliminary screening of 133 natural products against B [/fig_ref] : The chemical structure and formula of the five natural products: gossypol, xanthohumol, cryptotanshinone, honokiol and palmatine chloride, [fig_ref] Table 1: The summary of cytotoxicity of gossypol and xanthohumol on different cell lines [/fig_ref] : The details of the 133 natural products,: The raw data that presented in [fig_ref] Figure 1: The preliminary screening of 133 natural products against B [/fig_ref].
[fig] A 96 -: well flat-bottom plate was used for B. microti culture and drug screening. The culture conditions consisted of 25 µL of infected red blood cells (RBCs), 15 µL of uninfected normal RBCs (10% hematocrit), 110 µL of culture medium supplemented with 2% HB-101 (Irvine Scientific, Shanghai, China), 20% fetal bovine serum (FBS, ATLANTA Biologicals, Shanghai, China), 10 mg/L Albumax I (Gibco Life Technologies, Shanghai, China), 2 mm L-glutamine (Irvine Scientific, Shanghai, China), 2% Antibiotic/Antimycotic 100× (Corning, Shanghai, China), and hypoxanthine (200 µm)-thymidine (30 µm) (Sigma-Aldrich, St. Louis, MO, USA) [/fig]
[fig] Figure 1: The preliminary screening of 133 natural products against B. microti by in vitro culture. The asterisks indicate statistically significant differences between the treated and control groups (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05). (A) The natural products from A1 to B11. (B) The natural products from C1 to D11. (C) The natural products from E1 to F11. (D) The natural products from G1 to H8. (E) The natural products from H9 to 2E3. (F) The natural products from 2E4 to 2H5. [/fig]
[fig] Figure 2: The calculated inhibitory effects of 133 natural products on B. microti. The error bars indicate standard error of the means for each tested group. The hollow cylinders indicate the natural products with significant difference from DMSO that were subsequently used for rescreening. (A) The natural products from A1 to B11. (B) The natural products from C1 to D11. (C) The natural products from E1 to F11. (D) The natural products from G1 to H8. (E) The natural products from H9 to 2E3. (F) The natural products from 2E4 to 2H5. [/fig]
[fig] Figure 3: The rescreening of 47 natural products against B. microti by in vitro culture. The asterisks indicate statistically significant differences between the treated and control groups (**** p < 0.0001). (A) The natural products of 2A1, 2A5, 2B1, 2B6, 2D1, 2D2, 2D3, 2D4, 2D5 and 2D6. (B) The natural products of G4, A8, B8, 2F3, 2G2, 2H2, 2H4, 2H5, 2A4, 2B4, 2C3, 2E2, 2E3, A7, B4, B5 and B9. (C) The natural products of 2E5, 2E6, 2F1, 2F2, 2F5, 2G4, 2G5, 2H1, G3, G5, G8, H2, H4, H7, H8, E5, E9, E10, E11 and C10. [/fig]
[fig] Figure 4: The calculated inhibitory effects of 47 natural products on B. microti determined by microscopy. The error bars indicate standard error of the means for each tested group. Five drugs (2E6, G3, G5, 2F3, and B5) with significant differences from DMSO were selected as potential drugs against B. microti. The drugs were in the sequence of DMSO, DA, 2E5, 2E6, 2F1, 2F2, 2F5, 2G4, 2G5, 2H1, G3, G5, G8, H2, H4, H7, H8, E5, E9, E10, E11, C10, G4, A8, B8, 2F3, 2G2, 2H2, 2H4, 2H5, 2A4, 2B4, 2C3, 2E2, 2E3, A7, B4, B5, B9, 2A1, 2A5, 2B1, 2B6, 2D1, 2D2, 2D3, 2D4, 2D5, and 2D6. [/fig]
[fig] Figure 5: The inhibitory effects of 47 natural products on B. microti determined by flow cytometry. Two drugs (2E6 and B5) with significant differences from DMSO were finally selected as potential drugs against B. microti. The error bars indicate standard error of the means for each tested group. [/fig]
[fig] Figure 6: The cytotoxicity assay of xanthohumol (A) and gossypol (B). The negative control was treated with medium without drugs. [/fig]
[fig] Figure 7: The inhibitory effect of gossypol and xanthohumol on the in vitro culture of B. microti. The parasitemia for treatment with different concentrations of gossypol (A,B) as well as different concentrations of xanthohumol (C,D). ("ns": no significance, **** p < 0.0001, *** p < 0.001 and ** p < 0.01). [/fig]
[fig] Figure 8: The inhibitory effect of Gp (A) and Xn (B) on the in vivo culture of B. microti. [/fig]
[fig] Author: Contributions: Collected the blood samples: J.G. and X.L. Performed the experiments: J.G. and X.L. Participated in the data analysis: J.G., L.H., and S.W. Helped with the diagnostic assays: J.G. and X.L. Edited the manuscript: J.G., L.H., and J.Z. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This study was supported by the National Natural Science Foundation of China (Grant No. 31772729), the National Key Research and Development Program of China (Grant No. 2017YFD0501201) and the Natural Science Foundation of Hubei Province (Grant No. 2017CFA020). [/fig]
[table] Table 1: The summary of cytotoxicity of gossypol and xanthohumol on different cell lines. Ratio of the IC 50 on cell lines to the IC 50 of each drug on the in vitro culture of Babesia microti. KM: Kunming mice. [/table]
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10.3390/genes14071324
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CCBY
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10379332
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37510229
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s2orc_pubmed_articles
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A Porcine DNMT1 Variant: Molecular Cloning and Generation of Specific Polyclonal Antibody
DNA methyltransferase 1 (DNMT1), the first-identified DNA methyltransferase in mammals, has been well studied in the control of embryo development and somatic homeostasis in mice and humans. Accumulating reports have demonstrated that DNMT1 plays an important role in the regulation of differentiation and the activation of immune cells. However, little is known about the effects of porcine DNMT1 on such functional regulation, especially the regulation of the biological functions of immune cells. In this study, we report the cloning of DNMT1 (4833 bp in length) from porcine alveolar macrophages (PAMs). According to the sequence of the cloned DNMT1 gene, the deduced protein sequence contains a total of 1611 amino acids with a 2 amino acid insertion, a 1 amino acid deletion, and 12 single amino acid mutations in comparison to the reported DNMT1 protein. A polyclonal antibody based on a synthetic peptide was generated to study the expression of the porcine DNMT1. The polyclonal antibody only recognized the cloned porcine DNMT1 and not the previously reported protein due to a single amino acid difference in the antigenic peptide region. However, the polyclonal antibody recognized the endogenous DNMT1 in several porcine cells (PAM, PK15, ST, and PIEC) and the cells of other species MDBK, and MDCK cells). Moreover, our results demonstrated that all the detected tissues of piglet express DNMT1, which is the same as that in porcine alveolar macrophages. In summary, we have identified a porcine DNMT1 variant with sequence and expression analyses.
# Introduction
DNA methylation is a major epigenetic modification of the genome that plays a vital role in the regulation of gene expression [bib_ref] In vivo activity of murine de novo methyltransferases, Dnmt3a and Dnmt3b, Hsieh [/bib_ref] [bib_ref] DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and..., Okano [/bib_ref] [bib_ref] Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases, Okano [/bib_ref]. In mammals, there are three major DNA methyltransferases (DNMTs), which catalyze the covalent binding of a methyl group provided by S-adenosylmethionine to cytosine in DNA [bib_ref] rahu is a mutant allele of Dnmt3c, encoding a DNA methyltransferase homolog..., Jain [/bib_ref] [bib_ref] DNA methylation in mammals, Li [/bib_ref] [bib_ref] Bourc'his, D. The DNA methyltransferase DNMT3C protects male germ cells from transposon..., Barau [/bib_ref]. DNMT1, the first-identified DNA methyltransferase, is known to maintain DNA methylation patterns [bib_ref] New concepts in DNA methylation, Jeltsch [/bib_ref] [bib_ref] Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells...., Bestor [/bib_ref]. In contrast, DNMT3A and DNMT3B are de novo DNMTs, responsible for establishing methylation patterns [bib_ref] DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and..., Okano [/bib_ref]. Of these three DNMTs, DNMT1 is the most abundant methyltransferase in mammalian somatic cells [bib_ref] The human DNA methyltransferases (DNMTs) 1, 3a and 3b: Coordinate mRNA expression..., Robertson [/bib_ref] [bib_ref] Domain Structure of the Dnmt1, Dnmt3a, and Dnmt3b DNA Methyltransferases, Tajima [/bib_ref]. DNMT1 binds to the replication fork during DNA replication and has a high affinity for semi-methylated DNA double strands, which allows the enzyme to copy the methylation pattern after DNA replication [bib_ref] Targeted mutation of the DNA methyltransferase gene results in embryonic lethality, Li [/bib_ref] [bib_ref] DNA methyl transferase 1: Regulatory mechanisms and implications in health and disease, Dhe-Paganon [/bib_ref]. Accumulating evidence has implied that aberrant expression of DNMT1 causes alteration of the DNA methylation pattern, which is linked to the occurrence and development of several cancers [bib_ref] DNA methylation as a transcriptional regulator of the immune system, Morales-Nebreda [/bib_ref] [bib_ref] DNA methyltransferase 1 (DNMT1) protein expression in precancerous conditions and ductal carcinomas..., Peng [/bib_ref] [bib_ref] Increased DNA methyltransferase 1 protein expression correlates significantly with intestinal histological type..., Ksiaa [/bib_ref] [bib_ref] Dysregulation of p53/Sp1 control leads to DNA methyltransferase-1 overexpression in lung cancer, Lin [/bib_ref]. Therefore, DNMT1 has been identified as a therapeutic target for the treatment of some cancers [bib_ref] DNA methylation profiling in the clinic: Applications and challenges, Heyn [/bib_ref] [bib_ref] DNA methyltransferases: A novel target for prevention and therapy, Subramaniam [/bib_ref]. Because DNA methylation also regulates the differentiation and functions of immune cells [bib_ref] Azacytidine Treatment Inhibits the Progression of Herpes Stromal Keratitis by Enhancing Regulatory..., Varanasi [/bib_ref] , changes in DNA methylation patterns dysregulate these functions, causing immune system diseases. Therefore, controlling DNA methylation may offer an avenue for treating immune-related diseases [bib_ref] Understanding the Relevance of DNA Methylation Changes in Immune Differentiation and Disease, Calle-Fabregat [/bib_ref]. Macrophages are important components of the immune system, facilitating the maintenance of tissue homeostasis and preventing microbial infections [bib_ref] Co-stimulation with opposing macrophage polarization cues leads to orthogonal secretion programs in..., Munoz-Rojas [/bib_ref]. However, the phenotypes of macrophages can be altered by different stimuli to become classically activated (M1) or alternatively activated (M2) macrophages [bib_ref] Macrophage plasticity and polarization: In vivo veritas, Sica [/bib_ref] , leading to various diseases [bib_ref] Macrophage Polarization in Chronic Inflammatory Diseases: Killers or Builders?, Parisi [/bib_ref]. For example, M1-polarized macrophages promote the development of atherosclerosis. Although the underlying mechanism by which M1 macrophages are polarized requires further study, DNMT1 plays a key role in the regulation of M1 macrophage polarization by alteration of inflammation [bib_ref] DNA methyltransferase 1 and Kruppel-like factor 4 axis regulates macrophage inflammation and..., Tang [/bib_ref].
Epigenetic regulation by DNMT1 has been well studied in humans and mice, but the function of porcine DNMT1 is less well understood. We are particularly interested in the role of epigenetic regulation by DNA methylation in the immune response of porcine macrophages. In a previous study, we identified DNMT3B2 as the predominant isoform of DNMT3B in porcine alveolar macrophages and demonstrated its potential role in the regulation of lipopolysaccharide (LPS)-stimulated tumor necrosis factor α (TNF-α) expression [bib_ref] Identification of DNMT3B2 as the Predominant Isoform of DNMT3B in Porcine Alveolar..., Qiu [/bib_ref]. In this study, we characterized DNMT1 in porcine alveolar macrophages. Surprisingly, we identified a porcine DNMT1 variant with 1611 amino acids with sequencing and protein analyses.
# Materials and methods
## Cells and tissues
Pudong White piglets were purchased from the Shanghai Academy of Agricultural Sciences (Shanghai, China) at around 30 days of age, euthanized, and dissected to obtain porcine alveolar macrophages (PAMs) and various tissue samples (heart, liver, spleen, lung, kidney, small intestine, thymus, inguinal lymph nodes, submaxillary lymph nodes, mesenteric lymph nodes, tonsils, and hilar lymph nodes). The PAMs were isolated, as reported in our previous study [bib_ref] Nonstructural Protein 4 of Porcine Reproductive and Respiratory Syndrome Virus Modulates Cell..., Qi [/bib_ref]. Briefly, the pig lungs were washed with 400 mL of PBS containing 1 mM EDTA. The recovered cell suspension was spun at 500× g for 10 min, and the cell precipitate was resuspended in RPMI 1640 containing 10% fetal bovine serum (FBS), penicillin, streptomycin, and GlutaMAX™ Supplement (all purchased from Thermo Fisher Scientific, Shanghai, China). Furthermore, the tissue samples were freshly generated by using around 100 mg of each tissue (heart, liver, spleen, lung, kidney, small intestine, thymus, inguinal lymph nodes, submaxillary lymph nodes, mesenteric lymph nodes, tonsils, and hilar lymph nodes). Subsequently, the aliquoted tissues were homogenized and lysed for later Western blotting analysis. All the experiments were performed in accordance with procedures approved by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute (IACUC No: Shvri-po-2016060501), Chinese Academy of Agriculture Science. Human embryonic kidney (HEK-293T) cells, Marc-145 cells, mouse neuroblastoma (N2a) cells, baby hamster kidney (BHK-21) cells, Madin-Darby bovine kidney (MDBK) cells, Madin-Darby canine kidney (MDCK) cells, porcine kidney (PK)-15 cells, swine testicular (ST) cells, and porcine iliac artery endothelial cells (PIECs) were maintained in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) at 37 - C in a 5% CO 2 atmosphere.
## Cloning of porcine dnmt1 and sequence analysis
The total RNA was extracted from the PAMs (at least 1.0 × 10 6 cells) with TRIzol™ Reagent (Accurate Biotechnology, Changsha, China) [bib_ref] Total RNA extraction from tissues for microRNA and target gene expression analysis:..., Brown [/bib_ref] , and the cDNA was prepared with Super Script II Reverse Transcriptase (Thermo Fisher Scientific). Five pairs of primers for cloning the porcine DNMT1 gene (shown in were designed on the basis of the porcine DNMT1 cDNA sequence reported in the GenBank database (NM_001032355.1). All the PCR products were sequenced with gene-specific primers. All the images of agarose gel electrophoresis were captured with the Image Lab Software version 5.1 (Bio-Rad Laboratories, Hercules, CA, USA).
The amino acid sequences of the human (NP_001124295.1), mouse (NP_001300940.1), porcine (NP_001027526.1), and cloned porcine DNMT1 proteins were aligned with Clustal V and edited with Genedoc. A phylogenetic tree was constructed from the available DNMT1 proteins with the neighbor-joining method in MEGA version 6.06 [bib_ref] The neighbor-joining method: A new method for reconstructing phylogenetic trees, Saitou [/bib_ref].
## Generation of polyclonal anti-porcine dnmt1 antibody
A polyclonal antibody directed against porcine DNMT1 was generated as described in a previous study [bib_ref] An improved protocol for coupling synthetic peptides to carrier proteins for antibody..., Lateef [/bib_ref]. Briefly, antigenic peptides from the cloned porcine DNMT1 were predicted by using an online tool (https://novoprolabs.com/tools/peptide-antigen-design, accessed on 30 June 2020). According to the predicted structure of the cloned porcine DNMT1 (https://swissmodel.expasy.org/interactive, accessed on 30 June 2020), a peptide of 15 amino acids (SSPVKRPRKEPVDED) exposed outside was selected. The peptide was then synthesized chemically and conjugated with keyhole limpet hemocyanin (KLH) as the carrier protein.
## Plasmid transfection
The cloned porcine DNMT1 cDNA was inserted into the p3×Flag-CMV-14 vector (Sigma, St. Louis, MO, USA) and designated pFlag-DNMT1 (cloned). In the meantime, the reported porcine DNMT1 cDNA (NM_001032355.1) was synthesized and cloned into the p3×Flag-CMV-14 vector and the named pFlag-DNMT1 (NM_001032355.1). To determine the specificity of the anti-porcine DNMT1 antibody, HEK-293T cells were grown to 70-80% confluence and transfected with the pFlag-DNMT1 (cloned) or pFlag vector plasmid using Lipofectamine™ 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. To identify the porcine DNMT1 sequence recognized by the polyclonal anti-porcine DNMT1 antibody, BHK-21 cells were grown to 70-80% confluence and transfected with the plasmids pFlag-DNMT1 (cloned), pFlag-DNMT1 (NM_001032355.1), or pFlag-vector, as described above. After transfection for 24 h, the samples were collected for further analysis.
## Western blotting
Briefly, membranes containing the transferred protein samples were blocked with 5% skim milk for 2 h at room temperature. They were then incubated overnight at 4 - C with the individual primary antibodies: anti-Flag (M2, Sigma; diluted 1:2000), anti-porcine DNMT1 (generated in this study; diluted 1:2000), or anti-β-actin (Sigma; diluted 1:10,000). The membranes were then incubated for 1 h at room temperature with the appropriate secondary antibody: horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Abcam; diluted 1:5000) or HRP-conjugated goat anti-rabbit IgG (Abcam; diluted 1:10,000). The membrane was then treated with HRP substrate according to the instructions for Enhanced Chemiluminescence (ECL) Reagent (Pierce) and exposed to X-ray film. Images were captured with the Gel Doc™ EZ System (Bio-Rad Laboratories, USA).
## Polymerase chain reaction (pcr)
The total DNA from the PAM, PK15, ST, and PIEC cells was extracted with a cell DNA Isolation Mini Kit (Vazyme, Nanjing, China). DNA fragments containing antigenic peptides and inserted or deleted regions were amplified by PCR using the extracted DNA samples above. All PCR products were sequenced with gene-specific primers. All the images of agarose gel electrophoresis were captured with the Image Lab Software version 5.1 (Bio-Rad Laboratories, Hercules, CA, USA). The specific primers are shown in .
# Results
## Molecular cloning of porcine dnmt1 cdna and multiple sequence alignment of dnmt1 from different species
To characterize the expression profile of DNMT1 in porcine alveolar macrophages, we first cloned the DNMT1 cDNA with reverse transcription (RT)-PCR on the basis of the reported porcine DNMT1 sequence (NM_001032355.1) in the GenBank database. Five overlapping gene fragments covering the DNMT1 cDNA were obtained and sequenced [fig_ref] Figure 1: Multiple-sequence alignments of DNMT1 from different species [/fig_ref]. A 4833 bp full-length DNMT1 cDNA was identified, and the deduced protein sequence contained 1611 amino acids. In comparison, the reported porcine DNMT1 cDNA (NM_001032355.1) is 4830 bp long and encodes 1610 amino acids. Notably, the cloned DNMT1 cDNA contained a 6 bp nucleotide insertion at 3980-3981 bp and a 3 bp nucleotide deletion at 4812-4814 bp relative to the GenBank-reported porcine DNMT1 cDNA. We then assembled a multiple-sequence alignment of pig, human, and mouse DNMT1 proteins using full-length amino acid sequences. The cloned porcine DNMT1 was 99% identical to the previously reported porcine DNMT1 (NP_001027526.1), with a 2 amino acid insertion, a 1 amino acid deletion, and 12 single amino acid mutations [fig_ref] Figure 1: Multiple-sequence alignments of DNMT1 from different species [/fig_ref]. Furthermore, we compared the cloned porcine DNMT1 with the other eight porcine DNMT1s deposited in GenBank , which are predicted from the genomic sequence (NC_010444.4). Our results showed that the eight predicted porcine DNMT1s all had the same 2 insertion amino acids and 12 mutant amino acids as the cloned DNMT1; in comparison, the deleted region only existed in the 3 predicted porcine DNMT1 (XP_020937688.1, XP_020937695.1, and XP_020937699.1). Since the eight porcine DNMT1s remain predicted, we chose the cloned DNMT1, the reported porcine DNMT1 (NP_001027526.1), the human DNMT1 (NP_001124295.1), and the mouse DNMT1 (NP_001300940.1) for multiple sequence alignment. Moreover, our results showed that the cloned porcine DNMT1 was 88% and 75.3% identical to that of humans and of mice, respectively [fig_ref] Figure 1: Multiple-sequence alignments of DNMT1 from different species [/fig_ref]. Therefore, although the cloned porcine DNMT1 was highly homologous with the previously reported porcine DNMT1 (NP_001027526.1), the sequence length and the indicated insertion, deletion, and mutation amino acids suggested that the cloned DNMT1 differed from the reported porcine DNMT1.
## Phylogenetic analysis of dnmt1 protein sequences
To understand the relationships between the cloned porcine DNMT1 and those of other species, we performed a phylogenetic analysis of 20 deduced amino acid sequences from different species. Three clusters were identified: mammal, bird, and fish DNMT1. The cloned porcine DNMT1 clustered in the mammal group together with the DNMT1s of goats, sheep, cattle, horses, rabbits, humans, and monkeys but not with those of rodents. Therefore, consistent with the results of the multiple-sequence alignment, the two porcine DNMT1s were closer to human DNMT1 than to mouse DNMT1 [fig_ref] Figure 2: Phylogenetic analysis of DNMT1 proteins from various species [/fig_ref]. other species, we performed a phylogenetic analysis of 20 deduced amino acid sequences from different species. Three clusters were identified: mammal, bird, and fish DNMT1. The cloned porcine DNMT1 clustered in the mammal group together with the DNMT1s of goats, sheep, cattle, horses, rabbits, humans, and monkeys but not with those of rodents. Therefore, consistent with the results of the multiple-sequence alignment, the two porcine DNMT1s were closer to human DNMT1 than to mouse DNMT1 [fig_ref] Figure 2: Phylogenetic analysis of DNMT1 proteins from various species [/fig_ref].
## Generation of polyclonal antibody against porcine dnmt1 and cross-reactivity of the antibody against dnmt1 from different species
To determine the expression profile of DNMT1 in PAMs, we next developed a polyclonal antibody using a synthetic antigen peptide (SSPVKRPRKEPVDED), predicted and selected on the basis of the cloned porcine DNMT1 protein. The rabbits were immunized with this synthetic peptide, and antisera was prepared after seven immunizations. We used affinity chromatography to further purify the polyclonal antibody from the collected antisera. The enzyme-linked immunosorbent assay (ELISA) titer of the purified antibody was >10 5 . We used Western blotting to examine its reaction with the cloned porcine DNMT1 ectopically expressed in HEK-293T cells. Consistent with the results obtained with an anti-Flag antibody, the polyclonal antibody specifically detected the expression [fig_ref] Figure 2: Phylogenetic analysis of DNMT1 proteins from various species [/fig_ref]. Phylogenetic analysis of DNMT1 proteins from various species. The phylogenetic tree was constructed from available DNMT1 proteins with the neighbor-joining method in MEGA version 6.06. Scale bar indicates the genetic distance. The cloned DNMT1 is marked with black dots.
## Generation of polyclonal antibody against porcine dnmt1 and cross-reactivity of the antibody against dnmt1 from different species
To determine the expression profile of DNMT1 in PAMs, we next developed a polyclonal antibody using a synthetic antigen peptide (SSPVKRPRKEPVDED), predicted and selected on the basis of the cloned porcine DNMT1 protein. The rabbits were immunized with this synthetic peptide, and antisera was prepared after seven immunizations. We used affinity chromatography to further purify the polyclonal antibody from the collected antisera. The enzyme-linked immunosorbent assay (ELISA) titer of the purified antibody was >10 5 . We used Western blotting to examine its reaction with the cloned porcine DNMT1 ectopically expressed in HEK-293T cells. Consistent with the results obtained with an anti-Flag antibody, the polyclonal antibody specifically detected the expression of the cloned porcine DNMT1 [fig_ref] Figure 3: Cross-reactivity of the antibody against DNMT1 in various animal cells [/fig_ref] and specifically detected a band similar to the ectopically expressed porcine DNMT1 consistent with endogenous human DNMT1 in the vector-transfected group [fig_ref] Figure 3: Cross-reactivity of the antibody against DNMT1 in various animal cells [/fig_ref].
To detect the cross-reactivity of the antibody against DNMT1 from different species, we tested cell samples from humans, monkeys, pigs, mice, hamsters, cattle, and dogs with Western blotting. The polyclonal antibody recognized the endogenous DNMT1 in HEK-293T, Marc-145, PAM, MDBK, and MDCK cells, but not in N2a or BHK-21 cells [fig_ref] Figure 3: Cross-reactivity of the antibody against DNMT1 in various animal cells [/fig_ref]. To understand the different cross-reactivity of the antibody against DNMT1 proteins from various species, we further examined the peptide sequences of all the species described above. As shown in [fig_ref] Figure 3: Cross-reactivity of the antibody against DNMT1 in various animal cells [/fig_ref] , the cloned porcine DNMT1, human DNMT1, monkey DNMT1, and dog DNMT1 were 100% identical to the synthetic peptide. However, one amino acid differed in the synthetic peptide relative to the GenBank porcine DNMT1 (R6G) and bovine DNMT1 (D15A). Notably, the synthetic peptide differed from mouse DNMT1 and hamster DNMT1 in five amino acids, which resulted in its lack of cross-reaction with mouse and hamster DNMT1 [fig_ref] Figure 3: Cross-reactivity of the antibody against DNMT1 in various animal cells [/fig_ref]. Overall, our data indicate that a polyclonal antibody was successfully generated to detect the expression of cloned porcine DNMT1 and DNMT1 in several other species.
Genes 2023, 14, x FOR PEER REVIEW 7 of 12 of the cloned porcine DNMT1 [fig_ref] Figure 3: Cross-reactivity of the antibody against DNMT1 in various animal cells [/fig_ref] and specifically detected a band similar to the ectopically expressed porcine DNMT1 consistent with endogenous human DNMT1 in the vector-transfected group [fig_ref] Figure 3: Cross-reactivity of the antibody against DNMT1 in various animal cells [/fig_ref]. To detect the cross-reactivity of the antibody against DNMT1 from different species, we tested cell samples from humans, monkeys, pigs, mice, hamsters, cattle, and dogs with Western blotting. The polyclonal antibody recognized the endogenous DNMT1 in HEK-293T, Marc-145, PAM, MDBK, and MDCK cells, but not in N2a or BHK-21 cells [fig_ref] Figure 3: Cross-reactivity of the antibody against DNMT1 in various animal cells [/fig_ref]. To understand the different cross-reactivity of the antibody against DNMT1 proteins from various species, we further examined the peptide sequences of all the species described above. As shown in [fig_ref] Figure 3: Cross-reactivity of the antibody against DNMT1 in various animal cells [/fig_ref] , the cloned porcine DNMT1, human DNMT1, monkey DNMT1, and dog DNMT1 were 100% identical to the synthetic peptide. However, one amino acid differed in the synthetic peptide relative to the GenBank porcine DNMT1 (R6G) and bovine DNMT1 (D15A). Notably, the synthetic peptide differed from mouse DNMT1 and hamster DNMT1 in five amino acids, which resulted in its lack of cross-
## Identification of two different porcine dnmt1s with a single amino acid difference in the antigenic peptide region
Although the polyclonal antibody cross-reacted with bovine DNMT1, with which it shared one amino acid difference (D15A), it was unclear whether the antibody could be used to detect the reported porcine DNMT1 (R6G). Therefore, we examined the reactivity of the antibody against the reported porcine DNMT1 (NM_001032355.1) with Western blotting. To exclude the endogenous DNMT1, the BHK-21 cells were transiently transfected with plasmid pFlag-vector, pFlag-DNMT1 (NM_001032355.1), or pFlag-DNMT1 (cloned). Surprisingly, the polyclonal antibody only reacted with the cloned porcine DNMT1 and not with the reported porcine DNMT1 (NM_001032355.1) [fig_ref] Figure 4: Specific identification of two different porcine DNMT1s [/fig_ref]. These data indicate that the one-amino-acid difference (R6G) in the antigenic peptide region of the reported DNMT1 abolished its reactivity to our polyclonal antibody. of the antibody against the reported porcine DNMT1 (NM_001032355.1) with Western blotting. To exclude the endogenous DNMT1, the BHK-21 cells were transiently transfected with plasmid pFlag-vector, pFlag-DNMT1 (NM_001032355.1), or pFlag-DNMT1 (cloned). Surprisingly, the polyclonal antibody only reacted with the cloned porcine DNMT1 and not with the reported porcine DNMT1 (NM_001032355.1) [fig_ref] Figure 4: Specific identification of two different porcine DNMT1s [/fig_ref]. These data indicate that the one-amino-acid difference (R6G) in the antigenic peptide region of the reported DNMT1 abolished its reactivity to our polyclonal antibody. Approximately 100 mg of each tissue sample (heart, liver, spleen, lung, kidney, small intestine, thymus, inguinal lymph nodes, submaxillary lymph nodes, mesenteric lymph nodes, tonsils, and hilar lymph nodes) of piglets were obtained. Protein samples were prepared according to the material and method section to determine their DNMT1 expression by Western blotting.
## Identification of a porcine dnmt1 variant in different porcine cells and tissues
As shown in the results described above, our polyclonal antibody recognized the cloned porcine DNMT1 but not the previously reported protein. This result motivated us to determine the expression profile of DNMT1 in other porcine cells (PK15, ST, and PIEC) with Western blotting. Bands of the same size were detected in other porcine cells as were detected in PAMs [fig_ref] Figure 4: Specific identification of two different porcine DNMT1s [/fig_ref] , suggesting that the DNMT1 in those cells contained the same antigenic peptide. To confirm this result, we determined the sequence of the antigenic peptides in different porcine cells. Because the DNA sequence encoding the antigenic peptide is located in the same exon in all DNMT1 genes, we used PCR to amplify the sequence of interest from the DNA of different porcine cells. Sequence analysis showed that all the amplified sequences were 100% identical to the cloned DNMT1. Furthermore, the nucleotide sequences and deduced amino acids of the peptide region in those cells were 100% identical to those of the synthetic antigen peptide [fig_ref] Figure 4: Specific identification of two different porcine DNMT1s [/fig_ref]. We also examined the sequences of the inserted and deleted regions in the DNMT1 in those porcine cells.
Consistent with our previous findings, our results showed that the DNMT1 in all the porcine cells analyzed had a 2 amino acid insertion and a 1 amino acid deletion relative to the GenBank-reported porcine DNMT1 [fig_ref] Figure 4: Specific identification of two different porcine DNMT1s [/fig_ref]. Thus, the sequence and expression analyses revealed the presence of a porcine DNMT1 variant in these porcine cells that differed from the reported porcine DNMT1. Moreover, the expression profile of DNMT1 in different tissues of piglet was investigated by Western blotting. Our results showed that all the detected tissues express DNMT1, consistent with that in porcine alveolar macrophages [fig_ref] Figure 4: Specific identification of two different porcine DNMT1s [/fig_ref]. Collectively, our results identify a different DNMT1 variant, relative to reported DNMT1 (NP_001027526.1), in various porcine cells and tissues.
# Discussion
In this study, we identified a porcine DNMT1 variant containing 1611 amino acids, with a 2 amino acid insertion, a 1 amino acid deletion, and 12 single amino acid mutations relative to the previously reported porcine DNMT1 protein. A rabbit polyclonal antibody was generated using a 15-amino acid antigenic peptide, which contained an amino acid mutation relative to the reported porcine DNMT1 (NP_001027526.1). Our data demonstrate that the polyclonal antibody specifically recognized the cloned porcine DNMT1 but not the originally reported porcine DNMT1, which contained a single amino acid difference in the antigenic peptide region. Our data also show that this antibody recognized endogenous DNMT1 in other porcine cells and tissues, which contained the same amino acids as the synthetic peptide. This antibody also cross-reacted with the DNMT1 proteins of other species including humans, monkeys, dogs, and cattle. Collectively, these data confirm the identification of a DNMT1 variant from PAMs.
Because DNMT1 plays a crucial role in the regulation of the inflammatory response and macrophage polarization [bib_ref] DNA methyltransferase 1 and Kruppel-like factor 4 axis regulates macrophage inflammation and..., Tang [/bib_ref] , it is important to understand the characteristics of DNMT1 in PAMs. Therefore, we cloned the DNMT1 cDNA from PAMs. Unexpectedly, we detected a DNMT1 variant with 1611 amino acids, which has a 2 amino acid insertion, a 1 amino acid deletion, and 12 single amino acid mutations relative to the previously reported porcine DNMT1. In further analysis, we demonstrated the existence of the same insertion, deletion, and mutation amino acids in the other three deposited porcine DNMT1 (XP_020937688.1, XP_020937695.1, and XP_020937699.1), which are the predicted sequences from the genomic sequence (NC_010444.4). Although those predicted sequences need to be verified, this datum is important to our results.
Fortunately, we have generated a polyclonal antibody to identify two different porcine DNMT1s with a single amino acid difference in the antigenic peptide region. Western blotting assay has confirmed that PK15, ST, and PIEC cell lines all express the DNMT1 variant. We also demonstrated that the DNA sequences encoding the antigenic peptide and the inserted and deleted regions of DNMT1 in all the porcine cell lines were 100% identical to the sequences in the DNMT1 cloned from the PAMs. Therefore, this porcine DNMT1 variant also exists in all the porcine cell lines tested. Notably, we have demonstrated that the various tissues of piglets are also robust in expressing the DNMT1 variant. Thus, this antibody has become an important tool to detect porcine DNMT1.
In the meantime, this antibody is useful in detecting DNMT1 from other species including humans, monkeys, dogs, and cattle. However, it is not capable of recognizing the DNMT1 of rodents such as mice and hamsters. According to sequence analysis, it is understandable that either mouse DNMT1 or hamster DNMT1 contains five amino acid differences in the antigenic peptide. Interestingly, either mouse or hamster DNMT1 does not contain the sixth amino acid difference in antigenic peptide, but they do both contain the fifteenth amino acid difference. According to our results, the sixth amino acid is a key amino acid for determining the reactivity of this polyclonal antibody; in comparison, the fifteenth amino acid is not. Thus far, it is clear that the sixth amino acid is a key amino acid for deciding the reactivity of this polyclonal antibody. Whether or not there is another key amino acid needs further experimental analysis.
The porcine DNMT1 variant was identified in PAMs from Pudong White pigs, which historically originate in Shanghai, China [bib_ref] Whole-genome resequencing reveals genetic structure and introgression in Pudong White pigs, Huang [/bib_ref]. The indigenous Pudong White pig, which has the advantages of feed and production, has been identified as a distinctive genetic resource, with a unique genetic structure, and has been added to China's livestock and poultry genetic resources listed for conservation by the Ministry of Agriculture of China. In a phylogenetic analysis, Pudong White pigs clustered together with other Chinese pigs rather than with European pigs or the out-group [bib_ref] Whole-genome resequencing reveals genetic structure and introgression in Pudong White pigs, Huang [/bib_ref]. Therefore, to know the expression profile of this DNMT1 variant in other Chinese pigs, European pigs, and the out-group, further studies are needed.
In somatic cells, canonical full-length DNMT1 is highly conserved in the same species. Some mutation(s) in canonical DNMT1 may alter its functions, causing DNMT1-related disorders. Hereditary sensory and autonomic neuropathy 1E (HSAN1E) has been shown to be associated with mutations in DNMT1 [bib_ref] Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss, Klein [/bib_ref] , and the naturally occurring S878F DNMT1 mutation is reported to result in elevated fetal hemoglobin levels [bib_ref] A natural DNMT1 mutation elevates the fetal hemoglobin level via epigenetic derepression..., Gong [/bib_ref]. An investigation of the single nucleotide polymorphisms (SNPs) of DNMT1 showed that mutations of DNMT1 are related to a variety of diseases, including cancers, heart disease, and autoimmune diseases [bib_ref] A Meta-Analysis of the Association between DNMT1 Polymorphisms and Cancer Risk, Li [/bib_ref] [bib_ref] Association of DNMT1 Gene Polymorphisms with Congenital Heart Disease in Child Patients, Wang [/bib_ref] [bib_ref] Genetic variants in DNMT1 and the risk of cardiac autonomic neuropathy in..., Santos-Bezerra [/bib_ref] [bib_ref] Gene-gene and gene-sex epistatic interactions of DNMT1, DNMT3A and DNMT3B in autoimmune..., Cai [/bib_ref] [bib_ref] DNA methyltransferase haplotype is associated with Alzheimer's disease, Pezzi [/bib_ref]. Although there has been no report of DNMT1-related disorders in pigs, it is important to determine whether this variant contributes to any disease susceptibility or resistance in Pudong White pigs. It would also be interesting to know whether this variant contributes to the specific traits of feed and production in Pudong White pigs.
In this study, we have demonstrated a DNMT1 variant in the PAMs of Pudong White pigs. To the best of our knowledge, this is the first time that a DNMT1 variant has been identified in porcine somatic cells. How this variant functionally regulates the homeostasis of porcine somatic cells, such as macrophages, is still unclear, and future studies are necessary to address this issue.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes14071324/s1, [fig_ref] Figure 1: Multiple-sequence alignments of DNMT1 from different species [/fig_ref] : Cloning the DNMT1 cDNA from PAMs. Total RNA was extracted from the PAMs, and the DNMT1 cDNA was amplified with five primer pairs. Table S1: Sequence of primers. : The GenBank accession number of predicted porcine DNMT1s.
## Informed consent statement: not applicable.
# Data availability statement:
The data presented in this study are available on request from the corresponding author.
## Conflicts of interest:
The authors declare no conflict of interest.
[fig] Figure 1: Multiple-sequence alignments of DNMT1 from different species. Sequences of pig (NP_001027526.1), human (NP_001124295.1), mouse (NP_001300940.1), and the cloned porcine DNMT1 proteins were aligned to determine the levels of homology. Black and gray shading highlights the sequence consistency and similarity across all selected sequences, respectively. The DMAP binding (yellow line), RFD (orange line), -CXXC-type zinc finger (brown line), BAH-1 (green line), BAH-2 (blue line), and Dcm (purple line) domains relative to the porcine DNMT1 (NP_001027526.1) are labeled. The red dashed box indicates the DNMT1 antigenic peptide, which we used for anti-DNMT1 antibody development. Blue and orange dashed boxes indicate inserted and deleted regions relative to the reported porcine DNMT1 (NP_001027526.1). To note that each interval of 10 amino acids is indicated as an asterisk. [/fig]
[fig] Figure 2: Phylogenetic analysis of DNMT1 proteins from various species. The phylogenetic tree was constructed from available DNMT1 proteins with the neighbor-joining method in MEGA version 6.06. Scale bar indicates the genetic distance. The cloned DNMT1 is marked with black dots. [/fig]
[fig] Figure 3: Cross-reactivity of the antibody against DNMT1 in various animal cells. (A) HEK-293T cells were transiently transfected with pFlag14-Vector or pFlag14-DNMT1 (cloned) plasmid. Cell lysates were analyzed with anti-Flag or anti-DNMT1 antibodies by Western blot analysis. (B) DNMT1 abundance in the cells derived from various species was measured using Western blot analysis. (C) Amino acid homology with selected antigenic peptide sequences of DNMT1 from different species. The red color letters indicate amino acid sites that differ from the antigenic peptide sequence. [/fig]
[fig] Figure 4: Specific identification of two different porcine DNMT1s. (A) BHK-21 cells were transiently transfected with pFlag-DNMT1 (NM_001032355.1), pFlag14-Vector, or pFlag14-DNMT1 (cloned) plasmids. The cell lysates were collected for analysis with an anti-Flag antibody or anti-DNMT1 antibody in a Western blot analysis. (B) Lysates of porcine cell lines (PK15, ST, PIEC) and PAMs were harvested to determine their DNMT1 expression with Western blotting. (C) Sequencing analysis of various porcine cells based on the selected antigenic peptide sequences. (D) Sequencing analysis of the inserted and deleted regions in DNMT1 from various porcine cells compared with that in the previously reported porcine DNMT1 (used as the control). (E)Approximately 100 mg of each tissue sample (heart, liver, spleen, lung, kidney, small intestine, thymus, inguinal lymph nodes, submaxillary lymph nodes, mesenteric lymph nodes, tonsils, and hilar lymph nodes) of piglets were [/fig]
[fig] Author: Contributions: L.Z.: Conceptualization, Methodology, Writing-original daft, Visualization. J.W. (Jiayun Wang), Y.Z., X.X., K.L., J.W. (Jianchao Wei), Z.L., D.S., B.L.: Methodology. Z.M.: Methodology, Resource. Y.Q.: Conceptualization, Resource, Writing-review and editing, Supervision, Project administration. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported in part by the National Natural Science Foundation of China (32273024), the Natural Science Foundation of Shanghai (22ZR1475900), and the Elite Program of CAAS (to Y.Q). Institutional Review Board Statement: All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China (IACUC no. SHVRI-PO-2016060501). [/fig]
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What makes a good life: using theatrical performance to enhance communication about polygenic risk scores research in patient and public involvement
The aim of this patient and public involvement and engagement (PPIE) work was to explore improvised theatre as a tool for facilitating bi-directional dialogue between researchers and patients/members of the public on the topic of polygenic risk scores (PRS) use within primary or secondary care.PRS are a tool to quantify genetic risk for a heritable disease or trait and may be used to predict future health outcomes.In the United Kingdom (UK), they are often cited as a next-in-line public health tool to be implemented, and their use in consumer genetic testing as well as patient-facing settings is increasing.Despite their potential clinical utility, broader themes about how they might influence an individual's perception of disease risk and decision-making are an active area of research; however, this has mostly been in the setting of return of results to patients.We worked with a youth theatre group and patients involved in a PPIE group to develop two short plays about public perceptions of genetic risk information that could be captured by PRS.These plays were shared in a workshop with patients/members of the public to facilitate discussions about PRS and their perceived benefits, concerns and emotional reactions.Discussions with both performers and patients/public raised three key questions: (1) can the data be trusted?; (2) does knowing genetic risk actually help the patient?; and (3) what makes a life worthwhile?Creating and watching fictional narratives helped all participants explore the potential use of PRS in a clinical setting, informing future research considerations and improving communication between the researchers and lay members of the PPIE group.
# Introduction
Patient and public involvement and engagement (PPIE), which enhances the quality and appropriateness of research, is a core component of healthcare research in the United Kingdom (UK)."Involvement" is research carried out "with" or "by" members of the public rather than "to", "about" or "for" them (National Institute for Health and Care Research 2021)."Engagement" is where information and knowledge about research are provided and disseminated (National Institute for Health and Care Research 2021).In order for healthcare to be equitable, it is essential that data, participation in clinical trials and participation in research are representative of the population.Yet, the majority of the public is rarely exposed to healthcare research directly [bib_ref] Reconceptualizing nature-of-science education in the age of social media, Höttecke [/bib_ref] and thus it can be difficult for researchers to explain, and engage in meaningful dialogue, about their work.Researchers are also trained to use highly specific terminology and present their findings in formal ways, such as academic papers and conferences, which can be unhelpful and uninviting to the public.
Theatre communication is the antithesis of such carefully documented academic communication [bib_ref] The magic of theater: photographing a performative academic career, Vanover [/bib_ref] and may avoid some of the known drawbacks in traditional presentation styles [bib_ref] Slide presentations as speech suppressors: when and why learners miss oral information, Wecker [/bib_ref].One advantage is in the sustained emotional impact, both for the researcher and the audience [bib_ref] coproducing knowledge through post-show discussions of researchbased theatre, Hundt [/bib_ref] [bib_ref] The use of research-based theatre in a project related to metastatic breast..., Gray [/bib_ref].There is a rich history of using theatre to collect, disseminate and validate health research, engaging both the creator and the audience more concretely with the research findings [bib_ref] Use of applied theatre in health research dissemination and data validation: a..., Stuttaford [/bib_ref] [bib_ref] Staging data: theatre as a tool for analysis and knowledge transfer in..., Rossiter [/bib_ref] [bib_ref] I Shall Live and Not Die": using monologues based on the experiences..., Kerr [/bib_ref]. [bib_ref] Staging data: theatre as a tool for analysis and knowledge transfer in..., Rossiter [/bib_ref] report how theatrical research-based performance can use the creative power of theatre as an interpretive, analytic tool.Furthermore, they argue that theatrical research-based performance allows greater space for discussion by removing the restrictions imposed by communicating solely about facts.The paper also notes the lack of strong evidence of the efficacy of using theatre and attributes this to the inherent difficulty of evaluating artistic intervention.However, it is well documented that fictional narratives in media other than theatre can impact public health activity, with televised soap operas dramatically increasing health screening [bib_ref] The impact of a television soap opera on the NHS Cervical Screening..., Howe [/bib_ref] [bib_ref] The effect of 'Alma's' death on women attending for a cervical smear:..., Richardson [/bib_ref] , and future health choices [bib_ref] Smoking in movies and adolescent smoking initiation: a longitudinal study among Argentinian..., Mejia [/bib_ref] [bib_ref] Entertainment-education and HIV/AIDS prevention: a field experiment in Tanzania, Vaughan [/bib_ref]. [bib_ref] The human drama of genetics: 'hard' and 'soft' media representations of inherited..., Henderson [/bib_ref] report that dramatized personal accounts can often make more of an impression that factual news reporting.
Polygenic risk scores (PRS) are a method for using the information in DNA to predict future health outcomes (for an overview of their potential clinical uses, see [bib_ref] Towards clinical utility of polygenic risk scores, Lambert [/bib_ref] , and for a discussion about ethics and responsible use, see [bib_ref] Responsible use of polygenic risk scores in the clinic: potential benefits, risks..., Adeyemo [/bib_ref].PRS exist for a range of diseases (e.g., cardiovascular disease; [bib_ref] Polygenic risk scores in cardiovascular risk prediction: a cohort study and modelling..., Sun [/bib_ref] , as well as non-disease traits (e.g., musical ability; [bib_ref] Genome-wide association study of musical beat synchronization demonstrates high polygenicity, Niarchou [/bib_ref].The PRS are created by sequencing (determining the exact sequence of bases in a DNA molecule) or genotyping (determining which common genetic variants an individual carries) DNA from samples donated by many individuals together with information collected about their phenotypes (observable traits).In order to apply the PRS for individual predictive purposes, the patient would also need to obtain their genomic data.Consent for sequencing/genotyping is needed both in the research to create new PRS and in clinical practice to apply an existing PRS diagnostically to a given patient.
The use of PRS in the UK is increasing; for example, via home DNA testing kits, where a consumer pays to receive information about their ancestry, disease risk and other traits.Genetic data may also become routinely collected in studies such as Genomics England's pilot programme for screening all newborn babies through genetic sequencing (Genomics England 2021) or the HEART study piloting the use of PRS as part of the National Health Service (NHS) health checks [bib_ref] A polygenic risk score added to a QRISK2 cardiovascular risk calculator demonstrated..., Fuat [/bib_ref].The UK Government has produced a document exploring the potential uses for PRS from insurance to sport to education (Government Office for Science 2022), and the UK's new healthcare strategy has tasked the NHS with incorporating genetic sequencing into routine clinical care [bib_ref] The NHS long term plan, Alderwick [/bib_ref].However, despite the enthusiasm and potential utility of many PRS, they are not yet ready for implementation in the clinic [bib_ref] Polygenic scores, risk and cardiovascular disease, Moorthie [/bib_ref].One reason for this is the challenge to healthcare professionals to confidently and competently communicate genetic risk to colleagues and patients.A recent paper on the responsible use of PRS highlighted the importance of risk communication to avoid patients suffering physical, financial or psychosocial harm [bib_ref] Responsible use of polygenic risk scores in the clinic: potential benefits, risks..., Adeyemo [/bib_ref].
Inspired by the work "Research Usually Sits on Shelves, Through the Play It Was Shared.Co-producing Knowledge Through Post-show Discussions of Research-Based Theatre" [bib_ref] coproducing knowledge through post-show discussions of researchbased theatre, Hundt [/bib_ref] , we examined theatrical and fictional approaches to encourage bi-directional dialogue about PRS within a PPIE framework and promote the exchange of ideas over a one-way knowledge transfer from researchers to a lay audience.We explored, with a youth theatre group and patients/members of the public, the use of two improvised plays in shaping discussions around PRS being used in clinical settings.The plays were supplementary to a more traditional PPIE workshop featuring a PowerPoint presentation.The intention was not to explore theatre as a tool for impacting public health communication through knowledge transfer (as in [bib_ref] Use of applied theatre in health research dissemination and data validation: a..., Stuttaford [/bib_ref] [bib_ref] After the crash: research-based theater for knowledge transfer, Colantonio [/bib_ref] , but as a tool within the PPIE framework to enhance communication between researchers and patients/public with the intention of eliciting ideas and change in research priorities and culture.
## Creating the plays
"I haven't resolved what I would want to know.I haven't resolved if I would want to know."Genetic Destinies play monologue.
An improvisational theatre workshop was held in October 2021 with Babolin Theatre's youth group (ages 16-19), two patient advocates with Sickle Cell Disease (a genetic condition), a researcher (statistician working with genetic data) and a PPIE Lead from the Cardiovascular Epidemiology Unit of the University of Cambridge.There were two sessions.In the morning, there were warm-up exercises for all attendees, followed by a presentation of PRS background material.In separate rooms, the researcher and the PPIE Lead were asked questions by the theatre students in a press-conference style interrogation (focusing on positive vs negative arguments).This was used by the theatre students to write a draft of the piece "Genetic Destinies", while the patients, researcher and PPIE Lead discussed questions and ideas that had been raised during the morning session.In the afternoon, there were further warm-up exercises and a discussion of how PRS information is presented to patients, using the example of coronary artery disease risk.This discussion was used by the theatre students to create a second piece ("A Good Life").The two pieces were performed for a small audience and recorded.
The two pieces, "Genetic Destinies" and "A Good Life", can be viewed here: www.youtu be.com/ watch?v=HD3sE o6iY48 (starting at 57:28).
"Genetic Destinies" is a 4-minute monologue.The actor stands at a podium and attempts to discuss PRS, but is quickly lost in his own thoughts and worries.The piece was created out of responses from the researcher/PPIE Lead during a press-conference style interrogation by the theatre group and remixed by the actor into a more abstract portrayal of someone so paralysed by the potential benefits and harms that they are unable to communicate clearly.
"A Good Life" is a 15-minute reactive piece between six actors.It was created through a group discussion between all participants of how PRS information is presented to patients, using the example of coronary artery disease risk.This was followed by each student writing down their thoughts about the discussion and then meeting as a group to create the piece.In "A Good Life", each actor has a story of the life of "A", who discovers their PRS for cardiovascular disease and chooses to change, or not change, their lifestyle.The actors interrupt each other, disagreeing on what are the key components of well-being-healthy living, hedonism or more time spent with family.As each version of "A" dies, their narrator leaves the stage declaring "that was a good life".
## Using the plays in the ppie workshop
The theatre workshop researcher and PPIE Lead, and an additional researcher (a health data scientist specialising in polygenic scores), facilitated an online PPIE workshop with 25 patients/members of the public in March 2022 to reflect on attitudes towards the potential uses and utility of PRS using the theatre pieces as a communication tool.Several pages of pre-reading were sent in advance.During the workshop, a short PowerPoint presentation on the ideas in the pre-reading was delivered, and participants were shown videos of the two theatre pieces.Verbal feedback was gathered in breakout groups and 16 participants returned written evaluation forms after the workshop (Supplementary C).Further details of the composition of the groups and details of the workshop can be found in Supplementary A.
Use of theatre to communicate risks "Overall, I thought about [PRS] from more angles, which made me less sure about how to tackle it ethically.As I had read the material and had the talk before the plays, [the plays] didn't help my understanding as such, but they were wonderful at making me think!"Public participant written feedback following the PPIE workshop.
PPIE workshop members were strongly in favour of the use of theatre to explain PRS.Some felt the theatre pieces were sufficient in themselves, while others at the PPIE workshop felt that the pieces needed to be broadened and further explained to connect to a wider audience.There was a clear contrast in the quantitative feedback; the pre-reading and PowerPoint presentation were judged more informative (Supplementary D: graph 1), but the theatre pieces created larger shifts in whether public participants felt positively or negatively about PRS (Supplementary D: graph 2).While the former could be explained by the order of availability to the public participants, this would not account for the subsequent emotional changes in perceptions of these issues.This could also be driven by how the researchers chose different questions to start the discussions when using fictional narratives.Understanding about how the pre-reading, presentation and theatre pieces impacted the public participants' emotional reactions to PRS is important, as their reactions may influence their willingness to donate samples or participate in testing opportunities.The knowledge could also help governments, health agencies or researchers develop communications for asking the public to participate in these activities.It is unclear whether the theatrical performances caused a change in emotions by illuminating aspects about PRS that would have gone otherwise unconsidered or simply sped up their emotional conclusions.In either case, there is a value in researchers, PPIE Leads and patients/members of the public taking to the time to explore PRS in creative ways, as it allows everyone to introspect, process and discuss as a group.
The researchers found the experience positive.In previous PPIE workshops, one researcher felt under pressure to present only as an expert and talk only about facts she knew to be evidenced in research.In contrast, talking about the plays with the public participants allowed her to relax, and she found the experience "liberating".She noted that there was more freedom to talk openly about the potential negative impact of her work and of misinterpretations of her work, even where those fears might be unrealistic.This framing of the topic supplied the researchers with more targeted feedback in this area than neutral questions might have gained.Freeing academics to speculate and articulate worries is an interesting potential benefit of fictional narratives, as argued by [bib_ref] The impact of involvement on researchers: a learning experience, Staley [/bib_ref] that the impact of PPI on the behaviours and emotions of researchers may be of greater importance than its impact on a specific piece of research.In [bib_ref] The power of symbolic capital in patient and public involvement in health..., Locock [/bib_ref] , researchers noted the pressure to suppress their opinions in discussions with patients/members of the public and the perception by patients/public of researchers as robotic/unfeeling and needing PPIE activities to humanise their research.While the issue of hierarchy within PPIE is known to sometimes silence public participants [bib_ref] The power of symbolic capital in patient and public involvement in health..., Locock [/bib_ref] , it is possible that it also silences those with expertise, to the detriment of the overall exchange of ideas.
Previous workshops facilitated by the PPIE Lead, covering a variety of health and data topics, almost always exclusively used PowerPoint presentations.This project was a useful exploration of more creative ways to encourage discussion between all participants.The positive responses to the theatrical plays, and the discussions that they generated, highlighted the value-for-money in spending time and public funds creating less traditional materials, as well as the learning potential for researchers and patients/members of the public.Similar to the researcher, the PPIE Lead also noted that this format empowered her to contribute her own thoughts and feelings during the discussions, instead of participating as an external observer.This insight will be useful in creating future materials and workshops.
Theatre group members (ages 16-19) were overwhelmingly positive about learning complex genetic ideas through theatrical devising, describing it as "intense", "fun" and "interesting to learn about".Prior to the theatre workshop, they had identified genetic concepts (e.g., heritability, familial and individual risk) as something they would learn about from the news or science lessons, but did not expect to understand.The theatre group director had also been concerned that the strong technical nature would make it difficult to create a narrative.However, feedback from the actors showed that they were surprisingly pleased with how interesting they found the activity, with multiple references that it made them think more about the real-world ethical implications.
The review of the discussions and subsequent feedback strongly suggested that theatrical and creative storytelling added value to the experience for both the theatre group members and PPIE workshop members, just through creating and watching the short videos.The plays deepened the dialogue between the researchers and the other participants and enabled all parties to better understand our shared concerns about PRS.
Feedback about the theatre pieces and PPIE workshop also identified topics that were underexplored.PPIE workshop members felt more exploration was needed into how one's relationship to risk changes over a lifetime, particularly around issues like having children (a common reason people seek out genetic testing in the UK).Several also said that understanding the plays would be difficult without some familiarity of the underlying concepts.There were also multiple recommendations to include a glossary of terms found in the pre-reading and that the prereading should include bespoke images or a short video explanation.
We identified three key questions about concerns around PRS from both the theatre group and PPIE workshops.Aspects of these questions arose separately in the workshops, despite the different activities (creating the plays versus watching the plays).
- "Can the data be trusted?"included concerns around data security, accuracy and diversity.Negative emotions like anxiety and panic were associated with the storage of genetic data and how private companies might misuse it, while general practitioners (GPs) were seen as "safe holders" of knowledge.Accuracy was discussed in a very binary fashion; that data should be very accurate simply because it is DNA and that if there was not a high accuracy in results then PRS should not be used at all.A second aspect of accuracy arose around ethnicity and how a lack of diversity in research data might increase inequalities in health outcomes.This was a particular concern for the Black patients who joined the theatre group.- "Does knowing genetic risk actually help the patient?" debated if individuals would want to know their risk score and the potential impact on friends and family.Some did want to know PRS information, while others felt it would not be helpful unless there was new definitive action that could be taken beyond the known advice to make healthy choices.Stronger evidence of good outcomes in practice was wanted.The younger theatre group members more often discussed this as the impact on their own life, while the older PPIE group members discussed the potential fallout on family members.
- "What makes a life worthwhile?"explored how individuals evaluated what was a good life and the cost-tobenefit of PRS versus using the funds to benefit public health in more immediate ways.There was a clear desire for a holistic approach to the usefulness of PRS in terms of life improvement, not just with regard to a single health outcome or health outcomes in general.
Both groups expressed that learning their PRS may not change decisions already made about future health benefits versus denial of current pleasures, especially if health advice (eat well, exercise, etc) remained the same.
Detailed discussions about these key questions can be found in Supplementary B.
# Conclusion
"Maybe if they had exercised more or quit smoking completely, they would have lived for longer.But what would have been the point?"A Good Life play.
The two plays developed with the theatre group were quite simplistic.However, creating and viewing fictional narratives helped both the theatre group and PPIE workshop members explore their own feelings about the information rather than focusing on improving how much they learnt.The variety of responses induced by the plays and the large well of uncertainty and negative responses from the PPIE workshop members will help guide future PPIE activities, as well as PRS research, particularly in the rush to push PRS into clinical practice.Future workshops will explore the hesitancy and distrust of PRS and will use this knowledge to inform design and communication around implementing PRS research.
Further work on the plays is dependent on future funding, but collaborations using fiction and/or art to help the public explore their thoughts and concerns about PRS could potentially have a large value as these new health tools become more frequently and widely used.Creating narratives in collaboration with under-served groups should be prioritised to reduce the information and health inequalities in their communities [bib_ref] A culture of understanding: reflections and suggestions from a genomics research community..., Kaplan [/bib_ref] , such as our own deliberate inclusion of chronically ill and Black collaborators in this project.There are several existing, well-produced videos about PRS for lay audiences on YouTube , but all are factual pieces.We aim to employ a combination of fiction and non-fiction videos in future PPIE activities to help people become familiar with the material.
Theatre is just one approach to art-based communication in research; collage [bib_ref] Biographical collage as a tool in Inuit communitybased participatory research and capacity..., Dutton [/bib_ref] , sewing [bib_ref] Sewing Is Part of Our Tradition": a case study of sewing as..., Brubacher [/bib_ref] , poetryand other art forms may also have similar utility in encouraging and supporting dialogue within PPIE activities.We encourage other research teams to create a wider array of materials for discussing their work and to consider the utility of realistic fictional narratives and theatrical works for facilitating bi-directional dialogue with lay audiences.
AcknowledgementsWe thank Sam Plumb and Babolin Theatre (www.babol inthe atre.com) and the patients/members of the public for participating in this work.Supplementary InformationThe online version contains supplementary material available at https:// doi.org/ 10. 1007/ s12687-023-00635-1.Author contribution A.M.M. and S.F. facilitated the theatre workshop, and A.M.M., S.A.L. and S.F. facilitated the PPIE workshop.A.M.M, S.A.L. and S.F. created the pre-reading material and evaluation forms.A.M.M. and S.F. wrote the main manuscript text, and A.M.M. prepared graphs 1 and 2. All authors reviewed the manuscript.DeclarationsConflict of interest The authors declare no competing interests.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http:// creat iveco mmons.org/ licen ses/ by/4.0/.Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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CD4 and CD8 T Cell Responses to the M. tuberculosis Ag85B-TB10.4 Promoted by Adjuvanted Subunit, Adenovector or Heterologous Prime Boost Vaccination
Background: Although CD4 T cells are crucial for defense against M.tb, it is still not clear whether the optimal response against M.tb in fact involves both CD4 and CD8 T cells. To test this, we used a new vaccine strategy that generated a strong balanced T cell response consisting of both CD4 and CD8 T cells.Methods and Findings:To compare CD4 and CD8 responses against Ag85B-TB10.4 (H4), H4 was delivered as a subunit vaccine in cationic liposomes (CAF01), expressed in Ad5 (Ad-H4) or as a heterologous prime boost vaccination. H4/CAF01 induced primarily CD4 T cells and Ad-H4 gave predominantly a CD8 T cell response. In contrast, the heterologous prime boost combination resulted in augmentation of both the CD4 and CD8 response. The majority (.40%) of the CD4 T cells induced by the heterologous prime boost protocol were polyfunctional, and expressed IFN-c + , IL-2 + , and TNF-a + , whereas most of the CD8 T cells expressed IFN-c + and TNF-a + and possessed strong cytotoxic potential. The heterologous prime boost protocol also gave an increase in protective efficacy against M.tb challenge compared to H4/CAF01 and Ad-H4. Both the H4 specific CD4 and CD8 T cells were recruited to the site of infection, at the onset of infection. However, compared to CD8 T cells, CD4 T cells showed more extensive recruitment and were the main T cell subset proliferating at the site of infection.Conclusions/Significance: Heterologous prime boost based on H4, produced an additive effect on the priming of CD4 and CD8 cells and in terms of the protective capacity of the vaccine, and therefore represent an interesting new vaccine strategy against M.tb. However, CD4 and CD8 T cells respond very differently to live M.tb challenge, in a manner which supports the consensus that CD4 T cells do play the major role during the early stages of an M.tb infection.
# Introduction
Approximately one-third of the world's population is latently infected with Mycobacterium tuberculosis (M.tb), and understanding the immune mechanisms involved in controlling this chronic disease is of high priority. The major mechanisms of cell-mediated immunity include CD4 Th1-cell mediated activation of macrophages to destroy intracellular bacterial pathogens, and the central role of IFN-c in the control of tuberculosis has been clearly demonstrated by the susceptibility to mycobacterial infections in mice with a disrupted IFN-c gene and in humans with mutations in genes involved in the IFN-c and IL-12 pathways [bib_ref] Disseminated tuberculosis in interferon gamma gene-disrupted mice, Cooper [/bib_ref] [bib_ref] Interferon-gamma and interleukin-12 pathway defects and human disease, Dorman [/bib_ref] [bib_ref] An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection, Flynn [/bib_ref] [bib_ref] Protection against Mycobacterium tuberculosis infection by CD8+ T cells requires the production..., Tascon [/bib_ref].Unlike CD4 T cells, which are crucial in the defence against M.tb, the role of CD8 T cells is not fully resolved. However, numerous studies have indicated that cytotoxic CD8 T cell-mediated killing of infected host cells do play a role in the defence against a M.tb infection [bib_ref] Immunization with a DNA vaccine cocktail protects mice lacking CD4 cells against..., Derrick [/bib_ref] [bib_ref] CD8-and CD95/95L-dependent mechanisms of resistance in mice with chronic pulmonary tuberculosis, Turner [/bib_ref] [bib_ref] Control of latent Mycobacterium tuberculosis infection is dependent on CD8 T cells, Van Pinxteren [/bib_ref] [bib_ref] Impaired resistance to Mycobacterium tuberculosis infection after selective in vivo depletion of..., Muller [/bib_ref] [bib_ref] Major histocompatibility complex class I-restricted T cells are required for resistance to..., Flynn [/bib_ref] [bib_ref] Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium..., Behar [/bib_ref]. Other results in favour of the protective effect of CD8 T cells come from recent studies, showing that lack of the RD-1 region in BCG (compared to M.tb) is responsible for the reduced CD8 T cell response induced by BCG compared to M.tb, and that re-introducing RD-1 into BCG selectively restored the CD8 T cell response and importantly also increased the protective efficacy of the vaccine [bib_ref] Induction of CD8 T cells against a novel epitope in TB10.4: correlation..., Billeskov [/bib_ref] [bib_ref] Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines..., Pym [/bib_ref] [bib_ref] Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis, Pym [/bib_ref] [bib_ref] Enhanced protection against tuberculosis by vaccination with recombinant Mycobacterium microti vaccine that..., Brodin [/bib_ref]. As a consequence there has recently been increased focus on the CD8 T cells and several new vaccine strategies that target these T cells are currently pursued in many laboratories involved in TB vaccine research. Viral vectors, such as adeno-or vaccinia virus, trigger a Th1-dominated immune response, characterized by potent induction of IFN-c, and are also efficient in inducing a robust CD8 T cell response against their target antigens. One M.tb vaccine of this type is MVA-85A, a replication-deficient recombinant vaccinia virus, expressing antigen 85A from M.tb. This vaccine has performed well in animal models where it induces not only CD8 T cells, but also CD4 T cells, and significant protection against M.tb. Results from clinical trials indicate that it is also immunogenic in humans but with a response almost exclusively composed of CD4 cells [bib_ref] Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally..., Mcshane [/bib_ref] [bib_ref] Boosting BCG with MVA85A: the first candidate subunit vaccine for tuberculosis in..., Mcshane [/bib_ref]. Replication-deficient recombinant adenovirus for comparison are widely accepted as one of the most efficient delivery platforms for the induction of a CD8 T cell dominated response and adeno vectors have been evaluated in a number of studies of TB antigens. The H1 molecule (Ag85B-ESAT-6) expressed in Ad5 was demonstrated to promote a strong response consisting exclusively of CD8 cells directed to a few immunodominant epitopes but unable to protect against TB [bib_ref] Alteration of epitope recognition pattern in Ag85B and ESAT-6 has a profound..., Bennekov [/bib_ref]. Another virally-vectored vaccine is Aeras-402 Ad35, expressing a fusion of antigen 85A, antigen 85B, and TB10.4. All three antigens are present in M.tb and BCG, and all are highly immunogenic. The characterization of this vaccine in animal models has so far been limited but also this vaccine is clearly capable of inducing a strong CD8 T cell responses [bib_ref] Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in..., Radosevic [/bib_ref].
The antigens Ag85B and TB10. [bib_ref] Protection against Mycobacterium tuberculosis infection by CD8+ T cells requires the production..., Tascon [/bib_ref] have been extensively characterized [bib_ref] Induction of CD8 T cells against a novel epitope in TB10.4: correlation..., Billeskov [/bib_ref] [bib_ref] Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the..., Skjot [/bib_ref] [bib_ref] Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins..., Skjot [/bib_ref] [bib_ref] High Frequency of CD4+ T Cells Specific for the TB10.4 Protein Correlates..., Hervas-Stubbs [/bib_ref] [bib_ref] TB subunit vaccines-putting the pieces together, Andersen [/bib_ref] and a fusion protein consisting of both Ag85B-TB10.4 adjuvanted with cationic liposomes induced a strong protection against infection with M.tb [bib_ref] Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine:..., Dietrich [/bib_ref]. In the present study we have compared CD4 and CD8 responses against the H4 molecule delivered as a subunit vaccine in cationic liposomes (CAF01), expressed in Ad5 (Ad-H4) or as a heterologous prime boost vaccination. Our data demonstrate that the heterologous prime boost vaccination gives higher levels of both polyfunctional CD4 T cells, cytotoxic CD8 T cells and protection against M.tb challenge than Ad-H4 or rH4 on their own. When we monitored cellular infiltrate in the lung post M.tb challenge in mice vaccinated by the heterologous prime boost protocol we observed that whereas high numbers of both H4 specific CD4 and CD8 T cells were promoted by the vaccine, the CD4 T cells showed accelerated recruitment to the site of infection compared to CD8 cells and importantly CD4 cells were the main subset proliferating in the lung in response to M.tb infection in the vaccinated animals.
# Results
## Induction and characterization of the vaccine induced antigen specific t cell responses
We first compared the immune response promoted by H4 delivered in cationic liposomes (CAF01) expressed in an adeno5 vector or as a hetrologous pirme boost. In particular, we were interested in whether we could induce both CD4 and CD8 T cells, as well as in the quantity and phenotype of these cells. CB6F1 mice were vaccinated once with rH4 followed by a booster Ad-H4 vaccination. Non-vaccinated mice were also included but had no detectable T cells responses to the vaccine components (data not shown). One week after the last vaccination, mice were sacrificed and splenocytes were stimulated in vitro with Ag85B and TB10.4, and the production of IFN-c, IL-2, and TNF-a by CD4 and CD8 T cells was analyzed by intracellular FACS. We found that all three vaccines, Ad-H4, rH4, and rH4/Ad-H4, elicited Ag85B specific CD4 T cells producing all three cytokines [fig_ref] Figure 1: Cytokine frequencies and phenotypic profiles of specific CD4 T cells in Ad-H4,... [/fig_ref] and a significant number of these cells were moreover triple positive [bib_ref] CD4 T cells guarantee optimal competitive fitness of CD8 memory T cells, Johansen [/bib_ref] [bib_ref] Mucosal luminal manipulation of T cell geography switches on protective efficacy by..., Santosuosso [/bib_ref] , and 42%, respectively) [fig_ref] Figure 1: Cytokine frequencies and phenotypic profiles of specific CD4 T cells in Ad-H4,... [/fig_ref]. However, only rH4 and rH4/ Ad-H4 vaccinated mice contained TNF-a + IL-2 + double positive CD4 T cells [fig_ref] Figure 1: Cytokine frequencies and phenotypic profiles of specific CD4 T cells in Ad-H4,... [/fig_ref]. The triple positive and TNF-a + IL-2 + double positive T cell phenotypes generally represent effector memory-and central memory T cells, respectively [bib_ref] Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major, Darrah [/bib_ref] , indicating the induction of a more memory like CD4 T cell immune response by the vaccines rH4 and rH4/Ad-H4, as compared to Ad-H4. Most importantly, the highest number of antigen specific T cells was seen in mice vaccinated with rH4/Ad-H4. This vaccine induced a higher frequency of both IFN-c (1.1%60.3) and TNF-a (1.3%60.3) producing Ag85B specific CD4 T cells compared to Ad-H4 and rH4 (p,0.001 and 0.05, respectively) [fig_ref] Figure 1: Cytokine frequencies and phenotypic profiles of specific CD4 T cells in Ad-H4,... [/fig_ref] , and the phenotypic profile of the vaccine induced T cell response reflected the combination of Ad-H4 and rH4 vaccinations since it contained a high frequency of triple-and double positive memory CD4 T cells, as well as a significant amount of IFN-c + TNF-a + double positive (and single positive IFN-c + ) effector CD4 T cells [fig_ref] Figure 1: Cytokine frequencies and phenotypic profiles of specific CD4 T cells in Ad-H4,... [/fig_ref] [bib_ref] Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major, Darrah [/bib_ref] [bib_ref] T-cell quality in memory and protection: implications for vaccine design, Seder [/bib_ref]. In general, the level of TB10.4 specific CD4 T cells was found to be low in all three vaccinated groups, however, with a significant increase of IFN-c producing CD4 T cells in the rH4/Ad-H4 vaccinated mice [fig_ref] Figure 1: Cytokine frequencies and phenotypic profiles of specific CD4 T cells in Ad-H4,... [/fig_ref]. In comparison, BCG vaccinated mice showed low numbers of Ag85B specific CD4 T cells, but the highest numbers of TB10.4 specific T cells of which the major phenotypes were IFN-c + TNF-a + IL-2 + or IFNc + TNF-a + expressing CD4 T cells. In terms of CD8 T cells, we found that only the mice vaccinated with Ad-H4 or rH4/Ad-H4 had detectable numbers of antigen specific CD8 T cells and C), but by combining rH4 and Ad-H4 a larger CD8 T cell population was observed. We observed an increase in Ag85B specific IFN-c producing CD8 T cells from 1.1%60.1 in the Ad-H4 group to 6.8%61.9 in the rH4/Ad-H4 group (p,0.001), and an increase from 6.2%60.6 to 16%62.3 in the IFN-c producing CD8 T cells specific for TB10.4 (p,0.001) . Moreover, when summing up the T cells expressing all combinations of IFN-c, TNF-a, and IL-2 we found that in the rH4/Ad-H4 group 6.9% of all CD8 T cells were specific for Ag85B and 19.6% for TB10.4. Both vaccines induced IFNc + TNF-a + double positive CD8 T cells specific for both Ag85B and TB10.4, and interestingly also a minor, but still detectable, population of triple positive antigen specific CD8 T cells and D). Consistent with this, stimulation of splenocytes from the vaccinated mice in vitro for 72 hours (instead of 6 hours in the FACS analysis) followed by ELISA analysis of IFN-c secretion, also revealed significantly higher IFN-c production in the rH4/ Ad-H4 vaccine group, although mice from all vaccine groups gave measurable responses . Taken together, we found that heterologous prime boost based on rH4 and Ad-H4 had a clear additive effect on the number of vaccine antigen specific CD4 and CD8 T cells, inducing higher numbers of polyfunctional CD4 and CD8 T cells compared to rH4 and Ad-H4 vaccinations.
## Vaccine induced cytotoxicity
To further characterize the vaccine induced T cells we next analyzed the cytotoxic potential of the induced CD8 (and CD4) T cells. We used the recently published MHC class I restricted epitopes (specific for TB10.4 [bib_ref] An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection, Flynn [/bib_ref] [bib_ref] Protection against Mycobacterium tuberculosis infection by CD8+ T cells requires the production..., Tascon [/bib_ref] [bib_ref] Immunization with a DNA vaccine cocktail protects mice lacking CD4 cells against..., Derrick [/bib_ref] [bib_ref] CD8-and CD95/95L-dependent mechanisms of resistance in mice with chronic pulmonary tuberculosis, Turner [/bib_ref] [bib_ref] Control of latent Mycobacterium tuberculosis infection is dependent on CD8 T cells, Van Pinxteren [/bib_ref] [bib_ref] Impaired resistance to Mycobacterium tuberculosis infection after selective in vivo depletion of..., Muller [/bib_ref] [bib_ref] Major histocompatibility complex class I-restricted T cells are required for resistance to..., Flynn [/bib_ref] [bib_ref] Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium..., Behar [/bib_ref] [bib_ref] Induction of CD8 T cells against a novel epitope in TB10.4: correlation..., Billeskov [/bib_ref] and TB10.4 [bib_ref] Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins..., Skjot [/bib_ref] [bib_ref] High Frequency of CD4+ T Cells Specific for the TB10.4 Protein Correlates..., Hervas-Stubbs [/bib_ref] [bib_ref] TB subunit vaccines-putting the pieces together, Andersen [/bib_ref] [bib_ref] Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine:..., Dietrich [/bib_ref] [bib_ref] Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major, Darrah [/bib_ref] [bib_ref] T-cell quality in memory and protection: implications for vaccine design, Seder [/bib_ref] [bib_ref] CD8+-T-cell responses of Mycobacterium-infected mice to a newly identified major histocompatibility complex..., Majlessi [/bib_ref] [bib_ref] Control of latent Mycobacterium tuberculosis infection is dependent on CD8 T cells, Van Pinxteren [/bib_ref] [bib_ref] Dendritic cells infected with Mycobacterium bovis bacillus Calmette Guerin activate CD8(+) T..., Feng [/bib_ref] and one MHC class II restricted epitope (TB10.4 70-88 ) [bib_ref] Induction of CD8 T cells against a novel epitope in TB10.4: correlation..., Billeskov [/bib_ref] [bib_ref] High Frequency of CD4+ T Cells Specific for the TB10.4 Protein Correlates..., Hervas-Stubbs [/bib_ref] [bib_ref] CD8+-T-cell responses of Mycobacterium-infected mice to a newly identified major histocompatibility complex..., Majlessi [/bib_ref]. CFSE labeled splenocytes from naïve mice, pulsed (CFSE high) or not pulsed (CFSE low) with either of the two MHC class I restricted peptides or the MHC class II restricted epitope from TB10.4, were adoptively transferred into mice that had been vaccinated with rH4/Ad-H4 a week before. For comparison we included Ad-H4 and rH4 vaccinated mice. After 4 hours peptide specific lysis of the transferred cells was determined by FACS analysis of recipient splenocytes. We observed 78.8%60.2 and 80.6%60.2 specific killing of the TB10.4 3-11 loaded target cells (CFSE high) in Ad-H4 and rH4/Ad-H4 mice, respectively, compared to non-loaded target cells (CFSE low) (p,0.05). The rH4/Ad-H4 mice also demonstrated strong cytotoxic potential against TB10.4 20-28 loaded target cells with 70.5%62.3 specific killing, which was slightly higher than the TB10.4 20-28 specific killing in Ad-H4 mice (53.5%66.6, p,0.05) [fig_ref] Figure 3: Functional characterization of the vaccine induced T cells [/fig_ref] and C). In contrast, there was no killing of target cells loaded with the MHC II restricted epitope TB10.4 70-88 , and the rH4 group did not demonstrate any cytotoxic killing of the transferred target cells [fig_ref] Figure 3: Functional characterization of the vaccine induced T cells [/fig_ref]. Thus, both Ad-H4 and rH4/Ad-H4 induced CD8 T cells with a cytotoxic potential.
## The protective efficacy of the vaccine induced t cell responses
We next analyzed the vaccine induced protection. Vaccinated CB6F1 mice were challenged six weeks after final vaccination, and six weeks post challenge, the mice were sacrificed and the bacterial numbers were determined in the lungs. Ad-H4, rH4, the hetrologous prime boost combination, non-vaccinated, and BCG vaccinated mice were included. All the H4 based vaccines induced significant protection against M.tb challenge but as observed with the T cell response, combining the two vaccines, rH4 and Ad-H4, showed an additive effect in terms of protection. Thus, mice vaccinated with the heterologous prime boost combination of rH4/Ad-H4 showed a bacterial reduction of 1.2 Log 10 60.1 CFU, which was significantly higher than the protection in Ad-H4 and rH4 vaccinated mice (p,0.01 and p,0.001, respectively). The BCG-vaccinated mice showed a protection of 1.42 Log 10 60.1 CFU, which was not significantly different from rH4/Ad-H4 vaccinated mice [fig_ref] Figure 4: The protective efficacy and correlation of protection with infection driven immune response... [/fig_ref]. Interestingly, the CFU levels at six weeks post infection in the vaccine groups directly correlated with the ESAT-6 CD4 T cell response (R 2 = 0.78) [fig_ref] Figure 4: The protective efficacy and correlation of protection with infection driven immune response... [/fig_ref] , in agreement with previous work indicating that the ESAT-6 specific response can be used as a predictor for bacterial load [bib_ref] Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine:..., Dietrich [/bib_ref]. In the spleen, the highest protection was seen in BCG vaccinated mice, and in the groups vaccinated with either rH4 or rH4/Ad-H4.
## Recruitment of vaccine induced antigen specific cd4 and cd8 t cells to the site of infection
As we had observed an increase in both CD4 and CD8 T cells in the group receiving the heterologous prime boost combination of rH4/Ad-H4, and increased protection, it was important to compare how the vaccine primed CD4/CD8 T cells responded to an M.tb infection (in the group where they were both induced, e.g. the rH4/Ad-H4 group). As controls, non-vaccinated mice were included. Mice vaccinated with rH4/Ad-H4 (or non-vaccinated) were challenged with virulent M.tb six weeks after last vaccination, and two weeks after infection the mice were sacrificed. The two weeks post infection time point was chosen since the majority of cells in the lung at that time point are recalled T cells and not yet newly infection-primed T cells (Non vaccinated showed ,1% CD8 Ag85B or TB10.4 specific T cells and ,0.2% CD4 T cells) [bib_ref] Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine:..., Dietrich [/bib_ref] , which enabled us to measure recruitment of the pre-existing CD4 and CD8 T cells. Lung lymphocytes were stimulated in vitro with Ag85B and TB10.4, and expression of IFN-c, IL-2, and TNF-a in the CD4 and CD8 T cell subsets was analyzed by intracellular FACS. Two weeks post infection we observed a significant higher proportion of Ag85B and in particular of TB10.4 specific CD4 T cells in the lungs in the rH4/Ad-H4 group when compared to the non-vaccinated group (p,0.01) [fig_ref] Figure 5: Cytokine frequencies and phenotypic profiles of specific CD4 and CD8 T cells... [/fig_ref] and C). There were also increased levels of CD8 T cells in the lungs of rH4/Ad-H4 vaccinated mice, in particular of the IFN-c + TNF-a + effector phenotype [fig_ref] Figure 5: Cytokine frequencies and phenotypic profiles of specific CD4 and CD8 T cells... [/fig_ref] and D) [bib_ref] Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major, Darrah [/bib_ref] [bib_ref] T-cell quality in memory and protection: implications for vaccine design, Seder [/bib_ref]. Of all CD8 T cells 13.8% were specific for TB10.4 and 2.4% for Ag85B in the vaccinated group, and the numbers for CD4 T cells were 8.0% and 4.8%, respectively [fig_ref] Table 1: Frequency of T cell subsets [/fig_ref]. Thus, both CD4 and CD8 T cells were recruited to the lungs. Importantly, the CD8 T cells induced in the non-vaccinated mice showed a much higher frequency of single positive T cells, as compared to the vaccinated group, indicating the induction of terminally differentiated T cells . Cytokine frequencies and phenotypic profiles of specific CD8 T cells in Ad-H4, rH4, or rH4/Ad-H4 vaccinated mice one week after final vaccination. CB6F1 mice were vaccinated once with Ad-H4, twice with rH4 in cationic liposomes, or once with rH4 in cationic liposomes followed two weeks later by one Ad-H4 vaccination. Mice were sacrificed one week after the final vaccination and splenocytes were stimulated with Ag85B or TB10.4 peptides prior to staining with anti-CD8, -CD44, -IFN-c, -IL-2, and -TNF-a. (A and C) Frequencies represent IFN-c, IL-2, or TNF-a producing CD44 + CD8 + T cells out of total CD8 T cells specific for Ag85B (A) or TB10.4 (C). Background staining from cells stimulated with medium alone has been subtracted. Data represent the mean+SEM of a minimum of five mice per group with ***, p,0.001, **, p,0.01, and *, p,0.05 by the infection alone [fig_ref] Figure 5: Cytokine frequencies and phenotypic profiles of specific CD4 and CD8 T cells... [/fig_ref]. Interestingly, in the rH4/ Ad-H4 group more than 50% of all the cytokine producing CD4 T cells had a more memory-like phenotype (IFN-c + IL-2 + TNF-a + , IL-2 + TNF-a + or IL-2 + ) [bib_ref] Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major, Darrah [/bib_ref] [bib_ref] T-cell quality in memory and protection: implications for vaccine design, Seder [/bib_ref] , in comparison with only 10-20% of all the CD8 T cells [fig_ref] Table 1: Frequency of T cell subsets [/fig_ref].
To analyze whether there was a more extensive recruitment, or expansion, of CD4 versus CD8 T cells, we next quantified the cell proportions of Ag85B and TB10.4 specific CD4 or CD8 T cells before and after infection. To determine the numbers of the antigen specific cells, all the subpopulations were added to give the total number of T cells expressing any combination of IFN-c, TNF-a, and IL-2 [fig_ref] Table 1: Frequency of T cell subsets [/fig_ref]. Pre-infection (two weeks pre-infection) we observed equal amounts of Ag85B specific CD4 and CD8 T cells in the vaccinated group in the spleen [fig_ref] Figure 6: Recruitment of Ag85B and TB10 [/fig_ref]. However, 2 weeks post infection there were 2K times more Ag85B CD4 T cells than CD8 T cells in the lungs [fig_ref] Figure 6: Recruitment of Ag85B and TB10 [/fig_ref]. As for TB10.4 specific T cells, around 50 times more TB10.4 specific CD8 T cells were observed pre-infection in the spleen than CD4 T cells (TB10.4 CD4/CD8 ratio of 0.025) [fig_ref] Figure 6: Recruitment of Ag85B and TB10 [/fig_ref]. However, after infection we found almost the same numbers of TB10.4 CD4 and CD8 T cells in the lungs (CD4/CD8 ratio of 0.80) [fig_ref] Figure 6: Recruitment of Ag85B and TB10 [/fig_ref]. This demonstrated that the CD4 T cells in both the Ag85B and TB10.4 pool of T cells were initially recruited/expanded more than the CD8 T cells, even though both cell types were clearly recruited to the lung at the onset of the infection.
## The proliferative capacity of the recruited t cells at the site of infection
Since CD4 T cells were preferentially recruited to the infection site, we next evaluated if these infiltrating CD4 T cells were the result of an increased turn-over rate of CD4 versus CD8 T cells. The vaccinated and challenged mice were given BrdU in the drinking water three days before sacrifice to measure the degree of proliferation of CD4 and CD8 T cells at two weeks post infection. Of all CD4 T cells 31.5%67.3 had incorporated BrdU, whereas this number was 11.3%61.3 (p,0.05) for CD8 T cells . Stimulation with TB10.4 or Ag85B followed by gating on the IFNc positive antigen specific T cells showed that also for antigen specific T cells the CD4 T cell population showed the highest proliferation . Gating on all combinations of T cell subtypes expressing one or more of the cytokines IFN-c/IL-2/ TNF-a clearly demonstrated that the majority of proliferating CD4 cells were cytokine expressing, in contrast to the noncytokine producing CD4 T cell population, which did not proliferate (IFN-c 2 IL-2 2 TNF-a 2 , . In contrast, for the CD8 T cell population, the majority of the cytokine producing cells were non-proliferating . Moreover, when looking at the frequency of cytokine producing CD4 T cells, we found a clear tendency towards higher IFN-c, IL-2, and TNF-a production by the BrdU + CD4 T cells compared to the BrdU 2 CD4 T cell population . This tendency was less pronounced, however still present, in the cytokine producing antigen specific CD8 T cells . In conclusion, both the vaccine induced CD4 and CD8 T cells undergo cell division and were recruited to the site of infection, at the onset of the infection. However, the CD4 T cells showed a higher frequency of memory-like phenotypes, were recruited to the lungs at a higher rate, and proliferated more than CD8 T cells.
# Discussion
CD4 T cells are crucial for defense against M.tb. However, it is still not clear whether the optimal response against infection with M.tb in fact involves both CD4 and CD8 T cells and if these subsets play a role in distinct and different stages of the infection [bib_ref] Control of latent Mycobacterium tuberculosis infection is dependent on CD8 T cells, Van Pinxteren [/bib_ref]. In the present study we therefore compared the well characterized vaccine fusion molecule rH4 (Ag85B-TB10.4) delivered either as a subunit vaccine in cationic liposomes (rH4/ CAF01) expressed in an adeno5 vector (ad-H4) or as a heterologous prime boost combination. We clearly show that rH4/CAF01 induces primarily CD4 T cells, and Ad-H4 CD8 T cells, but the heterologous prime boost combination induces both CD4 and CD8 T cells.
Although CD8 T cells are a significant part of the immune response induced by the natural infection, the role of CD8 T cells in the defense against M.tb is not fully resolved. Lately, several vaccine strategies have made use of viral vectors to deliver the vaccine antigens, and to induce vaccine specific CD8 T cells [bib_ref] Dendritic cells infected with Mycobacterium bovis bacillus Calmette Guerin activate CD8(+) T..., Feng [/bib_ref] [bib_ref] Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent..., Wang [/bib_ref] [bib_ref] Protective immunity against Mycobacterium tuberculosis induced by dendritic cells pulsed with both..., Mcshane [/bib_ref]. These studies all point to a protective role for CD8 T cells, even though in most of these studies CD4 T cells were also primed. Studies using MVA as delivery of the vaccine antigens have indicated that this vector is particularly efficient when used as a booster vaccine, and boosting of both CD4 and CD8 T cells have been observed [bib_ref] Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally..., Mcshane [/bib_ref] [bib_ref] Enhanced Immunogenicity of CD4(+) T-Cell Responses and Protective Efficacy of a DNA-Modified..., Mcshane [/bib_ref]. Likewise, adenoviral vectors have been found to serve as efficient boosters of existing immune responses. Thus, adenovirus expressing Ag85A was able to boost BCG immunity, which in turn increased the protection against M.tb [bib_ref] Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium..., Santosuosso [/bib_ref]. As with MVA, adenovirus boosted both the CD4 and CD8 T cell activity, and priming of both these subsets was also observed when following immunization with adenovirus expressing mycobacterial antigens [bib_ref] Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in..., Radosevic [/bib_ref] [bib_ref] Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent..., Wang [/bib_ref] [bib_ref] Mucosal luminal manipulation of T cell geography switches on protective efficacy by..., Santosuosso [/bib_ref]. In other recent studies, in which only CD8 T cells to immunodominant antigens (such as ESAT-6 or TB10.4) were induced, only minor or no protection was observed [bib_ref] Alteration of epitope recognition pattern in Ag85B and ESAT-6 has a profound..., Bennekov [/bib_ref] [bib_ref] High Frequency of CD4+ T Cells Specific for the TB10.4 Protein Correlates..., Hervas-Stubbs [/bib_ref] , indicating that the contribution of CD8 T cells to protection may depend on, or increase inthe presence of, an ongoing CD4 T cell response. This is in line with studies showing that both CD8 T cell memory induction and maintenance can be influenced by CD4 T cells [bib_ref] CD4 T cells are required for CD8 T cell survival during both..., Novy [/bib_ref] [bib_ref] CD4 T cells guarantee optimal competitive fitness of CD8 memory T cells, Johansen [/bib_ref] [bib_ref] CD4+ T cells are required for secondary expansion and memory in CD8+..., Janssen [/bib_ref] [bib_ref] Requirement for CD4 T cell help in generating functional CD8 T cell..., Shedlock [/bib_ref] [bib_ref] Influence of strong CD4 epitope on long-term virus-specific cytotoxic T cell responses..., Sauzet [/bib_ref] [bib_ref] Exhaustion of CTL memory and recrudescence of viremia in lymphocytic choriomeningitis virusinfected..., Thomsen [/bib_ref] [bib_ref] High numbers of IL-2-producing CD8+ T cells during viral infection: correlation with..., Kristensen [/bib_ref]. However, it should be mentioned that in two studies using vaccination with a DNA vaccine encoding CFP10, or a mix of several TB proteins, the vaccine induced CD8 T cells were shown to induce protection, demonstrating that in some instances CD8 T cells do provide protection against acute M.tb infection in a manner that is not strictly dependent upon simultaneous induction of CD4 T cells [bib_ref] Immunization with a DNA vaccine cocktail protects mice lacking CD4 cells against..., Derrick [/bib_ref] [bib_ref] Vaccine-elicited 10-kilodalton culture filtrate protein-specific CD8+ T cells are sufficient to mediate..., Wu [/bib_ref].
In the present study we wanted to study the potential of a heterologous prime boost vaccine strategy that induce both CD4 and CD8 T cells, and to compare the phenotype of vaccine primed CD4 and CD8 T cells, as well as the recruitment and proliferation of these cells following infection with M.tb. We combined vaccination of a CD4 T cell inducer, rH4/CAF01, with a strong CD8 T cell inducer, Ad-H4. Mice vaccinated with the rH4/Ad-H4 combination showed an increase in CD4 T cell activity indicating that rH4 primed CD4 T cells can be boosted by Ad-H4 immunization. Compared to Ad-H4, rH4/Ad-H4 also induced significant higher numbers of cytokine producing CD8 T cells. In the rH4/Ad-H4 group approximately 27% of all CD8 T cells were specific for the vaccine antigens, with 7% being specific for Ag85B and 20% for TB10.4. Most of these (45-80%) CD8 T cells expressed IFN-c and TNF-a [fig_ref] Figure 1: Cytokine frequencies and phenotypic profiles of specific CD4 T cells in Ad-H4,... [/fig_ref] which suggests an effector phenotype. This clearly demonstrated that combining rH4 and Ad-H4 had a strong additive effect on priming for both the CD4 and CD8 T cell response. It can be speculated that the boosting of the CD4 T cells, which occurred in parallel with the priming of CD8 T cells, provided increased CD4 help for the CD8 T cells, thereby further promoting the activation and expansion of these cells. We also observed a slightly increased cytolytic function of the CD8 T cells in the rH4/Ad-H4 group, in particular against TB10.4 20-28 loaded target cells (17% increase in the specific killing of these cells in the rH4/Ad-H4 group cpmpared to the Ad-H4 group) [fig_ref] Figure 3: Functional characterization of the vaccine induced T cells [/fig_ref]. This may be due to increased CD8 T cell numbers, increased cytolytic capacity of the individual T cells, or that the CD8 T cells in the rH4/Ad-H4 group were primed in the presence of CD4 T cells, which have been suggested to increase both the persistence of CD8 T cells and the ability of the CD8 cells to respond to a secondary encounter with their antigen [bib_ref] CD4 T cells are required for CD8 T cell survival during both..., Novy [/bib_ref] [bib_ref] CD4 T cells guarantee optimal competitive fitness of CD8 memory T cells, Johansen [/bib_ref] [bib_ref] CD4+ T cells are required for secondary expansion and memory in CD8+..., Janssen [/bib_ref] [bib_ref] Requirement for CD4 T cell help in generating functional CD8 T cell..., Shedlock [/bib_ref] [bib_ref] Influence of strong CD4 epitope on long-term virus-specific cytotoxic T cell responses..., Sauzet [/bib_ref].
In terms of protection against infection with M.tb, we found, as observed with the numbers of vaccine specific T cells [fig_ref] Figure 1: Cytokine frequencies and phenotypic profiles of specific CD4 T cells in Ad-H4,... [/fig_ref] , that combining the two vaccine strategies also had a significant additive effect in the lung. Thus, mice vaccinated with rH4/Ad-H4 showed a protection of 1.2 Log 10 60.1 CFU, which was significantly higher than the protection observed in the Ad-H4 and rH4 mice [fig_ref] Figure 4: The protective efficacy and correlation of protection with infection driven immune response... [/fig_ref] and not significantly different from that observed in BCG vaccinated mice. The Ad-H4 group showed a protection of approximately 0.4 Log 10 CFU, which is in agreement with a recent study using Ad-35-Ag85A-Ag85B-TB10.4 [bib_ref] Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in..., Radosevic [/bib_ref]. Interestingly, in the spleen, the lowest protection was observed in the Ad-H4 group, which may indicate that prevention of dissemination from the lung to the spleen require CD4 T cells more than CD8 T cells [fig_ref] Figure 4: The protective efficacy and correlation of protection with infection driven immune response... [/fig_ref].
In the rH4/Ad-H4 group two weeks after infection we observed that 14% of all CD8 T cells in the lungs were specific for TB10.4 and 2-3% for Ag85B, indicating that the CD8 epitopes were indeed processed and presented in the lungs at the very onset of infection, and that CD8 T cells primed by Ad5 vector delivery, as well as the CD4 T cells, were recruited to the site of infection (preinfection the numbers in the lung were less than 0.1%, data not shown) [fig_ref] Figure 5: Cytokine frequencies and phenotypic profiles of specific CD4 and CD8 T cells... [/fig_ref]. For both the CD4 and CD8 T cell subsets we observed more polyfunctional cells (IFN-c + TNF-a + or IFNc + TNF-a + IL-2 + ) in the rH4/Ad-H4 group following M.tb challenge compared non-vaccinated mice. This could indicate that this vaccination strategy resulted in a pool of polyfunctional T cells that was maintained after challenge, whereas the natural infection of unprimed mice tended to drive the activated T cells toward a state of terminal differentiation (IFN-c + ) [fig_ref] Figure 5: Cytokine frequencies and phenotypic profiles of specific CD4 and CD8 T cells... [/fig_ref] [bib_ref] T-cell quality in memory and protection: implications for vaccine design, Seder [/bib_ref]. Interestingly, in the rH4/Ad-H4 vaccinated mice, we noted important differences between the CD4 and CD8 T cell recall responses. Thus, although we observed strong, local CD8 T cell memory, as measured by total CD8 T cell numbers, CD4 T cells preferentially accumulated (due to recruitment and/or expansion) at the site of infection [fig_ref] Figure 6: Recruitment of Ag85B and TB10 [/fig_ref]. In line with this, CD4 T cells with proliferative capacity showed higher accumulation at the site of infection than CD8 T cells two weeks post infection (although we can exclude that this bias in CD4 proliferation may in part reflect a bias inherent in the CD4 prime/boost mechanism). This was observed by analyzing the total pool of CD4 T cells, and also when studying TB10.4 and Ag85B antigen specific T cells . Moreover, as for the phenotype of the proliferating cells, the majority of the cytokine (any combination of IFN-c, TNF-a or IL-2) producing CD4 T cells belonged to the proliferating CD4 T cell population, in contrast to the cytokine producing CD8 T cells that where mainly non-proliferating . Taken together, these data revealed that although both CD4 and CD8 T cells were primed through vaccination, there was an increased recruitment and proliferation of the CD4 T cells as compared to the CD8 T cells suggesting that this subset may be primarily involved in the recall response that results in early control of bacterial replication in vaccinated mice, and that increasing the CD4 T cell numbers in this stage of the infection is important. We are presently testing H4 in new adjuvants specifically selected for their ability to induce a strong CD4 T cell response.
In conclusion, heterologous prime boost based on H4, produced an additive effect on the priming of CD4 and CD8 cells and in terms of the protective capacity of the vaccine, and therefore represent an interesting vaccine strategy against M.tb. However, compared to CD8 T cells, antigen specific CD4 cells were more polyfunctional, recruited at a higher rate, and proliferated more following infection with M.tb, indicating that although both CD4 and CD8 epitopes are clearly presented at the very onset of infection, CD4 and CD8 T cells respond very differently to live infection, in a manner which support the consensus that CD4 T cells play the major role during the early stages of an M.tb infection.
# Materials and methods
# Ethics statement
The handling of mice were conducted in accordance with the regulations set forward by the Danish Ministry of Justice and animal protection committees by Danish Animal Experiments Inspectorate, and in compliance with European Community Directive 86/609 and the U.S. Association for Laboratory Animal Care recommendations for the care and use of laboratory animals.
## Animals
Studies were performed with six-eight week-old female CB6F1 (C57BL/66BALB/c) mice from Harland Netherlands. Non-infected mice were housed in cages in appropriate animal facilities at Statens Serum Institut or at the Panum Institute, University of Copenhagen. Infected animals were housed in cages contained within laminar flow safety enclosures (Scantainer from Scanbur, Denmark) in a separate biosafety level 3 facility at Statens Serum Institut. All mice were fed radiation sterilized 2016 Global Rodent Maintenance diet (Harlan, Scandinavia) and water ad libitum. All animals were allowed a oneweek rest before initiation of the experiments.
## Brdu
In experiments measuring proliferation, BrdU (0.8 mg/ml) was added to the drinking water to the mice for three days before sacrifice.
## Bacteria
M.tb Erdman was grown at 37uC in suspension in Sauton medium (BD Pharmingen) enriched with 0.5% sodium pyruvate, 0.5% glucose, and 0.2% Tween 80. All bacteria were stored at and B) or per lung post infection (C and D). Background staining from cells stimulated with medium alone has been subtracted. Data represent the mean+SEM of four mice per group with **, p,0.01 (2-way ANOVA with Bonferroni posttest). (E and F) The ratio between Ag85B (E) and TB10.4 (F) specific CD4 and CD8 T cells was calculated from A-D. doi:10.1371/journal.pone.0005139.g006 . The proliferative capacity and frequency of the recruited Ag85B and TB10.4 specific CD4 and CD8 T cells to the lungs. CB6F1 mice were vaccinated once with rH4 in cationic liposomes followed two weeks later by one Ad-H4 vaccination, and challenged by the aerosol route with virulent M.tb at week 10. The last three days before killing, BrdU (0.8 mg/ml) was added to the drinking water. Two weeks after challenge, mice were sacrificed and lung lymphocytes were stimulated with Ag85B or TB10.4 peptides prior to staining with anti-CD4, -CD8, -BrdU, and -IFNc (A-C). (A) Frequencies represent the percentage of BrdU + CD4 or CD8 T cells out of total CD4 or CD8 T cells. Data represent the mean of +SEM of four mice per group with *, p,0.05 (Unpaired t-test, two-tailed). Dot plots, representative of four mice, are also shown depicting BrdU + CD4 or CD8 T cells. (B and C) Frequencies represent the percentage of BrdU + CD4 or CD8 T cells out of total IFN-c + CD4 or CD8 T cells specific for either Ag85B (B) or TB10.4 (C). Data represent the mean+SEM of two mice per group with **, p,0.01 (Unpaired t-test, two-tailed). Background staining from cells stimulated with medium alone has been subtracted. (D and E) CB6F1 mice were vaccinated once with rH4 in cationic liposomes followed two weeks later by one Ad-H4 vaccination, and challenged by the aerosol route with virulent M.tb at week 10. The last three days before sacrificing the animals, BrdU (0.8 mg/ml) was added to the drinking water. Two weeks after challenge mice were sacrificed and lung lymphocytes were stimulated with TB10.4 peptides prior to staining with anti-CD4, -CD8, -BrdU, -IFN-c, TNF-a, and IL-2. The BrdU histograms represent a normalized overlay of all the cytokine producing CD4 (D) and CD8 (E) T cells, (IFN-c + IL-2 + TNF-a + , IFN-c + TNF-a + , IFN-c + IL-2 + , IFN-c + , IL-2 + TNF-a + , IL-2 + , and TNF-a + ) (solid line), and all the non-cytokine producing CD4 and CD8 T cells (IFN-c 2 IL-2 2 TNF-a 2 ) (dashed line), and the percentages shown are the percentages of cytokine producing or cytokine non-producing T cells out of total T cells. Instead of displaying counts in the FACS histogram overlays, each histogram in the overlay has been normalized to its maximum counts (''% of MAX'' counts). Data represent the mean of +SEM of three mice per group. Background staining from cells stimulated with medium alone has been subtracted. doi:10.1371/journal.pone.0005139.g007 280uC in growth medium at ,5610 8 CFU/ml. Bacteria were thawed, sonicated, washed, and diluted in phosphate-buffered saline (PBS) for immunizations and infections. All bacterial work was performed at the Statens Serum Institut by authorized personnel.
## Construction of recombinant replication-deficient adenoviral 5 based vaccine ad-h4
The M.tb fusion protein H4 consisting of the two antigens Ag85B and TB10.4 was amplified by overlapping PCR from plasmid template and cloned into a pacCMV-based shuttle vector. From the shuttle plasmid, human type 5 recombinant non-replicating adenovirus vectors were produced from homologous recombination by standard methods [bib_ref] Use of recombinant adenovirus for metabolic engineering of mammalian cells, Becker [/bib_ref]. After purification, adenoviral stocks were immediately aliquoted and frozen at 280uC in 10% glycerol, and the infectivity of the adenoviral stocks was determined with Adeno-X Rapid Titer kit (BD Clontech).
## Immunization
Mice immunized with Ad-H4 were anesthetized and injected once with 2610 7 infectious units (IFU) s.c. in the right hind footpad. Mice immunized with the Ag85B-TB10.4 fusion protein (rH4) were immunized twice at two-week intervals s.c. at the base of the tail with a total volume of 200 ml containing 5 mg rH4 formulated in cationic liposomes (CAF01). For boosting studies, mice received a single dose of rH4 injected s.c. at the base of the tail and two weeks later the mice were given one booster vaccination of Ad-H4 in the right footpad. Non-vaccinated control mice received NaCl s.c. at the base of the tail, and the BCG control mice received a single dose of BCG Danish 1331 (2.5610 5 CFU) injected s.c. at the base of the tail.
## Experimental infections
When challenged by the aerosol route, the animals were infected with ,50 CFU of M.tb Erdman/mouse with an inhalation exposure system (Glas-Col, Indiana, USA). Mice were challenged eight weeks after the first vaccination and killed either two weeks post-challenge for immune surveillance or six weeks post-challenge for bacterial counts. Numbers of bacteria in the lung were determined by serial three-fold dilutions of individual whole-organ homogenates in duplicate on 7H11 medium. Colonies were counted after two-three weeks of incubation at 37uC. Protective efficacies were expressed as log 10 bacterial CFU.
## Lymphocyte preparation
Splenocytes and lung lymphocytes were obtained by passage of the organs through a 100 mm nylon cell strainer (BD Pharmingen, USA) followed by two washing procedures using RPMI 1640. Cells in each experiment were cultured in sterile microtiter wells (96-well plates; Nunc, Denmark) containing ,2610 6 cells in 200 ml of RPMI 1640 supplemented with 1% (v/v) premixed penicillin-streptomycin solution (Invitrogen Life Technologies), 1 mM glutamine, and 5% (v/v) fetal calve serum (FCS) at 37uC/ 5% CO 2 . The lymphocyte cultures were further used for ELISA and FACS analysis.
## Antigens for in vitro stimulation
Synthetic overlapping peptides (9-and 18-mers) covering the complete primary structure of Ag85B, TB10.4, and ESAT-6 were synthesized by standard solid-phase methods on a SyRo peptide synthesizer (MultiSynTech, New England) at the JPT Peptide Technologies (Berlin, Germany), or at Schafer-N (Copenhagen, Denmark). Peptides were lyophilized and stored dry at 220uC until reconstitution in PBS.
## Ifn-c enzyme-linked immunosorbent assay (elisa)
Microtiter plates (96 well; Maxisorb; Nunc, Denmark) were coated with 1 mg/ml monoclonal rat anti-murine IFN-c (clone R4-6A2; BD Pharmingen). Free binding sites were blocked with 2% (w/v) milk powder in PBS. Culture supernatants were harvested from lymphocyte cultures after 72 hours of in vitro antigen stimulation and tested in triplicates. IFN-c was detected with a 0.1 mg/ml biotin-labeled rat anti-murine antibody (mAb; clone XMG1.2; BD Pharmingen, USA) and 0.35 mg/ml horseradish peroxidase-conjugated streptavidin (Zymed, Invitrogen, USA). The enzyme reaction was developed with 3.39, 5.59tetramethylbenzidine, hydrogen peroxide (TMB plus; Kementec, Denmark) and stopped with 0.2 M H 2 SO 4. rIFN-c (BD Pharmingen, USA) was used as a standard. Plates were read at 450 nm with an ELISA-reader and analyzed with KC4 3.03 Rev 4 software.
# Flow cytometric analysis
Intracellular cytokine staining procedure: Splenocytes or lung lymphocytes were stimulated for 1 h with 2 mg/ml antigen at 37uC/5% CO 2 and subsequently incubated for 5 h at 37uC with 10 mg/ml brefeldin A (Sigma-Aldrich, USA) at 37uC. The cells were washed in FACS buffer (PBS containing 0.1% sodium azide and 1% FCS) before surface staining with rat anti-mouse antibodies. Cells were washed with FACS buffer before fixation and permeabilization using the BD Cytofix/Cytoperm TM (BD, San Diego, CA, USA) according to the manufacturer's protocol followed by intracellular staining. When staining with FITC-anti-BrdU an extra fixation and permabilisation step, as well as DNAse treatment, was introduced before the intracellular staining. Cells were washed and resuspended in formaldehyde solution 4% (w/v) pH 7.0 (Bie & Berntsen, Denmark) and samples were analyzed on a six-color BD FACSCanto flow cytometer (BD Biosciences, USA). Data analysis was done on FlowJo Software (ß Tree Star, Asland, OR, USA). The following antibodies were used for surface staining: PerCP-Cy.5.5-anti-CD8a (53-6.7), APC-Cy7-anti-CD4 (GK1.5), and FITC-anti-CD44. For intracellular staining the following antibodies were used: PE-anti-TNF-a, APC-anti-IL-2, PE-Cy7-anti-IFN-c, and FITC-anti-BrdU. All antibodies were purchased from BD Pharmingen (San Diego, USA) or eBiosciences (San Diego, USA).
## In vivo cytotoxicity assessed by adoptive transfer of cfse-labeled target cells
Single splenocyte suspensions of CB6F1 mice were obtained by passage through a fine 100 mm nylon cell strainer (BD Pharmingen, USA). Erythrocytes were depleted by lysis in ammonium chloride solution, washed in PBS before resuspension in incomplete RPMI and stained with 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (Sigma-Aldrich, San Louis, USA) at CFSE high (40 mM) or CFSE low (4 mM) concentration for 10 min at 37uC. Excess CFSE was quenched with RPMI containing 10% FCS and subsequently washed in medium without FCS. Next, CFSE high labeled cells were pulsed with TB10.4 3-11, TB10.4 20-28 , or TB10.4 74-88 peptides at a concentration of 10 mg/ml for 1.5 hr at 37uC. After being washed and resuspended in PBS the CFSE high and CFSE low suspensions for each peptide was mixed at equal volumes. Finally, 10610 6 cells per target peptide per mouse were given intravenously into naïve mice and mice vaccinated one week before. Four hours later, adoptively transferred mice were sacrificed. Spleens were removed, homogenized and resuspended in formaldehyde before acquisition on a BD FACSCanto flowcytometer (BD Biosciences, USA). To evaluate the frequency of specific lysis, the ratio of CFSE high and CFSE low of infected mice were compared to naïve control mice and was calculated using the formula (12(%CFSEhigh cells/%CFSE low cells)6100%). For the immunized and naïve groups two mice were used respectively.
[fig] Figure 1: Cytokine frequencies and phenotypic profiles of specific CD4 T cells in Ad-H4, rH4, or rH4/Ad-H4 vaccinated mice one week after final vaccination. CB6F1 mice were vaccinated once with Ad-H4, twice with rH4 in cationic liposomes, or once with rH4 in cationic liposomes followed two weeks later by one Ad-H4 vaccination. Mice were sacrificed one week after the final vaccination and splenocytes were stimulated with Ag85B or TB10.4 peptides prior to staining with anti-CD4, -CD44, -IFN-c, -IL-2, and -TNF-a. (A and C) Frequencies represent IFN-c, IL-2, or TNF-a producing CD44 + CD4 + T cells out of total CD4 T cells specific for Ag85B (A) or TB10.4 (C). Background staining from cells stimulated with medium alone has been subtracted. Data represent the mean+SEM of a minimum of five mice per group with ***, p,0.001, **, p,0.01, and *, p,0.05(2-way ANOVA with Bonferroni posttest). (B and D) IFN-c, IL-2, and TNF-a cytokine profiles of Ag85B (B) or TB10.4 (D) specific CD4 T cells shown as triple, double, or single positive CD4 T cells. The color code for the pies is shown in (E). Dot plots representative of five mice are also shown in (B) and (D), depicting IFN-c expressed by CD4 + CD44 + cells after stimulation with Ag85B (B) or TB10.4 (D). doi:10.1371/journal.pone.0005139.g001 [/fig]
[fig] Figure 3: Functional characterization of the vaccine induced T cells. The specific lysis of TB10.4 3-11 , TB10.4 20-28 , and TB10.4 70-88 loaded cells were determined in an in vivo cytotoxicity assay. Unloaded (CFSE low ) and TB10.4 3-11 , TB10.4 20-28 , or TB10.4 70-88 (CFSE high ) loaded splenocytes from naïve mice were transferred into Ad-H4 (A), rH4 (B), or rH4/Ad-H4 (C) vaccinated mice. (A-C)The amount of splenocytes killed in vivo by cytotoxic T cells specific for either of the TB10.4 target peptides was observed as a reduction in the CFSE high population Percent specific lysis was calculated and is shown in the far right graphs. Non-specific cytotoxicity from non-vaccinated mice has been subtracted (Non-specific killing in non-vaccinated mice of either CFSEhigh or CFSElow cells were from 5-15%). doi:10.1371/journal.pone.0005139.g003 [/fig]
[fig] Figure 4: The protective efficacy and correlation of protection with infection driven immune response in the vaccinated and challenged mice. (A and B) Protection in vaccinated mice (expressed as Log 10 CFU reduction) compared to unvaccinated and BCG vaccinated controls challenged by the aerosol route with virulent M.tb. six weeks after the final vaccination. Six weeks post-challenge, the mice were sacrificed and the bacterial burden (CFU) was measured in the lung or spleen. Data represent the mean+SEM of a minimum of five mice per group in two individual experiments with ***, p,0.001 and **, p,0.01 (2-way ANOVA with Bonferroni posttest). (C) Relation between protection (CFU) and ESAT-6 specific IFN-c producing CD4 T cells is shown as a fitted regression line with the correlation coefficient (R 2 ). Six weeks post challenge, the mice were sacrificed and the lung cells were stimulated with ESAT-6 overlapping peptides prior to staining with anti-CD4, -CD44, and -IFN-c. Frequencies represent IFN-c producing ESAT-6 specific CD44 + CD4 + T cells out of total CD4 T cells. Background staining from cells stimulated with medium alone has been subtracted. Data represent the mean of +SEM of a minimum of five mice per group in two individual experiments. doi:10.1371/journal.pone.0005139.g004 [/fig]
[fig] Figure 5: Cytokine frequencies and phenotypic profiles of specific CD4 and CD8 T cells in non-vaccinated or rH4/Ad-H4 vaccinated mice two weeks after challenge. CB6F1 mice were vaccinated once with rH4 in cationic liposomes followed two weeks later by one Ad-H4 vaccination, and challenged by the aerosol route with virulent M.tb six weeks after the final vaccination. Two weeks after challenge the mice were sacrificed and lung lymphocytes were stimulated with Ag85B or TB10.4 peptides prior to staining with anti-CD4, -CD8, -CD44, -IFN-c, -IL-2, and -TNFa. Frequencies represent IFN-c, IL-2, or TNF-a producing CD44 + CD4 + or CD44 + CD8 + T cells specific for Ag85B (A and B) or TB10.4 (C and D). Background staining from cells stimulated with medium alone has been subtracted. Data represent the mean+SEM of four mice per group with **, p,0.01 (2-way ANOVA with Bonferroni posttest). IFN-c, IL-2, and TNF-a cytokine profiles of Ag85B or TB10.4 specific CD4 and CD8 T cells are shown in pies as triple, double, or single positive CD4 or CD8 T cells. The color code for the pies is the same as shown inFig. 1E. doi:10.1371/journal.pone.0005139.g005 [/fig]
[fig] Figure 6: Recruitment of Ag85B and TB10.4 CD4 and CD8 T cells to the lung. Antigen specific CD4 and CD8 T cells in rH4/Ad-H4 vaccinated mice were measured two weeks before and after challenge. CB6F1 mice were vaccinated once with rH4 in cationic liposomes followed two weeks later by one Ad-H4 vaccination, and challenged by the aerosol route with virulent M.tb six weeks after the final vaccination. Two weeks before challenge and two weeks after challenge mice were sacrificed, and splenocytes and lung lymphocytes, respectively, were stimulated with Ag85B or TB10.4 peptides prior to staining with anti-CD4, -CD8, -CD44, -IFN-c, -IL-2, and -TNF-a. (A-D) The number of cytokine producing T cells is a total number of triple, double, and single positive IFN-c, TNF-a, and IL-2 producing antigen specific CD4 or CD8 T cells either per spleen pre-infection (A [/fig]
[table] Table 1: Frequency of T cell subsets. [/table]
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10.1186/1743-422X-9-125
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CCBY
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3463466
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22721418
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s2orc_pubmed_articles
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Co-circulation of two genotypes of dengue virus serotype 3 in Guangzhou, China, 2009
Dengue is emerging as the most important mosquito borne viral disease in the world. In mainland China, sporadic and large outbreaks of dengue illness caused by the four serotypes of dengue virus (DENV-1 to DENV-4) have been well documented. Guangdong province is the major affected area in China, and DENV-1 has dominantly circulated in Guangdong for a long time. In this study, a family cluster of DENV-3 infection in Guangzhou was described. Three cases were diagnosed as dengue fever based on clinical manifestation, serological and RT-PCR assays. Two DENV-3 strains were isolated in C6/36 cells and the complete genome sequences were determined. Phylogenetic analysis revealed that the new DENV-3 isolates from the family cluster were grouped within genotype III. Considering the fact that several DENV-3 strains within genotype V were also identified in Guangzhou in 2009, at least two genotypes of DENV-3 co-circulated in Guangzhou. Careful investigation and virological analysis should be warranted in the future.
# Background
Dengue is increasing in both frequency and magnitude worldwide, posing a heavy public health and economic burden especially in tropical and subtropical countries. Today, dengue ranks as the most important mosquitoborne viral disease in the world. Annually, up to 50 million human infections occur with 22, 000 deaths mainly in children. Even, population growth, urbanization, international travel, and global warming continuously enhance vector transmission and disease outbreaks [bib_ref] Present and future arboviral threats, Weaver [/bib_ref]. Dengue virus (DENV) contains four serotypes, and each of them can cause a wide spectrum of clinical manifestations, including mild dengue fever (DF), severe dengue haemorrhagic fever (DHF) and deadly dengue shock syndrome (DSS). Although intensive efforts have been made for decades, no preventive vaccines or antiviral drugs is currently available. The pathogenesis of DHF and DSS remains poorly understood. However, secondary infection with another DENV serotypes clearly increased the risk of severe diseases via the mechanism of antibody dependent enhancement (ADE) [bib_ref] Serotype-specific differences in the risk of dengue hemorrhagic fever: an analysis of..., Fried [/bib_ref] [bib_ref] Neutralization and antibody-dependent enhancement of dengue viruses, Halstead [/bib_ref] [bib_ref] Association of increased plateletassociated immunoglobulins with thrombocytopenia and the severity of disease..., Saito [/bib_ref]. Epidemiological and in vivo data also indicated that anti-DENV antibodies mediated pathogenesis of a second heterotypic DENV infection [bib_ref] Cross-reacting antibodies enhance dengue virus infection in humans, Dejnirattisai [/bib_ref] [bib_ref] Relationship of preexisting dengue virus (DV) neutralizing antibody levels to viremia and..., Endy [/bib_ref].
Mainland China has experienced large outbreaks of DF during World War II, after that dengue disappeared for about 30 years. Since 1978, mainland China has seen a resurgence of dengue, epidemics involving hundreds of thousands of people have occurred in many provinces of Southern China, including Hainan, Guangdong, Guangxi, Fujian, Yunnan and Zhejiang provinces [bib_ref] Dengue Fever in mainland China, Wu [/bib_ref] [bib_ref] Molecular epidemiology of dengue viruses in southern China from 1978 to, Wu [/bib_ref] [bib_ref] Dengue in China: a clinical review, Qiu [/bib_ref] [bib_ref] Etiologic and serologic investigations of the 1980 epidemic of dengue fever on..., Li [/bib_ref] [bib_ref] Epidemic situation of dengue fever outbreak in Guangdong province, China, He [/bib_ref] [bib_ref] Current situation and surveillance on dengue fever in China, Wang [/bib_ref]. Currently, DF is listed as the notifiable infectious disease by the Ministry of Health, China. The recent epidemiology of dengue in China is characterized by a 3-5 year cycle. Most cases are DF, and only a few DHF or DSS cases have been reported over the last decade in mainland China [bib_ref] Dengue Fever in mainland China, Wu [/bib_ref] [bib_ref] Molecular epidemiology of dengue viruses in southern China from 1978 to, Wu [/bib_ref] [bib_ref] Epidemic situation of dengue fever outbreak in Guangdong province, China, He [/bib_ref].
In dengue endemic country, the presence of four serotypes of DENV is common, and co-circulation of multiple dengue serotypes in the same area has been well documented [bib_ref] Concurrent infections by all four dengue virus serotypes during an outbreak of..., Bharaj [/bib_ref] [bib_ref] Importation and co-circulation of multiple serotypes of dengue virus in Sarawak, Malaysia, Holmes [/bib_ref] [bib_ref] Common occurrence of concurrent infections by multiple dengue virus serotypes, Lorono-Pino [/bib_ref]. Guangdong province has been recognized as the major affected area of China. Although all four serotypes of DENV have been isolated in China, the dominant serotype circulating in Guangdong is DENV-1, no other serotypes has been recorded since 2001 [bib_ref] Dengue Fever in mainland China, Wu [/bib_ref] [bib_ref] Molecular epidemiology of dengue viruses in southern China from 1978 to, Wu [/bib_ref] [bib_ref] Epidemic situation of dengue fever outbreak in Guangdong province, China, He [/bib_ref] [bib_ref] The origin of dengue viruses caused the DF outbreak in Guangdong province, Chen [/bib_ref]. Large DF outbreaks involving more than 1000 cases caused by DENV-1 have been described in Guangdong, China in 2002 and 2006, respectively [bib_ref] Epidemic situation of dengue fever outbreak in Guangdong province, China, He [/bib_ref] [bib_ref] Molecular characterization of the E gene of dengue virus type 1 isolated..., Zheng [/bib_ref].
In this study, we sought to determine the cause of a family cluster of DF in Guangzhou, Guangdong province, China in 2009, and analyze the possible origin of these emerging isolates responsible for the epidemic.
# Materials and methods
## Case description
On Aug 6, 2009, three adult family members admitted to Guangzhou No.8 People's Hospital as suspected DF cases. The 30-year-old son firstly had a sudden fever with headache, then his father (56-year-old) and mother (50-year-old) fell ill subsequently in the following two days. All the three cases developed typical DF symptoms, including fever, headache, chills, rash, muscle and joint pain, and anorexia. The couples developed diarrhoea, and none of them showed vomiting. The tourniquet tests were all positive. All patients recovered uneventfully and discharged on .
# Ethics statement
The research was approved by the Review Board of Guangzhou No.8 People's Hospital and the Ethical Committee of State Key Laboratory of Pathogen and Biosecurity. Informed consent was obtained from patients.
## Serological assay and rt-pcr
Acute phrase sera were subjected to serological assays using IgM and IgG capture ELISA kit (PanBio, Queensland, Australia) according to the manufacturer's instruction. RT-PCR assays were performed to detect and typing of DENVs as previously described [bib_ref] Molecular evolution and epidemiology of dengue-3 viruses, Lanciotti [/bib_ref].
## Virus isolation and identification
Acute phase sera from the three patients were inoculated in C6/36 mosquito cells (Aedesalbopitus clone) and maintained in 1640 medium (Life Technologies, CA, USA) supplement with 2% fetal bovine serum (Life Technologies) at 28°C in 5% CO 2 . When complete cytopathic effects (CPE) were observed, culture supernatants from positive samples were collected and stored at −70°C until use. Indirect immunofluscence assay (IFA) was performed as previously described [bib_ref] A broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within..., Deng [/bib_ref].
## Sequencing of complete genome of denv-3 isolates
The viral RNA was extracted from 200 μl of DENV-3 infected C6/36 culture supernatant using Purelink RNA mini kit (Life Technologies) in accordance with the manufacturer's instructions. A total of 11 overlapping amplicons spanning the complete genomic region were amplified using 11 pairs of primers. The PCR products were sequenced and assembled. The 5' and 3' untranslated regions (UTRs) of viral genome of each isolate were determined using a rapid amplification of either 5' or 3' cDNA ends (RACE) kit (Roche, Mannheim, Germany) followed the manufacturer's recommendation. All primers can be found in [fig_ref] Table 1: Primers used for sequencing reactions Primer location refers to DENV-3 strain 80-2... [/fig_ref].
## Sequence alignment and phylogenetic analysis
The complete nucleotide sequences of the complete sequences of coding region or envelope (E) gene of global DENV-3 strains were retrieved from GenBank [fig_ref] Table 2: DENV-3 isolates investigated in this study [/fig_ref]. Multiple sequence alignment was carried out employing the CLUSTAL W program [bib_ref] Clustal W and Clustal X version 2.0, Larkin [/bib_ref]. Phylogenetic analyses based on the nucleotide sequence of complete coding region of 44 DENV-3 or complete envelope gene
# Results
All three family members were diagnosed as DF according to the new guideline of World Health Organization. Laboratory tests disclosed low WBC and lymphocytes counts for all the three cases. Normal platelet counts were recorded for two cases, while that of the mother was low. None of the patients presented plasma leakage, severe bleeding, or severe organ involvement. All cases recovered in a week post admission. The acute phase sera from all the three family members were positive for dengue IgM antibody, but negative for IgG antibody. Two of the three cases were positive for DENV-specific RT-PCR. DNA sequencing of the PCR products and blast analysis revealed closely homologous with DENV-3. Considering the fact that DENV-3 has not been described in Guangdong for many years, all the three samples were inoculated into C6/36 cells to isolate the viruses. Typical CPE were observed six or seven days post inoculation for two of three samples. After another passage in C6/36 cells, two strains were isolated and named with GZ1D3 and GZ2D3, respectively. Both strains were further confirmed by IFA using dengue specific monoclonal antibody.
Finally, the complete genome sequences of the isolates were determined, assembled and submitted to GenBank [GenBank: GU363549; JN662391]. Both strains were highly homologous (99.9%) with only three nucleotide differences. Phylogenetic analysis based on the complete envelope gene classified DENV-3 isolates into five genotypes , which was confirmed by the Bayesian method (Additional file 1: . Phylogenetic tree based on the complete sequence of coding region of DENV-3 genome showed the same genotype classification . The newly isolated DENV-3 strains , which indicated that two genotypes of DENV-3 were co-circulating in Guangdong, 2009.
# Discussion
In the present study, a family cluster of DENV-3 infections in Guangzhou, China was described. Three family members were diagnosed as DF, and all recovered finally. All the three family members recalled mosquito biting before illness, and none of them went aboard or on trip recently. Although family cluster of vector-borne diseases have been intensively described [bib_ref] Crimean-Congo hemorrhagic fever in Albania, Papa [/bib_ref] [bib_ref] Family cluster of Rocky Mountain spotted fever, Jones [/bib_ref] , information regarding DF family cluster is limited. There is no doubt that any cluster of cases is of great concern and should be thoroughly investigated. Dengue can cause both the large epidemics and sporadic infections. The recognition of clustering of disease is important for medical providers and public health personnel in treating and controlling the disease, because multiple infections can occur simultaneously or following an index case. In this study, strict mosquito control measures were initiated immediately after confirmation of the DF cases, and no further cases were reported nearby thereafter.
Whether dengue is endemic in Guangdong remains disputable. Most dengue epidemics in Guangdong were initiated by the introduction of virus from imported cases [bib_ref] Epidemic situation of dengue fever outbreak in Guangdong province, China, He [/bib_ref] [bib_ref] The origin of dengue viruses caused the DF outbreak in Guangdong province, Chen [/bib_ref] [bib_ref] Molecular characterization of the E gene of dengue virus type 1 isolated..., Zheng [/bib_ref] [bib_ref] Epidemiological analysis of imported cases of dengue fever in Guangdong province and..., Yang [/bib_ref] , however, in this study none of the family member travelled aboard. Further epidemiology investigation also did not identify imported case nearby either. The origin of these DENV-3 isolate is really interesting. Since DENV-1 has circulated in Guangzhou for about ten years, the new DENV-3 has potential to increase the rate of secondary heterotypic infection. Furthermore, the previous studies showed that epidemic DHF has appeared in association with DENV-3 [bib_ref] Potential risk for dengue hemorrhagic fever: the isolation of serotype dengue-3 in..., Briseno-Garcia [/bib_ref] [bib_ref] Emergence and global spread of a dengue serotype 3, subtype III virus, Messer [/bib_ref] [bib_ref] Comparative complete genome analysis of dengue virus type 3 circulating in India..., Sharma [/bib_ref]. In the Americas, DENV-3 presented greater epidemic potential and virulence [bib_ref] Molecular evolution and epidemiology of dengue-3 viruses, Lanciotti [/bib_ref]. Whatever, the emerging DENV-3 in Guangzhou might represent a risk factor for severe dengue illness, careful investigation and surveillance should be warranted in the future.
(See figure on previous page.) Phylogenetic tree based on the complete envelope gene from 58 DENV-3 strains sampled globally. The evolutionary history was inferred using the neighbor-joining method with MEGA 5 software [bib_ref] MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum..., Tamura [/bib_ref]. Each strain is abbreviated with strain name and country of origin followed by the year of isolation. Bootstrap values greater than 0.9 based on 1000 replicates are shown for key nodes. The tree was rooted using DENV-1 strain Nauru, DENV-2 strain New Guinea C and DENV-4 strain B5 as outgroups. The newly DENV-3 isolates in the study are marked with red squares and other Chinese DENV-3 isolates taken for comparison are marked with blue squares.
Most importantly, phylogenetic analysis demonstrated that at least two different genotypes of DENV-3 were cocirculated in Guangdong, China in 2009, which partly agree with the findings of the previous study. Five genotypes of DENV-3 have been reported [bib_ref] Molecular evolution and epidemiology of dengue-3 viruses, Lanciotti [/bib_ref] [bib_ref] Extinction and rapid emergence of strains of dengue 3 virus during an..., Wittke [/bib_ref]. The newly isolates in Guangdong form a distinct cluster with other Chinese isolates sampled from Zhejiang province in 2009 [bib_ref] Dengue virus serotype 3 subtype III, Sun [/bib_ref]. All these genotype III DENV-3 strains were closely related to those sampled in India in 2007 and 2008, suggesting these Chinese isolates might be imported from India. Previously, the introduction of new genotype III of DENV-3 has been recognized as one of the factors leading to the emergence of DHF in Pakistan and India [bib_ref] Comparative complete genome analysis of dengue virus type 3 circulating in India..., Sharma [/bib_ref] [bib_ref] Cocirculations of two genotypes of dengue virus in 2006 out-break of dengue..., Khan [/bib_ref]. These strains are therefore interesting and their virological characterization and virulence analyses are currently underway.
However, three additional DENV-3 strains (09/GZ/1081, 09GZ/1483, and 09/GZ/10806), belonging to genotype V, were also identified in Guangzhou, 2009 in a separate study. In addition, two strains (07CSHL001 and DTID-ZJU04) sampled from China, were grouped within genotype II. The origin of these isolates is difficult to determine without further information. The situation that multiple genotypes of DENV-3 co-circulated in Guangzhou, China deserves close concern and careful investigation.
## Additional file
Additional file 1: . Phylogenetic tree based on the complete envelope gene from 58 DENV-3 strains by Bayesian method. The evolutionary history was inferred using BEAST 1.7.1 software. The tree was rooted using DENV-1 strain Nauru, DENV-2 strain New Guinea C and DENV-4 strain B5 as outgroups. The newly DENV-3 isolates in the study are marked with red squares and other Chinese DENV-3 isolates taken for comparison are marked with blue squares.
(See figure on previous page.) Phylogenetic tree based on complete sequences of coding region of viral genome from 44 DENV-3 strains sampled globally. The evolutionary history was inferred using the neighbor-joining method with MEGA 5 software [bib_ref] MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum..., Tamura [/bib_ref]. Bootstrap values greater than 0.9 based on 1000 replicates are shown for key nodes. The tree was rooted using DENV-1 strain Nauru, DENV-2 strain New Guinea C and DENV-4 strain B5 as outgroups. The newly DENV-3 isolates in the study are marked with red squares and other Chinese DENV-3 isolates taken for comparison are marked with blue squares.
[table] Table 1: Primers used for sequencing reactions Primer location refers to DENV-3 strain 80-2 [GenBank: AF317645]. b Primer F11 was also used as 3'RACE primer. [/table]
[table] Table 2: DENV-3 isolates investigated in this study [/table]
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A comparison of owner perceived and measured body condition, feeding and exercise in sport and pet dogs
Dog obesity is a significant problem in the US and elsewhere.The purpose was to evaluate factors contributing to pet obesity in sport and pet dog owners.Owners were recruited over social media to answer a questionnaire regarding demographics, health, body condition, feeding, exercise and dog related expenses.Owners identified as pet or sport dog owners.We asked owners to measure the pelvic circumference and hock to stifle length in their dogs in order to calculate percent fat.Owners reported that their dogs were in "ideal" body condition.However, percent fat calculated from owner measurements was significantly different between groups (Sport: 16 ± 10%fat; Pet: 24 ± 10% fat; p < 0.05) and revealed that over 50% of the dogs were over fat.Owners reported feeding dogs a range of 413 to 1,133 Kilocalories (Kcal) per day that correlated well with dog size (R = 0.58; p < 0.05).The size of the treats fed was smaller in the Sport dogs (treat was pinky nail to thumbnail sized) than in Pet dogs (treat was bigger than thumb to larger than palm).Owners reported walking their dogs on leash every day for 15-45 min per session.Overall, owners did a poor job in identifying correct body condition of their dogs.This is concerning because 50% of the dogs were over fat.Better understanding of correct body condition and feeding for the level of physical activity is still a critical issue in controlling obesity in pet dogs.
# Introduction
Research in the US, Australia and Europe estimates that 40-60% of pet dogs are obese resulting in lifelong health problems including arthritis, diabetes, poor quality of life and reduced longevity [bib_ref] Quality of life is reduced in obese dogs but improves after successful..., German [/bib_ref].A number of factors likely contribute to this problem, including owner's knowledge of correct body condition, over-feeding meals and treats and lack of regular exercise [bib_ref] Perceptions of body condition, diet and exercise by sports dog owners and..., Kluess [/bib_ref].Previous work from this lab suggested that owner knowledge of correct body condition was a significant barrier to intervening in the dog's body condition with food and exercise [bib_ref] Perceptions of body condition, diet and exercise by sports dog owners and..., Kluess [/bib_ref].In the current study we wished to see if we could improve owners' identification of their dog's body condition using several strategies including categories (ideal, overweight, etc), a Purina 9 point scale with descriptions, but without the category labels and having the person measure their own dog.We also investigated feeding, exercise and expenditures related to dog ownership.This study focused specifically on dogs that participate regularly in canine sports compared to pet dogs that do not participate in sports.Dogs that compete in canine sports have a high level of physical activity during competitions and presumably when practicing for the event.Canine Sports can include companion 10.3389/fvets. Frontiers in Veterinary Science 02 frontiersin.orgsports where the dog and person perform the sport activity together; examples include obedience, rally obedience, agility, Internationale Prufungs-Ordung (IPO) and tracking.Performance sports involve the person, but the dog does most of the physical activity in frisbee sports, scentwork, field trials, herding, lure coursing, and dock diving.Participation in canine sports is increasing in popularity with the American Kennel Club reporting 3 million entries in 22,000 events last year.We hypothesized that sports dog owners would more correctly estimate body condition, sports dogs would have more appropriate feeding regimes, Sports dogs would exercise more than pet dogs and spend more on their dogs.
# Materials and methods
This study was approved by the Auburn University Institutional Review Board and the IACUC.Owners were recruited over social media to answer a Qualtrics questionnaire that included the Dogs and Walking Survey (DAWGS) ( 5) that was open from February 2021 to Dec 2021.The current study is part of a larger set of data.Owners self-identified as pet owners with no sports participation, previous sport owners or current sport dog owners.
## Dog demographics and health
Dog demographics included the age, relative size, breed, and sterilization status.We asked about previous diagnoses for temporary or permanent health conditions including orthopedic problems, cardiorespiratory problems, and metabolic disease.We also asked about the number of times they visited the veterinarian in the past year.
## Dog body condition
We asked owners to evaluate body condition using a scale that described visual cues to determine body condition (Purina Body Condition Scale).The scale usually includes labels for each of the categories ("too thin, " "ideal, " and "too heavy") but we removed those so that owners would use the descriptions and images of the scale and not the labels to judge their dog's body condition.We also asked them how they would qualitatively rate their dogs body condition (underweight, ideal, overweight, obese).To quantitate body condition, we asked owners to measure the pelvic circumference and hock to stifle length in their dogs (see Figure [fig_ref] FIGURE 1: Questions for owners regarding the body condition of their dog [/fig_ref].We used this data to calculate percent fat using the following equations ( 6 The anthropometric measurements for percent fat are validated against DEXA and the Purina Body condition scale.
## Dog feeding
For this section, we asked owners about the type of food (dry, canned, raw, combination, etc.), the brand of food, the formula of food, the number of kilocalories per cup, the amount fed per day, and the number of times fed per day.We used this information to calculate the kilocalories per day for the kibble fed dogs.For treats, we asked about the number of treats, the type of treat, the number of treats given when actively training the dog, and the size of the treat (based on a person's hand; for example: pinky nail size, thumbnail sized, etc).
## Dog exercise
The amount of exercise a dog received was determined using some basic questions and the DAWGS survey [bib_ref] Development and psychometric testing of the dogs and WalkinG survey (DAWGS), Richards [/bib_ref].The basic questions included how many times a week they exercised the dog, the main type of exercise, the length of time per session, and the
## Sport dog exercise
For owners that indicated that they currently participate in sports, we asked additional questions regarding their sports experience and the number of hours per week they spend on sports-specific training, conditioning or foundation work, walking, running or fetch.We also asked how many days per week the dog was involved in sports or physical activity.
# Dog related expenses
We asked about the amount of money spent on dog care like food, treats, toys, training classes/lessons, and equipment like collars, leashes, etc.
# Data analysis
We used G*Power 3.1.9.7 to calculate the sample size for a MANOVA (power = 0.95, effect size f 2 V = 0.0625).We estimated a total sample size of 172 was sufficient for the power required with 4 groups.We ran a GLM MANOVA with dependent variables percent fat, age, sex/sterility status, size of treats and minutes walked per week using IBM SPSS 29.0.0.0.We did a Tukey post hoc test when appropriate.All descriptive data were summarized using mean ± standard deviation.For some variables, we ran a Pearson product-moment correlation using GraphPad Prism 9.4.1.We also ran 1-way ANOVAS with a Tukey post hoc test when appropriate.The alpha level was set a priori at 0.05.
# Results
## Dog demographics
Overall, 284 self-reported dog owners responded to our online survey.251 owners responded to the questions regarding location and 246 were from the United States, four from Canada, and one from Europe.Owners from the USA represented 36 states with the largest percent from Alabama, Florida, and Georgia (50%) (see Figure [fig_ref] FIGURE 2: Regional distribution of respondents to the internet survey [/fig_ref] for distribution by state).We asked owners if they were current dog sports participants (Sport), a pet owner (Pet) or a previous sport dog owner (Previous Sport).Participants that did not answer the question were categorized as "Undeclared." Owners identifying as sport dog owners reported participating in an average of 4 ± 2 dog sports (range: 1-11 sports).
The MANOVA results indicated that we had a main effect of percent body fat (p < 0.001, F = 21.27),age (p = 0.007, F = 27.98), and size of the treats (p = 0.002, F = 15.96).The average age of the dogs in the undeclared, sport group and the previous sport group was 5 ± 2 years, but it was 6 ± 3 years for the pet group (p = 0.007, F = 5.109).The sterilization status of the dogs by group is presented in Figure [fig_ref] FIGURE 3: Sterilization status of dog in each group [/fig_ref].The Undeclared (74%, 87/120 answered), Pet (91%, 58/58 answered) and Previous Sport owners (88%, 16/16 answered) reported the highest percentage of neutered or spayed dogs.The Sport group (90/90 answered) had the highest percentage of intact dogs (42%).The entire sample represented 68 breeds (including mixed breed as a category).The percent of owner-reported pure breed dogs was 66% (n = 87) for Undeclared, 74% (n = 90) for Sport, 34% (n = 58) for Pet and 10% (n = 15) for Previous Sport groups.The owners reported the average size of their dog as medium sized.
## Dog health
Owners were asked about previous temporary and permanent diagnoses.Most owners reported no previous health problems in their Reported physical activities performed with the dog by group.
dogs (Undeclared: 87%; Sport: 89%; Pet: 84%; Previous Sport: 72%).Some temporary orthopedic injuries were reported (Undeclared: 9%; Sport: 8%; Pet: 12%; Previous Sport: 11%).Four dogs were reported as having permanent orthopedic injuries, five had other chronic conditions, one had a permanent cardiorespiratory condition.When asked about the number of vet visits last year, most owners reported between 1-3 vet visits (see Figure.A small percentage of owners in the Undeclared (13%; n = 11/87), Pet (7%, n = 4/58) and Previous Sport (6%, n = 1/16) groups reported no vet visits in the last year.
## Dog body condition
We asked owners about their dog's body condition using several methods.We asked if they considered their dog underweight, ideal, overweight or obese.All groups reported, on average, their dogs were in the "ideal" category.We also asked participants to look at a Purina Body condition scale without the weight categories (underweight, ideal etc).Owners in all groups reported a 5 ± 1 score (ideal on Purina scale).Participants also measured their dog's pelvic circumference and hock to stifle length.In the Undeclared group, only three owners answered these questions, so this group was excluded from the analysis.We calculated percent fat from these measures.Percent fat calculated from owner measurements was significantly different between groups (Sport: 16 ± 10%fat, n = 71; Pet: 24 ± 10% fat, n = 46; Previous Sport: 23 ± 7% fat, n = 13; Sport different from Pet p < 0.001; F = 21.27).Figure [fig_ref] FIGURE 5: Calculated body fat percentage per group organized by ranges of percent fat [/fig_ref] shows the percentage of dogs that fell into percent fat categories.The undeclared group was left out of the analysis because of a large number of missing values.The correlation (Pearson Product Moment) was r = 0.25 (p < 0.05) for the owner-determined Purina Body condition score and calculated percent fat.Overall, 55% of the Sports dog owners correctly identified their dogs body condition (categories vs. % fat), but only 13% of the pet dog owners correctly identified the body condition of their dog.We investigated the possibility that spay/neuter status played a role in the pet versus sport differences in body condition.The calculated percent fat for intact males was 15 ± 7% fat, neutered males were 18 ± 9% fat, intact females were 23 ± 16% fat, neutered females were 22 ± 10% fat.There were no significant differences between intact and spay/neutered dogs for percent fat and, when sex was taken into account, there were also no significant differences (p = 0.15, F = 1.92).
## Dog exercise
Days per week an owner exercised their dog was similar in the Undeclared (9 ± 6 times/week, n = 30/120), Sport (7 ± 4 times/week, n = 72/90), Pet groups (8 ± 6 times/week, n = 58/58) and Previous Sport groups (8 ± 5 times/week, n = 11/16) (F = 0.72, p = 0.54).The types of exercise by group are summarized in Figure.The main type of exercise was walking on a lead.Playing fetch and walking or running off leash was also similar between the groups.The Sport group had a The number of veterinary visits per group.The Undeclared group was not analyzed because these questions were not answered.
## Dog feeding
Most owners fed dry kibble food (Undeclared: 60%; Sport: 52%; Pet: 74%; Previous Sport: 63%).The most popular brand was Purina (Undeclared: 22%; Sport: 19%; Pet: 29%; Previous Sport: 31%).Feeding a raw diet was more common in the Sport group (Undeclared: 10%; Sport: 15%; Pet: 2%; Previous Sport: 0%). Figure [fig_ref] FIGURE 7: Pictoral description of reported feeding methods in the Pet and the Sport... [/fig_ref] is a summary of diets used by the Sport and Pet groups.Sport owners (kibble fed) reported feeding dogs a range of 497 ± 196 kcal (small dogs) to 1,109 ± 465 kcal (large dogs) and pet owners (kibble fed) reported 413 ± 158 kcal (small dogs) to 1,133 ± 434 kcal (large dogs) (Undeclared: 447 to 926; Previous Sport: 373 to 1,249 kcal small to large).The correlation between dog size and Kcal was R = 0.58 (n = 105; p < 0.05).Most owners fed the dog 2x/day (Undeclared: 77%; Sport: 86%; Pet: 60%; Previous Sport: 71%).Free feeding was more common in the Pet group (undeclared: 8%; pet:18%; sport: 1%; previous sport: 0%).Owners reported feeding treats as snacks 1-2x/day (Undeclared: 47%; Sport: 41%, Pet: 47%; Previous Sport: 31%).Undeclared (18%), Sport owners (12%) and Previous Sport (19%) reported feeding no treats as snacks compared to Pet owners (3%).The size of the treat also was smaller in the Undeclared (pinky nail to thumbnail sized; 68%) and Sport dogs (60%) than in Pet dogs (28%) and Previous Sport dogs (25%).Pet owners reported feeding larger sized treats (bigger than thumb to larger than palm; 34%) compared to Sport owners (12%) (Undeclared: 0%, Previous Sport: 13%) (p = 0.002, F = 6.426).
# Dog related expenses
We looked at total dog costs per month, undeclared ($419 ± 505) and Sport groups ($321 ± 167) spent more than the Pet ($123 ± 82; p < 0.05 different from Undeclared and Sport; F = 12.26, p < 0.01).
# Discussion
A strength of this study was we were able to sample dog owners around the US and a small number in Canada.We also included actual measurements of the dog so body fatness could be directly assessed.The most important finding was that most owners perceived their dogs to be in an "ideal" body condition, but measurements revealed that over 50% of the dogs were over fat.In general, pet dogs were considerably more over-fat compared to sports dogs.Dog exercise was consistent across groups and suggested that owners walked with their dogs most days of the week for 15 to 45 min.The most common type of food fed was dry kibble for many dogs and the calories that the owners reported feeding was consistent across groups.The number of treats fed was consistent across groups, but the pet group reported feeding a larger size of treat.The Sport group spent more money on their dogs including food, toys, leashes and training.Despite considerable commitment by the owners to maintain their dog's health and well-being, many of the dogs were over fat and owner's assessment of their dog's body condition was very flawed in the sport and pet groups.
## Dog demographics and health
The sample represented around 38 states and two regions of Canada.The largest representation was on the east coast of the US.The dogs were on average middle aged, spayed/neutered and medium sized.We did see an effect of age among groups, with the pet group having a slightly higher age than the other groups.However, this is a statistical difference without meaning because the means were 5 years versus 6 years.We had a large representation of breeds with no one breed being predominant in any owner category.Owners reported that their dogs were generally healthy and visited the vet at least 1-3 times per year.This finding was consistent with previous research suggesting that at least 40% of pet owners visited the veterinarian at least once per year (7)(AVMA 2017-2018).We reported that 7% of Pet owners reported no contact with the veterinarian.This was higher than reported by Bir et al. [bib_ref] Familiarity and use of veterinary services by US resident dog and cat..., Bir [/bib_ref] , but lower than reported by the AVMA US Pet Ownership Statistics 2017-2018.
## Dog body condition
One of the most concerning findings in this study was that 55% of the Sport dog owners correctly identified their dogs body condition, but only 13% of the pet dog owners correctly identified the body condition of their dog.The percent correctly identifying body condition in the Sports dog owners was consistent with our previous work (4) and the work of others (8-10).Thirteen percent correct in the Pet owner group was very low and of considerable concern because many of these dogs were significantly over fat according to the owner reported measurements.Our hopes that owners would be able to use the Purina scale descriptions effectively was not realized, since most owners reported their dogs had" ideal" body condition.The finding that good descriptions of body condition do not overcome owners preconceived notions about their dog's body condition was consistent with Eastland-Jones et al. [bib_ref] Owner misperception of canine body condition persists despite use of a body..., Eastland-Jones [/bib_ref].It appears that more education is required regarding the use of the Purina scale or other measurements to optimize assessment of body condition by pet owners.It is unlikely that an owner would implement a change in feeding or exercise if they could not identify their dog as overfat.
## Dog exercise
In general, exercise was very comparable between Pet and Sport dogs.The most common exercise in all groups was walking and most owners reported walking their dogs every day for 15-45 min per session.This is consistent with the recommendations for physical activity for dogs and humans (11).Our participants reported walking with their dog more than participants in Christian et al.and a similar amount compared to Hoerster et al. [bib_ref] Jog with your dog: dog owner exercise routines predict dog exercise routines..., Hoerster [/bib_ref] and Banton et al. ( 14), but less than Kinsman et al. .Based on a write in question from our previous study (4), we asked several questions about activities that the dog did without the owner, but found no difference for time reported for activities without the owner.The Sport group spent 1-3 h sport training per week and 0-3 h on conditioning/foundation exercises.This emphasizes that sport dogs get a reasonable amount of exercise that is specific to their sport, in addition to traditional activities like walking [bib_ref] Epidemiology of injury among handlers and dogs competing in the sport of..., Kerr [/bib_ref].Using the DAWGS survey, the overall positivity about dog walking and positivity about the outcomes from dog walking was similar among groups and consistent with other studies [bib_ref] Influences of dog attachment and dog walking on reducing loneliness during the..., Lee [/bib_ref] [bib_ref] Dogs, physical activity, and walking (dogs PAW): acceptability and feasibility of a..., Richards [/bib_ref].
## Dog feeding
Most owners fed dry kibble food and the most popular brand was Purina.Feeding a raw diet was more common in the Sport group.Dinallo et al. [bib_ref] Internet survey of feeding, dietary supplement, and rehabilitative medical management use in..., Dinallo [/bib_ref] reported that 61% of agility competitors fed commercial dry kibble and 25% fed raw or other homemade food.This was similar in flyball competitors (33% fed raw; 61% fed kibble) (20).Sport and Pet owners reported feeding dogs a range of approximately 400 to 1,130 kilocalories (Kcal).There was a moderate correlation between dog size and kilocalories fed.This correlation is good because it suggests at least some adherence to the idea that a smaller dog requires fewer kilocalories.Most owners fed the dog two times per day.This practice is consistent with good feeding practices from studies comparing obese to non-obese dogs [bib_ref] Owner misperception of canine body condition persists despite use of a body..., Eastland-Jones [/bib_ref] [bib_ref] / jn/136.7.1951S 23. Robertson ID. The association of exercise, diet and other..., Bland [/bib_ref].Free feeding was more common in the Pet group but practiced minimally in the Sport group.Feeding of treats was an area that was different between Sport and Pet owners.We specifically asked about treats as snacks versus treats used for training.Most owners reported feeding treats as snacks one to two times per day, but a reasonable percent of Sports owners said they did not feed treats as snacks.We asked about the size of the treat using a person's hand as a reference.The majority of Sports dog owners reported a treat of a pinky nail to thumbnail sized.However, Pet owners reported feeding treats that were bigger than thumb to larger than palm.A popular large sized treat can add 125 kilocalories or more to a dog's daily intake.It is well understood that snack feeding can contribute to obesity in dogs [bib_ref] / jn/136.7.1951S 23. Robertson ID. The association of exercise, diet and other..., Bland [/bib_ref] [bib_ref] A survey of dog Owners' attitudes toward treats, Morelli [/bib_ref].Therefore, it is important to counsel owners to curb this practice of using treats as snacks for their dog.
## Dog expenses
Overall, the Sport group spent more than twice as much in total dog costs per month compared to the Pet group.This is supported by the idea that owners participating in dog sports activities have a "culture of commitment" that often involves considerable sacrifice of money and time for the dog [bib_ref] If it weren't for my hobby, I'd have a life: dog sports,..., Gillespie [/bib_ref].However, spending money on the dog's comfort and well-being is not exclusive to sport and often relates to an owner's perceived level of dog-human companionship [bib_ref] Understanding dog-human companionship, Dotson [/bib_ref] [bib_ref] Equations based on anthropometric measurements for adipose tissue, body fat, or body..., Holbrook [/bib_ref].
# Limitations
The anthropometric measurements in this study were taken by the owner rather than a researcher due to the questionnaire based nature of the study.It is possible that owners made measurement errors in determining the pelvic circumference and hock to stifle length.However, it is unlikely that the error was subjectively biased because the owner was not aware of the results of the percent fat calculation from the measurements.It is not common to report the training of people taking anthropometric measurements in humans (28) and dogs [bib_ref] Supplementing five-point body condition score with body fat percentage increases the sensitivity..., Li [/bib_ref] [bib_ref] Effect of breed on body composition and comparison between various methods to..., Jeusette [/bib_ref] because it is generally considered a fairly simple measure.Another limitation was the assessment of intensity of exercise in the dogs.We were unable to assess relative intensity (low, moderate, vigorous) from the questionnaire information.In humans, exercise intensity is well documented for a variety of activities, but, in dogs, this literature dos not exist and is likely complicated by the large variation in dog size in different breeds.In the future, the use of wearables for dogs may improve the estimation of exercise intensity.
# Conclusion
Despite considerable time and financial commitment to their dogs, owners in the Sport and Pet groups did a poor job at correctly assessing their dogs body condition.This is a concerning finding since it is unlikely that a person that perceives their dogs body condition to be "ideal" will implement changes in feeding or exercise.The owners in this study generally did a creditable job in regularly exercising their dogs and most fed their dogs using accepted practices to maintain body condition.However, the size of treats was larger in the most over fat dogs.Better understanding of correct feeding for the level of physical activity is still a critical issue in controlling obesity in pet dogs.
[fig] FIGURE 1: Questions for owners regarding the body condition of their dog.(A).The Purina 9-point scale with the categories removed.(B).Instructions and diagram for owner measurements of their dog. [/fig]
[fig] FIGURE 2: Regional distribution of respondents to the internet survey.The number indicates the number of respondents per state.33 respondents did not indicate their geographical location. [/fig]
[fig] FIGURE 5: Calculated body fat percentage per group organized by ranges of percent fat.*p < 0.05 mean differences between the Pet group and Sport group. [/fig]
[fig] FIGURE 3: Sterilization status of dog in each group. [/fig]
[fig] FIGURE 7: Pictoral description of reported feeding methods in the Pet and the Sport group. [/fig]
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10.1155/2017/2318476
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CCBY
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5514319
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28744462
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s2orc_pubmed_articles
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Prevalence and Genetic Characterization of Cryptosporidium Infection in Java Sparrows (Lonchura oryzivora) in Northern China
Cryptosporidiosis is a cosmopolitan parasitosis that affects a wide range of hosts including birds. As information concerning Cryptosporidium in birds is limited, the present study examined the prevalence and genotypes of Cryptosporidium in Java sparrows in Beijing and Shangqiu, northern China. Three hundred and fifty fecal samples were collected from Java sparrows (Lonchura oryzivora, 225 white Java sparrows and 125 gray Java sparrows) in Beijing and Shangqiu in October 2015, and the samples were examined by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene. The overall Cryptosporidium prevalence is 13.42% (47/350), with 16.44% (37/225) in white Java sparrows and 8.00% (10/125) in gray Java sparrows. Cryptosporidium prevalence was 9.82% (16/163) in Java sparrows from Beijing and 16.58% (31/187) in Java sparrows from Shangqiu. The prevalence of Cryptosporidium in females and males was 40.63% (26/64) and 7.34% (21/286), respectively. The Cryptosporidium prevalence in Java sparrows of different ages varied from 10.47% to 16.33%. Sequence analysis of the SSU rRNA gene revealed that all the samples represented C. baileyi. This is the first report of Cryptosporidium in gray Java sparrows in China, which extend the host range for C. baileyi. These results provide baseline information for further studies of molecular epidemiology and control of Cryptosporidium infection in poultry in China.
# Introduction
Enteric parasite Cryptosporidium is one of the most important causative agents of gastrointestinal illnesses in humans and animals [bib_ref] Molecular investigation of Cryptosporidium in small caged pets in northeast China: host..., Li [/bib_ref] [bib_ref] Prevalence and genotyping of Cryptosporidium infection in pet parrots in North China, Zhang [/bib_ref] [bib_ref] First report of zoonotic Cryptosporidium spp, Zhao [/bib_ref]. Food and water contaminated by Cryptosporidium oocysts are the most important resources for human infection [bib_ref] First report of zoonotic Cryptosporidium spp, Zhao [/bib_ref] [bib_ref] Zoonotic potential and molecular epidemiology of Giardia species and giardiasis, Feng [/bib_ref] [bib_ref] Molecular epidemiology of cryptosporidiosis: an update, Xiao [/bib_ref]. Moreover, it can be transmitted to humans by direct contact with infected animals [bib_ref] First report of zoonotic Cryptosporidium spp, Zhao [/bib_ref] [bib_ref] Cryptosporidiosis and Cryptosporidium species in animals and humans: a thirty colour rainbow?, Šlapeta [/bib_ref]. Human infection with Cryptosporidium may show symptoms of diarrheal illness and some other severe diseases. Cryptosporidium infection in birds may present symptoms of respiratory disease, digestive symptoms, and renal disease [bib_ref] Cryptosporidium in birds, fish and amphibians, Ryan [/bib_ref] [bib_ref] Cryptosporidium Taxonomy: recent advances and implications for public health, Xiao [/bib_ref].
Cryptosporidium infection in birds was firstly recorded in 1929. So far, over 30 avian species have been reported as the hosts of Cryptosporidium in the world [bib_ref] Cryptosporidium in birds, fish and amphibians, Ryan [/bib_ref]. Respiratory symptoms frequently occur in birds, and this infection leads to higher morbidity and mortality in birds, especially in broiler chickens [bib_ref] Cryptosporidium in birds, fish and amphibians, Ryan [/bib_ref]. Recently, more than 17 Cryptosporidium species/genotypes (C. galli, C. baileyi, C. meleagridis, C. hominis, C. parvum, C. muris, Cryptosporidium avian I-V, goose genotypes I-IV, duck genotype, and Eurasian woodcock genotype) have been recorded in birds worldwide [bib_ref] Cryptosporidium in birds, fish and amphibians, Ryan [/bib_ref] [bib_ref] Cryptosporidium spp. parasitize exotic birds that are commercialized in markets, commercial aviaries,..., Gomes [/bib_ref] [bib_ref] Cryptosporidium infections in birds -a review, Nakamura [/bib_ref] [bib_ref] Molecular characterization of Cryptosporidium spp. from fecal samples of birds kept in..., Nakamura [/bib_ref] [bib_ref] Cryptosporidium spp. in pet birds: genetic diversity and potential public health significance, Qi [/bib_ref] [bib_ref] Identification of novel Cryptosporidium genotypes from the Czech Republic, Ryan [/bib_ref]. Of these, C. hominis, C. parvum, and C. meleagridis are wellknown causative agents of human infection [bib_ref] Cryptosporidium spp. in pet birds: genetic diversity and potential public health significance, Qi [/bib_ref]. In view of such severe situations, a number of studies concerning Cryptosporidium prevalence and genotypes in birds have been conducted worldwide. In China, a few studies on Cryptosporidium infection in birds have been reported in Henan [bib_ref] Cryptosporidium spp. in pet birds: genetic diversity and potential public health significance, Qi [/bib_ref] , Qinghai [bib_ref] Cryptosporidium spp. in quails (Coturnix coturnix japonica) in Henan, China: molecular characterization..., Wang [/bib_ref] , and Zhejiang [bib_ref] Cryptosporidiosis in broiler chickens in Zhejiang Province, China: molecular BioMed Research International..., Wang [/bib_ref] provinces. A preliminary survey indicated that Cryptosporidium could infect white Java sparrows (Lonchura oryzivora) [bib_ref] Cryptosporidium spp. in pet birds: genetic diversity and potential public health significance, Qi [/bib_ref] , but the number of sampled birds is too small ( = 25), which may not reflect the actual prevalence of Cryptosporidium in this bird. Also, no information is available about the prevalence of Cryptosporidium in gray Java sparrows in China. Therefore, we investigated the Cryptosporidium prevalence in Java sparrows (Lonchura oryzivora) in China and assessed its associated risk factors.
# Materials and methods
## The investigated sites. the survey was conducted in
Beijing and Shangqiu cities (two main locations of birds' production), northern China. Beijing city (39 ∘ 26 -41 ∘ 03 N, 115 ∘ 25 -117 ∘ 30 E) lies to the south of the Yanshan Mountains with an average altitude of 43.5 m, annual precipitation of 626 mm, and average annual temperature of 12.6 ∘ C. Shangqiu city (114 ∘ 82 -116 ∘ 45 E, 33 ∘ 98 -34 ∘ 80 N) is located in Henan province and has a subhumid warm temperate continental monsoonal climate. The altitude of Shangqiu city ranges from 30 m to 70 m, and the average annual temperature is 14.2 ∘ C.
## Collection and preparation of samples.
A total of 350 fecal samples were collected randomly from 225 white Java sparrows (Lonchura oryzivora) and 125 gray Java sparrows (Lonchura oryzivora), of which 163 were obtained from Beijing and 187 were obtained from Shangqiu from pet shops in 2015. Fecal samples were collected by using aseptic cotton and then filtered (0.3 mm wire mesh), and the filtrate was transferred into a 1.5 mL tube. All the samples were stored at 4 ∘ C until used for further analysis. Genomic DNA was extracted from feces using a Stool DNA kit (OMEGA, USA) according to the manufacturer's instructions. The DNA samples were stored at −20 ∘ C until used. Information about species, origin, gender, and age of Java sparrows was obtained and listed in [fig_ref] Table 1: Factors associated with prevalence of Cryptosporidium in Java sparrows in Beijing and... [/fig_ref].
## Sequencing analyses.
The positive PCR products of Java sparrows were sequenced by GenScript Company (Nanjing, China). Cryptosporidium species were determined by alignment with known reference sequences available in Gen-Bank using BLAST (https://www.ncbi.nlm.nih.gov/BLAST/). Representative nucleotide sequences were deposited in Gen-Bank under accession numbers KY034418-KY034427 and KY962159.
## Statistical analysis. the variation in
Cryptosporidium prevalence ( ) of Java sparrows of different geographical location ( 1), age ( 2), species ( 3), and gender ( 4) was analyzed by 2 test using SPSS 19.0. In the multivariable regression analysis, each of these variables was included in the binary logit model as an independent variable. The best model was judged by Fisher's scoring algorithm. All tests were two-sided, and values of probability ( ) < 0.05 were considered statistically significant. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were estimated to explore the strength of the association between Cryptosporidium positivity and the conditions tested.
# Results and discussion
Forty-seven out of 350 Java sparrows (13.42%) were tested to be Cryptosporidium-positive by nested PCR amplification of the SSU rRNA gene [fig_ref] Table 1: Factors associated with prevalence of Cryptosporidium in Java sparrows in Beijing and... [/fig_ref]. Cryptosporidium prevalence was 9.82% (16/163) in Beijing and 16.58% (31/187) in Shangqiu [fig_ref] Table 1: Factors associated with prevalence of Cryptosporidium in Java sparrows in Beijing and... [/fig_ref]. The prevalence in white Java sparrows and gray Java sparrows was 16.44% (37/225) and 8.00% (10/125), respectively [fig_ref] Table 1: Factors associated with prevalence of Cryptosporidium in Java sparrows in Beijing and... [/fig_ref]. Moreover, female Java sparrows (40.63%, 26/64) had a statistically higher Cryptosporidium prevalence than males (7.34%, 21/286) [fig_ref] Table 1: Factors associated with prevalence of Cryptosporidium in Java sparrows in Beijing and... [/fig_ref]. The infection rates of Cryptosporidium in the different age ranged from 10.47% to 16.33%, with the highest prevalence in the 6-8-month group [fig_ref] Table 1: Factors associated with prevalence of Cryptosporidium in Java sparrows in Beijing and... [/fig_ref] , but the difference was not statistically significant among age groups ( > 0.05). Species and sex of Java sparrows were considered as main risk factors to influence the positive rate significantly.
Analyses of the obtained SSU rRNA sequences indicated that all of the 47 isolates represented C. baileyi. These sequences were deposited in GenBank under accession numbers KY034418-KY034427 and KY962159. Comparative analyses of all the obtained SSU rRNA sequences with that of the two known reference sequences (C. baileyi, GU816040 and C. baileyi, AF262324) indicated that no variation was found among them.
In this study, the prevalence of Cryptosporidium in Java sparrows is 13.42%, which is higher than the 4.86% prevalence in birds in Brazil by PCR [bib_ref] Molecular characterization of Cryptosporidium spp. from fecal samples of birds kept in..., Nakamura [/bib_ref] , 8.1% in white Java sparrows in China by Sheather's sugar flotation technique [bib_ref] Cryptosporidium spp. in pet birds: genetic diversity and potential public health significance, Qi [/bib_ref] , and 3.22% in parrots in China by ELISA [bib_ref] Prevalence and genotyping of Cryptosporidium infection in pet parrots in North China, Zhang [/bib_ref]. Moreover, the Cryptosporidium prevalence of the present study was lower than that in pigeons in Thailand (25%) [bib_ref] Molecular identification of Cryptosporidium spp. in seagulls, pigeons, dogs, and cats in..., Koompapong [/bib_ref]. Different age distributions, test methods, geological environment conditions, and the breeding density may contribute to the difference of Cryptosporidium prevalence [bib_ref] Prevalence and genotyping of Cryptosporidium infection in pet parrots in North China, Zhang [/bib_ref] [bib_ref] Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in dairy..., Huang [/bib_ref].
In the present study, all of the obtained 47 Cryptosporidium isolates belong to C. baileyi. C. baileyi is approximately the most common avian Cryptosporidium [bib_ref] Genotypic characterization and phylogenetic analysis of Cryptosporidium sp. from domestic animals in..., Huber [/bib_ref] [bib_ref] Large-scale survey of Cryptosporidium spp. in chickens and Pekin ducks (Anas platyrhynchos)..., Wang [/bib_ref] because it has been reported in a wide range of avian hosts including chickens, turkeys, ducks, black-billed magpie, common myna, crested lark, Gouldian finch, red-billed leiothrix, mixed-bred falcons, zebra finch, rose-ringed parakeet, white Java sparrow, grey partridge, saffron finch, and ruddy shelducks [bib_ref] Cryptosporidium in birds, fish and amphibians, Ryan [/bib_ref] [bib_ref] Cryptosporidium spp. in pet birds: genetic diversity and potential public health significance, Qi [/bib_ref] [bib_ref] Genotypic characterization and phylogenetic analysis of Cryptosporidium sp. from domestic animals in..., Huber [/bib_ref] [bib_ref] Large-scale survey of Cryptosporidium spp. in chickens and Pekin ducks (Anas platyrhynchos)..., Wang [/bib_ref] [bib_ref] Upper respiratory tract infection caused by Cryptosporidium baileyi in three mixed-bred falcons..., Van Zeeland [/bib_ref].
# Conclusion
The present study revealed overall Cryptosporidium prevalence of 13.42% (47/350) with 16.44% (37/225) in white Java sparrows and 8.00% (10/125) in gray Java sparrows. This is the first report of C. baileyi in gray Java sparrows in China, which extend the host for C. baileyi. These results provide baseline information for further studies of molecular epidemiology and control of Cryptosporidium infection in poultry in China.
## Conflicts of interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
[fig] 2. 3: PCR Amplification. Cryptosporidium species/genotypes were determined by nested PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene using primers F1 (5 -CCCATTTCCTTCGAAACAGGA-3 ) and R1 (5 -TTC-TAGAGCTAATACATGCG-3 ) for primary amplification and F2 (5 -AAGGAGTAAGGAACAACCTCCA-3 ) and R2 (5 -GGAAGGGTTGTATTTATTAGATAAAG-3 ) for secondary amplification [16, 17]. PCR reaction (25 L) was composed of 1x PCR buffer (Mg 2+ -free), 2 mM MgCl 2 , 200 M of each deoxyribonucleoside triphosphate (dNTP), 0.4 M of each primer, 0.2 U of HotStart Taq DNA polymerase (Takara, Dalian, China), and 2 L of DNA template. The cycling conditions were 5 min at 95 ∘ C for initial denaturation and then 35 cycles of 45 s at 94 ∘ C, 45 s at 55 ∘ C, and 1 min at 72 ∘ C, followed by final extension at 72 ∘ C for 10 min. Both positive and negative controls were included in each test. PCR products were observed under UV light after electrophoresis in 1.5% agarose gel containing GoldView6 (Solarbio, Beijing, China). [/fig]
[table] Table 1: Factors associated with prevalence of Cryptosporidium in Java sparrows in Beijing and Shangqiu, northern China. [/table]
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10.1055/s-0041-1731591
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CCBY
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8890917
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34303315
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s2orc_pubmed_articles
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Novel Indigenous Probiotic Lactobacillus reuteri Strain Produces Anti-biofilm Reuterin against Pathogenic Periodontal Bacteria
Objective The aim of this study was to evaluate the effect of reuterin produced by a novel probiotic strain of Lactobacillus reuteri against periodontal biofilms. Materials and Methods L. reuteri LC382415 (an indigenous Indonesian strain) was cultured in Man, Rogosa, and Sharpe (MRS) agar in anaerobic conditions for 24 hours. To isolate reuterin, L. reuteri was suspended in 300-mM glycerol in MRS broth and incubated under anaerobic conditions for 3 hours, and the supernatant fraction was filtered. The presence of reuterin was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its concentration was determined. The effect of reuterin on Porphyromonas gingivalis ATCC 33277 and T. denticola ATCC 35405 biofilms was evaluated using biofilm assays. Biofilms were formed by incubating bacteria in 96-well microplates for 48 hours. A dose-dependent experiment was performed with reuterin concentrations of 12.5, 25, 50, and 100 μg/mL on biofilms. The inhibitory effect was measured at 1, 3, 6, and 24 hours. The biofilm masses were measured at 490 nm. Statistical analysis was using one-way ANOVA. Results The SDS-PAGE assay confirmed the presence of reuterin (52 kDa) in the culture supernatant of the L. reuteri strain. Reuterin in a concentration as low as 12.5 μg/mL significantly inhibited single-and mixed-species biofilms (p < 0.05). Conclusions This is the first study to demonstrate the promising effect of reuterin isolated from L. reuteri LC382415 against periodontal bacteria. Further studies are warranted to explore the mechanism of this active component.AbstractDOI https://doi.
# Introduction
Periodontitis is considered one of the most prevalent diseases worldwide and the main cause of adult tooth loss. 1 It is also associated with systemic pathologies, such as cardiovascular disease, obesity, and diabetes. [bib_ref] Periodontal disease and systemic conditions: a bidirectional relationship, Kim [/bib_ref] The etiologic agents of periodontal disease are pathogenic dental plaque biofilms. [bib_ref] Periodontitis and dental caries occur together, Merchant [/bib_ref] Under certain conditions, these biofilms harbor gram-negative bacterial pathogens such as Porphyromonas gingivalis and Treponema denticola, which are closely associated with periodontitis. [bib_ref] Porphyromonas gingivalis: an overview of periodontopathic pathogen below the gum line, How [/bib_ref] These bacteria produce virulence factors that evoke an exacerbated immune-inflammatory response, leading to the destruction of periodontal tissues. Traditionally, periodontitis is treated by mechanical debridement of dental plaque combined with chemical plaque control measures. However, periodontal therapy is costly and may not Novel Indigenous Probiotic Lactobacillus reuteri Indonesian Strain Against Periodontitis Widyarman,Theodorea. be sufficiently effective. Therefore, numerous studies have explored alternative therapies for periodontal disease. [bib_ref] Alendronate treatment of naturally-occurring periodontitis in beagle dogs, Reddy [/bib_ref] [bib_ref] Photodynamic therapy for periodontal diseases: state of the art, Meisel [/bib_ref] Probiotics have been shown to provide numerous benefits as in treatments for various human diseases, including allergic diseases (atopic dermatitis), bacterial vaginosis, urinary tract infections, and gastrointestinal disorders, and as preventives of dental caries. [bib_ref] Use of probiotics in gastrointestinal disorders: what to recommend?, Verna [/bib_ref] [bib_ref] Microorganisms with claimed probiotic properties: an overview of recent literature, Fijan [/bib_ref] [bib_ref] New perspectives on probiotics in health and disease, Daliri [/bib_ref] [bib_ref] Effects of probiotics, prebiotics, and synbiotics on human health, Markowiak [/bib_ref] According to the World Health Organization, the term "probiotics" refers to "live microorganisms (that) when administered in adequate amounts confer a health benefit on the host."Probiotics have a long history of use in health promotion and are generally considered safe. [bib_ref] Probiotics for periodontal health: a review of the literature, Raff [/bib_ref] Lactobacillus spp. and Bifidobacterium spp. are commonly used probiotic bacteria. [bib_ref] Probiotics: a promising role in dental health, Mahasneh [/bib_ref] Previous studies have attempted to examine the role of probiotics in various oral diseases, including periodontitis. [bib_ref] Probiotics in the treatment of periodontal disease: a systematic review, Jayaram [/bib_ref] Lactobacillus spp. produce antimicrobial substances, such as bacteriocins. Laboratory strains of Lactobacillus reuteri are known to produce an antimicrobial compound called reuterin, which is a low molecular class of bacteriocin. [bib_ref] Therapeutic application of synbiotics, a fusion of probiotics and prebiotics, and biogenics..., Ohshima [/bib_ref] However, there are no studies on the antibacterial effect of clinical strains of reuterin isolated from L. reuteri, particularly in periodontitis. To fill this research gap, this first study attempted to investigate the antibacterial properties of L. reuteri LC382415 against pathogenic periodontal bacteria. Moreover, we isolated reuterin and examined its activity against pathogenic periodontal biofilms.
# Materials and methods
## Isolation and purification of reuterin
Previous study done by Widyarman and colleagues in 2018 succeeded in isolating an indigenous Indonesian probiotic L. reuteri strain (LC382415) from a healthy young adult after a comprehensive screening assay. [bib_ref] Isolation and identification of Indonesian Lactobacillus reuteri strain from saliva of young..., Widyarman [/bib_ref] At first we compare the presence of reuterin isolated from L. reuteri LC382415 and L. reuteri ATCC 55730 (as a control) with the molecular weight 52 kDa.
In this in vitro experimental study, strain L. reuteri LC382415 was inoculated on de Man, Rogosa, and Sharpe (MRS) agar plate and incubated in anaerobic conditions at 37°C for 72 hours. This colony was then harvested via centrifugation at 4,000 × g for 10 minutes and washed twice with phosphate-buffered saline (Oxoid) (pH 7.4).
Reuterin was isolated following a protocol used by Chen et al and Spinler et al with some modifications. [bib_ref] Glycerol induces reuterin production and decreases Escherichia coli population in an in..., Cleusix [/bib_ref] [bib_ref] Human-derived probiotic Lactobacillus reuteri demonstrate antimicrobial activities targeting diverse enteric bacterial pathogens, Spinler [/bib_ref] In brief, an L. reuteri LC382415 culture was harvested at 1.5 × 10 10 CFU/mL and resuspended in 5 mL of 300-mM glycerol in MRS broth. This inoculum was incubated under anaerobic conditions at 37°C for 3 hours. After fermentation, the cells were collected via centrifugation at 4,000 × g for 10 minutes. The supernatant fraction was filtered using a filter membrane (pore size: 0.22 μm; Millipore) and stored at 4°C. The presence of reuterin in the medium was determined by analyzing its molecular weight using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. Four concentrations of reuterin , and 100 μg/mL) were prepared using the Bradford method. [bib_ref] A rapid and sensitive method for the quantitation of microgram quantities of..., Bradford [/bib_ref] The Bradford assay showed a mean reuterin concentration of 208.06 μg/mL.
## Confirmation of reuterin by sds-page
SDS-PAGE was used to confirm the presence of reuterin in the samples using a protocol published by Costas with modifications. [bib_ref] The analysis of bacterial proteins by SDS polyacrylamide gel electrophoresis, Costas [/bib_ref] The polyacrylamide gel was prepared by assembling gel cassettes using clean glass plates. We combined 30 mL of acrylamide stock solution, 22.5 mL of separating gel buffer, and 36.15 mL of deionized water in a side-arm flask and degassed the solution using a vacuum pump for 3 minutes. While the solution was being gently swirled to ensure adequate mixing, 0.9 mL of 10% (w/v) SDS stock was added, along with 0.45 mL of 10% ammonium peroxydisulfate and 45 μL of tetramethylethylenediamine (TEMED). Two gels were poured, delivering 10 mL volumes of acrylamide/buffer mixture. When the mixture reached 30 mm from the top of the plates, polytetrafluoroethylene (PTFE)-coated combs were carefully inserted, projecting 35 mm into the gel cassettes. We prepared a stacking gel mix in a side-arm flask for two gels (20 mL). This gave a final concentration of 5% acrylamide. The mixture was subsequently degassed, and 200 μL of 10% (w/v) SDS was then added, along with 100 μL of 10% (w/v) ammonium peroxydisulfate and 20 μL of TEMED. PTFE combs were inserted in 15 to 20 wells 25 mm into the assembly, leaving 10 mm for the stacking gel. The gel was left to set for 1 hour. The combs were then removed, and the sample wells were washed thoroughly with deionized water and filled with reservoir buffer.
To prepare the sample, we added 10 μL of native buffer into 30 μL of sample proteins and heated the mixture in a thermoblock machine (BioSan) at 100°C for 5 minutes. After that, 20 μL of the protein extract was loaded into each well using a micropipette. The gels were then removed from the assembly/pouring stand, placed in a running apparatus, and filled with tank buffer. The gels were electrophoresed at a constant current of 80 mA (50 V, 30 W) and a constant temperature of 15°C for 2 hours. Coomassie Blue staining solutions were then added. [bib_ref] The analysis of bacterial proteins by SDS polyacrylamide gel electrophoresis, Costas [/bib_ref] Bradford assays were performed to visualize the reuterin bands using a Coomassie Plus Bradford Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. A standard curve was used to determine the protein concentration of each sample. Bovine serum albumin (Thermo Fisher Scientific) was used as a control sample.
## Reuterin on pathogenic oral bacterial biofilms
The activity of reuterin isolated from L. reuteri LC382415 against oral pathogenic bacterial biofilms was assessed using monospecies and dual-species biofilms of P. gingivalis ATCC 33277 and T. denticola ATCC 35405. The bacteria were cultured on Brain Heart Infusion (BHI) (Sigma Aldrich) broth using the anaerobic GasPak jar system and incubated at 37°C for 48 hours. [bib_ref] Effect of culture medium and carbon dioxide concentration on growth of anaerobic..., Stalons [/bib_ref] For monospecies biofilms, 200 μL of 10 7 -CFU/mL P. gingivalis or T. denticola inoculum was added to 96-well plates. For dual species biofilms, 100 μL of 10 7 -CFU/ mL P. gingivalis inoculum and 100 μL of 10 7 -CFU/mL T. denticola inoculum were added into 96-well plates (NEST). Both monospecies and dual species inocula were incubated under anaerobic conditions at 37°C for 48 hours to form biofilms. Four different concentrations of reuterin , and 100 μg/mL) were then added to the biofilms to evaluate its antibacterial activity. The effects were assessed at 1, 3, 6, and 24 hours. [bib_ref] A streptococcal effector protein that inhibits Porphyromonas gingivalis biofilm development, Christopher [/bib_ref] [bib_ref] Strawberry extract's effects on Enterococcus faecalis and Porphyromonas gingivalis biofilms in vitro, Widyarman [/bib_ref] Chlorhexidine 0.2% was used as a positive control. Wells containing biofilms without reuterin were used as negative controls.
## Biofilm examination
The first examination was conducted using crystal violet analysis. The next examination was conducted using total plate count for enumeration of CFUs.
Crystal violet (0.5% w/v) was added to all wells, incubated for 15 minutes, and then removed. Absolute ethanol (200 μL) was also added to all wells to dissolve remaining crystal violet-biofilm complex. Dissolved crystal violet within each well was measured and considered as biofilm mass.
After crystal violet staining procedure, the photos of biofilm mass were observed by Carl Zeiss Primovert Inverted Microscope (Germany) at a 40x objective. Furthermore the absorbance measurements were performed using a microplate reader (SAFAS) at a wavelength of 595 nm. All treatments were performed in independently triplicate experiments.
For the total plate count, after biofilms were washed with PBS and dried at room temperature, the formed biofilm layer at the base of well was collected and diluted 10-fold. Ten microliters of the dilution solution with concentrations of 10 −4 to 10 −7 were poured on each MRS agar medium plate and were incubated anaerobically at 37°C (2 × 24 h), and then the CFU count was performed manually
## Standard curve for absorbance and biofilm quantification
To quantify absorbance (optical density) for bacterial concentration measurement, a standard curve was used to demonstrate the relationship between optical density and colony count, P. gingivalis and T. denticola inocula at 1 Log10 CFU/mL were diluted in BHI (Sigma Aldrich) broth in a 1:10 dilution series to achieve six dilutions (10-10 6 ) [fig_ref] Figure 1: Reuterin isolate from L [/fig_ref] , available in the online version only). Biofilms were grown under static conditions in sterile 96-well microplates, with each well containing 200 μL, at 37°C for 24 hours. Upon removal from incubation, the biofilms were heat fixed and stained with crystal violet. The absorbance was measured at a 595-nm excitation wavelength. 24
# Statistical analysis
The obtained results were statistically analyzed using one-way analysis of variance (ANOVA) to assess differences in biofilm reduction. The level of significance was set to p < 0.05. The Shapiro-Wilk test was used to assess normality, and Levene's test was used to assess the homogeneity of variance. The statistical analysis was performed using IBM SPSS Statistics version 20 (IBM Corp.) for Windows. 25
# Results
## Isolation of reuterin, sds-page, and bradford assay
The presence of reuterin isolated from L. reuteri LC382415 and ATCC 55730 is shown in ►Fig. 1. SDS-PAGE of the LC382415 culture supernatants showed a 52-kDa band, confirming the presence of reuterin. 26
## Activity of reuterin against pathogenic periodontal biofilms
The photographs of the biofilm lining 96-well plates of reuterin against each monospecies P. gingivalis and T. denticola were taken after staining with crystal violet. The biofilms were observed using Carl Zeiss Primovert Inverted Microscope at a 40x objective [fig_ref] Figure 2: Inverted optical images of Porphyromonas gingivalis biofilms after treatment with various concentrations... [/fig_ref].
The biofilm assay by using crystal violet showed that reuterin significantly inhibited monospecies P. gingivalis and T. denticola in dose dependent activity and incubation time. Reuterin in a concentration as low as 12.5 μg/mL significantly inhibited single-and mixed-species biofilms (p <0.05). The most effective of reuterin concentration in the monospecies biofilm of P. gingivalis and T. denticola was 100 μg/mL due to which it can inhibit up to 50 and 77% reduction, respectively, compared with negative control (►Figs. 4 and 5) in 1 hour and 100 μg/mL reuterin concentration showed significantly inhibited monospecies of those bacteria in prolonged incubation time compared with negative control. Moreover, reuterin exhibited a significant dose-dependent activity against mixed-species P. gingivalis and T. denticola biofilms in a concentration as low as 12.5 μg/mL (►Fig. 6) within 3 hours. Incubation for up to 24 hours with higher concentrations (25-100 μg/mL) showed an even more significant anti-biofilm activity.
In the total plate count analysis of CFUs, the presence of reuterin isolated from L. reuteri LC382415 on each monospecies biofilm of P. gingivalis and T. denticola showed anti-biofilm activity of concentration-dependent reuterin at various incubation time [fig_ref] Figure 7: CFU counts at various incubation times were done to determine the anti-biofilm... [/fig_ref].
Overall, there was effect of reuterin to decrease each monospecies biofilm of P. gingivalis and T. denticola in dose-dependent activity and incubation time.
# Discussion
Periodontal diseases represent a significant health burden, with an estimated annual cost of USD 8 billion, particularly Novel Indigenous Probiotic Lactobacillus reuteri Indonesian Strain Against Periodontitis Widyarman,Theodorea.
in Malaysia. [bib_ref] National economic burden associated with management of periodontitis in Malaysia, Dom [/bib_ref] Therefore, considerable efforts have been made to explore new therapeutic strategies against periodontal bacteria. In this study, we found that novel clinical isolates of an indigenous Indonesian L. reuteri strain show promising properties against periodontal pathogens through a reuterin-based mechanism. Importantly, we also found that reuterin can inhibit biofilm formation of periodontal bacteria.
The antimicrobial activity of probiotics is associated with the production of organic acids, peptides (bacteriocins), carbon dioxide, hydrogen peroxide, ethanol, and diacetyl. [bib_ref] Microbiological, technological and therapeutic properties of kefir: a natural probiotic beverage, De Oliveira Leite [/bib_ref] Treatment strategies based on probiotics exert antibacterial activities through two mechanisms: (1) inhibition of specific organisms by interfering with adhesion, colonization, and biofilm formation and (2) inhibition of pathogen growth via various substances, such as organic acids, hydrogen peroxide, and bacteriocins. [bib_ref] The battle of probiotics and their derivatives against biofilms, Barzegari [/bib_ref] Through these mechanisms, probiotics can inhibit the growth of dental plaque biofilms, which is in line with the findings of this study. Haukioja, in 2010 reported that patients with gingivitis, periodontitis, and pregnancy gingivitis locally treated with a culture supernatant of an L. acidophilus strain showed significant recovery. [bib_ref] Probiotics and oral health, Haukioja [/bib_ref] Other studies examining L. reuteri strains and L. brevis have also reported improved gingival health, as measured by decreased gum bleeding and inflammation. [bib_ref] Decreased gum bleeding and reduced gingivitis by the probiotic Lactobacillus reuteri, Krasse [/bib_ref] [bib_ref] Short-term effect of chewing gums containing probiotic Lactobacillus reuteri on the levels..., Twetman [/bib_ref] [bib_ref] The Anti-inflammatory effects of glycerol-supplemented probiotic Lactobacillus reuteri on infected epithelial cells..., Widyarman [/bib_ref] In this study, we also assess reuterin efficacy as antibiofilm agent against mixed-species biofilm from two periodontitis-causing bacteria. This is done to mimic the condition of major pathogens that cause periodontitis in vitro. It can be inferred from ►Fig. 5 that higher concentration of reuterin better reduces the biofilm mass concentration. It can be postulated that reuterin has the property of antimicrobial effects by inhibiting the production of bacterial ribonucleotide reductase. This is an enzyme that catalyzes the first step in DNA synthesis via competition (HPA-dimer) with ribonucleotides for binding sites or via reaction (3-HPA) with the unstable sulfhydryl groups of ribonucleotide reductase or thioredoxin. This inhibitory activity may explain the broad-spectrum activity of reuterin against periodontal bacteria. [bib_ref] Inhibitory activity spectrum of reuterin produced by Lactobacillus reuteri against intestinal bacteria, Cleusix [/bib_ref] Thus, the higher concentration of the reuterin may give higher efficacy in reducing bacterial biofilm mass. This study provides encouraging evidence for the treatment of periodontal disease using probiotic-derived product, such as reuterin. Further studies are warranted to explore the effect of reuterin in vivo.
# Conclusions
This study is the first to demonstrate the activity of reuterin derived from clinical isolates of L. reuteri against monospecies and dual-species periodontal bacterial biofilms. The Graphs showing the inhibition of Porphyromonas gingivalis biofilm growth after treatment with reuterin at different concentrations (μg/mL) and incubation times, as well as comparisons with positive and negative controls. P. gingivalis treated with bacterial culture medium as a negative control; P. gingivalis treated with 0.2% chlorhexidine as a positive control. Each graph shows the average of independently triplicate experiments.
## Fig. 5
Graphs showing the inhibition of Treponema denticola biofilm growth after treatment with reuterin at different concentrations (μg/mL) and incubation times, as well as comparisons with positive and negative controls. Treponema denticola treated with bacterial culture medium as a negative control; Treponema denticola treated with 0.2% chlorhexidine as a positive control. Each graph shows the average of independently triplicate experiments. Graphs showing the inhibition of mixed-biofilm growth after treatment with reuterin at different concentrations (μg/mL) and incubation times, as well as comparisons with positive and negative controls. Each graph shows the average of independently triplicate experiments. (A) Treponema denticola treated with bacterial culture medium as a negative control; (B) Treponema denticola treated with 12.5 µg/mL reuterin; (C) Treponema denticola treated with 25 µg/mL reuterin; (D) Treponema denticola treated with 50 µg/mL reuterin; (E) Treponema denticola treated with 100 µg/mL reuterin; (F) Treponema denticola treated with 0.2% chlorhexidine as a positive control.
[fig] Figure 1: Reuterin isolate from L. reuteri in 12% polyacrylamide gel. M, marker protein (molecular weight: 15-100 kDa); bands 1: reuterin isolate from L. reuteri ATCC 55730; bands 2-3: reuterin isolate from Indonesian LC382415 strain (52 kDa). [/fig]
[fig] Figure 2: Inverted optical images of Porphyromonas gingivalis biofilms after treatment with various concentrations of reuterin and control: (A) Treated with bacterial culture medium as a negative control; (B) Treated with 12.5 µg/mL reuterin; (C) Treated with 25 µg/mL reuterin; (D) Treated with 50 µg/mL reuterin; (E) Treated with 100 µg/mL reuterin; (F) Treated with 0.2% chlorhexidine as a positive control. The biofilms were observed using Carl Zeiss Primovert Inverted Microscope at a 40x objective. [/fig]
[fig] Figure 3: Inverted optical images of Treponema denticola biofilms after treatment with various concentrations of reuterin and control: (A) Treated with bacterial culture medium as a negative control; (B) Treated with 12.5 µg/mL reuterin; (C) Treated with 25 µg/mL reuterin; (D) Treated with 50 µg/mL reuterin; (E) Treated with 100 µg/mL reuterin; (F) Treated with 0.2% chlorhexidine as a positive control. The biofilms were observed using Carl Zeiss Primovert Inverted Microscope at a 40x objective. [/fig]
[fig] Figure 7: CFU counts at various incubation times were done to determine the anti-biofilm activity of concentration-dependent reuterin. (A) Porphyromonas gingivalis treated with bacterial culture medium as a negative control; (B) P. gingivalis treated with 12.5 µg/mL reuterin; (C) P. gingivalis treated with 25 µg/mL reuterin; (D) P. gingivalis treated with 50 µg/mL reuterin; (E) P. gingivalis treated with 100 µg/mL reuterin; (F) P. gingivalis treated with 0.2% chlorhexidine as a positive control. [/fig]
[fig] Figure 8: CFU counts at various incubation times were done to determine the anti-biofilm activity of concentration-dependent reuterin. [/fig]
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10.1002/ece3.4611
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CCBY
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6303718
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30598760
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s2orc_pubmed_articles
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Asymmetric response of different functional insect groups to low‐grazing pressure in Eurasian steppe in Ningxia
In recent years, the continued loss and fragmentation of steppe has caused decreased ecosystem functions and species losses in insect diversity. In the 2000s, the Chinese government developed a series of national projects, such as the construction of enclosures, to conserve natural ecosystems, including steppe. However, the effects of these enclosures on steppe arthropod community are largely unknown. In the present study, we selected enclosed and low-grazing regions at eight National Grassland Fixed Monitoring Stations to examine the compositional differences in four insect functional groups and their associated ecological functions. The results showed that diversity significantly differed between the enclosed and low-grazing regions, with the number of insect families being significantly higher in enclosed regions than in regions with low-grazing pressure.The responses of the insect community to steppe management also varied among the four groups (herbivores, predators, parasitoids, and pollinators). The abundances of herbivores, predators, and parasitoids were higher in enclosed regions than in low-grazing regions, while there was no significant difference in pollinators. Additionally, there were no significant differences in the predator/prey ratio between enclosed regions and low-grazing regions in any of the steppe types. The parasitic wasp/prey ratio was higher in enclosed regions than in low-grazing regions in meadow steppe and typical steppe, while there were no significant differences between the enclosed and low-grazing regions in desert steppe and steppe desert. Herbivores were observed to benefit much more from enclosures than predators, parasitoids, and pollinators. Therefore, we recommend lowgrazing should be considered in steppe conservation, which could conserve biodiversity and achieve biocontrol functions of arthropod community. K E Y W O R D S community, diversity, ecological function, population density, predator S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article: Zhao Z, Wei J, Zhang K, et al. Asymmetric response of different functional insect groups to low-grazing pressure in Eurasian steppe in Ningxia. Ecol Evol.
## | introduc ti on
Steppe is an important habitat type in northwest China, harboring a highly diverse insect community and threatened ecosystems [bib_ref] Effects of timing and frequency of mowing on the threatened scarce large..., Korosi [/bib_ref] [bib_ref] Local and landscape factors affecting communities of plants and diurnal Lepidoptera in..., Tropek [/bib_ref].
However, the continued loss and fragmentation of natural habitats has caused the degeneration of steppe and associated ecosystem function in the past few decades, which has resulted in substantial concern around the world [bib_ref] Multifunctional floodplain management and biodiversity effects: A knowledge synthesis for six European..., Schindler [/bib_ref] [bib_ref] The sustainable development of grasslandlivestock systems on the Tibetan plateau: Problems, strategies..., Shang [/bib_ref]. Furthermore, the changes in landscape pattern (land cover conversion) have caused severe biodiversity loss in steppe habitats across China [bib_ref] Urban expansion dynamics and natural habitat loss in China: A multiscale landscape..., He [/bib_ref] [bib_ref] Landscape changes have greater effects than climate changes on six insect pests..., Zhao [/bib_ref]. On the one hand, the increasing demand for grains and vegetables has led to the rapid expansion of arable land, which occupies a large amount of steppe and increasing natural and semi-natural habitats [bib_ref] Are both agricultural intensification and farmland abandonment threats to biodiversity? A test..., Katayama [/bib_ref] [bib_ref] Farmland abandonment: Threat or opportunity for biodiversity conservation? A global review, Queiroz [/bib_ref]. On the other hand, the abandonment of poor arable lands and marginal lands often results in the biological invasion of small bushes or other alien plants, which directly causes secondary succession in natural steppe [bib_ref] Biodiversity of Palaearctic grasslands: A synthesis, Dengler [/bib_ref] [bib_ref] Black locust (Robinia pseudoacacia) beloved and despised: A story of an invasive..., Vitkova [/bib_ref].
In the 2000s, the Chinese government developed a series of national projects to conserve natural ecosystems, including steppe [bib_ref] Managing China's pastoral lands: Current problems and future prospects, Hua [/bib_ref]. In northwest China, both fencing and grazing restriction strategies have been conducted to restore steppe ecosystems through the "Tianbao" project [bib_ref] Effects of precipitation on grassland ecosystem restoration under grazing exclusion in Inner..., Hao [/bib_ref]. Many enclosed areas have been established to enhance both insect and plant biodiversity [bib_ref] Exotic plants promote pollination niche overlap in an agroecosystem, Marrero [/bib_ref] [bib_ref] Traditional grazing regimes promote biodiversity and increase nectar production in Tibetan alpine..., Mu [/bib_ref] , and the plant diversity associated with aboveground net primary productivity (ANPP) has been greatly enhanced as a result. The insect fauna is also an important component in steppe, accounting for approximately 60% of all living species (plants 15% and vertebrates 4%) [bib_ref] Grazing history influences biodiversity: A case study on ground-dwelling arachnids (Arachnida: Araneae,..., Paschetta [/bib_ref]. Insects are characterized by high diversity due to their immense species richness and various life forms, including herbivores, pollinators, parasitoids, and predators, making insect communities an important part of terrestrial ecosystems, especially in steppe [bib_ref] Logging legacies affect insect pollinator communities in southern appalachian forests, Jackson [/bib_ref] [bib_ref] Impacts of invasive plants on resident animals across ecosystems, taxa, and feeding..., Schirmel [/bib_ref].
For example, herbivores could shift the plant community composition by feeding on different components of the plant community and disturbing interspecific relationships and can even affect the reproductive success of native plants [bib_ref] Plant biotic interactions in the sonoran desert: Current knowledge and future research..., Franklin [/bib_ref] [bib_ref] Concurrent biotic interactions influence plant performance at their altitudinal distribution margins, Kaarlejarvi [/bib_ref]. Additionally, pollinators can benefit plants and enhance seed dispersal through the mutualistic interactions between these insects and the plants they pollinate [bib_ref] Ant-aphid interactions increase ant floral visitation and reduce plant reproduction via decreased..., Levan [/bib_ref].
Until now, the effects of fencing and grazing prevention (based on enclosure strategies) on insect communities have been largely unknown [bib_ref] Dynamics and resilience of rangelands and pastoral peoples around the globe, Reid [/bib_ref] , and the response of insect communities to fencing and the prevention of grazing appears to vary at different spatial scales [bib_ref] Herbivore effects on productivity vary by guild: Cattle increase mean productivity while..., Charles [/bib_ref]. At the local scale, the enclosure of steppe causes changes in soil quality and microenvironments, which mediate the composition of plants associated with the invertebrate community [bib_ref] Identifying qualitative effects of different grazing types on below-ground communities and function..., Macdonald [/bib_ref] [bib_ref] Impacts of invasive plants on resident animals across ecosystems, taxa, and feeding..., Schirmel [/bib_ref]. At the landscape scale, fencing and grazing prevention resulting from landscape simplification often negatively affect diversity and the abundance of various taxonomic groups, such as invertebrates [bib_ref] Local and landscape management drive traitmediated biodiversity of nine taxa on small..., Kormann [/bib_ref].
Ecological processes, the species pool, and diversity patterns depend on habitat composition, microenvironments, and landscape patterns [bib_ref] The impacts of biologically induced micro-environments on biodiversity in a dry Mediterranean..., Alhamad [/bib_ref] [bib_ref] Arable plant diversity on conventional cropland-The role of crop species, management and..., Seifert [/bib_ref]. In particular, plant community associated with the landscape matrix could affect the mobility of organisms, which could also influence the structure of the insect community [bib_ref] Response of native insect communities to invasive plants, Bezemer [/bib_ref] [bib_ref] Contrasting effects of habitat area and connectivity on evenness of pollinator communities, Marini [/bib_ref]. Thus, exploring the effects of ecological restoration measures on insect biodiversity and determining how to develop conservation strategies to enhance ecological functions are key topics in ecological conservation and reconstruction [bib_ref] Conservation paleobiology: Leveraging knowledge of the past to inform conservation and restoration, Dietl [/bib_ref] [bib_ref] Contribution of genetics to ecological restoration, Mijangos [/bib_ref].
In northwest China, steppe and shrub steppe are the most species-rich ecosystems and can be divided into several main steppe types [bib_ref] Predicting invasion in grassland ecosystems: Is exotic dominance the real embarrassment of..., Seabloom [/bib_ref] [bib_ref] Modern pollen and vegetation relationships in the Yili Basin, Zhao [/bib_ref]. At present, the steppes have evolved into climax communities that are also facing several challenges under global change [bib_ref] Plant population differentiation and climate change: Responses of grassland species along an..., Frei [/bib_ref] [bib_ref] Biodiversity and climate change: Integrating evolutionary and ecological responses of species and..., Lavergne [/bib_ref]. One of the most prevalent disturbances in steppe is livestock grazing, which can change plant community composition, soil compactness, and nutrient cycling [bib_ref] Soil food web stability in response to grazing in a semi-arid prairie:..., Andres [/bib_ref] [bib_ref] Habitat type plays a greater role than livestock grazing in structuring shrubsteppe..., Elwell [/bib_ref]. In the past, overstocking was very common in China due to the increasing demand for production [bib_ref] Herders' opinions about desirable stocking rates and overstocking in the rangelands of..., Hou [/bib_ref].
Grazers can also indirectly impact other grassland organisms, such as invertebrates and birds, through structural changes to the habitat caused by frequent herbivory and trampling [bib_ref] Light grazing of saltmarshes is a direct and indirect cause of nest..., Sharps [/bib_ref]. Many invertebrate groups have critical ecosystem functions in steppe ecosystems, and plants benefit from many of them through pollination and seed dispersal by insects [bib_ref] Effects of large herbivores on grassland arthropod diversity, Van Klink [/bib_ref].
The predators and parasitic wasps that attack herbivores and pollinators could make the plant-insect interactions more complex [bib_ref] Effects of plant neighborhoods on plant-herbivore interactions: Resource dilution and associational effects, Hamback [/bib_ref]. However, invertebrates, particularly pollinators, have been given less attention in grazing studies, especially in northwest China.
Many strategies (fencing and grazing prevention) have been developed to restore the vegetation cover and soil structure and recover steppe health [bib_ref] Effect of vegetation type on microstructure of soil aggregates on the Loess..., Zhao [/bib_ref]. Across the steppe of Ningxia, fencing, reduced grazing, and reseeding have been applied to restore ecosystem health in steppe [bib_ref] Effect of prohibiting grazing policy in northern China: A case study of..., Chen [/bib_ref]. In addition, conservation areas (fully enclosed areas) have been established in different steppe types to improve steppe biodiversity in China. Recent research has shown that conservation strategies could effectively enhance plant diversity and the abundance of endangered species [bib_ref] Improving conservation outcomes with a new paradigm for understanding species' fundamental and..., Beever [/bib_ref] [bib_ref] Remote sensing for conservation monitoring: Assessing protected areas, habitat extent, habitat condition,..., Nagendra [/bib_ref]. In terms of invertebrate functional groups, there have been few experiments exploring the interaction between the conservation strategy used and the insect community [bib_ref] Landscape impacts on pollinator communities in temperate systems: Evidence and knowledge gaps, Senapathi [/bib_ref]. Therefore, based on the above literature, two questions were addressed: (a) Does the conservation strategy used in Chinese steppe (enclosures) increase the diversity and richness of the insect community in different steppe types? (b) Could the different functional groups of the insect community associated with different ecological functioning be enhanced under this conservation strategy compared with control conditions?
## | material s & me thods
## | study area
The study area was located in Ningxia Hui Autonomous Region of northwest China, which was a part of in Eurasian Steppe. Totally, there were four steppe types (meadow steppe (MS), typical steppe (TS), desert steppe (DS), and steppe desert (SD)) in Ningxia, which were widely distributed in Eurasian Steppe [bib_ref] Primary production and rain use efficiency across a precipitation gradient on the..., Bai [/bib_ref] [bib_ref] Plant cover associated with aboveground net primary productivity (ANPP) mediates insect community..., Zhao [/bib_ref]. The four steppe types accounted for more than 90% of total steppe in Ningxia, which also had significant differences of plant biomass and species composition (see Supporting Information
## | insect collection
Sticky traps (yellow) were used to collect insect samples in the studied regions. Five cards were placed at each NGMS to capture insects using a 5-point sampling method, which is an empirical method used for insect collection [bib_ref] Habitat heterogeneity stabilizes the spatial and temporal interactions between cereal aphids and..., Zhao [/bib_ref]. Each point was a replicate, and there were five replicates within each NGMS. The same 5-point sampling method was used to collect insect samples in the adjacent regions with low-grazing pressure. The sticky cards were randomly placed throughout the enclosed and low-grazing regions for 5 days, following which all sticky cards were transported back to the laboratory for insect identification. The collection periods were 20-25 July in 2016 and 20-25 July in 2017 at all sampling locations.
# | statistical analysis
The number of insect individuals captured in the field was counted for each card, and the mean values ± SE were then calculated. Based on family level, Shannon-Wiener index (H = − ∑ k i=1 (P i )(l n P i )) was used to compute the diversity of insect arthropods in four steppe types, respectively [bib_ref] Plant cover associated with aboveground net primary productivity (ANPP) mediates insect community..., Zhao [/bib_ref]. The insects were then classified into four functional groups (herbivore, pollinator, predator, and parasitic wasp). For each functional group, multiple comparisons and tests of the insect community across the two different treatments (enclosure regions and low-grazing regions) and four different steppe types were examined to identify significant differences.
We conducted split-plot analysis, as our designed experiments have different treatments applied to plots of different sizes. The steppe types were treatments, and sampling points within each steppe type were replicates. Mixed-effects models were used to facility to deal with complicated error structures and hence avoid the pitfalls of pseudoreplication. The function lme is called because the explanatory variables are a mixture of fixed effects (management treatment: enclosure regions and low-grazing regions) and random effects (steppe types). All analyses were performed using the statistical software R 3.3.1 (R Development Core Team, 2016) with the "vegan" and "lmer" packages.
## | re sults
Three sticky traps (sampling points) of both enclosure and low-grazing regions in meadow steppe could cover more than 85% species and four sticky traps could account for more than 95% species . Additionally, three sticky traps could also include 89%, 87%, and 91% species in typical steppe, desert steppe, and steppe desert, respectively . Additionally, more than 95% species could be collected by four sticky traps in all steppe types .
The responses of different groups to the enclosure regions (ER) and low-grazing regions (LGR) were varied due to species-specific . Diversity significantly differed between the enclosure regions (ER) and low-grazing regions (LGR) (t 1,9 = 6.59, p = 0.006, in meadow steppe. The ER also had higher diversity than that in LGR in typical steppe (t 1,19 = 8.37, p < 0.001), desert steppe (t 1,39 = 3.76, p = 0.01), and steppe desert (t 1,9 = 4.09, p = 0.01, . Similarly, the number of insect families in ER in meadow steppe, typical steppe, desert steppe, and steppe desert was higher than that in LGR (MS, t 1,9 = 9.68, p < 0.001; TS, F I G U R E 3 Insect community response (diversity (a) and number of insect families (b)) to different management patterns (enclosures and low-grazing pressure) in four steppe types (meadow steppe, typical steppe, desert steppe, and steppe desert) in Ningxia, northwest China. Asterisks above the bars indicate differences between enclosure regions (ER) and low-grazing regions (LGR) ( *** p < 0.001; ** p < 0.01; * p < 0.05). Black columns are enclosure regions, and white columns are low-grazing regions t 1,19 = 7.35, p < 0.001; DS, t 1,39 = 2.68, p = 0.03; SD, t 1,9 = 4.95, p = 0.01, .
Herbivore abundance in the ER of meadow steppe, typical steppe, and steppe desert was higher than that in LGR (MS, t 1,9 = 2.09, p = 0.09; TS, t 1,19 = 10.21, p < 0.001; SD, t 1,39 = 3.92, p = 0.01, . However, there was no significant difference in the abundance of herbivores between ER and LGR in desert steppe (t 1,9 = 2.35, p = 0.07, .
In terms of the other functional groups, the abundance of predators in the ER of typical steppe and desert steppe was higher than that in LGR (TS, t 1,19 = 4.29, p = 0.04; DS, t 1,39 = 6.28, p = 0.012, , while there was no significant difference in the abundance of predators between ER and LGR in both meadow steppe and steppe desert (MS, t 1,9 = 1.39, p = 0.14; SD, t 1,9 = 1.16, p = 0.38, . The abundance of parasitoid wasps in the ER of meadow steppe and steppe desert was significantly higher than that in LGR (MS, t 1,9 = 5.29, p = 0.01; SD, t 1,9 = 8.92, p = 0.002, , while there was no significant difference in the other two steppe types (typical steppe and desert steppe) (TS, t 1,19 = 1.08, p = 0.36; DS, t 1,39 = 0.68, p = 0.78, .
There were no differences in pollinator abundance between ER and LGR in any of the steppe types (MS, t 1,9 = 0.69, p = 0.67; TS, t 1,19 = 0.43, p = 0.91; DS, t 1,39 = 1.24, F = 0.38; SD, t 1,9 = 0.68, p = 0.62, .
## F i g u r e 4
The abundances of different functional groups under different management patterns (enclosures and low-grazing pressure) in four steppe types (meadow steppe, typical steppe, desert steppe, and steppe desert) in Ningxia, northwest China ((a), herbivores; (b), predators; (c), parasitoid wasps; (d), pollinators). Asterisks above the bars indicate differences in mean values among different steppe types ( *** p < 0.001; ** p < 0.01; * p < 0.05). Black columns are enclosure regions, and white columns are low-grazing regions F I G U R E 5 Effects of different management patterns (enclosures and low-grazing pressure) on the biocontrol function (predator/herbivore ratio (a) and parasitoid wasp/herbivore ratio (b)) in different steppe types (meadow steppe, typical steppe, desert steppe, and steppe desert) in Ningxia, northwest China. Asterisks above the bars indicate differences in mean values among the different steppe types ( *** p < 0.001; ** p < 0.01; * p < 0.05). Black columns are enclosure regions, and white columns are low-grazing regions
The predator/herbivore ratio in the ER of all steppe types was not significantly different from that in LGR (MS, t 1,9 = 2.38, p = 0.09; TS, t 1,19 = 1.67, p = 0.62; DS, t 1,39 = 1.38, p = 0.59; SD, t 1,9 = 1.53, p = 0.28, . In contrast, the parasitoid wasp/herbivore ratio in LGR in meadow steppe, typical steppe, and desert steppe was significantly higher than that in ER (MS, t 1,9 = 3.92, p = 0.02; TS, t 1,19 = 6.92, p < 0.001; DS, t 1,39 = 3.24, p = 0.04), while there was no significant difference in steppe desert (t 1,9 = 0.42, p = 0.79, .
## | d iscuss i on
Since the middle of the 20th century, a series of changes has occurred in steppe use in China: (a) large-scale landscape modification of natural environments with changes in land cover, (b) afforestation of "bare" lands, (c) abandonment of infertile arable lands, and (d) overstocking [bib_ref] An overview of biodiversity and conservation status of steppes of the Anatolian..., Ambarli [/bib_ref]. To face the challenges of biodiversity loss and ecosystem degradation under global change, many conservation strategies have been implemented to restore ecosystems. In general, the abundance and diversity of insects could be influenced by the intensity of management (enclosures), especially in grasslands [bib_ref] Global effects of land use on local terrestrial biodiversity, Newbold [/bib_ref] [bib_ref] Beyond global warming -Ecology and global change, Vitousek [/bib_ref]. However, insect richness was found to be largely unaffected by land use intensity (grazing and mowing frequency) across several groups [bib_ref] A literature review of insect responses to fire, compared to other conservation..., Swengel [/bib_ref]. [bib_ref] Contrasting effects of grassland management modes on species-abundance distributions of multiple groups, Simons [/bib_ref] also found that the intensity of land use affected the taxonomic richness of only plants and herbivores, while grazing intensity affected the taxonomic richness of all groups [bib_ref] Contrasting effects of grassland management modes on species-abundance distributions of multiple groups, Simons [/bib_ref].
Unmanaged steppe could enhance the abundance and diversity of Orthoptera assemblages (herbivores) compared with managed grasslands in Mediterranean steppe rangeland [bib_ref] Effects of ecological restoration on Orthoptera assemblages in a Mediterranean steppe rangeland, Alignan [/bib_ref]. However, [bib_ref] Managing calcareous grassland for the declining Duke of Burgundy Hamearis lucina butterfly:..., Goodenough [/bib_ref] also found that moderate grazing intensity in both autumn and winter could enhance the abundance of butterflies while having disadvantageous effects on plants in winter [bib_ref] Managing calcareous grassland for the declining Duke of Burgundy Hamearis lucina butterfly:..., Goodenough [/bib_ref]. McIver and Macke (2014) even found an increase in the species richness and abundance of the butterfly community in steppe after artificial disturbances (fire or mechanical treatments) [bib_ref] Short-term butterfly response to sagebrush steppe restoration Treatments, Mciver [/bib_ref]. Therefore, low-grazing pressure and disturbance could facilitate most insect taxa while having no effects on other species [bib_ref] Moderation is best: Effects of grazing intensity on plant-flower visitor networks in..., Lazaro [/bib_ref]. Light grazing resulted in larger local populations of butterflies compared to heavy grazing or no grazing at all [bib_ref] Population dynamics and future persistence of the clouded Apollo butterfly in southern..., Johansson [/bib_ref] ; thus, it is possible to considerably reverse the negative trends and reduce extinction risk through conservation actions. Furthermore, the abundance and richness of herbivores could be greatly increased through effective enclosure strategies, which was well supported in our present experiment.
For predators and parasitic wasps, there have been fewer experiments examining the effects of grazing on natural enemy richness or diversity. [bib_ref] Effects of land use and landscape patterns on Orthoptera communities in the..., Weking [/bib_ref] reported that the abundance and diversity of herbivores (Orthoptera) could be enhanced by grazing across western Siberia [bib_ref] Effects of land use and landscape patterns on Orthoptera communities in the..., Weking [/bib_ref]. However, different functional groups of cursorial spiders (Aranei) and true bugs (Heteroptera) in northeastern Ukraine had varied responses to management intensity via the gully terrain (slope or bottom) [bib_ref] The impact of cattle grazing on cursorial spiders (Aranei) and true bugs..., Polchaninova [/bib_ref]. In our experiment, the abundance of predators was higher in ER than in LGR only in typical and desert steppe, while the abundance of parasitic wasps was higher in ER than in LGR only in meadow and steppe desert.
Therefore, different functional groups have different responses to the management pattern, and the nature of these responses depends on species-specific characteristics. [bib_ref] A multiscale approach for identifying conservation needs of two threatened sympatric steppe..., Benitez-Lopez [/bib_ref] found that low levels of management (the rotation of plowing and fallows and a reduction in the frequency and intensity of plowing) could benefit sandgrouses (steppe birds) and other steppe species, while both leaving land fallow (no disturbance) and highly intense agriculture (arable lands) have detrimental effects on bird conservation [bib_ref] A multiscale approach for identifying conservation needs of two threatened sympatric steppe..., Benitez-Lopez [/bib_ref].
In our experiment, we found that pollinators showed no significant response to unmanaged steppe, which indicates that a complete enclosure strategy could not effectively conserve pollinators. [bib_ref] Effects of grazing management on biodiversity across trophic levels-The importance of livestock..., Van Klink [/bib_ref] found that low stocking densities favored high abundances of voles, pollinators, and flowers (van [bib_ref] Effects of grazing management on biodiversity across trophic levels-The importance of livestock..., Van Klink [/bib_ref]. However, the bird community showed no significant responses to the grazing level [bib_ref] Birds of a feather flock together: Using trait-groups to understand the effect..., Howland [/bib_ref]. Biocontrol functions (predator/herbivore and parasitic wasp/herbivore ratios) were not also enhanced by the enclosure strategy in the present experiment, which indicates that complete enclosures can impede the sustainable management of steppe. The varied responses of different groups to the management pattern in steppe were an important reason for this phenomenon. In general, herbivores benefited more from the enclosures than their natural enemies (predator and parasitic wasps). The homogeneous vegetation structure in the enclosed regions may not be attractive to predators (ladybeetles) or parasitic wasps (Aphidiidae), which have been reported to be sensitive to the management activity in steppe [bib_ref] Plant-insect interactions from early Permian (Kungurian) Colwell Creek Pond, Schachat [/bib_ref]. In contrast, low-grazing pressure caused patchy vegetation cover, including areas containing different plant species or puddles [bib_ref] Contrasting effects of grassland management modes on species-abundance distributions of multiple groups, Simons [/bib_ref]. The abundance of some insects, including dung beetles and flies, could be increased by the feces of grazing animals [bib_ref] The application of an ecosystem services framework to estimate the economic value..., Beynon [/bib_ref]. Grazing can indirectly enhance biodiversity via changing vegetation cover and hence improve biocontrol functions in regions with low-grazing pressure [bib_ref] Effects of large herbivores on grassland arthropod diversity, Van Klink [/bib_ref]. Therefore, different steppe use patterns have district effects on different insect functional groups and need to be considered separately when studying the effects of steppe use on ecological communities [bib_ref] Identifying qualitative effects of different grazing types on below-ground communities and function..., Macdonald [/bib_ref]. Additionally, the use of common and rare species as additional parameters describing the overall composition of the insect community will shed light on the potential mechanisms behind the effects of different steppe use patterns [bib_ref] Contrasting effects of grassland management modes on species-abundance distributions of multiple groups, Simons [/bib_ref].
## | con clus ion
Enclosures in the Ningxia steppe enhanced the diversity and number of insect families. However, the responses of different insect functional groups to the enclosures varied due to their varied feeding characteristics and other species-specific factors. Enclosures could increase the abundance of herbivores while having no effect on pollinators. Furthermore, full enclosures reduced the parasitoid wasp/herbivore ratio and impeded the service of biocontrol.
Biocontrol functions can be greatly enhanced in steppe by optimizing grassland utility via grazing intensity. Conservation measures that are focused on enclosures cannot achieve the aim of biodiversity conservation [bib_ref] Local and landscape management drive traitmediated biodiversity of nine taxa on small..., Kormann [/bib_ref]. Therefore, light grazing should be considered on increase biocontrol functions have been newly considered to conserve biodiversity and achieve sustainable management [bib_ref] The ecosystem approach to fisheries: Management at the dynamic interface between biodiversity..., Jennings [/bib_ref] [bib_ref] Effects of land use and landscape patterns on Orthoptera communities in the..., Weking [/bib_ref].
## Ack n owled g m ents
We would like to thank Qi Jiang, Zhanjun Wang, Yun Li, Feng Wang, for their technical assistance of data collection in the field. We also thank Huanli Xu, Chaodong Zhu, Zeqing Niu, Hu Li, and Xingyue Liu for the insect identification. We would also thank the anonymous reviewers whose comments have significantly improved the qual-
## Co n fli c t o f i nte r e s t
None declared.
## Auth o r s co ntr i b uti o n s
## Data acce ss i b i lit y
Insect species composition in enclosure and low-grazing regions of steppe: Dryad https://doi.org/10.5061/dryad.50sk900.
## O rci d
## Zihua zhao
http://orcid.org/0000-0003-2353-2862
[fig] F: I G U R E 2 Species accumulation of sampling points in a site in enclosure regions (ER) and low-grazing regions (LGR) four steppe types (a) meadow steppe; (b) typical steppe; (c) desert steppe; and (d) steppe desert. Solid circles are enclosure regions, and empty circles are lowgrazing regions [/fig]
[fig] Z: hao and R hang designed the experiments. hao, S Wei, Mg hu, and W Huang performed the experiments and collected the data in the field. hao analyzed the data and wrote the first draft. hao, J Wei, K hang, H Li, X Pan, and R hang revised the manuscript and approved the final version. [/fig]
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10.1074/jbc.M011240200
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CCBY
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11278865
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s2orc_pubmed_articles
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Enhanced Antiviral and Antiproliferative Properties of a STAT1 Mutant Unable to Interact with the Protein Kinase PKR*
We have previously reported a physical association between STAT1 and the protein kinase double-stranded RNA-activated protein kinase (PKR). PKR inhibited STAT1 function in a manner independent of PKR kinase activity. In this report, we have further characterized the properties of both molecules by mapping the sites of their interaction. A STAT1 mutant unable to interact with PKR displays enhanced interferon ␥ (IFN-␥)-induced transactivation capacity compared with STAT1. This effect appears to be mediated by the higher capacity of STAT1 mutant to heterodimerize with STAT3. Furthermore, expression of STAT1 mutant in STAT1 ؊/؊ cells enhances both the antiviral and antiproliferative effects of IFNs as opposed to STAT1. We also provide evidence that STAT1 functions as an inhibitor of PKR in vitro and in vivo. That is, phosphorylation of eIF-2␣ is enhanced in STAT1 ؊/؊ than STAT1 ؉/؉ cells in vivo, and this correlates with higher activation capacity of PKR in STAT1 ؊/؊ cells. Genetic experiments in yeast demonstrate the inhibition of PKR activation and eIF-2␣ phosphorylation by STAT1 but not by STAT1 mutant. These data substantiate our previous findings on the inhibitory effects of PKR on STAT1 and implicate STAT1 in translational control through the modulation of PKR activation and eIF-2␣ phosphorylation.
Cytokines and growth factors exert a diverse range of biological activities, from host defense, growth regulation, to immunomodulation. Upon ligand binding to cell-surface receptors, JAK 1 kinases are activated and proceed to phosphorylate the receptor on tyrosine residues, which then function as docking sites for cytoplasmic transcription factors of the STAT family. STATs are subsequently activated by tyrosine phosphorylation, dimerize by phosphotyrosyl⅐SH2 interactions, and translocate to the nucleus to induce transcription of cytokineresponsive genes. A single tyrosine phosphorylation site in the carboxyl-terminal activation domain is absolutely essential for STAT dimerization and DNA binding (3), whereas phosphorylation of a serine residue in this region is important for transactivational activity.
One of the major STATs intimately involved in both the innate and acquired immune responses is STAT1. Upon virus infection or exposure to interferons (IFNs), STAT1 is found in protein complexes that bind specific DNA sequences upstream to genes responsible for host resistance. For instance, IFN-␣/ induces formation of the heterodimeric ISGF3, whereas IFN-␥ induces binding of homodimeric STAT1. Moreover, dsRNA, an intermediate produced during virus replication, can also activate STAT1 DNA binding. The non-redundant role of STAT1 in the antiviral response is further appreciated by findings that stat1 null mice (STAT1 Ϫ/Ϫ ) are highly susceptible to microbial infection. IFN signaling leads to the expression of a number of genes, one of which encodes for the dsRNA-dependent protein kinase, PKR. PKR is a serine/ threonine protein kinase that displays two distinct activities: (i) autophosphorylation upon dsRNA bindingand (ii) phosphorylation of the eukaryotic translation initiation factor eIF-2␣ (9, 10), a modification resulting in inhibition of protein synthesis. Several studies with cultured cells provide evidence for antiviral, antiproliferative, and tumor suppressor functions of PKR. However, pkr null (PKR Ϫ/Ϫ ) mice exhibit a modest susceptibility to viral infection and show no signs of tumor formation, suggesting that the lack of PKR can be compensated by other PKR-like molecules. This hypothesis is supported by the recent identification of the PKR-related genes, PERK/PEKand the mouse homolog of the yeast eIF-2␣ kinase, GCN2.
We previously described an association between PKR and STAT1. This interaction takes place in unstimulated cells and diminishes upon treatment with IFNs or dsRNA. Increased levels of PKR⅐STAT1 complex have a negative effect on STAT1 DNA-binding and transactivation capacities. In this report we have mapped the interaction sites between the two proteins and have identified a novel function of STAT1. Specifically, we demonstrate that STAT1 functions as an inhibitor of PKR activation and eIF-2␣ phosphorylation in vitro and in vivo. A mutant of STAT1, which was unable to interact with PKR, could not inhibit PKR function in yeast and was better able to mediate the transcriptional, antiviral, and antiproliferative responses of IFNs compared with STAT1. Taken together, these findings not only support our previous observations, but they also provide strong evidence for tight regulation of PKR and STAT1 functions by virtue of their interaction. In addition, our data suggest that STAT1 has a dual role in regulation of gene expression by functioning as a transcriptional factor and possibly as translational regulator through PKR activation and eIF-2␣ phosphorylation.
# Materials and methods
Cell Culture and Transfections-HeLa S3, U3A, STAT1 ϩ/ϩ , STAT1 Ϫ/Ϫ , PKR ϩ/ϩ , and PKR Ϫ/Ϫ cells were maintained in Dulbecco's modification of Eagle's medium supplemented with 10% calf serum, 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin (Life Technologies). For IFN treatment, cells were incubated with 1000 IU/ml of recombinant murine IFN-␣/ (Lee Biomolecules) or 100 IU/ml of IFN-␥ (PharMingen). Double-stranded RNA transfections were performed as previously described. Transient transfections were performed with LipofectAMINE Plus reagent (Life Technologies). T7 vaccinia virus transient transfections were carried out using the recombinant vaccinia virus, vTF7-3, encoding the bacteriophage T7 RNA polymerase. In vivo [ 35 S]methionine experiments were performed as previously described. pBABE, pBABE-HA-STAT1␣ WT, or TM STAT1 Ϫ/Ϫ cells were generated as previously described. Cell cycle analyses and CPE assays were performed as described in previous studies (Refs. 23 and 24, respectively).
Plasmid Construction-GST-PKRK296R, GST-PKR 1-262 (PKR N), and GST-PKR K296R 263-551 (PKR C) were generated as previously described. GST-PKRLS4K296R was generated by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene) with the following primer pairs ( Cell Extract Preparation, Immunoprecipitation, and Immunoblot Analysis-Cell extract preparation, immunoprecipitation, and immunoblotting were performed as previously described. The following antibodies were used: STAT1␣ (Santa Cruz Biotechnology); HA (12CA5, Roche Molecular Biochemicals); STAT2 (Upstate Biotechnology Inc.); STAT3 (Santa Cruz); PKR; GST (Amersham Pharmacia Biotech); Myc (9E10, Roche Molecular Biochemicals); eIF-2␣; phosphoserine 51 of eIF-2␣ (Research Genetics Inc.); FLAG (M2, Kodak); HA horseradish peroxidase antibody (3F10, Roche Molecular Biochemicals); phosphotyrosine (4G10/PY20, UBI and Transduction Laboratories); and phosphoserine 727 of STAT1␣. Proteins were visualized by ECL (Amersham Pharmacia Biotech).
DNA Binding and Transactivation Assays-Electrophoretic mobility shift analysis was performed using the dsDNA c-Fos c-sis-inducible element (SIE, 5Ј-GATCGTGCATTTCCCGTAAATCTTGTCTACAATT-C-3Ј) according to protocols previously described. The Dual Luciferase system (Promega) was used to assess the transactivation potential of STAT1. Briefly, STAT1 Ϫ/Ϫ cells or cells expressing STAT1 WT or TM were transfected with Renilla luciferase (pRL-TK) and pGL-2XIFP53 GAS luciferase. Twenty-four hours after transfection, cells were replated and treated with IFN-␥ for 18 h before harvesting. The results presented represent quadruplicate experiments where GAS luciferase activity was normalized to Renilla luciferase activity.
Isoelectric Focusing and PKR in Vitro Kinase Assays-Isoelectric focusing and immunoblot analysis of yeast eIF-2␣ were performed as previously described. PKR in vitro kinase assays were carried as previously described.
GST Pull-down Assays-Protein production and extraction were performed according to previously described protocols. Normalized GST fusion proteins were co-incubated with HeLa whole cell lysates or [ 35 S]methionine in vitro translated proteins, washed, subjected to SDS-PAGE, and visualized by fluorography.
Yeast Plasmids, Transformations, Growth Protocols, and Protein Extractions-Wild-type and mutants of HA-STAT1 1-413 were subcloned by restriction digest of BamHI-NotI sites into a modified form of the yeast expression vector, pEMBL/yex4, containing a NotI site in the multiple cloning site. Transformation of yeast strains H2544 and J110 and growth analyses were performed as previously described.
# Results
The catalytic domain of PKR specifically associates with the DNA-binding domain of STAT1. To map the PKR⅐STAT1 interaction we performed a series of binding assays using full-length GST-PKRK296R mutant (GST-PKR WT could not be overexpressed in bacteria (32)) or truncations of PKR bearing either the dsRNA-binding (GST-PKR N, amino acids 1-262) or catalytic (GST-PKR C, amino acids 263-551) domain [fig_ref] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro [/fig_ref]. HeLa S3 extracts expressing human HA-STAT1␣ were incubated with GST-PKR fusion proteins, subjected to SDS-PAGE, and immunoblotted with an anti-HA antibody. As shown in [fig_ref] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro [/fig_ref] , both full-length GST-PKR (lane 5) and the carboxyl terminus of PKR (lane 4) interacted with STAT1 whereas binding to the amino terminus was not detectable (lane 3). Furthermore, this interaction is specific for STAT1, because we could not detect binding of GST-PKR proteins with other STATs [fig_ref] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro [/fig_ref].
We previously reported that binding of PKR to STAT1 is independent of RNA but requires an intact RNA-binding domain, because the dsRNA-binding-defective mutant PKRLS4 (Arg 58 -Ser 59 -Lys 60 to Gly 58 -Ala 59 -Leu 60 ) (33) does not interact with mouse STAT1 in NIH3T3 cell extracts stably expressing this PKR mutant. Based on this, we proposed that the integrity of the dsRNA-binding domain of PKR plays a role in PKR⅐STAT1 interaction in vivo. The observation, however, that the carboxyl terminus of PKR is required for binding to STAT1 in vitro [fig_ref] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro [/fig_ref] prompted us to examine the interaction of STAT1 with GST-PKR bearing either the LS4 or the LS9 mutation (Ala 66 -Ala 68 to Gly 66 -Pro 68 ), which also abolishes RNA binding (33) [fig_ref] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro [/fig_ref]. The RNA-binding-defective mutants were expressed and purified in the GST-PKRK296R background, because expression of LS4 and LS9 in the GST-PKR WT background could not be achieved (data not shown). HeLa S3 extracts containing HA-STAT1␣ were incubated with GST-PKRK296R, GST-PKRK296RLS4, or GST-PKRK296RLS9, and GST-PKR bound proteins were subjected to immunoblotting with anti-HA antibody. All GST-PKR mutants interacted with STAT1 equally well [fig_ref] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro [/fig_ref] , suggesting that mutations within the dsRNA-binding domain do not interfere with PKR binding to STAT1 in vitro.
To map the region on STAT1 that facilitates its interaction with PKR, we used truncated STAT1 proteins corresponding to its amino-terminal, DNA-binding, linker, SH2, or transactiva-tion domains [fig_ref] FIG. 2: Mapping of amino acids on STAT1 required for PKR-binding [/fig_ref]. The pull-down assays were performed with GST-PKR C, because this protein binds to STAT1 [fig_ref] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro [/fig_ref] and is more stable than GST-PKRK296R (data not shown). We observed that GST-PKR C specifically interacted with the DNA-binding domain of STAT1 Mutations of STAT1 That Affect Binding to PKR-To identify amino acids in STAT1 that form contacts with PKR, we next constructed mutations within amino acids 343-348 of HA-STAT1 1-413 that abolished binding to PKR. Alanine-scan mutagenesis of each of the six amino acids did not yield a point mutant of HA-STAT1 1-413 that disrupted interaction with PKR (data not shown). However, a three-amino acid substitution (TM; Arg 346 -Leu 347 -Leu 348 to Ala 346 -Asp 347 -Asp 348 ) within this region disrupted the ability of STAT1 to interact with GST-PKR C [fig_ref] FIG. 2: Mapping of amino acids on STAT1 required for PKR-binding [/fig_ref]. Interestingly, HA-STAT1 1-413 TM possessed faster mobility on SDS-PAGE gels compared with WT most likely through changes in the overall charge of the molecule.
To further characterize the interaction of full-length HA-STAT1␣ TM with PKR, we utilized the human fibrosarcoma, U3A cell line, which lacks endogenous STAT1. HA-STAT1␣ WT or TM were co-transfected with PKRK296R into U3A cells, after which, the protein extracts were immunoprecipitated against PKR and immunoblotted with HA antibodies.
As seen in [fig_ref] FIG. 2: Mapping of amino acids on STAT1 required for PKR-binding [/fig_ref] , STAT1␣ WT associated with both transfected and endogenous PKR (upper panel, lanes 2 and 5). Conversely, STAT1 TM binding with endogenous PKR was completely abolished (lane 3) and displayed very marginal binding to transfected PKR, which was detectable only after long exposures (lane 6). In contrast to HA-STAT1 1-413 TM, full-length STAT1␣ TM did not display any difference in its migration pattern compared with STAT1␣ WT. These in vitro findings were also verified in vivo by the yeast two-hybrid assay (data not shown). Taken together, it appears that the DNA-binding domain of STAT1 interacts with PKR in vitro and in vivo.
DNA Binding and Transcriptional Properties of STAT1 TM-To test the ability of STAT1 TM to respond to IFN-␥ treatment, we performed transient transfection assays in STAT1 Ϫ/Ϫ cells using STAT1 WT or STAT1 TM and a luciferase reporter construct driven by two copies of the GAS element from the IFP53 gene. As shown in [fig_ref] FIG. 3: STAT1 TM displays elevated transcriptionalproperties [/fig_ref] , we observed that luciferase expression in cells transfected with STAT1 WT was induced by IFN-␥ treatment. However, in cells transfected with STAT1 TM, we observed, after normalization to Renilla luciferase, a much higher basal luciferase activity (ϳ5-fold) that could be slightly induced by IFN-␥ stimulation.
To better characterize STAT1 TM, we infected STAT1 Ϫ/Ϫ fibroblasts with retroviruses harboring the puromycin-resistant gene and HA-STAT1␣ WT or HA-STAT1␣ TM. As a control, retroviruses containing only the puromycin-resistant gene were used. After puromycin selection, polyclonal populations of STAT1 WT-expressing cells showed ϳ5-fold greater expression over STAT1 TM pools [fig_ref] FIG. 2: Mapping of amino acids on STAT1 required for PKR-binding [/fig_ref]. Transactivation assays using the 2XIFP53-GAS luciferase reporter correlated with our findings in transient transfection experiments that STAT1 TM confers higher basal reporter activity, which can be induced by IFN-␥ treatment [fig_ref] FIG. 3: STAT1 TM displays elevated transcriptionalproperties [/fig_ref]. We next tested whether STAT1 TM could bind DNA after IFN treatment. Although we could not detect ISGF3 formation in STAT1 TM cells in response to IFN-␣/ (data not shown), IFN-␥ stimulation resulted in the formation of DNA-binding complexes consisting of either STAT1⅐STAT3 heterodimers or STAT3 homodimers, but not that of STAT1 homodimers [fig_ref] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro [/fig_ref]. This finding is consistent with previous re-ports that STAT3 is activated following IFN treatment (3). Moreover, the intensity of the STAT3 homodimer appears to be higher compared with control or STAT1 WT cells (compare [fig_ref] FIG. 2: Mapping of amino acids on STAT1 required for PKR-binding [/fig_ref].
The ability of STAT1 to be phosphorylated upon IFN stimulation was also examined [fig_ref] FIG. 3: STAT1 TM displays elevated transcriptionalproperties [/fig_ref]. To compare STAT1 phosphorylation per equal amounts of STAT1 protein, we used a 5-fold higher amount of STAT1 TM extracts versus STAT1 WT before and after IFN stimulation. These reactions were also normalized to total protein concentration by the addition of treated or untreated STAT1 Ϫ/Ϫ control extracts. Although STAT1 WT was tyrosine-phosphorylated following IFN-␣/ or IFN-␥ treatment (top panel, [fig_ref] FIG. 5: STAT1 inhibits PKR activity in vitro [/fig_ref] , we failed to detect STAT1 TM tyrosine phosphorylation . In contrast, phosphorylation of serine 727 did not significantly differ between STAT1 WT and STAT1 TM after IFN treatment (middle panel, [fig_ref] FIG. 5: STAT1 inhibits PKR activity in vitro [/fig_ref]. Reprobing of the membrane with antibodies to HA revealed the hypo-and hyperphosphorylated forms of STAT1 usually observed after IFN treatment (lower panel). Because STAT1 is also known to form heterodimers with STAT3 following IFN stimulation (3), we tested whether STAT1 TM could associate with STAT3. A much higher amount of STAT3 co-precipitated with STAT1 TM before and after IFN treatment [fig_ref] FIG. 3: STAT1 TM displays elevated transcriptionalproperties [/fig_ref] , although STAT1 protein levels were approximately equal (bottom panel). We also analyzed expression and activation of STAT3 in the same protein extracts used for the STAT1/STAT3 co-immunoprecipitation. STAT3 phosphorylation was slightly elevated (ϳ50%) in cells expressing STAT1 TM before or after treatment with either IFN-␣/ or IFN-␥ [fig_ref] FIG. 3: STAT1 TM displays elevated transcriptionalproperties [/fig_ref]. This increase in STAT3 activity may account for increased STAT3 DNA binding in STAT1 TM cells.
STAT1 TM Enhances the Antiviral and Antiproliferative Effects of IFNs-The biological effects of STAT1 TM activation were examined by cell cycle analysis after treatment with IFN-␣/ or IFN-␥ [fig_ref] FIG. 4: STAT1 TM has enhanced antiproliferative and antiviral properties [/fig_ref]. A greater proportion of STAT1 TMexpressing cells (IFN-␣/, 6 -8%; IFN-␥, 10 -11%) were arrested in G 0 /G 1 phase after treatment with either type I or type II IFNs (right panel) compared with control (left panel) or STAT1 WT-expressing (middle panel) cells. In addition, the ability of STAT1 TM cells to resist virus infection was also investigated. Control, STAT1 WT, and STAT1 TM cells were primed with IFNs and subsequently infected with serially diluted VSV. The amount of virus needed to induce CPE was qualitatively measured. As shown in [fig_ref] FIG. 4: STAT1 TM has enhanced antiproliferative and antiviral properties [/fig_ref] , STAT1 TM cells that were treated with IFN-␥ were ϳ50-fold more resistant to VSV infection compared with STAT1 WT cells, and ϳ10 4 -fold more resistant versus control STAT1 Ϫ/Ϫ cells. In contrast, IFN-␣/-treated STAT1 TM cells were 10-fold more susceptible to VSV CPE compared with control, STAT1 WT, and STAT1 ϩ/ϩ cells. Interestingly, even untreated STAT1 TM cells provided a greater degree of protection compared with STAT1 WT and STAT1 ϩ/ϩ cells. This enhanced ability of STAT1 TM cells to resist virus infection was also observed after encephalomyelocarditis virus infection (data not shown). Western blotting against STAT1␣ revealed that STAT1 TM is expressed at much lower levels than STAT1 WT and endogenous STAT1 from STAT1 ϩ/ϩ cells (bottom panel). Taken together, these data suggest that STAT1 TM enhances the antiproliferative and antiviral effects of IFNs on a per molecule basis.
STAT1 Functions as a PKR Inhibitor in Vitro-To gain better insight into the PKR⅐STAT1 interaction, we next mapped Transformants were streaked on control 10% glucose (upper plate) or 10% galactose agar plates and monitored for slow growth phenotype. B, transformants were grown in galactose medium, and relative growth rates were monitored at the indicated times by trypan blue cell counting. The upper graph represents the growth curves of control J110 transformants whereas the bottom graph shows the growth curves of H2544 transformants. C, J110 and H2544 were grown in galactose medium to induce HA-STAT1 1-413 WT and TM expression. 2 mg of protein extracts were immunoprecipitated with HA antibodies and immunoblotted with HA antibodies. D, J110 and H2544 were grown in galactose medium to induce PKR expression. Total protein extracts were subjected to immunoblot analysis with rabbit antisera to human PKR. The upper band, which is present in J110 extracts lacking PKR, is nonspecific (NS). E, extracts from J110 control and H2544 strains transformed with vector alone, K3L, HA-STAT1 1-413 WT, or HA-STAT1 1-413 TM were subjected to isoelectric focusing after induction with galactose and probed with antibodies against yeast eIF-2␣. the STAT1-binding site on PKR [fig_ref] FIG. 5: STAT1 inhibits PKR activity in vitro [/fig_ref]. In agreement with earlier results, the full-length kinase domain (lane 4), but not the dsRNA-binding domain (lane 3), bound PKR. Truncation of the kinase domain from either the amino or carboxyl terminus [fig_ref] FIG. 5: STAT1 inhibits PKR activity in vitro [/fig_ref] defined a region critical for STAT1 binding: amino acids 367-415 (lanes 5-9). Interestingly, this corresponds to the same region in the large lobe of the PKR kinase domain, to which the vaccinia virus K3L protein binds to block PKR activation. In view of this fact, we tested whether the two proteins could compete for binding to PKR. Bacterially expressed FLAG-K3L was co-incubated with GST-PKR C and extracts from HeLa cells expressing increasing amounts of HA-STAT1␣. Immunoblotting was performed to detect either binding of HA-STAT1␣ or FLAG-K3L to GST-PKR C. As seen in [fig_ref] FIG. 5: STAT1 inhibits PKR activity in vitro [/fig_ref] , STAT1 displaced K3L from PKR in a dose-dependent manner (bottom panel, lanes , indicating that K3L and STAT1 bind to the same region on PKR.
The PKR pseudosubstrate, K3L, has been shown to bind PKR and block access of eIF-2␣ to the catalytic pocket of PKR. To address the importance of STAT1 binding to PKR, we investigated the ability of STAT1 to act as a cellular inhibitor of PKR. A series of in vitro assays were performed to assess the effect of STAT1 on PKR activation. Human PKR was activated by reovirus dsRNA in vitro from HeLa S3 cells in the presence of increasing amounts of recombinant full-length STAT1 [fig_ref] FIG. 5: STAT1 inhibits PKR activity in vitro [/fig_ref]. We observed that PKR autophosphorylation diminished in a dose-dependent manner by the addition of STAT1 (top panel), although PKR protein levels were equal (bottom panel).
## Stat1 inhibits the antiproliferative properties of pkr in
Yeast-To date, the best approach to test for the translational function of PKR is in Saccharomyces cerevisiae. It has been shown that high levels of PKR expression in yeast are toxic due to inhibition of general translation. However, at lower levels of expression PKR can substitute the function of GCN2, the only eIF-2␣ kinase known to exist in S. cerevisiae, by phosphorylating eIF-2␣ on serine 51 to inhibit protein synthesis. Through this approach, a number of PKR inhibitors have been identified and characterized. Given that PKR expression in yeast results in inhibition of cell growth, we wanted to analyze whether STAT1 could block this effect when co-expressed with PKR. Yeast strain H2544, which lacks the yeast eIF-2␣ kinase GCN2, contains a stable integration of human PKR WT cDNA downstream to a galactose-inducible promoter, whereas the isogenic strain J110 is identical except that the PKR cDNA was not inserted. Previous studies have shown that induction of PKR expression in H2544 results in inhibition of cell growth through phosphorylation of eIF-2␣. Conversely, co-expression of a PKR inhibitor, such as K3L, leads to rescue of PKR-mediated growth inhibition. To test the inhibitory activity of STAT1 on PKR, strains J110 and H2544 were transformed with vector alone, K3L, HA-STAT1 1-413 WT, or HA-STAT1 1-413 TM. Transformants were streaked onto minimal media plates containing either glucose or galactose as a carbon source, and the effect of each of these proteins on PKR-mediated growth inhibition was monitored. All transformants of the isogenic J110 strain grew well in either glucose or galactose indicating that expression of these exogenous proteins did not perturb normal yeast growth characteristics [fig_ref] FIG. 6: STAT1 inhibits PKR activity in yeast [/fig_ref] , upper graph). H2544 transformants containing only empty plasmid DNA demonstrated a slow-growth phenotype after PKR induction [fig_ref] FIG. 6: STAT1 inhibits PKR activity in yeast [/fig_ref]. However, expression of K3L reversed this growth inhibitory phenotype (lower plate, labeled K3L). Likewise, expression of STAT1 1-413 WT also rescued yeast growth (lower plate, labeled WT), although in contrast, the interaction mutant of STAT1 was unable to counteract the growth inhibitory effects of PKR (lower plate, labeled TM). Growth curves to assess the degree of rescue showed that the ability of HA-STAT1 1-413 WT transformants to rescue growth was half as potent relative to K3L [fig_ref] FIG. 6: STAT1 inhibits PKR activity in yeast [/fig_ref] , lower graph). Because it was not possible to quantify the relative levels of K3L and STAT1 1-413 WT expression, the degree of rescue may be dependent on their different levels of expression. Efforts to rescue growth by full-length HA-STAT1␣ were unsuccessful, because we could not detect HA-STAT1␣ expression in yeast (data not shown). However, the truncated HA-STAT1 1-413 proteins were readily detectable in yeast protein extracts [fig_ref] FIG. 6: STAT1 inhibits PKR activity in yeast [/fig_ref] as was the expression of human PKR in H2544 transformants [fig_ref] FIG. 5: STAT1 inhibits PKR activity in vitro [/fig_ref]. In correlation with the growth curves, the extent of eIF-2␣ phosphorylation, as assessed by isoelectric focusing experiments, was diminished in H2544 transformants expressing either K3L or HA-STAT1 1-413 WT [fig_ref] FIG. 3: STAT1 TM displays elevated transcriptionalproperties [/fig_ref]. The induction of PKR levels in [fig_ref] FIG. 6: STAT1 inhibits PKR activity in yeast [/fig_ref] , was probably translational in nature as a result of inhibition of eIF-2␣ phosphorylation and up-regulation of PKR protein synthesis by K3L [fig_ref] FIG. 3: STAT1 TM displays elevated transcriptionalproperties [/fig_ref]. This PKR up-regulation was not evident in lane 7 most likely due to the weaker inhibitory effect of STAT1 1-413 WT on eIF-2␣ phosphorylation compared with K3L [fig_ref] FIG. 3: STAT1 TM displays elevated transcriptionalproperties [/fig_ref].
## Increased pkr activation and eif-2␣ phosphorylation in stat1
Ϫ/Ϫ Cells-Next we investigated whether the loss of STAT1 would augment PKR activity. To do so, protein extracts from untreated or IFN-treated STAT1 ϩ/ϩ and STAT1 Ϫ/Ϫ cells were used to assess PKR activity in vitro. We noticed that the basal activity of PKR was ϳ5-fold higher in STAT1 Ϫ/Ϫ cells compared with STAT1 ϩ/ϩ cells [fig_ref] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro [/fig_ref] , although PKR protein levels were equivalent, as assessed by in vivo [ 35 S]methionine labeling (lower panel, compare [fig_ref] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro [/fig_ref]. The increase in PKR activity after IFN treatment in STAT1 ϩ/ϩ cells (top panel) reflects increased PKR expression, whereas no such increase in PKR activity/protein was observed in STAT1 Ϫ/Ϫ cells. This is consistent with the notion that transcriptional up-regulation of PKR after IFN treatment is dependent on the JAK/STAT pathway (upper panel, [fig_ref] FIG. 2: Mapping of amino acids on STAT1 required for PKR-binding [/fig_ref]. To further substantiate our findings that PKR activity is elevated in STAT1 Ϫ/Ϫ cells, we compared the levels of eIF-2␣ phosphorylation in STAT1 ϩ/ϩ and STAT1 Ϫ/Ϫ cells in vivo. The effect of STAT1 on eIF-2␣ phosphorylation in STAT1 ϩ/ϩ and STAT1 Ϫ/Ϫ cells was examined by treatment with dsRNA and immunoblotting with a phosphospecific antibody to phosphoserine 51 of eIF-2␣ [fig_ref] FIG. 7: PKR activity and eIF-2␣ phosphorylation are elevated in STAT1 ؊/؊ cells [/fig_ref]. These experiments showed that a higher amount of eIF-2␣ was phosphorylated in STAT1 Ϫ/Ϫ cells compared with STAT1 ϩ/ϩ [fig_ref] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro [/fig_ref] and that this phosphorylation was more highly induced after dsRNA treatment (compare [fig_ref] FIG. 2: Mapping of amino acids on STAT1 required for PKR-binding [/fig_ref]. Taken together, the inhibition of PKR activity by STAT1 in mammalian and yeast cells supports our findings that STAT1 can inhibit PKR activity in vitro and in vivo. We have mapped the sites of interaction between PKR and STAT1 in vitro using GST pull-down assays and also in vivo. We found that STAT1 interacts with PKR on amino acids 367-415, an area located within the large lobe of the kinase domain of PKR that is also bound by the vaccinia virus encoded PKR inhibitor, K3L. Sequence comparison with other eIF-2␣ family members, GCN2, HRI, and the newly discovered PERK/PEK revealed that this part of the kinase domain is highly conserved and suggests that these members might be capable of interacting with STAT1. Our previous observations that the dsRNA-binding mutant PKRLS4 was unable to associate with STAT1 in vivo, at first, appear to be challenged by our in vitro studies herein, which show that the carboxyl terminus of PKR is required for binding to STAT1 and that GST-PKRK296RLS4 can interact with STAT1 [fig_ref] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro [/fig_ref]. One possible explanation for this difference may be inferred, based on structural data of the amino terminus of PKRand also on previous mutational studies, that the LS4 and LS9 mutations could introduce conformational changes that would affect its interaction with STAT1 in vivo. Such conformational changes may not take place when GST-PKRK296RLS4 and GST-PKRK296RLS9 are purified from bacteria. Another conceivable explanation is that the association of PKR with STAT1 in vivo is facilitated by the presence of other protein(s) whose action is modulated by the amino terminus of PKR and may become limited when the interaction is tested in vitro.
In the case of STAT1, amino acids 343-348 provide a major site of interaction with PKR. This region of STAT1 corresponds to -sheet 3, a structure located on the back side of the DNAbinding domain of STAT1. Mutation of three amino acids within this region (STAT1 TM; Arg 346 -Leu 347 -Leu 348 to Ala 346 -Asp 347 -Asp 348 ) abolished binding to PKR in vitro and in vivo. Paradoxically, sequence alignment of this three-residue stretch of wild-type STAT1 with other STAT members showed almost complete conservation. Similarly, residues in -sheet 3 of the DNA-binding domain of STAT3 responsible for specific interaction with c-Jun are also nearly identical between STAT members; however, c-Jun specifically interacts with STAT3 (42). Thus, our observation that PKR specifically interacts with STAT1 can probably only be explained in the context of the presentation of -sheet 3 in STAT1 compared with other STAT molecules. In the end, structural analysis might be necessary to determine the exact points of contact between PKR and STAT1.
In light of our previous observations that the ability of PKR to interact with STAT1 can block STAT1 DNA binding (6), we examined whether release of STAT1 from this inhibitory mechanism might augment its transcriptional activity. In contrast to STAT1 WT, we were unable to observe tyrosine phosphorylation of STAT1 TM following IFN stimulation, and as a result, ISGF3 and STAT1 homodimer formation could not be detected in DNA binding experiments. We believe that the inability of STAT1 TM to be phosphorylated cannot be the result of a misfolded protein, because this mutant is phosphorylated on serine 727 after IFN treatment. Rather, it appears that some undefined negative regulatory mechanism is responsible for this phenomenon. The protein kinase that directly phosphorylates serine 727 of STAT1 in vivo is not as yet known. It is unlikely to be PKR because STAT1 is not phosphorylated by PKR in vitro. Interestingly, a recent report shows a defective serine 727 phosphorylation of STAT1 in PKR Ϫ/Ϫ fibroblasts after IFN stimulation providing evidence for an indirect role of the kinase in this process. Although unable to bind DNA as a homodimer, STAT1 TM was competent and more capable in forming DNA-binding complexes with STAT3 relative to STAT1 WT cells. Interestingly, tyrosine phosphorylation of STAT3 was elevated by 50% before and after IFN treatment in polyclonal cell populations expressing STAT1 TM. The mechanism behind this finding is not clear at this time, but it might be possible that recruitment of STAT3 to the JAK⅐receptor complex is more efficient in STAT1 TM cells compared with STAT1 WT cells. Nevertheless, the net effect of increased STAT1⅐STAT3 heterodimer and STAT3 homodimer formation probably contributes to up-regulation of STAT1⅐STAT3 DNAbinding and GAS-dependent transactivation. Because STAT3 homodimers appear to play a minimal role in the transactivation of IFN-dependent genes by IFN-␥ (44), it is likely that the transcriptional activity observed in our reporter assays is contributed by the STAT1⅐STAT3 heterodimer. This is the second instance where a transcriptional role of STAT1 has been shown to require serine 727 but not tyrosine 701 phosphorylation. An earlier study demonstrated that mutation of serine 727 to alanine, but not tyrosine 701 to phenylalanine, on STAT1 significantly ablated the TNF-␣-dependent induction of caspase genes. It was speculated that STAT1 could potentially participate in signaling pathways independent of its tyrosine phosphorylation state; such a hypothesis is supported by our findings in here.
The antiproliferative and antiviral effects of IFNs were also investigated, because a number of cell cycle regulatory proteins are regulated at the transcriptional level through the JAK/ STAT signaling pathway. IFN-dependent cell cycle arrest, as measured by FACS analysis, was significantly higher in cells expressing STAT1␣ TM versus control cells or cells expressing STAT1␣ WT. In addition, the ability of STAT1 TM cells to resist VSV infection was ϳ10-fold greater than control or STAT1 WT cells following IFN-␥ stimulation. These differences become more significant when the variation in the expression levels between STAT1 WT and TM is considered (i.e. 5-fold higher expression of WT than STAT1 TM; [fig_ref] FIG. 2: Mapping of amino acids on STAT1 required for PKR-binding [/fig_ref]. Interestingly, even untreated STAT1 TM cells provided greater protection relative to control and STAT1 WT cells, suggesting that the anti-viral responses available within STAT1 TM cells are already enhanced prior to IFN pretreatment.
Given the fact that STAT1 and K3L, a pseudosubstrate in-hibitor of PKR, can compete for binding with PKR, we next analyzed whether STAT1 could inhibit PKR activation. We observed that recombinant STAT1 inhibited PKR activation in vitro in a dose-dependent manner. However, it is unlikely that STAT1 is a substrate or pseudosubstrate for PKR, because sequence alignment of STAT1 with K3L and eIF-2␣ did not reveal any significant similarities, findings that coincide with our earlier observations that PKR does not phosphorylate STAT1 in vitro or in vivo. Instead, the interaction of the two proteins might prevent opening of the cleft that separates the two lobes of PKR's kinase domain, thus inhibiting both PKR activation and eIF-2␣ phosphorylation. The capacity of STAT1 to neutralize PKR activity is further appreciated from in vivo studies. PKR exhibits the same substrate specificity as the yeast GCN2 kinase that regulates protein synthesis by phosphorylating eIF-2␣ to inhibit cell growth and replacement of GCN2 with PKR leads to inhibition of translation and yeast growth. As such, this system has been consistently used as a means to probe for PKR activation, as well as for characterizing inhibitors of PKR, like K3L. The observation that HA-STAT1 1-413 WT, but not the interaction mutant, TM, was able to block PKR activity and subsequently relieve the growth inhibitory effects of PKR, supports our model whereby PKR activity is tightly regulated by its interaction with STAT1. These results from yeast were further substantiated in STAT1 Ϫ/Ϫ cells where we observed augmentation of both PKR activity and in vivo eIF-2␣ phosphorylation in comparison to wild-type cells.
We rationalize that the interaction of PKR and STAT1 modulates cellular proliferation in normally growing cells and in cells challenged by viruses. In unstimulated cells, the balance of PKR⅐STAT1 heterodimer formation versus their free monomers may dictate cellular proliferative capacity. For example, perturbing this equilibrium by the expression of catalytic mutants of PKR would sequester a larger fraction of STAT1and could contribute to increased virus susceptibility and to the transformed phenotype of cells expressing such mutants of PKR. Conversely, loss of PKR's repressive activity in PKR Ϫ/Ϫ mice would favor the antiviral and antiproliferative functions of STAT1. As such, PKR Ϫ/Ϫ mice are still capable of providing host resistance to many viruses. It would thus appear that STAT1 provides a first line of defense against virus infection, whereas the role of PKR in viral resistance appears to be secondary. This is illustrated by the observation that STAT1 Ϫ/Ϫ mice are exquisitely sensitive to a variety of pathogens, whereas PKR Ϫ/Ϫ mice exhibit a modest sensitivity. Increased eIF-2␣ phosphorylation in STAT1 Ϫ/Ϫ cells could provide a compensatory mechanism for the loss of STAT1 and may play a role in differences in the susceptibility of STAT1 Ϫ/Ϫ mice to virus infection. Therefore, eIF-2␣ phosphorylation may play a role in the differential sensitivity of various viruses to interferon treatment, an effect that could be mediated through the interaction of STAT1 with PKR and other eIF-2␣ kinases, because they share high sequence homology in the STAT1 binding region. Generation of mice lacking multiple members of this family of kinases 2 may be useful in determining such virus-specific responses.
[fig] FIG. 1: The kinase domain of PKR specifically interacts with STAT1 in vitro. A, GST alone, GST-PKR N, GST-PKR C, and GST-PKR FL were subjected to SDS-PAGE and visualized by Coomassie Blue staining. B, GST-PKR fusion proteins were co-incubated with protein extracts of HeLa S3 cells transfected with HA-STAT1␣ (top), STAT2 (second panel), STAT3 (third panel), or myc-STAT5B (bottom panel). Bound proteins were revealed by immunoblotting with HA (top panel), STAT2 (second panel), STAT3 (third panel), or Myc (bottom panel) antibodies. C, GST alone, GST-PKRK296R, GST-PKRK296RLS4, and GST-PKRK296RLS9 were co-incubated with protein extracts from HeLa S3 cells transfected with HA-STAT1␣. After GST-pull down, proteins were detected by immunoblotting with HA (top panel) whereas the GST fusion proteins were visualized by Coomassie Blue staining (bottom panel). [/fig]
[fig] FIG. 2: Mapping of amino acids on STAT1 required for PKR-binding. A, STAT1 was truncated into five portions represented by the various shades of the molecule depicted. B, GST alone or GST-PKR C was co-incubated with [ 35 S]methionine-labeled, in vitro translated STAT1 proteins and subjected to SDS-PAGE. C, GST alone or GST-PKR C was incubated with in vitro translated STAT1 proteins truncated from either the amino or carboxyl terminus and subjected to SDS-PAGE. D, similar pull-down assays were performed using GST or GST-PKR C with in vitro translated STAT1 proteins further truncated from the carboxyl terminus. E, GST alone or GST-PKR C was co-precipitated with in vitro translated STAT1 1-413 wild-type (WT) or triplemutant (TM). F, U3A cells were transiently transfected with HA-STAT1␣ WT or TM in the presence or absence of PKRK296R. Extracts were immunoprecipitated with anti-PKR antibodies and immunoblotted with HA (top panel) and PKR (bottom panel) antibodies. [/fig]
[fig] FIG. 3: STAT1 TM displays elevated transcriptionalproperties.A, STAT1 Ϫ/Ϫ cells were transiently transfected with pRL-TK, pGL-2XIFP53-GAS, and control vector, HA-STAT1␣ WT, or HA-STAT1␣ TM. Transfected cells were left untreated (white bars) or stimulated with IFN-␥ for 18 h (black bars) and harvested. B, STAT1 Ϫ/Ϫ cells stably transduced with control vector, HA-STAT1␣ WT, or HA-STAT1␣ TM were transfected with pRL-TK and pGL-2ϫIFP53-GAS. Transfected cells were left untreated (white bars) or stimulated with IFN-␥ for 18 h (black bars) and harvested. Luciferase activity was measured and normalized to Renilla luciferase activity. The data represent the average of quadruplicate experiments. The relative protein levels of STAT1 WT and STAT1 TM were compared by immunoblotting with antibodies against HA. C, control, WT, and TM cells were treated with IFN-␥ for 30 min or left untreated. Protein extracts were incubated with 32 P-radiolabeled csis-inducible element from the c-Fos promoter and subjected to DNA binding assays. Competition reactions were performed using a 200-fold excess of unlabeled oligonucleotide. Supershift experiments were performed using non-reactive mouse IgG 1 , STAT1␣, or STAT3 antibodies. D, control, STAT1 WT, and STAT1 TM cells were left untreated, or stimulated with IFN-␣/ or IFN-␥ for 30 min. STAT1 WT and TM protein levels were normalized with the appropriately stimulated or unstimulated control cell extracts (STAT1 Ϫ/Ϫ ) and subsequently immunoprecipitated with STAT1␣ antibody and immunoblotted against phosphotyrosine (top panel), STAT1␣ phosphoserine 727 (middle panel), and HA (bottom panel) antibodies. E, normalized protein extracts were immunoprecipitated with antibodies against STAT1 and immunoblotted with STAT3 (top panel) or HA (bottom panel) antibodies. F, the above extracts were immunoprecipitated with antibodies against STAT3 and immunoblotted against phosphotyrosine (top panel) or STAT3 (bottom panel). [/fig]
[fig] FIG. 4: STAT1 TM has enhanced antiproliferative and antiviral properties. A, polyclonal STAT1 Ϫ/Ϫ cell lines transduced with control vector, HA-STAT1␣ WT, or HA-STAT1␣ TM were synchronized by serum starvation for 48 h and stimulated with IFN-␣/ or IFN-␥ for 12 and 24 h. Cells were fixed, stained with propidium iodide, and subjected to cell cycle analysis. The data were processed using the WINMDI v2.8 application. The relative cell-cycle distributions presented in the table represent two averaged experiments. B, cells were pretreated with IFN-␣/ or IFN-␥ for 18 h before being infected with serially diluted VSV for 24 h, after which viable cells were stained with crystal violet (top panel). Whole-cell extracts from the cell lines were immunoblotted with antibodies against STAT1␣ and immunoblotted with an antibody against the HA epitope (bottom panel). [/fig]
[fig] FIG. 5: STAT1 inhibits PKR activity in vitro.A, GST-PKR truncated proteins were generated according to the schematic shown. GST-PKR proteins were co-incubated with extracts expressing HA-ST-AT1␣ and binding to PKR was determined by immunoblotting with STAT1␣ antibodies (top panel) and reprobed with GST antibody (bottom panel) to determine the levels of fusion protein. B, GST alone or GST-PKR C was incubated with a constant amount of recombinant FLAG-K3L and cell extracts expressing increasing amounts of HA-STAT1␣ (mock, 0.1 g, 0.5 g, and 5 g of DNA). SDS-PAGE was performed followed by immunoblot analysis with HA (top panel) or FLAG (bottom panel) antibodies. C, PKR was immunoprecipitated from 50 g of HeLa S3 protein extracts, and PKR was activated in vitro in the presence of activator reovirus dsRNA, [␥-32 P]ATP, and 500 ng of GST protein or increasing amounts of purified STAT1␣ protein (5, 50, and 500 ng of STAT1␣; lanes 4, 5, and 6, respectively). The reactions were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes, exposed (top panel), and immunoblotted with anti-STAT1␣ (middle panel) or anti-PKR (bottom panel) antibodies. [/fig]
[fig] FIG. 6: STAT1 inhibits PKR activity in yeast. A, strains J110 (bottom half of both plates) and H2544 (top half of both plates) were transformed with control vector (labeled C), K3L (labeled K3L), HA-STAT1 1-413 WT (labeled WT), or HA-STAT1 1-413 TM (labeled TM). [/fig]
[fig] FIG. 7: PKR activity and eIF-2␣ phosphorylation are elevated in STAT1 ؊/؊ cells. A, STAT1 ϩ/ϩ (top panel) and STAT1 Ϫ/Ϫ MEFs were treated with IFN-␣/ overnight or left untreated. Protein extracts were immunoprecipitated against mouse PKR, and in vitro kinase assays were performed in the presence of activator reovirus dsRNA and [␥-32 P]ATP. To monitor PKR protein levels, [ 35 S]methionine in vivo labeling was performed (bottom panel) in STAT1 ϩ/ϩ , STAT1 Ϫ/Ϫ , PKR ϩ/ϩ , and PKR Ϫ/Ϫ MEFs. Mouse PKR was immunoprecipitated with a rabbit anti-mouse PKR polyclonal antibody, subjected to SDS-PAGE, and visualized by fluorography. B, STAT1 ϩ/ϩ or STAT1 Ϫ/Ϫ cells were left untreated or transfected with dsRNA (i.e. poly(rI⅐rC), 100 g/ml), and protein extracts were subjected to SDS-PAGE analysis. Immunoblotting was performed with antibodies against phosphoserine 51 of eIF-2␣ (top panel) or eIF-2␣ (bottom panel). [/fig]
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10.1038/s41467-021-23726-4
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CCBY
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8203779
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34127662
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s2orc_pubmed_articles
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Distinct mechanisms of the human mitoribosome recycling and antibiotic resistance
Ribosomes are recycled for a new round of translation initiation by dissociation of ribosomal subunits, messenger RNA and transfer RNA from their translational post-termination complex. Here we present cryo-EM structures of the human 55S mitochondrial ribosome (mitoribosome) and the mitoribosomal large 39S subunit in complex with mitoribosome recycling factor (RRF mt ) and a recycling-specific homolog of elongation factor G (EF-G2 mt ). These structures clarify an unusual role of a mitochondria-specific segment of RRF mt , identify the structural distinctions that confer functional specificity to EF-G2 mt , and show that the deacylated tRNA remains with the dissociated 39S subunit, suggesting a distinct sequence of events in mitoribosome recycling. Furthermore, biochemical and structural analyses reveal that the molecular mechanism of antibiotic fusidic acid resistance for EF-G2 mt is markedly different from that of mitochondrial elongation factor EF-G1 mt , suggesting that the two human EF-G mt s have evolved diversely to negate the effect of a bacterial antibiotic.
T he process of protein synthesis in all living cells is orchestrated by highly complex macromolecular assemblies called ribosomes, in coordination with mRNA, tRNAs, and multiple translational factors. Mitochondrial ribosomes (mitoribosomes) and their associated translation machinery are distinct from those in the cytoplasm and display features reminiscent of prokaryotic translation 1 , in line with the assumption that mitochondria have evolved from endocytosis of an α-proteobacterium by an ancestral eukaryotic cell 2 . However, cryo-electron microscopy (cryo-EM) structures have revealed that the mammalian mitoribosomes have diverged considerably from bacterial ribosomes and acquired several unique features [bib_ref] The structure of the human mitochondrial ribosome, Amunts [/bib_ref] [bib_ref] The complete structure of the 55S mammalian mitochondrial ribosome, Greber [/bib_ref] [bib_ref] Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome, Kaushal [/bib_ref] [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref] [bib_ref] Structural insights into unique features of the human mitochondrial ribosome recycling, Koripella [/bib_ref] [bib_ref] Structure of the mammalian mitochondrial ribosome reveals an expanded functional role for..., Sharma [/bib_ref]. A striking difference is the reversal in the protein to RNA ratio, as the bacterial ribosomes are high in ribosomal RNA (rRNA) whereas the mammalian mitoribosomes are high in protein. The increase in protein mass is the result of acquisition of multiple mito-specific ribosomal proteins (MRPs) and addition of extensions to many MRPs, that are homologous to bacterial ribosomal proteins. Though the steps of mitochondrial translation closely resemble those for prokaryotic translation in the general sequence of events and the homologous accessory protein factors involved, they also show significant structural and functional differences [bib_ref] Mechanism of protein biosynthesis in mammalian mitochondria, Christian [/bib_ref].
The complex process of protein synthesis is accomplished in four essential steps of initiation, elongation, termination, and the ribosome recycling. The transition between translation termination and ribosome recycling is well characterized in eubacteria. During translation termination, the nascent polypeptide chain attached to the peptidyl tRNA is released from the ribosome with the help of a class I release factor (RF) that interacts with the stop codon exposed at the ribosomal decoding site, or aminoacyl-tRNA binding site (A site) [bib_ref] Termination of translation: interplay of mRNA, rRNAs and release factors?, Kisselev [/bib_ref] [bib_ref] Release factors and their role as decoding proteins: specificity and fidelity for..., Poole [/bib_ref]. Subsequently, the class I RF is dissociated from the ribosome with the help of a class II RF in a GTP hydrolysis-dependent manner [bib_ref] Release factor RF3 in E.coli accelerates the dissociation of release factors RF1..., Freistroffer [/bib_ref] [bib_ref] Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome..., Gao [/bib_ref]. At the end of the termination, the translated mRNA and the deacylated tRNA remain associated with the ribosome [bib_ref] The fourth step of protein synthesis: disassembly of the posttermination complex is..., Kaji [/bib_ref] [bib_ref] Novel roles for classical factors at the interface between translation termination and..., Karimi [/bib_ref] , a state referred to as the posttermination complex (PoTC). In order to initiate a new round of protein synthesis, the ribosome must be split into its two subunits and its bound ligands must be removed. In eubacteria, the disassembly of the PoTC requires the concerted action of two protein factors, the ribosome recycling factor (RRF) and the elongation factor G (EF-G) [bib_ref] The fourth step of protein synthesis: disassembly of the posttermination complex is..., Kaji [/bib_ref] [bib_ref] Specific interaction between the ribosome recycling factor and the elongation factor G..., Rao [/bib_ref] [bib_ref] Splitting of the posttermination ribosome into subunits by the concerted action of..., Zavialov [/bib_ref]. RRF binds to the PoTC as the 70S ribosome adopts a ratcheted conformation [bib_ref] Progression of the ribosome recycling factor through the ribosome dissociates the two..., Barat [/bib_ref] [bib_ref] Mechanism for the disassembly of the posttermination complex inferred from cryo-EM studies, Gao [/bib_ref] , in which the small (30S) subunit of the ribosome rotates in an anticlockwise direction with respect to the large (50S) subunit [bib_ref] A ratchet-like inter-subunit reorganization of the ribosome during translocation, Frank [/bib_ref]. This is followed by the binding of EF-G in conjugation with guanosine 5′triphosphate (GTP) to the RRF-bound PoTC and the dissociation of the 70S ribosome into its large and small subunits, a process that requires the hydrolysis of GTP on EF-G [bib_ref] Complete kinetic mechanism for recycling of the bacterial ribosome, Borg [/bib_ref] [bib_ref] The role of ribosome recycling factor in dissociation of 70S ribosomes into..., Hirokawa [/bib_ref] [bib_ref] Sequence of steps in ribosome recycling as defined by kinetic analysis, Peske [/bib_ref]. Though the involvement of a third factor, initiation factor 3 (IF3) in the recycling process is generally agreed upon, its precise function has been debated [bib_ref] Novel roles for classical factors at the interface between translation termination and..., Karimi [/bib_ref] [bib_ref] Splitting of the posttermination ribosome into subunits by the concerted action of..., Zavialov [/bib_ref] [bib_ref] The role of ribosome recycling factor in dissociation of 70S ribosomes into..., Hirokawa [/bib_ref].
Unlike most eubacteria, where a single form of EF-G is known to participate in both the elongation and ribosome recycling steps [bib_ref] Complete kinetic mechanism for recycling of the bacterial ribosome, Borg [/bib_ref] [bib_ref] Distinct functions of elongation factor G in ribosome recycling and translocation, Savelsbergh [/bib_ref] , mammalian mitochondria utilize two isoforms of EF-G, EF-G1 mt , and EF-G2 [bib_ref] Identification and characterization of two novel human mitochondrial elongation factor genes, hEFG2..., Hammarsund [/bib_ref] [bib_ref] EF-G2mt is an exclusive recycling factor in mammalian mitochondrial protein synthesis, Tsuboi [/bib_ref]. While EF-G1 mt specifically functions as a translocase during the polypeptide elongation step [bib_ref] Expression and characterization of isoform 1 of human mitochondrial elongation factor G, Bhargava [/bib_ref] , EF-G2 mt has been reported to act exclusively as a second recycling factor together with RRF mt [bib_ref] EF-G2mt is an exclusive recycling factor in mammalian mitochondrial protein synthesis, Tsuboi [/bib_ref]. Human RRF mt is about 25-30% identical to its eubacterial homologs but carries an additional 79 amino acids (aa) long extension at its N-terminus [bib_ref] Identification and cloning of human mitochondrial translational release factor 1 and the..., Zhang [/bib_ref]. The recent high-resolution cryo-EM structures of RRF mt bound to the 55S mitoribosomes [bib_ref] Structural insights into unique features of the human mitochondrial ribosome recycling, Koripella [/bib_ref] , and an in vivo formed mitoribosomal complex [bib_ref] Structural basis of mitochondrial translation, Aibara [/bib_ref] revealed that the structurally conserved segment of the RRF mt is similar to its bacterial analog on [bib_ref] Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome..., Gao [/bib_ref] [bib_ref] Progression of the ribosome recycling factor through the ribosome dissociates the two..., Barat [/bib_ref] [bib_ref] Mechanism for the disassembly of the posttermination complex inferred from cryo-EM studies, Gao [/bib_ref] [bib_ref] Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: functional implications, Agrawal [/bib_ref] [bib_ref] Structures of the bacterial ribosome in classical and hybrid states of tRNA..., Dunkle [/bib_ref] [bib_ref] Crystal structure of the ribosome recycling factor bound to the ribosome, Weixlbaumer [/bib_ref] [bib_ref] Structural insights into initial and intermediate steps of the ribosome-recycling process, Yokoyama [/bib_ref] [bib_ref] Structural basis for ribosome recycling by RRF and tRNA, Zhou [/bib_ref] [bib_ref] Key intermediates in ribosome recycling visualized by timeresolved cryoelectron microscopy, Fu [/bib_ref] and off [bib_ref] Crystal structure of Thermotoga maritima ribosome recycling factor: a tRNA mimic, Selmer [/bib_ref] [bib_ref] Solution structure of the ribosome recycling factor from Aquifex aeolicus, Yoshida [/bib_ref] the 70S ribosome in terms of its overall size and domain composition. However, the unique mito-specific N-terminal extension (NTE) in RRF mt extends towards the GTPase-associated center and interacts with the functionally important 16S rRNA elements of the mitoribosomal 39S subunit, including the rRNA helices 89 (H89), H90, and H92 [bib_ref] Structural insights into unique features of the human mitochondrial ribosome recycling, Koripella [/bib_ref].
Valuable mechanistic inferences about the bacterial ribosome recycling process were made from the structures of the 70S- RRF (e.g., Agrawal et al. [bib_ref] Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: functional implications, Agrawal [/bib_ref] and dissociated 50S- RRF- EF-G 14,20 complexes. Capturing the simultaneous binding of both factors on the 70S ribosome is challenging, however, owing to the rapid rate of 70S ribosomes dissociation into subunits by the combined action of RRF and EF-G [bib_ref] Complete kinetic mechanism for recycling of the bacterial ribosome, Borg [/bib_ref]. To slow down this reaction, a heterologous system with T. thermophilus RRF and E. coli EF-G was used to capture both factors on the 70S ribosome by cryo-EM [bib_ref] Structural insights into initial and intermediate steps of the ribosome-recycling process, Yokoyama [/bib_ref]. Subsequently, a time-resolved cryo-EM study was also able to capture various 70S- RRF- EF-G functional intermediates, albeit at low resolution [bib_ref] Key intermediates in ribosome recycling visualized by timeresolved cryoelectron microscopy, Fu [/bib_ref]. More recently, a bacterial ribosome recycling complex containing both RRF and EF-G was obtained by X-ray crystallography by stabilizing EF-G on the 70S ribosome through a fusion between EF-G and ribosomal protein bL9 [bib_ref] Structural basis for ribosome recycling by RRF and tRNA, Zhou [/bib_ref]. All these structures conclude that binding of EF-G to the 70S- RRF complex induces rotation of RRF domain II towards the helix 44 (h44) region of the 30S subunit, destabilizing the crucial intersubunit bridges B2a and B3, and thereby facilitating the dissociation of the 70S ribosome into its two subunits.
With a molecular weight of 87 kD, human EF-G2 mt is slightly larger than EF-G1 mt (83 kD), as well as the two isoforms of bacterial EF-Gs, EF-G (78 kD), and EF-G2 (73 kD), identified in certain bacterial species but without known function for the second bacterial isoform [bib_ref] Structural basis for interaction of the ribosome with the switch regions of..., Connell [/bib_ref] [bib_ref] Phylogenetic distribution of translational GTPases in bacteria, Margus [/bib_ref] [bib_ref] Analysis of the fusA2 locus encoding EFG2 in Mycobacterium smegmatis, Seshadri [/bib_ref] , except in case of a spirochete 42 . EF-G2 mt has about 36% aa sequence identity to EF-G1 mt and about 30% aa identity to both its bacterial homologs. Some mammalian mitochondrial translation steps are now better understood through determination of the cryo-EM structures of the initiation [bib_ref] Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM, Kummer [/bib_ref] [bib_ref] Insertion domain within mammalian mitochondrial translation initiation factor 2 serves the role..., Yassin [/bib_ref] and the elongation 6,46 complexes at highresolution. Our previous study of the human mitoribosome recycling complex of the RRF mt -bound 55S 7 provided useful insights into the mito-specific aspects of the recycling process, but a complete 55S mitoribosomal recycling complex comprising both RRF mt and EF-G mt remained elusive.
In this work, we investigate the role of mito-specific aa segments of RRF mt and EF-G2 mt in recycling the 55S mitoribosome PoTC by determining cryo-EM structures of the key intermediate mitoribosome- RRF mt - EF-G2 mt complexes, and determine the effect of fusidic acid, an antibiotic that is known to inhibit the GTPase activity of bacterial EF-G 47 , on the GTPase activity of EF-G2 mt . Our study reveals distinct features of the mechanism of human mitoribosome recycling and of the mechanism of fusidicacid resistance shown by EF-G1 mt and EF-G2 mt .
# Results and discussion
Structure of the human mitoribosome recycling complex. To investigate the molecular mechanism of ribosome recycling in mammalian mitochondria, we first prepared a model posttermination complex (PoTC) by incubating the human 55S mitoribosome with puromycin 7,48 . The model PoTC was briefly incubated with human RRF mt and human EF-G2 mt -GMPPCP to obtain the mitoribosome recycling complex. (see "Methods" section). Single-particle cryo-EM analysis on this complex yielded three major classes that each represent a major functional state formed during human mitoribosome recycling, referred henceforth to as Class I, Class II, and Class III [fig_ref] Figure 1: Cryo-EM structures of the human mitochondrial recycling complexes in three functional states [/fig_ref]. Class I corresponds to the intact 55S mitoribosome that carries a strong density for RRF mt, and was refined to 3.5 Å (Figs. 1 and S1a). Class II, a relatively small class with only 28,929 particle images, corresponds to the 55S mitoribosome that carries both RRF mt and EF-G2 mt , and was refined to 3.9 Å . In this class, the densities corresponding to the large (39S) mitoribosomal subunit, RRF mt , and EF-G2 mt are well resolved, but the small (28S) mitoribosomal subunit appears to be loosely bound and present in multiple poses (Figs. 1 and S2). The most populated Class III corresponds to the dissociated 39S subunits that carry both RRF mt and EF-G2 mt , and was refined to 3.15 Å (Figs. 1 and S2c). Class II likely represents an ensemble of low-population intermediate states of mitoribosome recycling that occur between the states represented by Classes I and III. In addition to these three recycling complexes, we have also obtained a class of particles consisting of 55S mitoribosomes without either of the two factors, where the 28S subunit was rotated by about 8°around its long axis such that its shoulder side moves closer to the 39S subunit while its platform side moves away from it [fig_ref] Figure 3: Direct involvement of RRF mt domain II and EF-G1 mt domain IV... [/fig_ref]. A similar orientation for the small subunit relative to the large subunit, termed as "subunit rolling" has been reported earlier for the 80S ribosomes 49 and the 55S mitoribosomes [bib_ref] The structure of the human mitochondrial ribosome, Amunts [/bib_ref] [bib_ref] Structural insights into unique features of the human mitochondrial ribosome recycling, Koripella [/bib_ref].
RRF mt binding stabilizes the rotated state of the 28S subunit in the 55S mitoribosome. Superimposition of our Class I complex with the RRF mt -unbound human 3 , bovine [bib_ref] Structure of the mammalian mitochondrial ribosome reveals an expanded functional role for..., Sharma [/bib_ref] and porcine 4 55S mitoribosomes showed that the small 28S subunit was rotated counter-clockwise by about 8.5°with respect to the large 39S subunit [fig_ref] Figure 3: Direct involvement of RRF mt domain II and EF-G1 mt domain IV... [/fig_ref] , similar to the "ratchet-like inter-subunit rotation" observed in the bacterial 70S ribosome 21 and the 55S mitoribosomes complexed with translational factors [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref] [bib_ref] Structural insights into unique features of the human mitochondrial ribosome recycling, Koripella [/bib_ref] [bib_ref] Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM, Kummer [/bib_ref] [bib_ref] Structural insights into mammalian mitochondrial translation elongation catalyzed by mtEFG1, Kummer [/bib_ref]. In addition to the inter-subunit rotation, the head domain of the 28S subunit is rotated by about 4°towards the tRNA exit (E) site in a direction roughly orthogonal to the inter-subunit motion [fig_ref] Figure 3: Direct involvement of RRF mt domain II and EF-G1 mt domain IV... [/fig_ref] , similar to "head swiveling" in the bacterial 70S ribosomes [bib_ref] Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA..., Ratje [/bib_ref] [bib_ref] Structures of the bacterial ribosome at 3.5 A resolution, Schuwirth [/bib_ref]. As expected, the structure of the Class I complex matches the previously published 3.9 Å resolution map of the analogous 55S- RRF mt complex 7 .
The Class I map showed the characteristic "L" shaped RRF mt density and a density corresponding to a pe/E-state tRNA within the inter-mitoribosomal subunit space. The overall positioning and domain arrangement of RRF mt in the Class I map is similar to the bacterial RRF on [bib_ref] Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome..., Gao [/bib_ref] [bib_ref] Progression of the ribosome recycling factor through the ribosome dissociates the two..., Barat [/bib_ref] [bib_ref] Mechanism for the disassembly of the posttermination complex inferred from cryo-EM studies, Gao [/bib_ref] [bib_ref] Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: functional implications, Agrawal [/bib_ref] [bib_ref] Structures of the bacterial ribosome in classical and hybrid states of tRNA..., Dunkle [/bib_ref] [bib_ref] Crystal structure of the ribosome recycling factor bound to the ribosome, Weixlbaumer [/bib_ref] [bib_ref] Structural insights into initial and intermediate steps of the ribosome-recycling process, Yokoyama [/bib_ref] [bib_ref] Structural basis for ribosome recycling by RRF and tRNA, Zhou [/bib_ref] [bib_ref] Key intermediates in ribosome recycling visualized by timeresolved cryoelectron microscopy, Fu [/bib_ref] and off 37 the 70S ribosomes, and also to the structures of RRF bound to the 70S chloroplast ribosome [bib_ref] Structure of the chloroplast ribosome with chl-RRF and hibernation-promoting factor, Boerema [/bib_ref] [bib_ref] Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional..., Sharma [/bib_ref] , and RRF mt bound to the human mitochondrial 55S in our previous study 7 . As observed in bacteria, domain I is positioned close to the peptidyl transferase center (PTC) and extends towards the α-sarcin-ricin loop (SRL). A striking difference between the human RRF mt and its bacterial counterpart is the presence of a 79 aa long N-terminal extension (NTE) in RRF mt. We could model the last 14 aa residues of the NTE of RRF mt into an additional density contiguous with the α-helix1 from domain I. As discussed in our previous study 7 , the NTE is strategically positioned in the intersubunit space between domain I and several functionally important 16S rRNA structural elements such as H89, H90, H92 (A-loop), and MRP L16 [fig_ref] Figure 4: Stabilization of the E-site tRNA on the dissociated 39S subunit [/fig_ref] and interacts with several nucleotides (nts) and aa residues in its vicinity [bib_ref] Structural insights into unique features of the human mitochondrial ribosome recycling, Koripella [/bib_ref]. Interestingly, unlike the α-helical nature inferred for the part of this segment of NTE 7 , we find that its higher resolution density to be partially unstructured. A similar observation of a relatively unstructured NTE has been recently , and c 39S- RRF mt - EF-G2 mt - GMPPCP complex (Class III). In these three panels, the 28S subunit is shown in yellow, the 39S subunit in blue, E-tRNA in orchid, RRF mt in green and EF-G2 mt in red. A lighter shade of yellow differentiates the 28S ribosomal proteins from the 12S rRNA, while a lighter shade of blue differentiates the 39S ribosomal proteins from the 16S rRNA. Landmarks of the 28S subunit: h, head; b, body. Landmarks of the 39S subunit: CP central protuberance, Sb stalk base; L1, MRP uL1m. d Cryo-EM density of RRF mt extracted from the Class I complex. e Cryo-EM density of RRF mt extracted from the Class III complex. f Cryo-EM density of EF-G2 mt extracted from Class III complex. See Figs. S1 and S2, for overall and local resolutions, respectively, of densities corresponding to the mitoribosome, RRF mt , and EF-G2 mt in these complexes. reported in an in vivo state complex [bib_ref] Structural basis of mitochondrial translation, Aibara [/bib_ref]. However, the mitoribosomal components interacting with RRF mt 7 essentially remain unaltered.
We found a small density in a tight pocket surrounded by the outer bend of the junction between domains I and II of RRF mt , MRP uS12m and the small subunit's 12S rRNA helix h44, and the large subunit's 16S rRNA helices H69 and H71 . Except for our previous lower resolution map of the 55S- RRF mt complex 7 , this additional density is not observed in any of the available 55S mitoribosomal structures, whether complexed with other translational factors [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref] [bib_ref] Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM, Kummer [/bib_ref] [bib_ref] Structural insights into mammalian mitochondrial translation elongation catalyzed by mtEFG1, Kummer [/bib_ref] or not [bib_ref] The structure of the human mitochondrial ribosome, Amunts [/bib_ref] [bib_ref] The complete structure of the 55S mammalian mitochondrial ribosome, Greber [/bib_ref]. Since our complex was reconstituted from purified components, this additional density should correspond to an RRF mt NTE segment, that has been stabilized through interactions with multiple mitoribosomal components in its vicinity. Though bacterial and mitochondrial ribosomes exhibit significant differences in their overall shape, composition, and conformation, their internal rRNA core regions are largely conserved [bib_ref] The structure of the human mitochondrial ribosome, Amunts [/bib_ref] [bib_ref] The complete structure of the 55S mammalian mitochondrial ribosome, Greber [/bib_ref] [bib_ref] Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome, Kaushal [/bib_ref] [bib_ref] Structure of the mammalian mitochondrial ribosome reveals an expanded functional role for..., Sharma [/bib_ref]. Comparison of the RRF binding sites between the bacterial and mitochondrial ribosomes reveals that the H69 of 16S rRNA is slightly shorter in the mammalian mitoribosomes . This minor shortening of H69 is critical because it directly impacts the interaction of H69 with domain II of RRF mt . The small density, most likely corresponding to Nterminus segment of the mito-specific NTE, appears to compensate for the shortened H69 by mediating the interactions between RRF mt and H69 . We generated a model that would place 10 aa residues (Ala2-Val11) at the N-terminus of NTE into this density . This model was guided by the observed sidechain density for two consecutive large side chains of Phe8 and Arg9 within the first 11 aa . Since there are other possibilities for two consecutive aa residues with large sidechains in the remaining 54 aa residues in the unmodelled segment of NTE, further confirmation of this assignment may need a well-resolved density for the entire NTE.
It should be noted that the RRF mt -bound 55S mitoribosomes (present work and ref. [bib_ref] Structural insights into unique features of the human mitochondrial ribosome recycling, Koripella [/bib_ref] were never observed in the unrotated state, suggesting that the RRF mt binding locks the ribosome in a fully rotated state. This is in contrast to the bacterial 70S- RRF complexes that were found both in their rotated and unrotated conformational states [bib_ref] Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome..., Gao [/bib_ref] [bib_ref] Progression of the ribosome recycling factor through the ribosome dissociates the two..., Barat [/bib_ref] [bib_ref] Mechanism for the disassembly of the posttermination complex inferred from cryo-EM studies, Gao [/bib_ref] [bib_ref] Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: functional implications, Agrawal [/bib_ref] [bib_ref] Structures of the bacterial ribosome in classical and hybrid states of tRNA..., Dunkle [/bib_ref] [bib_ref] Crystal structure of the ribosome recycling factor bound to the ribosome, Weixlbaumer [/bib_ref] [bib_ref] Structural insights into initial and intermediate steps of the ribosome-recycling process, Yokoyama [/bib_ref] [bib_ref] Structural basis for ribosome recycling by RRF and tRNA, Zhou [/bib_ref] [bib_ref] Key intermediates in ribosome recycling visualized by timeresolved cryoelectron microscopy, Fu [/bib_ref]. The rotated conformation of the 55S mitoribosome seems to prime subunit dissociation by either destabilizing or completely breaking seven out of fifteen inter-subunit bridges in the unrotated 55S mitoribosome [bib_ref] The structure of the human mitochondrial ribosome, Amunts [/bib_ref] [bib_ref] Structure of the mammalian mitochondrial ribosome reveals an expanded functional role for..., Sharma [/bib_ref]. The simultaneous interactions of N-terminus segment of the RRF mt NTE with RRF mt 's structurally conserved domain I, MRP uS12m, h44, H69, and H71 likely help prevent the back-rotation of the small 28S subunit. The rotated state of the 55S mitoribosome could serve as an ideal substrate for the subsequent binding of EF-G2 mt to complete subunit dissociation. In this context, it is important to note that the entire 79 aa long NTE is an integral part of the mature protein and is known to be essential for RRF mt function during the mitoribosome recycling [bib_ref] Identification and cloning of human mitochondrial translational release factor 1 and the..., Zhang [/bib_ref] [bib_ref] The human mitochondrial ribosome recycling factor is essential for cell viability, Rorbach [/bib_ref].
RRF mt domain II motion helps split the 55S mitoribosome into its two subunits. Using fast-kinetics, it has been shown that the splitting/recycling of the 70S ribosome by the concerted action of RRF and EF-G happens in the sub-second time scale [bib_ref] Complete kinetic mechanism for recycling of the bacterial ribosome, Borg [/bib_ref]. Both RRF and EF-G have been captured on the 70S ribosome with timeresolved cryo-EM [bib_ref] Key intermediates in ribosome recycling visualized by timeresolved cryoelectron microscopy, Fu [/bib_ref]. It is more challenging without time-resolved techniques, but RRF and EF-G were also captured on the 70S ribosomes by using the factors from different species [bib_ref] Structural insights into initial and intermediate steps of the ribosome-recycling process, Yokoyama [/bib_ref] or by crosslinking the EF-G with one of the ribosomal proteins [bib_ref] Structural basis for ribosome recycling by RRF and tRNA, Zhou [/bib_ref]. In the present work, collection of very large cryo-EM datasets (altogether 21,752 micrographs [fig_ref] Figure 1: Cryo-EM structures of the human mitochondrial recycling complexes in three functional states [/fig_ref] , enabled the isolation of a small subset of 55S particles (Class II) that contained both RRF mt and EF-G2 mt [fig_ref] Figure 1: Cryo-EM structures of the human mitochondrial recycling complexes in three functional states [/fig_ref]. However, the 28 S subunit density was found to be weak and present in multiple destabilized conformations relative to the 39S subunit in this complex.
Both Class II and Class III maps showed readily recognizable densities corresponding to RRF mt , EF-G2 mt ., and E-site tRNA. The Class III complex had superior resolution, which enabled more accurate analysis of molecular-level interactions between the two factors and the mitoribosome, and their functional implications, while the Class II map was useful for interpreting large-scale conformational changes. In line with the bacterial 70S/ 50S- RRF- EFG complexes [bib_ref] Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome..., Gao [/bib_ref] [bib_ref] Mechanism for the disassembly of the posttermination complex inferred from cryo-EM studies, Gao [/bib_ref] [bib_ref] Structural insights into initial and intermediate steps of the ribosome-recycling process, Yokoyama [/bib_ref] [bib_ref] Structural basis for ribosome recycling by RRF and tRNA, Zhou [/bib_ref] [bib_ref] Key intermediates in ribosome recycling visualized by timeresolved cryoelectron microscopy, Fu [/bib_ref] , the conformation of RRF mt is substantially different between the Class I and Class III recycling complexes. The conformation of RRF mt domain I remains unchanged among all three classes. In the Class II and III maps, domain II was rotated by about 45°towards the small subunit compared to its position in the Class I complex [fig_ref] Figure 3: Direct involvement of RRF mt domain II and EF-G1 mt domain IV... [/fig_ref]. This large conformational change is enabled by a highly flexible hinge regions between domain I and domain II in RRF mt . Due to this rotation, the tip of domain II moved by about 40Å towards the h44 of 12S rRNA. When the maps of Class II and III were superimposed, this motion resulted in a major steric clash between the RRF mt domain II and the 28S subunit elements h44 and MRP uS12m [fig_ref] Figure 3: Direct involvement of RRF mt domain II and EF-G1 mt domain IV... [/fig_ref]. In the 55S mitoribosome, h44 is involved in the formation of two intersubunit bridges B2a and B3 by pairing with H69 and H71, respectively [bib_ref] The structure of the human mitochondrial ribosome, Amunts [/bib_ref] [bib_ref] Structure of the mammalian mitochondrial ribosome reveals an expanded functional role for..., Sharma [/bib_ref]. By displacing h44, Structures of two NTE segments of RRF mt . a Density corresponding to the N-terminus segment of RRF mt NTE (pink) observed in the intersubunit space sandwiched between the 28S SSU components, including MRP uS12m (cyan) and the 12S rRNA helix h44 (brown), the 39S LSU components, including 16S rRNA helices H69 (medium blue) and H71 (dark blue), and the outer bent of the junction between domains I and II of RRF mt . The dotted line (pink) depicts the connection between two structurally stabilized segments of the RRF mt 's NTE. b H69 superimposition from bacterial (gray) [bib_ref] Crystal structure of the ribosome recycling factor bound to the ribosome, Weixlbaumer [/bib_ref] and human mitochondrial ribosomes (blue) reveals the shortening of H69 in the mitoribosome, also depicted in secondary structures of H69 on the lower left. Domain II of RRF mt interacts with the shorter H69 through its strategically positioned N-terminus of NTE (pink). c Sequence of NTE showing two modeled regions (pink). Thumbnails to the left depicts an overall orientation of the 55S mitoribosome, with semitransparent 28S (yellow) and 39S (blue) subunits, and overlaid positions of ligands. Landmarks on the thumbnail: h, head, and b, body of the 28S subunit, and CP central protuberance, Sb stalk base of the 39S subunit.
RRF mt disrupts these crucial intersubunit bridges, thereby splitting the 55S mitoribosome into its two subunits.
A well-defined 28S structure was not seen within the Class II 55S mitoribosomal complex, but several inter-subunit bridges do appear to be destabilized or broken, and the small subunit seems to be dissociating from the large subunit. To remove any possible large subunit contamination from the Class II map, extensive reference-based 3D classification was employed, but this class of 55S mitoribosomal particles with a well-resolved 39S subunit and a poorly resolved 28S subunit remained unchanged. This supports it being an ensemble of authentic, short-lived functional intermediates of mitoribosome recycling, where the small subunit is captured in multiple positions during its separation from the large subunit.
EF-G2 mt binding induces conformational changes in RRF mt . The 55S- RRF mt complex (Class I) undergoes large conformational changes upon binding to EF-G2 mt (Classes II and III). EF-G2 mt would not be able to access its binding site on the mitoribosome without the significant movement seen in domain II of RRF mt in Class II and Class III complexes. This movement eliminates the direct spatial conflicts of EF-G2 mt domains III, IV, and V with the initial position of RRF mt domain II in the Class I complex (Figs. 3a, b and [fig_ref] Figure 5: EF-G2 mt binding induces large-scale conformational changes in the uL11m stalk-base region... [/fig_ref]. This change could be induced either during or upon binding of EF-G2 mt . The domain II of RRF mt is repositioned into a cavity created by domains III, IV, and V of EF-G2 mt and an extensive network of interactions are formed between the two mitochondrial recycling factors. This is also in agreement with the location of RRF domain II reported in the bacterial 50S [bib_ref] Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome..., Gao [/bib_ref] and 70S complexes [bib_ref] Structural insights into initial and intermediate steps of the ribosome-recycling process, Yokoyama [/bib_ref] [bib_ref] Key intermediates in ribosome recycling visualized by timeresolved cryoelectron microscopy, Fu [/bib_ref]. A majority of the interactions are formed between the hinge regions that connect the two domains of RRF mt and the loop regions from domain III of EF-G2 mt . Several aa residues from the RRF mt hinge region (Pro183-Thr186) interact with EF-G2 mt domain III residues Glu495-Leu499 via hydrogen bonds and hydrophobic interactions. Arg187 from the α-helix following the hinge region of RRF mt domain II has close hydrogen-bonding interactions with Tyr556 of EF-G2 mt domain III . A second set of contacts formed between the two factors involves residues Ile109-Arg110 from the second hinge region of RRF mt and residues Ser527-Gln529 from the domain III of EF-G2 mt .
Domain IV of EF-G2 mt presses against domain II of RRF mt through multiple interactions. The surface residues of α-helix 1 (Asn629, Ser633, and Leu636) and α-helix 2 (Thr664, Met665, Ser667, and Ala668) from domain IV of EF-G2 mt interact with residues in β-strand 3 (Ser134-Met138), and its adjoining loop region (Gln132 and Ile133) from domain II of RRF mt through a combination of electrostatic and hydrophobic interactions . Gln637 from the α-helix 1 of EF-G2 mt also shares a hydrogen bond with Ser112 from the hinge region of RRF mt . Contacts are observed between the C-terminal α-helix of EF-G2 mt domain IV and the α-helix 3 from the triple-helix bundle of RRF mt domain I. In bacteria, the analogous C-terminal α-helix of EF-G is often considered as part of domain V though the first atomic models of EF-G 55,56 grouped it with domain IV. While residues Ser776-Leu778 from EF-G2 mt domain IV pair with residues Arg251 and Val255 from RRF mt domain I through hydrogen bonds , Arg775 from EF-G2 mt domain IV strongly interacts with Glu259 from RRF mt domain I through a salt-bridge . Direct interactions of EF-G2 mt domain III with RRF mt domain II at its hinge regions, known to confer interdomain flexibility to the bacterial factor [bib_ref] Solution structure of the ribosome recycling factor from Aquifex aeolicus, Yoshida [/bib_ref] , likely help trigger the dissociation of 55S mitoribosomes into subunits by enabling the repositioning of RRF mt domain II. At the same time, the Direct involvement of RRF mt domain II and EF-G1 mt domain IV in human mitoribosome recycling. a Comparison of the overall conformation of RRF mt between the Class I (gray) and Class III (green) complexes revealed that domain II rotates by about 45°towards the 28S subunit. Such a rotation would sterically clash with the 12S rRNA helix 44 (h44, brown) and MRP uS12m (cyan) and destabilize a crucial inter-subunit bridge, B2a (yellow). Inset shows the magnified view of the inter-subunit bridge B2a with RRF mt domain II residues V121-K127 (shown with density in magenta) disrupting B2a by inserting between h44 residues (C1491 and A1492) and H69 residues (A2581 and A2582). b Superimposition of the Class III complex with the Class I complex shows a direct overlap between the loop1 region (dark cyan) of EF-G2 mt domain IV (red) and the intersubunit bridge B2a (yellow) formed between h44 (brown) and H69 (blue). Inset shows the magnified view of EF-G2 mt domain IV residues L582-R585 (shown with density in dark cyan) that will disrupt the bridge B2a by inserting between h44 residues (G1559 and U1560) and H69 residue (A2576) and pushing the 28S subunit (h44) away. Thumbnails to the left depict an overall orientation of the 55S mitoribosome, with semitransparent 28S (yellow) and 39S (blue) subunits, and overlaid position RRF mt . Landmarks on the thumbnails are same as in .
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23726-4 ARTICLE multiple interactions between domain IV of EF-G2 mt and domain II of RRF mt appear to stabilize the RRF mt domain II in the altered position, which would push the 28S subunit away from the 39S subunit and prevent domain II from reverting back to its previous orientation, in order to maintain the 39S- RRF mt - EF-G2 mt complex in a dissociated state.
In addition to aiding to the function of RRF mt , EF-G2 mt plays a direct role in destabilizing the 55S mitoribosome. Superimposition of the maps of Class I and Class III complexes reveals a direct steric clash between the loop1 region of EF-G2 mt domain IV and the 28S subunit component (rRNA h44), that participates in the formation of the inter-subunit bridge B2a [fig_ref] Figure 3: Direct involvement of RRF mt domain II and EF-G1 mt domain IV... [/fig_ref]. More importantly, the orientation of domain IV loop1 seems to be unique to EF-G2 mt since the analogous region in EF-G1 mt is positioned away from the intersubunit bridge B2a towards the decoding center [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref] [bib_ref] Structural insights into mammalian mitochondrial translation elongation catalyzed by mtEFG1, Kummer [/bib_ref].
Role of mito-specific MRPs in stabilizing the deacylated tRNA. The presence of E-site tRNA in Class III complex of the dissociated 39S subunit suggests a markedly different mechanism of deacylated tRNA removal during recycling of mitochondrial PoTC as compared to that in the bacteria, where the deacylated tRNA goes with the small ribosomal subunit during the 70S ribosome splitting [bib_ref] Key intermediates in ribosome recycling visualized by timeresolved cryoelectron microscopy, Fu [/bib_ref]. This observation is particularly intriguing since a majority (11 of 12) of the rRNA segments in bacterial 23S rRNA that are known to interact with the E-site tRNA are absent in the mito-16S rRNA [bib_ref] A structural model for the large subunit of the mammalian mitochondrial ribosome, Mears [/bib_ref]. We find that the E-site tRNA in the 39S complex is stabilized by multiple interactions with Arg-rich and Lys-rich electro-positive segments of MRPs, including mL64 6 , uL33m, and uL1m 58 [fig_ref] Figure 4: Stabilization of the E-site tRNA on the dissociated 39S subunit [/fig_ref]. With the tightly held body of the deacylated tRNA through conserved interaction of its CCA end with the rRNA H88 and multiple new interactions with MRPs, transition of its anticodon end from a pe/E state 7 in Class I complex to E/E-state in Class II and III complexes could in part facilitate the dissociation of the two mitoribosomal subunits. Moreover, the mechanism of subsequent release of deacylated tRNA from the 39S subunit remains unknown.
Dynamic interactions among uL11m, CTD of uL12m, and EF-G2 mt . EF-G2 mt binding resulted in a prominent conformational change in the uL11m stalk-base region of the 39S subunit. The uL11m stalk-base region moved towards the CTD of uL12m, a component of the L10-L12 stalk, and assumed a unique conformation not reported previously [bib_ref] The structure of the human mitochondrial ribosome, Amunts [/bib_ref] [bib_ref] The complete structure of the 55S mammalian mitochondrial ribosome, Greber [/bib_ref] [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref] [bib_ref] Structural insights into unique features of the human mitochondrial ribosome recycling, Koripella [/bib_ref] [bib_ref] Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM, Kummer [/bib_ref]. The 16S rRNA H43 of the uL11m stalk-base moved by 3 Å towards the CTD of uL12m, oriented parallel to the domain V of EF-G2 mt , while the NTD of MRP uL11m moved about 5 Å away from the EF-G2 mt domain V [fig_ref] Figure 5: EF-G2 mt binding induces large-scale conformational changes in the uL11m stalk-base region... [/fig_ref]. This is in sharp contrast to the 55S- EF-G1 mt translocation complexes [bib_ref] The structure of the human mitochondrial ribosome, Amunts [/bib_ref] [bib_ref] The complete structure of the 55S mammalian mitochondrial ribosome, Greber [/bib_ref] [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref] [bib_ref] Structural insights into unique features of the human mitochondrial ribosome recycling, Koripella [/bib_ref] [bib_ref] Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM, Kummer [/bib_ref] [bib_ref] Structural insights into mammalian mitochondrial translation elongation catalyzed by mtEFG1, Kummer [/bib_ref] , where the uL11m stalk-base region was observed to move 5 Å closer towards the domain V of EF-G1 mt [fig_ref] Figure 5: EF-G2 mt binding induces large-scale conformational changes in the uL11m stalk-base region... [/fig_ref]. The movement of uL11m away from the EF-G2 mt domain V and towards the CTD of uL12m is essential for the binding of EF-G2 mt in the present conformation, to avoid the steric clash between the NTD of uL11m and domain V of EF-G mt [fig_ref] Figure 5: EF-G2 mt binding induces large-scale conformational changes in the uL11m stalk-base region... [/fig_ref]. It is also possible that the conformation of EF-G2 mt observed in our 39S- EF-G2 mt complex (Class III) was attained after the dissociation of 39S subunit from the 55S complex (Class II).
In addition to the unique conformation of the uL11m stalk-base region, the CTD of uL12m was also observed in a distinct conformation. In the 55S- EF-G1 mt complexes [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref] [bib_ref] Structural insights into mammalian mitochondrial translation elongation catalyzed by mtEFG1, Kummer [/bib_ref] , the CTD of uL12m is positioned close to EF-G1 mt so that α-helices 1 and 2 of uL12m CTD would have close interactions with the G′ subdomain of EF-G1 mt [fig_ref] Figure 5: EF-G2 mt binding induces large-scale conformational changes in the uL11m stalk-base region... [/fig_ref] , thereby providing stability to the otherwise flexible uL12m CTD. In contrast, the CTD of uL12m rotates by about 60°and shifts away by about 7 Å from the G′ subdomain of EF-G2 mt in the 39S- EF-G2 mt complex [fig_ref] Figure 5: EF-G2 mt binding induces large-scale conformational changes in the uL11m stalk-base region... [/fig_ref]. As a result of this large rotational movement, interactions between the uL12m CTD α-helix 2 and the G′ subdomain of EF-G2 mt are lost, while the contacts between the α-helix 1 and the G′ subdomain of EF-G2 mt are maintained [fig_ref] Figure 5: EF-G2 mt binding induces large-scale conformational changes in the uL11m stalk-base region... [/fig_ref]. Since protein uL12 is known to play a central role in the recruitment of translational factors to the bacterial ribosome [bib_ref] Structural basis for the function of the ribosomal L7/12 stalk in factor..., Diaconu [/bib_ref] [bib_ref] The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3..., Helgstrand [/bib_ref] [bib_ref] Interaction of the G' domain of elongation factor G and the C-terminal..., Datta [/bib_ref] [bib_ref] Direct visualization of translational GTPase factor pool formed around the archaeal ribosomal..., Imai [/bib_ref] ; the semi-stable conformation of uL12m observed in the 39S- EF-G2 mt complex represents a late-stage conformation of uL12m prior to its detachment from the EF-G2 mt as EF-G2 mt 's participation in the 55S ribosome recycling process nears completion. Structural basis for use of EF-G2 mt in mitoribosomal recycling instead of EF-G1 mt . In most bacterial species, a single EF-G is involved in both translocation and ribosome recycling. Mammalian mitoribosomes have evolved to utilize the two isoforms EF-G1 mt and EF-G2 mt to cope with the significantly altered environment in mitochondria as compared to the bacterial cytoplasm [bib_ref] Identification and characterization of two novel human mitochondrial elongation factor genes, hEFG2..., Hammarsund [/bib_ref] [bib_ref] EF-G2mt is an exclusive recycling factor in mammalian mitochondrial protein synthesis, Tsuboi [/bib_ref] and perform two separate functions [bib_ref] Identification and characterization of two novel human mitochondrial elongation factor genes, hEFG2..., Hammarsund [/bib_ref] [bib_ref] EF-G2mt is an exclusive recycling factor in mammalian mitochondrial protein synthesis, Tsuboi [/bib_ref]. EF-G1 mt is used during translation elongation while EF-G2 mt is used along with RRF mt for the recycling of the 55S mitoribosome. The overall position and domain arrangement of EF-G2 mt is similar to that of EF-G1 mt in the recently published human 6 and porcine 46 translocational complexes . There are, however, some specific structural distinctions between the two factors that assign them specialized functional roles. The most striking of these is the presence of a C-terminal extension (CTE) in EF-G1 mt domain IV [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref]. Besides its CTE, the size of the conserved C-terminal αhelix of EF-G1 mt domain IV is substantially longer (16 aa) than the C-terminal α-helix (12 aa) of EF-G2 mt domain IV .
The sturdier and longer C-terminal α-helix of EF-G1 mt domain IV and its 11 aa CTE would not permit the coexistence of RRF mt on the mitoribosome due to a major steric clash between domain I of RRF mt and the C-terminal region of EF-G1 mt . Even a reorientation of the C-terminal α-helix and its CTE away from the domain I of RRF mt would not resolve the problem as they would then clash with H89 of the 16S rRNA and the NTE of RRF mt , that has been positioned in the inter subunit space between the domain I of RRF mt and 16S rRNA helix, H89 . Structural analysis of the bacterial EF-Gs from various species [bib_ref] The structure of the ribosome with elongation factor G trapped in the..., Gao [/bib_ref] [bib_ref] Staphylococcus aureus elongation factor G-structure and analysis of a target for fusidic..., Chen [/bib_ref] [bib_ref] Control of ribosomal subunit rotation by elongation factor G, Pulk [/bib_ref] has revealed that the length of their C-terminal αhelices are about 12 aa long, suggesting that only EF-G2 mt can function alongside RRF mt during subunit splitting. Interestingly, the C-terminal α-helices of EF-G2 from T. thermophilus 39 and EF-G2 mt are similar in size though the function of EF-G2 in T. thermophilus is not understood. Moreover, the interaction between the C-terminal regions of EF-G2 mt domain IV and RRF mt domain I is essential for stabilizing the bound RRF mt . This tight anchoring of RRF mt domain I to the mitoribosome would prevent RRF mt dissociation from the mitoribosome, when its domain II undergoes substantial rotation to displace the h44 region of the 28S subunit. This agrees with the observation that deletion of the last few aa residues from the Cterminal region of bacterial RRF adversely effects its function during 70S ribosome recycling [bib_ref] Amber mutations in ribosome recycling factors of Escherichia coli and Thermus thermophilus:..., Fujiwara [/bib_ref]. Now the question is why EF-G1 mt mediates the translocation step during mitoribosomal elongation and not EF-G2 mt ? The most probable answer is that the CTE in the domain IV of EF-G1 mt that limits its ability to participate in the mitochondrial ribosome recycling, is directly involved in the elongation step of mammalian mitochondrial protein synthesis [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref]. By contacting the inner bend of the A-site tRNA, the CTE of EF-G1 mt can help in translocating the CCA arm of the A-site tRNA into the P site, and by interacting with a 16S rRNA helix, H71, it prevents the P-site tRNA from reverting back into the A site [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref]. This analysis is further supported by the fact that bacterial EF-Gs, which lack the CTE, are inactive on the 55S mitochondrial ribosomes while EF-G1 mt is active on the 70S ribosomes [bib_ref] Bovine mitochondrial ribosomes. Elongation factor specificity, Eberly [/bib_ref].
Specific sequence differences in a critical region (loop1) within the domain IV of EF-G2 mt can also make it ineffective in driving translocation. During EF-G-catalyzed translocation in bacteria, the presence of two universally conserved glycine residues at the tip of domain IV (loop 1) region facilitate the insertion of domain IV into the decoding center (DC) [bib_ref] The structure of the ribosome with elongation factor G trapped in the..., Gao [/bib_ref]. In the DC, the loop 1 of domain IV destabilizes the codon-anticodon interactions of the In the 39S- RRF mt - EF-G2 mt complex, the uL11m stalk-base region (blue) moves towards the CTD of uL12m and away from the domain V of EF-G2 mt , as compared to its position in the EF-G2 mt -unbound 55S mitoribosome (light gray) [bib_ref] The structure of the human mitochondrial ribosome, Amunts [/bib_ref]. b In sharp contrast, in the presence of EF-G1 mt , the uL11m stalk-base region was found to move towards the domain V of EF-G1 mt in the translocation complex (dark gray) [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref]. c In the 55S- EF-G1 mt complex 6 , the CTD of uL12m (gray) is positioned in such a way that its α-helices 1 and 2 directly interact with the G′ subdomain of EF-G1 mt (yellow). d In our 39S- RRF mt - EF-G2 mt complex, the CTD of uL12m (blue) is rotated by about 60°and shifted away by about 7 Å from the G′ subdomain of EF-G2 mt , resulting in the loss of contacts between its α-helix 2 and G′ subdomain of EF-G2 mt while a new set of interactions is formed between its α-helix 1 and G′ subdomain of EF-G2 mt . Thumbnails to the left depict an overall orientation of the 39S subunit (semitransparent blue), and overlaid positions of the ligands. Landmarks on the thumbnail: CP central protuberance, Sb stalk base, L1 MRP uL1m. mRNA-tRNA duplex with the universally conserved 16S rRNA bases A1492 and A1493 in the 30S A site thereby aiding the A-site tRNA along with its associated codon to translocate into the P site [bib_ref] How the ribosome hands the A-site tRNA to the P site during..., Zhou [/bib_ref]. The loop 1 region is conserved in EF-G1 mt but is significantly altered in EF-G2 mt . EF-G1 mt retains both the glycine residues (Gly544 and Gly545) in its domain IV loop 1 region whereas the second glycine is replaced by an aspartic acid in EF-G2 mt , which alters the conformation of the tip of domain IV rendering it structurally unfavorable for insertion into the grove between the P-site tRNA and the associated codon . A similar observation has been made in case of the spirochaete B. burgdorferi that utilizes EF-G2 exclusively for ribosome recycling [bib_ref] A bacterial elongation factor G homologue exclusively functions in ribosome recycling in..., Suematsu [/bib_ref] , where one of these conserved loop1 glycines is substituted by an alanine rendering it inactive for promoting the translocation step. Interestingly, however, the T. thermophilus EF-G2 carries both the conserved glycines. Moreover, the Ala546 and Gly547 residues that follow the conserved glycines of loop 1 in EF-G1 mt are substituted by the large polar sidechain residues lysine and arginine, respectively, in the corresponding region of EF-G2 mt , thereby significantly altering the hydrophobicity of the loop 1 tip. Point mutations and deletions at the loop 1 tip region are known to have a pronounced effect on the function of EF-G during the elongation step of bacterial protein synthesis [bib_ref] Mutational analysis of the functional role of the loop region in the..., Kolesnikov [/bib_ref] , but have a negligible effect on the activity of EF-G in bacterial ribosome disassembly [bib_ref] New insights into the enzymatic role of EF-G in ribosome recycling, Zhang [/bib_ref]. Our study thus provides a structural rationale for the biochemical finding that mammalian mitoribosomes utilize two distinct EF-G-like factors during the translation elongation and recycling phases [bib_ref] EF-G2mt is an exclusive recycling factor in mammalian mitochondrial protein synthesis, Tsuboi [/bib_ref].
Mammalian 55S mitoribosome recycling does not require GTP hydrolysis by EF-G2 mt . The overall G domain structure in the 39S- RRF mt - EF-G2 mt complex (Class III map) is similar to the G domain in the 55S- EF-G1 mt complex [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref] [bib_ref] Structural insights into mammalian mitochondrial translation elongation catalyzed by mtEFG1, Kummer [/bib_ref]. The well-ordered density in our maps of highly conserved translational GTPase consensus motifs such as the P-loop, switch I and switch II regions, allowed complete modeling of these essential regions. A welldefined density corresponding to a bound GMPPCP molecule is also readily identifiable in the nucleotide binding pocket. GMPPCP is stabilized through interactions with universally conserved aa residues, such as Asp80 and Lys83 of P-loop, Thr122 of switch I and His145 of switch II [fig_ref] Figure 8: Sequence of main events in human mitochondrial ribosome recycling suggested by structures... [/fig_ref]. As in the 55S- EF-G1 mt complex 6 , a crucial Mg 2+ ion is positioned near the γ phosphate of GMPPCP and is coordinated by Thr84 and Thr122 from the P-loop and switch I regions, respectively [fig_ref] Figure 8: Sequence of main events in human mitochondrial ribosome recycling suggested by structures... [/fig_ref]. The catalytic His145 (His124 in EF-G1 mt ), known to play a central role in the hydrolysis of the bound nucleotide [bib_ref] Structure of EF-G-ribosome complex in a pretranslocation state, Chen [/bib_ref] [bib_ref] Elongation factor G bound to the ribosome in an intermediate state of..., Tourigny [/bib_ref] , is found oriented towards the γ phosphate of the bound GMPPCP [fig_ref] Figure 8: Sequence of main events in human mitochondrial ribosome recycling suggested by structures... [/fig_ref] , suggesting an active conformation of the factor prior to GTP hydrolysis.
The highly conserved α-sarcin-ricin stem-loop (SRL) region is known to be essential for the GTPase activity of all the translational G proteins [bib_ref] The structure of the ribosome with elongation factor G trapped in the..., Gao [/bib_ref] [bib_ref] Structure of EF-G-ribosome complex in a pretranslocation state, Chen [/bib_ref] [bib_ref] Elongation factor G bound to the ribosome in an intermediate state of..., Tourigny [/bib_ref]. Base A3129 from the SRL was Structural basis for the exclusive roles of EF-G1 mt in tRNA translocation and EF-G2 mt in mitoribosome recycling. a The presence of a substantially longer C-terminal α-helix (salmon) along with the presence of a unique CTE (blue), which is required for mitochondria tRNA translocation 6 , prevents the simultaneous binding of EF-G1 mt and RRF mt (green) on the mitoribosome due to a major steric clash. Furthermore, any conformational repositioning the Cterminal α-helix of EF-G1 mt will result in direct steric clash with the H89 (orange) of 16S rRNA and the NTE of RRF mt (pink). b Having a shorter C-terminal α-helix (red) and the absence of CTE allows the simultaneous binding of EF-G2 mt and RRF mt (green) on the mitoribosome. c In EF-G1 mt 6 , the presence of two universally conserved glycine residues (dark green) at the tip of domain IV loop1 region (salmon) facilitates its insertion between the mRNA-tRNA duplex at the decoding center (DC) during EF-G1 mt -catalyzed translocation. d In EF-G2 mt, the second glycine of domain IV loop 1 region (red) is substituted by an aspartic acid (dark green) altering its conformation and flexibility, and thereby making its insertion into the DC during translocation unfavorable. e The boxed aa sequence corresponds to the loop 1 situated at the tip of domain IV, which is conserved between EF-G1 mt and the T. thermophilus EF-G. First of the two universally conserved loop 1 glycine residues (green) is substituted by an alanine in B. burgdorferi EF-G2, while the second one is replaced by an aspartic acid in EF-G2 mt .
found to be contacting the Switch II His145 through hydrogen bonding interactions and thereby stabilizing His145 in its activated conformation poised to perform the hydrolysis reaction [fig_ref] Figure 8: Sequence of main events in human mitochondrial ribosome recycling suggested by structures... [/fig_ref]. While EF-G-dependent GTP hydrolysis is essential for an efficient splitting of the 70S ribosome into its subunits [bib_ref] Novel roles for classical factors at the interface between translation termination and..., Karimi [/bib_ref] [bib_ref] Splitting of the posttermination ribosome into subunits by the concerted action of..., Zavialov [/bib_ref] [bib_ref] Complete kinetic mechanism for recycling of the bacterial ribosome, Borg [/bib_ref] , dissociation of the mammalian 55S mitoribosomes does not require EF-G2 mt -dependent GTP hydrolysis, which is only needed for the release of EF-G2 mt from the dissociated large subunit [bib_ref] EF-G2mt is an exclusive recycling factor in mammalian mitochondrial protein synthesis, Tsuboi [/bib_ref]. Our structural results strongly corroborate the Tsuboi and coworkers 27 observation as a significant proportion (82%) of the EF-G2 mt was found complexed to the 39S subunits as compared to the small proportion (18%), that remained associated with the 55S mitoribosomes (see [fig_ref] Figure 1: Cryo-EM structures of the human mitochondrial recycling complexes in three functional states [/fig_ref]. Even though GTP hydrolysis by EF-G2 mt is not necessary for 55S mitoribosome splitting, the presence of GTP or its non-hydrolysable analogs GDPNP/GMPPCP in the nucleotide binding pocket is essential, as the presence of GDP or the absence of any nucleotide does not split the 55S mitoribosome [bib_ref] EF-G2mt is an exclusive recycling factor in mammalian mitochondrial protein synthesis, Tsuboi [/bib_ref].
Divergent mechanisms of fusidic acid (FA) resistance by EF-G1 mt and EF-G2 mt . FA is a fusidane class antibiotic that is used to treat bacterial skin infections along with chronic bone and joint infections. It is effective against several species of gram-positive bacteria and is clinically used to treat methicillin-resistant S. aureus (MRSA). FA prevents the release of EF-G- GDP from the 70S ribosome after GTP hydrolysis by preventing the switch II from attaining its GDP-bound conformation [bib_ref] The structure of the ribosome with elongation factor G trapped in the..., Gao [/bib_ref]. Prior structural studies have demonstrated that FA binds in an interdomain pocket between the G domain, domain II, and domain III of EF-G [bib_ref] The structure of the ribosome with elongation factor G trapped in the..., Gao [/bib_ref] [bib_ref] Crystal structures of EF-G-ribosome complexes trapped in intermediate states of translocation, Zhou [/bib_ref]. Stable binding of FA requires the switch I region to be disordered [bib_ref] The structure of the ribosome with elongation factor G trapped in the..., Gao [/bib_ref] , because an ordered switch I would overlap with the binding site of FA. Biochemical studies have shown that a substantially higher concentration (10-fold to 100-fold) of FA is needed to inhibit the activity of EF-G1 mt during mitochondrial elongation [bib_ref] Expression and characterization of isoform 1 of human mitochondrial elongation factor G, Bhargava [/bib_ref] [bib_ref] Purification and characterization of elongation factor G from bovine liver mitochondria, Chung [/bib_ref]. Recent cryo-EM structures of EF-G1 mt bound to the 55S mitoribosomes have presented the switch I in a welldefined conformation [bib_ref] Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features..., Koripella [/bib_ref] [bib_ref] Structural insights into mammalian mitochondrial translation elongation catalyzed by mtEFG1, Kummer [/bib_ref] , in contrast to bacterial 70S- EF-G complexes where the density for switch I has been consistently poorly resolved [bib_ref] The structure of the ribosome with elongation factor G trapped in the..., Gao [/bib_ref] [bib_ref] How the ribosome hands the A-site tRNA to the P site during..., Zhou [/bib_ref] [bib_ref] Structure of EF-G-ribosome complex in a pretranslocation state, Chen [/bib_ref] [bib_ref] Elongation factor G bound to the ribosome in an intermediate state of..., Tourigny [/bib_ref] [bib_ref] Crystal structures of EF-G-ribosome complexes trapped in intermediate states of translocation, Zhou [/bib_ref]. It was proposed that the increased resistance observed for EF-G1 mt towards FA resulted from a small insertion in the switch I region of EF-G1 mt [bib_ref] Structural insights into mammalian mitochondrial translation elongation catalyzed by mtEFG1, Kummer [/bib_ref]. The two positively charged lysine residues (Lys80 and Lys82) in this insertion form salt bridges with the negatively charged phosphate backbone of the SRL from the 39S subunit , and hence were hypothesized to confer additional stability to the switch I of EF-G1 mt [bib_ref] Structural insights into mammalian mitochondrial translation elongation catalyzed by mtEFG1, Kummer [/bib_ref]. Biochemical evidence showed EF-G1 mt to be more resistant towards FA than its bacterial counterpart [bib_ref] Expression and characterization of isoform 1 of human mitochondrial elongation factor G, Bhargava [/bib_ref] [bib_ref] Purification and characterization of elongation factor G from bovine liver mitochondria, Chung [/bib_ref].
FA is known to inhibit both the translocation and the ribosome recycling steps in bacteria, though there is conflicting evidence on which step of translation is primarily targeted by FA [bib_ref] Complete kinetic mechanism for recycling of the bacterial ribosome, Borg [/bib_ref] [bib_ref] Distinct functions of elongation factor G in ribosome recycling and translocation, Savelsbergh [/bib_ref]. EF-G1 mt and EF-G2 mt follow diverse mechanisms to render resistance to the antibiotic fusidic acid (FA). a Stabilized Switch I region (salmon) 6 in EF-G1 mt blocks FA (dark blue) from accessing its binding site. The Switch I region sterically overlapping with FA is colored in magenta. Switch I stability is achieved by the presence of two unique lysine residues (K80 and K82) that strongly interact with the phosphates of SRL by forming salt bridges (light blue) [bib_ref] Structural insights into mammalian mitochondrial translation elongation catalyzed by mtEFG1, Kummer [/bib_ref]. b The absence of these lysine residues in the Switch I region (dark cyan) of bacterial EF-G disorders the Switch I 47 and hence makes it susceptible to FA binding and inhibition. c In EF-G2 mt , the primary aa sequence of switch I (green) is highly altered enabling the formation of three salt bridges within its switch I, thereby stabilizing it. Furthermore, strong interactions are observed between the switch I and domain II (purple) in EF-G2 mt . The Switch I region sterically overlapping with FA is colored in red. d EF-G1 mt (salmon) and EF-G2 mt (green) show strong resistance even at high concentrations of FA, while the bacterial EF-G (dark cyan) is susceptible to FA inhibition even at low concentrations. Each point in the plots represents the average of normalized data from two independent sets of experiments. Raw luminescence values obtained from the GTPase-Glo™ Assay are shown in Supplementary . e The aa sequence alignment of the switch I region (boxed) in three EF-Gs. The lysine residues shown in salmon are unique to mammalian EF-G1 mt and confer stability to switch I by interacting with the SRL. Residues shown in green (except R117) are unique to EF-G2 mt and confer stability to switch I by forming salt bridges within the switch I region. Comparison of the FA binding pocket between the bacterial EF-G, EF-G1 mt , and EF-G2 mt revealed that key aa residues reported to be necessary for the stable binding of FA 47,72 are highly conserved , thereby suggesting a similar binding mechanism for FA for all the three EF-Gs. The three aa insertion in EF-G1 mt that confers resistance to FA is not present in EF-G2 mt and the corresponding region in EF-G2 mt does not contain any positively charged aa residues , that could strongly interact with the SRL. However, the cryo-EM map of EF-G2 mt shows a well-resolved density for the switch I region and enabled its modeling , indicating an alternative mechanism for the stabilization of switch I in EF-G2 mt . Sequence alignment showed that the switch I region composition in EF-G2 mt is significantly different as compared to EF-G1 mt and the bacterial EF-G . Three new salt bridge interactions were identified within the switch I of EF-G2 mt . Arg98 forms the first two salt bridges by pairing with Asp105 and Asp107, respectively, while Arg117 and Asp104 are involved in the formation of the third salt bridge . Furthermore, stronger interactions are observed between the switch I and domain II in EF-G2 mt compared to EF-G1 mt . Thr110 from switch I is placed in the close proximity of Asp419 of domain II with the possibility of hydrogen bond formation , while the corresponding interaction in EF-G1 mt is between Asp404 and Val88, a much weaker interaction with no possibility of hydrogen bond formation. There is also potential for a tight T-stacking interaction between Phe417 and Tyr96 in EF-G2 mt , while the corresponding residues in EF-G1 mt being His402 and Arg72 offer no such interaction. Overall, through a combination of internal salt bridges and additional contacts with domain II, switch I gets highly stabilized in EF-G2 mt . Since a stabilized switch I region occludes the binding site of FA , EF-G2 mt is expected to exhibit strong resistance towards FA in the lines of EF-G1 mt . To test this hypothesis, we measured the GTPase activity of EF-G2 mt alongside EF-G1 mt and E. coli EF-G in the presence of FA under multiple-turnover conditions (see Methods). Our data shows that while 1 µM FA has no effect on the GTPase activity in E. coli, significant inhibition is observed at higher concentrations of FA. In contrast, FA has almost no effect on the GTPase activity of either EF-G1 mt or EF-G2 mt even up to 10 mM FA. Our results are consistent with the earlier finding that EF-G1 mt is highly resistant to FA compared to the bacterial EF-G [bib_ref] Expression and characterization of isoform 1 of human mitochondrial elongation factor G, Bhargava [/bib_ref] [bib_ref] Purification and characterization of elongation factor G from bovine liver mitochondria, Chung [/bib_ref] , and also consistent with recent finding that showed EF-G2 mt is not susceptible to inhibition by FA [bib_ref] Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long..., Lee [/bib_ref]. Our results and analysis suggest FA resistance in EF-G2 mt occurs by a structural mechanism fundamentally different from that in EF-G1 mt .
In conclusion, structures of three distinct functional states formed during the process of human mitoribosome recycling are presented [fig_ref] Figure 1: Cryo-EM structures of the human mitochondrial recycling complexes in three functional states [/fig_ref]. A previous biochemical finding that GTP hydrolysis is not required for the RRF mt - EF-G2 mt -mediated splitting of the post-termination mitoribosomal complex is corroborated. We also show that a mito-specific segment of the RRF mt 's NTE compensates for the slightly reduced size of the H69 within the 16S rRNA of the mitoribosomal large subunit . This is the first evidence showing a translational factor compensating for an rRNA segment lost, perhaps during the evolution of the mammalian mitochondrial translation machinery. Our structures reveal how RRF mt 's domain II and EF-G2 mt domain IV directly help in disrupting the central inter-subunit bridge, B2a [fig_ref] Figure 3: Direct involvement of RRF mt domain II and EF-G1 mt domain IV... [/fig_ref] , how the deacylated tRNA is held with the dissociated 39S subunit components [fig_ref] Figure 4: Stabilization of the E-site tRNA on the dissociated 39S subunit [/fig_ref] , and suggest how the dynamics of the interactions among the uL11m, CTD of uL12m, and the G'domain of EF-G2 mt alters between elongation and recycling steps [fig_ref] Figure 5: EF-G2 mt binding induces large-scale conformational changes in the uL11m stalk-base region... [/fig_ref]. Structural analysis of domain IV of EF-G1 mt and EF-G2 mt explains their specific roles in two distinct steps of elongation and recycling, respectively . Analysis of their GTPase domains complemented by GTPase assays reveal two distinct mechanisms of antibiotic fusidic acid resistance adopted by two homologous GTPases . These observations allow us to highlight distinct features of the main steps of human mitoribosomal recycling [fig_ref] Figure 8: Sequence of main events in human mitochondrial ribosome recycling suggested by structures... [/fig_ref]. Future studies using timeresolved cryo-EM should help further resolve the short-lived intermediates that form during the transition from Class I to Class III states.
# Methods
Purification of 55S mitoribosomes. The source of mitochondrial ribosomes was human embryonic kidney cells lacking N-acetyl-glucosaminyltransferase I (HEK293S GnTI) that were cultured in roller bottles using FreeStyle TM 293 media (Gibco, Life Technologies) supplemented with 5% fetal bovine serum (Gibco, Life . b Binding of RRF mt locks the mitoribosome in a partially destabilized state with rotated 28S subunit [fig_ref] Figure 1: Cryo-EM structures of the human mitochondrial recycling complexes in three functional states [/fig_ref]. The mito-specific N-terminus of the RRF mt 's NTE occupies a unique site near the bridge B2a region . c Subsequent binding of EF-G2 mt further destabilizes the 55S complex with disruption of additional inter-subunit bridges, in a fast reaction leading to multiple short-lived intermediate states, with the 28S subunit present in multiple orientations relative to the 39S subunit [fig_ref] Figure 1: Cryo-EM structures of the human mitochondrial recycling complexes in three functional states [/fig_ref]. d 28S and 39S subunits are dissociated. While the 28S subunit still carries mRNA, the 39S subunit carries RRF mt , EF-G2 mt , and surprisingly a deacylated tRNA mt in the 39S subunit's E-site region [fig_ref] Figure 1: Cryo-EM structures of the human mitochondrial recycling complexes in three functional states [/fig_ref]. This is in sharp contrast to the tRNA dissociation mechanism in eubacterial ribosome recycling, where tRNA goes with the small 30S ribosomal subunit. e Depiction of final recycling products, steps of ligand release between d and e are yet to be characterized.
Technologies). After centrifugation at 1000 × g for 7 min, the HEK293S GnTI cellpellet was transferred to a glass homogenizer and resuspended in buffer containing 50 mM HEPES-KOH pH 7.5, 10 mM KCL, 1.5 mM MgOAc, 70 mM sucrose, 210 mM mannitol, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF. The cells were homogenized by applying 120 strokes and the supernatant was separated from the cell debris by spinning at 950 × g for 15 min. The supernatant was then spun at 11,000 × g for 15 min, and the resulting pellet that contains crude mitochondria was resuspended in SEM buffer (250 mM sucrose, 20 mM HEPES-KOH pH 7.5, 1 mM EDTA, and 1 mM EGTA). RNase-free DNase I (3 units/ml) was added to the crude mitochondria and incubated at 4°C for 1 h in a rocking platform to allow gentle mixing. A discontinuous gradient was prepared in a Beckman polyallomer tube by layering 2.5 ml of 60%, 4 ml of 32%, 1 ml of 23%, and 1 ml of 15% sucrose solutions in buffer containing 10 mM HEPES-KOH pH 7.5 and 1 mM EDTA. DNase-treated sample was loaded on the discontinuous gradient and centrifuged for 1 h at 135,000 × g using Ti70 rotor in Beckman ultracentrifuge. The brownish-orange layer containing pure mitochondria was carefully separated and re-suspended in SEM buffer.
Four volumes of lysis buffer (25 mM HEPES-KOH pH 7.5, 100 mM KCl, 25 mM MgOAc, 1.7% Triton X-100, 2 mM DTT and 1 mM PMSF) was added to the mitochondrial-pellet and then incubated for 15 min at 4°C. The sample was centrifuged at 30,000 × g for 20 min and the supernatant was loaded on top of 1 M sucrose cushion in buffer (20 mM HEPES-KOH pH 7.5, 100 mM KCl, 20 mM MgOAc, 1% Triton X-100 and 2 mM DTT). After centrifugation for 17 h at 90,000 × g using Ti70 rotor in Beckman ultracentrifuge, a minimal volume of Mitobuffer (20 mM HEPES-KOH pH 7.5, 100 mM KCl, 20 mM MgOAc, and 2 mM DTT) enough to dissolve the pellet was added. 10-30% continuous sucrose density gradients were prepared in Mitobuffer, using the gradient making apparatus (C.B.S. Scientific Co.). The resuspended pellet was subjected to 10-30% continuous sucrose density gradient centrifugation at 60,000 × g for 17 h using Sw32 rotor in Beckman ultracentrifuge. The gradient was fractionated on ISCO gradient analyzer (Teledyne ISCO, Inc), and the fractions corresponding to the mitoribosomes were collected and pooled. Finally, the pooled mitoribosomes were concentrated by spinning them at 130,000 × g for 6 h using Ti70 rotor, and the pellet was resuspended in Polymix buffer (5 mM HEPES-KOH pH 7.5, 100 mM KCl, 20 mM MgOAc, 5 mM NH 4 Cl, 0.5 mM CaCl 2 , 1 mM DTT, 1 mM spermidine, and 8 mM putrescine).
Overexpression and purification of RRF2 mt . RRF mt clone was a gift from Prof. Linda Spremulli, University of North Carolina. The SUMO-tagged RRF mt was over-expressed in Rosetta2 cell lines and lysis buffer (500 mM NaCl, 1× PBS, 4 mM ß-mercaptoethanol, 1 mM PMSF, and 10 mM imidazole) was added to the pelleted cells. After sonication, the lysate was treated with DNase and then centrifuged for 30 min at 16,000 × g. The supernatant was applied to a His-trap Ni 2+ column and the SUMO-tagged protein was eluted from the column using elution buffer (250 mM NaCl, 1× PBS, 4 mM ß-mercaptoethanol, and 300 mM imidazole) using standard affinity purification protocols. The purified protein was dialyzed in buffer (20 mM Tris-HCl pH 8.0, 250 mM NaCl, 4 mM ß-mercaptoethanol, and 5% glycerol) and then the SUMO tag was cleaved from RRF mt by incubation with SUMO express protease for 1 h at 30°C. Finally, the SUMO tag and the SUMO express protease were separated from the RRF mt by passing through a His-trap Ni 2+ column that specifically adsorbs the SUMO tag and the SUMO express protease while pure RRF mt was released into the column flow-through.
Overexpression and purification of EF-G2 mt . The GST-tagged EF-G2 mt was cloned into pGEX6.1 vector and over-expressed in Rossetta (pLysS + RARE) cell lines. Cells were grown in LB media with 100 µg/ml ampicillin until 0.6 O.D. and protein over-expression was induced by adding 100 mM isopropyl-1-thio-Dgalactopyranoside (IPTG). The cell culture was left overnight at 16°C for optimal protein yields. The cells were pelleted in a JLA 10.5 rotor at 5000 rpm for 30 min and were shock-frozen in liquid nitrogen and stored at −80°C. The frozen cells were resuspended in lysis buffer (1× PBS-pH7.5, 10 mM MgCl 2 , 1 mM phenylmethyl sulphonyl fluoride (PMSF) and 1 mM DTT). After sonication and DNase I (5 μg/ml) treatment, the lysate were centrifuged at 14,000 rpm for 30 min to remove cell debris. The supernatant was passed through GSTrap™ HP column equilibrated with binding buffer 1× PBS-pH7.5 and 1 mM DTT. The GST-tag was cleaved from EF-G2 mt by loading 3C precision protease (gifted by Dr. Hongmin Li's lab, Wadsworth center, USA) along with cleavage buffer (50 mM Tris-HCl-pH 7.5, 150 mM NaCl, and 1 mM DTT). After incubating the EF-G2 mt with 3C precision protease overnight, relatively pure EF-G2 mt was eluted by passing the elution buffer (50 mM Tris-HCl-pH 7.5, 150 mM NaCl and 1 mM DTT). To obtain highlevel purity, EF-G2 mt was further passed through an anion exchange HiTrap Q HP column (GE healthcare, USA) and pure protein was eluted by running a gradient with 20 mM Tris-HCl-pH 7.5 and 500 mM NaCl.
Preparation of the 55S- RRF mt - EF-G2 mt - GMPPCP complex. Fifty micromolar of puromycin and 150 nM 55S mitoribosomes were mixed in HEPES polymix buffer (5 mM HEPES-KOH pH 7.5, 100 mM KCl, 20 mM MgOAc, 5 mM NH 4 Cl, 0.5 mM CaCl 2 , 1 mM DTT, 1 mM spermidine, and 8 mM putrescine) and incubated for 10 min at 37°C to obtain the model post-termination (PoTC) complex. Fifteen micromolar of RRF mt was added to this reaction mixture of PoTC and incubated for an additional 5 min at 37°C to obtain the 55S- RRF mt complex as described earlier [bib_ref] Structural insights into unique features of the human mitochondrial ribosome recycling, Koripella [/bib_ref]. Five micromolar of EF-G2 mt, together with 200 µM GMPPCP, was added to the 55S- RRF mt complex and incubated for various timepoints (5 s, 30 s and 2 min) at 37°C to obtain the 55S- RRF mt - EF-G2 mt - GMPPCP complex, which was immediately utilized for the cryo-EM grid preparation.
GTPase-Glo assay. GTPase activity was measured using the GTPase-Glo™ Assay by Promega and carried out as described [bib_ref] A homogenous bioluminescent system for measuring GTPase, GTPase activating protein, and guanine..., Mondal [/bib_ref]. In brief, a 10 µl reaction consisting of 0.1 µM ribosomes, 0.25 µM either bacterial (E. coli) EF-G or EF-G1 mt or EF-G2 mt and 1 µM GTP were incubated at room temperature in GTPase-Glo™ buffer in the absence and presence of varying amounts of fusidic acid (FA) for 1 h. Ten microliter of Reconstituted GTPase-Glo™ reagent was added to the reaction and left shaking at room temperature for 30 min. Finally, 20 µL of detection reagent was added, incubated for 10 min at room temperature and luminescence was measured in a Turner Biosystems Veritas™ Microplate Luminometer after 10 min. A negative control that contained only 1 µM GTP and a positive control that contained no FA were used each time. Luminescence was measured at FA concentrations of 1, 10, 100, and 1000 µM. Data was collected in duplicates and each duplicate dataset was normalized between 0 and 1 using the equation:
[formula] x norm ¼ x À x min x max À x min :ð1Þ [/formula]
As GTPase activity and luminescence measured had an inverse relationship, % GTPase activity was calculated with the formula %GTPase activity = (1x norm ) × 100. The values at each point were then averaged and standard deviation was calculated. The raw luminescence values are presented in . The plot was generated using Python library Matplotlib.
Cryo-electron microscopy and image processing. Home-made carbon was coated as a continuous layer (~50 Å thick) onto Quantifoil holey carbon 1.2/1.3 copper grids, which were then glow-discharged for 30 s on a plasma sterilizer. Four microliter of the sample was loaded to each of the multiple grids, incubated for 15 s at 4°C and 100% humidity, and then blotted for 4 s before flash-freezing into the liquid ethane using a Vitrobot IV (FEI). Data was acquired on a Titan Krios electron microscope equipped with a Gatan K2 summit direct-electron detecting camera at 300 KV. A defocus range of −1.0 to −3.0 µm was used at a calibrated magnification of 22,500×, yielding a pixel size of 1.0732 Å on the object scale. A dose rate of 8.25 electrons per pixel per second and an exposure time of 10 seconds resulted in a total dose of 71.6 eÅ −2 . CryoSPARC 3.0.1 [bib_ref] cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination, Punjani [/bib_ref] was employed for all the subsequent data processing steps. After applying full-frame motion correction to all 50 movie frames corresponding to each of the 21,752 micrographs that were collected, 142 bad micrographs were deselected after determining their contrast transfer function (CTF) using CTFFIND4 [bib_ref] CTFFIND4: fast and accurate defocus estimation from electron micrographs, Rohou [/bib_ref]. From the remaining 21,610 micrographs, a total of 5,046,906 particles were autopicked, which were then subjected to local motion correction and then 4,049,952 particles were retained. This step was followed by reference-free 2D classification which allowed us to further separate the good particles (1,140,751) from the bad particles (2,909,201) based on the 2D averages. Reference-based 3D classification was employed first to separate the particles into intact 55S mitoribosomes (162,354 particles), 39S subunits (419,699 particles), 28S subunits (235,896 particles) and poorly aligned particles (322,802 particles). To obtain more homogenous sub-populations, particles corresponding to the 55S mitoribosomes, 39S and 28S subunits were each subjected to additional rounds of 3D classification that finally yielded three stable classes representing three distinct functional states formed during the human mitoribosome recycling process. Two 55S mitoribosome classes, Class I (93,212 particles) and Class II (28,929 particles), were finally refined to 3.49 and 3.91 Å, respectively, while the 39S subunit Class III (132,008 particles) was refined to 3.15 Å.
Model building and optimization. Coordinates corresponding to the small and large subunits from our published human mitoribosome structures 6 (PDB ID: 6VLZ) were docked independently as rigid bodies into the corresponding cryo-EM density maps of the Class I, Class II, and Class III complexes using Chimera 1.14 . To obtain optimal fitting into our cryo-EM densities, the models were subsequently refined in PHENIX 1.14 (Adams PD 2010) using the "real-space refinement" function. Coordinates belonging to the human RRF mt (Koripella et al.were placed independently into the corresponding cryo-EM densities of all the three maps as rigid bodies using Chimera 1.14 78 and the models were further real-space refined in PHENIX 1.14 79 to achieve optimal accommodation into the cryo-EM densities. The primary aa sequence of EF-G2 mt was submitted to the I-TASSER serverto generate the initial EF-G2 mt homology model that was used to interpret the corresponding cryo-EM density. Segments in the homology model that do not fully accommodate into the corresponding EF-G2 mt density were modeled de novo using Chimera 1.14 [bib_ref] UCSF Chimera-a visualization system for exploratory research and analysis, Pettersen [/bib_ref] and COOT 0.9.5 [bib_ref] Features and development of Coot, Emsley [/bib_ref]. For the final optimization of the model into the cryo-EM density, the "Real-space refinement" function in PHENIX 1.14 79 was utilized. Validation reports for all the models were obtained from the Molprobity server 82 and the overall statistics of EM reconstruction and molecular modeling are listed in . All the figures in the manuscript were generated using ChimeraX 1.1.1 [bib_ref] UCSF ChimeraX: meeting modern challenges in visualization and analysis, Goddard [/bib_ref].
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.
## Data availability
[fig] Figure 1: Cryo-EM structures of the human mitochondrial recycling complexes in three functional states. Segmented cryo-EM maps of the mitoribosomal a 55S•RRF mt complex (Class I), b 55S•RRF mt •EF-G2 mt •GMPPCP complex [/fig]
[fig] Figure 3: Direct involvement of RRF mt domain II and EF-G1 mt domain IV in human mitoribosome recycling. a Comparison of the overall conformation of RRF mt between the Class I (gray) and Class III (green) complexes revealed that domain II rotates by about 45°towards the 28S subunit. Such a rotation would sterically clash with the 12S rRNA helix 44 (h44, brown) and MRP uS12m (cyan) and destabilize a crucial inter-subunit bridge, B2a (yellow). Inset shows the magnified view of the inter-subunit bridge B2a with RRF mt domain II residues V121-K127 (shown with density in magenta) disrupting B2a by inserting between h44 residues (C1491 and A1492) and H69 residues (A2581 and A2582). b Superimposition of the Class III complex with the Class I complex shows a direct overlap between the loop1 region (dark cyan) of EF-G2 mt domain IV (red) and the intersubunit bridge B2a (yellow) formed between h44 (brown) and H69 (blue). Inset shows the magnified view of EF-G2 mt domain IV residues L582-R585 (shown with density in dark cyan) that will disrupt the bridge B2a by inserting between h44 residues (G1559 and U1560) and H69 residue (A2576) and pushing the 28S subunit (h44) away. Thumbnails to the left depict an overall orientation of the 55S mitoribosome, with semitransparent 28S (yellow) and 39S (blue) subunits, and overlaid position RRF mt . Landmarks on the thumbnails are same as in Fig. 2. [/fig]
[fig] Figure 4: Stabilization of the E-site tRNA on the dissociated 39S subunit. a Extracted cryo-EM densities corresponding to E-site tRNA and interacting 39S subunit components. The E-site tRNA (orchid) is stabilized on the 39S subunit through multiple interactions with the mitoribosomal components. The elbow region of the E-site tRNA is sandwiched between 39S MRPs uL1m (cyan) and the mitospecific mL64 (blue) while the acceptor arm and the CCA end have strong interactions with uL33m and the 16S rRNA helix H88, respectively. b Atomic models for the densities shown in a. Multiple MRP segments bearing positively charged aa residues (Arg and Lys; red) interact with the negatively charged phosphates of the E-site tRNA molecule. To better display the location of the positively charged aa residues at the interface of tRNA and MRPs, the b orientation is obtained by applying an upward rotation around a horizontal axis to the panel a orientation. AC and CCA refer to anticodon and CCA ends of the tRNA. Thumbnails to the left depict an overall orientation of the 39S subunit (semitransparent blue), with overlaid highlighted mitoribosomal components and ligands. Landmarks on the thumbnail: CP central protuberance, Sb stalk base. [/fig]
[fig] Figure 5: EF-G2 mt binding induces large-scale conformational changes in the uL11m stalk-base region and the CTD of uL12m. a [/fig]
[fig] Figure 6: Structural basis for the exclusive roles of EF-G1 mt in tRNA translocation and EF-G2 mt in mitoribosome recycling. a The presence of a substantially longer C-terminal α-helix (salmon) along with the presence of a unique CTE (blue), which is required for mitochondria tRNA translocation 6 , prevents the simultaneous binding of EF-G1 mt and RRF mt (green) on the mitoribosome due to a major steric clash. Furthermore, any conformational repositioning the Cterminal α-helix of EF-G1 mt will result in direct steric clash with the H89 (orange) of 16S rRNA and the NTE of RRF mt (pink). b Having a shorter C-terminal α-helix (red) and the absence of CTE allows the simultaneous binding of EF-G2 mt and RRF mt (green) on the mitoribosome. c In EF-G1 mt 6 , the presence of two universally conserved glycine residues (dark green) at the tip of domain IV loop1 region (salmon) facilitates its insertion between the mRNA-tRNA duplex at the decoding center (DC) during EF-G1 mt -catalyzed translocation. d In EF-G2 mt, the second glycine of domain IV loop 1 region (red) is substituted by an aspartic acid (dark green) altering its conformation and flexibility, and thereby making its insertion into the DC during translocation unfavorable. e The boxed aa sequence corresponds to the loop 1 situated at the tip of domain IV, which is conserved between EF-G1 mt and the T. thermophilus EF-G. First of the two universally conserved loop 1 glycine residues (green) is substituted by an alanine in B. burgdorferi EF-G2, while the second one is replaced by an aspartic acid in EF-G2 mt . [/fig]
[fig] Figure 8: Sequence of main events in human mitochondrial ribosome recycling suggested by structures in this study. a The model post-termination mitoribosomal complex (PoTC), with mRNA and pe/E-state tRNA mt [/fig]
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Antineutrophilic Cytoplasmic Antibody Positive Vasculitis Associated with Methimazole Use
ANCA-associated vasculitis (AAV) is a rare and potentially life threatening complication associated with antithyroid drug use. It is more commonly reported with propylthiouracil, with fewer cases reported with methimazole use. We present the case of a 55-year-old man with toxic multinodular goiter which was treated with methimazole for 6 months. He developed ANCA positive leukocytoclastic vasculitis with hemorrhagic and necrotic bullous lesions of lower extremities. The vasculitis was initially thought to be secondary to recent cephalosporin use; however, the skin lesions progressed despite stopping the cephalosporin and treatment with steroids, and he developed osteomyelitis. His vasculitis resolved after cessation of methimazole use. This case highlights the importance of careful monitoring for variable manifestations of AAV in patients treated with methimazole.
# Introduction
Methimazole (MMI) and propylthiouracil (PTU) are thionamide drugs, which are commonly used as first-line therapy in the treatment of hyperthyroidism due to Grave's disease and toxic nodular goiter in the United States. These medications are not without risk, however, and are associated with potential adverse reactions such as fever, rash, agranulocytosis, and hepatitis. These reactions usually occur within the first few months of initiating treatment [bib_ref] Antithyroid drugs, Cooper [/bib_ref] , although agranulocytosis can occur idiosyncratically at any time during treatment. ANCA positive vasculitis is a serious but lesser known complication of thionamides. Despite being previously described in the literature, there is a lower incidence of reported ANCA positive vasculitis with MMI use as compared to PTU [bib_ref] Antithyroid drugs and antincutrophil cytoplasmic antibody positive vasculitis. A case report and..., Gunton [/bib_ref] [bib_ref] Clinical characteristics of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis caused by antithyroid drugs, Noh [/bib_ref]. We report a patient who developed ANCA positive leukocytoclastic vasculitis after six months of MMI treatment.
## Case
A 55-year-old male was diagnosed with hyperthyroidism by his primary care physician. Thyroid sonogram showed a multinodular goiter. FNA biopsies of the dominant nodules were benign, and he was started on methimazole 20 mg twice a day for toxic nodular goiter. Six months later, he presented to the emergency department with bilateral lower extremity pain, redness, and swelling. He was diagnosed with cellulitis and discharged home on oral cephalexin; however, his lower extremity lesions progressed over the next month, and he was admitted to the hospital for further management.
During that admission, the patient was noted to have hemorrhagic and necrotic bullous lesions on the anterior aspect of the bilateral lower legs and dorsal aspect of the feet. Laboratory data showed elevated C-reactive protein suggestive of an inflammatory reaction, but without leukocytosis or eosinophilia. He had normal levels of rheumatoid factor, ribonucleoprotein antibody, and Sjogren SSA and SSB antibodies. Serum complement C3 and C4 levels were high; C3 was 180 mg/dL (<90 mg/dL) and C4 was 50 mg/dL (6-47 mg/dL). Antinuclear antibody (ANA) was positive in titres of 1 : 80 with a speckled pattern. ANCA screen as measured with indirect immunofluorescence was positive for p-ANCA and detected high MPO antibodies at 5.6 AI (normal < 1 AI). Work-up for HIV, hepatitis B, and hepatitis C was negative. Urinalysis was unremarkable. Skin biopsy of the lesions 2 Case Reports in Endocrinology Based on this work-up, the vasculitis was attributed to cephalexin. The patient was treated with high dose prednisone for 2 weeks in the hospital and discharged home with an additional 2 weeks of tapering glucocorticoids. He presented again 2 months later with persistent bilateral lower extremity skin lesions and suppurative discharge from the left foot. MRI and bone biopsy were consistent with acute osteomyelitis.
The endocrinology team was consulted during this readmission because of high TSH while being on methimazole. On examination, he had no lid lag or exophthalmos. Thyroid was nodular and enlarged about three times the normal size, with left lobe bigger than right. CXR showed an enlarged left thyroid lobe deviating the upper trachea to the right side. Thyroid antibodies were not elevated: thyroid peroxidase antibody was 14 IU/mL (<35 IU/mL), thyroglobulin antibody was <20 IU/mL (<20 IU/mL), and thyroid stimulating immunoglobulin was 125% (<140%).
The lower extremity lesions did not resolve despite stopping cephalexin and completing month-long course of steroids; therefore, we considered the possibility of methimazoleinduced leukocytoclastic vasculitis. Methimazole was discontinued. We then recommended total thyroidectomy for definitive management of a toxic multinodular goiter that was also causing tracheal deviation. Surgical pathology showed nodular hyperplasia with focal Hurthle cell features and calcifications with ossification. He was started on levothyroxine replacement therapy and antibiotics for osteomyelitis and discharged home. On 1-month follow-up in clinic, the patient's skin lesions were largely resolved and he was clinically well.
# Discussion
ANCA-associated vasculitis (AAV) is a group of small vessel vasculitides that consist of autoantibodies directed against the lysosomal enzymes of neutrophils. These autoantibodies are divided into two main groups: cytoplasmic (c-ANCA) which confers antigen specificity for proteinase 3 and is associated with Wegener's granulomatosis and perinuclear (p-ANCA) which reacts against myeloperoxidase (MPO) and is mainly associated with microscopic polyangiitis (MPA) and Churg-Strauss syndrome. AAV may cause a variety of constitutional symptoms including fever, myalgia, arthralgia, and flu like syndrome. Multisystem involvement can be seen, with the kidneys most commonly affected followed by skin and respiratory tract [bib_ref] Clinical characteristics of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis caused by antithyroid drugs, Noh [/bib_ref]. Vessels in the joints, eyes, skeletal muscle, gastrointestinal tract, and peripheral nerves may also be involved. The most common cutaneous lesion is leukocytoclastic vasculitis, which preferentially affects the lower extremities [bib_ref] Perinuclear antineutrophil cytoplasmic antibody-associated vasculitis in a patient with Graves' disease treated..., Martin [/bib_ref].
AAV in association with antithyroid drugs is a relatively uncommon entity, and the pathogenesis of vasculitis associated with ATDs is not well understood. There have been reports of cutaneous and systemic AAV, with most of the literature describing p-ANCA vasculitis in the setting of PTU use, particularly in Asian individuals [bib_ref] Antithyroid drugs and antincutrophil cytoplasmic antibody positive vasculitis. A case report and..., Gunton [/bib_ref] [bib_ref] Clinical characteristics of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis caused by antithyroid drugs, Noh [/bib_ref] [bib_ref] Perinuclear antineutrophil cytoplasmic antibody-associated vasculitis in a patient with Graves' disease treated..., Martin [/bib_ref] [bib_ref] Propylthiouracil-induced cutaneous vaculitis, Gammeltoft [/bib_ref] [bib_ref] ANCA-associated vasculitis and lupus-like syndrome caused by methimazole, Kawachi [/bib_ref] [bib_ref] Methimazoleinduced antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis and lupus-like syndrome with a cutaneous..., Thong [/bib_ref] [bib_ref] Central nervous system vasculitis after starting methimazole in a woman with Graves'..., Tripodi [/bib_ref] [bib_ref] Methimazole-induced antineutrophil cytoplasmic antibodyassociated diffuse alveolar haemorrhage in a Chinese woman with..., Lau [/bib_ref] [bib_ref] ANCA-associated vasculitis with central retinal artery occlusion developing during treatment with methimazole, Yasude [/bib_ref]. It has been postulated that PTU binds to myeloperoxidase and alters its structure, leading to formation of autoantibodies in susceptible individuals [bib_ref] Accumulation of 2-[14C]propylthiouracil in human polymorphonuclear leukocytes, Lam [/bib_ref]. There are no published data that suggest that methimazole can also alter the configuration of myeloperoxidase. Data from Gao et al. [bib_ref] The target antigens of antineutrophil cytoplasmic antibodies (ANCA) induced by propylthiouracil, Gao [/bib_ref] indicate that activated neutrophils produce increased amounts of myeloperoxidase which oxidize the thionamides into reactive intermediates. These intermediates then activate immunocompetent cells such as lymphocytes via covalently binding to self-proteins, leading to production of MPO-ANCA and hence causing vascular injury.
AAV is a rare complication of MMI use, with few cases described in the literature. The first case of MMI-induced AAV was described by . AAV is also less frequently described in toxic MNG compared to Grave's disease. In 1996, Gunton et al. assembled 27 cases of ATDinduced ANCA positive vasculitis in which only one case was related to MMI and only 1 case had underlying toxic MNG as the etiology of hyperthyroidism [bib_ref] Antithyroid drugs and antincutrophil cytoplasmic antibody positive vasculitis. A case report and..., Gunton [/bib_ref].
Noh et al. have reported that the incidence for PTUrelated AAV is about 39 times that for MMI [bib_ref] Clinical characteristics of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis caused by antithyroid drugs, Noh [/bib_ref]. Despite being associated with long term antithyroid drug treatment, with median onset time 42 months, it can also occur within a few months of starting the treatment. MPO-AAV can occur even at low doses for both MMI and PTU. It is reported to occur more frequently in women, although this may just reflect the female preponderance of thyroid disease. Interestingly, the appearance of p-ANCA antibodies does not always predict the development of clinical vasculitis [bib_ref] Prevalence of positive anti-neutrophil cytoplasmic antibody (ANCA) in patients receiving anti-thyroid medication, Gunton [/bib_ref] , and there is also no correlation between the MPO-ANCA titer and the severity of vasculitis [bib_ref] Clinical characteristics of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis caused by antithyroid drugs, Noh [/bib_ref].
Importantly, p-ANCA-associated vasculitis improves and has a good prognosis if antithyroid drugs are discontinued. Some patients may require steroids and/or immunosuppressive drugs depending upon the severity of the disease. Serious complications (pulmonary hemorrhage, acute kidney failure, and nonhealing ulcers) have been reported when ATDs have been continued due to unawareness of this uncommon adverse reaction.
Our patient had leukocytoclastic vasculitis with cutaneous lesions and no systemic manifestations. His vasculitis was initially thought to be secondary to cephalosporin use, but the skin lesions did not heal despite stopping cephalosporin and completing a course of prednisone. The patient in fact had progression of the lower extremity ulcers and developed osteomyelitis. After MMI was discontinued, the skin lesions resolved. This clinical course along with the presence of high p-ANCA titres suggested that the biopsyproven leukocytoclastic vasculitis was likely an AAV due to MMI use. We did not recommend PTU therapy after stopping MMI due to the higher reported incidence of vasculitis with PTU use and reports of cross reactivity between PTU and MMI [bib_ref] Antineutrophil cytoplasmic antibody-positive vasculitis in a patient with Graves disease: cross-reaction between..., Ahmed [/bib_ref] in AAV.
In conclusion, we suggest that clinicians be aware of this uncommon adverse reaction of MMI treatment. Patients treated with MMI should be carefully monitored for manifestations of AAV such as fever, skin involvement, myalgias, arthralgias, glomerulonephritis, and pulmonary hemorrhage, regardless of the period of administration of MMI. Early recognition of this serious adverse effect is crucial as immediate cessation of MMI is needed, along with possible administration of corticosteroids, in order to prevent progression of the disease and its serious sequelae.
[fig] Figure 1: Skin biopsy in low power field showing leukocytoclastic vasculitis. [/fig]
[fig] Figure 2: Skin biopsy in high power field showing leukocytoclastic vasculitis.revealed leukocytoclastic vasculitis with fibrin thrombi. No immune deposits were detected(Figures 1 and 2). [/fig]
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10.1017/S0033291723000454
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CCBY
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10600825
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36878891
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s2orc_pubmed_articles
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In-person 1-day cognitive behavioral therapy-based workshops for postpartum depression: a randomized controlled trial
In-person 1-day cognitive behavioral therapy-based workshops for postpartum depression: a randomized controlled trial.
Postpartum depression (PPD) is among the most common complications of delivery, affecting as many as one in five mothers and birthing parents [bib_ref] Perinatal depression: A systematic review of prevalence and incidence, Gavin [/bib_ref] [bib_ref] Prevalence and characteristics of postpartum depression symptomatology among Canadian women: A crosssectional..., Lanes [/bib_ref].It is associated with an increased risk of future depressive episodes [bib_ref] Consequences of maternal postpartum depression: A systematic review of maternal and infant..., Slomian [/bib_ref] , and more cognitive, emotional, and behavioral problems in offspring [bib_ref] Risk for psychopathology in the children of depressed mothers: A developmental model..., Goodman [/bib_ref] [bib_ref] Maternal postnatal depression and the development of depression in offspring up to..., Murray [/bib_ref] [bib_ref] Association of persistent and severe postnatal depression with child outcomes, Netsi [/bib_ref].When those with elevated levels of depressive symptoms are counted, as many as one in three individuals are affected [bib_ref] Perinatal maternal depressive symptoms as an issue for population health, Meaney [/bib_ref].Those with subsyndromal symptom levels also have poor outcomes as they often fail to seek treatment and are ineligible for clinical services, though many have parenting and offspring outcomes similar to those with formal major depressive disorder (MDD) diagnoses [bib_ref] The experience of seeking help for postnatal depression, Holopainen [/bib_ref] [bib_ref] Maternal depression and parenting behavior: A meta-analytic review, Lovejoy [/bib_ref].
While treating PPD can reduce its adverse effects [bib_ref] Changes in infant emotion regulation following maternal cognitive behavioral therapy for postpartum..., Krzeczkowski [/bib_ref] [bib_ref] The effect of perinatal depression treatment for mothers on parenting and child..., Letourneau [/bib_ref] , timely and accessible interventions are essential for optimizing outcomes.Healthcare systems are poorly equipped to treat problems requiring urgent psychotherapy like PPD, and as few as one in 10 sufferers receive evidencebased care [bib_ref] Patterns of depression and treatment in pregnant and postpartum women, Bowen [/bib_ref] [bib_ref] The perinatal depression treatment cascade: Baby steps toward improving outcomes, Cox [/bib_ref].Barriers to treatment include a lack of time and preferences for psychotherapy over medication [bib_ref] Women's views of antidepressants in the treatment of postnatal depression, Boath [/bib_ref] [bib_ref] Strategies for improving perinatal depression treatment in North American outpatient obstetric settings, Byatt [/bib_ref] [bib_ref] Women's attitudes, preferences, and perceived barriers to treatment for perinatal depression, Goodman [/bib_ref] [bib_ref] Help seeking in the perinatal period: A review of barriers and facilitators, Jones [/bib_ref].
Clinical practice guidelines recommend evidence-based psychotherapies (e.g.cognitive behavioral therapy (CBT)) for the vast majority of those with PPD [bib_ref] Canadian network for mood and anxiety treatments (CANMAT) 2016 clinical guidelines for..., Macqueen [/bib_ref].However, psychological treatments suffer from serious problems of capacity and waitlists can be long.To address this, there needs to be either a massive increase in the number of therapists or substantial changes to the way we treat PPD.
Even though international practice guidelines suggest that those with PPD should be a priority population for psychotherapy access, virtually no low-intensity interventions have proven effective in this population.While standard self-help approaches could be effective in those with PPD [bib_ref] An exploratory parallel-group randomised controlled trial of antenatal guided self-help (plus usual..., Trevillion [/bib_ref] , they may not be ideally suited for this group.Many mothers and birthing parents also lack the time to engage in self-help exercises which are quite burdensome when added to existing role demands.Moreover, despite their convenience and ease of use, mobile applications (apps) have not proven clinically effective for PPD [bib_ref] Evaluating the effectiveness and quality of mobile applications for perinatal depression and..., Tsai [/bib_ref].
One low-intensity approach to treating PPD is 1-day psychotherapy workshops.The delivery of psychotherapy in large groups (i.e. up to 30 participants) [bib_ref] One-day cognitivebehavioural therapy self-confidence workshops for people with depression: Randomised controlled trial, Horrell [/bib_ref] [bib_ref] Evaluating the feasibility of an innovative self-confidence webinar intervention for depression in..., Yunus [/bib_ref] may be capable of addressing the needs of those with PPD, as well as treating it on the scale required to address its prevalence.These 1-day interventions contain the core content of more comprehensive, evidence-based treatments, but their brevity and scalability make them easier to disseminate.
During the COVID-19 pandemic, a large (n = 403) randomized controlled trial (RCT) of 1-day CBT-based workshops for PPD delivered online led to reductions in PPD and anxiety, as well as improvements in the mother-infant relationship and infant temperament [bib_ref] Effect of online 1-day cognitive behavioral therapy-based workshops plus usual care vs..., Van Lieshout [/bib_ref].However, some participants expressed a preference for in-person workshops citing difficulties participating online while minding an infant, concerns about privacy, and the benefits of face-to-face social support.The effectiveness of these workshops outside of the pandemic context also remains unclear, their impact on other children in the home is not well understood, and despite their efficiency, their cost-effectiveness is not known.
A few RCTs of treatments for PPD that have examined costeffectiveness suggest that effective interventions are usually more expensive than treatment as usual (TAU).One trial of telephonebased peer-support showed that it was more effective than TAU and cost-effective (with an incremental cost-effectiveness ratio (ICER) of CAD$ 10 009), but the cost was higher in the intervention group [bib_ref] Prospective economic evaluation of a peer support intervention for prevention of postpartum..., Dukhovny [/bib_ref].Similarly, a facilitated self-help-based program for PPD was more effective but more costly than TAU at £13 324 per quality-adjusted life-year (QALY) (National Collaborating Centre for Mental Health, 2014).A probabilistic treatment model based on a National Institute for Health and Care Excellence (NICE) guideline found that structured psychological therapy was cost effective compared to TAU at an ICER of £17 481 [bib_ref] Methods to identify postnatal depression in primary care: An integrated evidence synthesis..., Hewitt [/bib_ref] which is just below the cost-effectiveness threshold of less than £20 000 recommended by.Given the potential scalability of 1-day CBT-based workshops for PPD, understanding its costeffectiveness will help determine their potential to treat PPD.
Given this background, we examined the effectiveness of in-person 1-day CBT-based workshops for PPD at improving depression, anxiety, the mother-infant relationship, and offspring behavior, as well as health-related quality of life and cost-effectiveness.
# Method
## Trial design and procedures
This parallel-group, multi-site, RCT took place in Ontario, Canada from 17 July 2018 to 28 February 2020, recruiting from seven geographic regions representing most of the provinces of Ontario's local health integration networks: Elgin county, Simcoe county, Prince Edward county, the city of Toronto, Huron county, Kitchener-Waterloo, and Halton regions.Community organizations, midwives, and public health units in these regions assisted with recruitment and provided workshop sites.
Participants in each region were centrally randomized in a 1:1 ratio to the experimental or control group.Individuals assigned to the experimental group received TAU and participated in the 1-day workshop.Participants in the control group also received TAU but were put on a waitlist to receive the CBT workshop 12 weeks later.In Ontario, healthcare is universally available, so TAU could involve care from a physician, nurse practitioner, and/or midwife, as well as pharmacotherapy and/or psychotherapy from a provincially funded program.They could also utilize the services of any private therapist.
Centralized randomization that used 77 blocks of size of four, six, and eight was used to allocate participants to experimental and control arms.The randomization scheme was created in R version 3.4.3(R Core Team, n.d.) and implemented by the study coordinator in REDCap [bib_ref] Research electronic data capture (REDCap) -A metadata-driven methodology and workflow process for..., Harris [/bib_ref] , enabling concealment of allocation sequence until group assignment.Staff making reminder calls and/or collecting data and the data analyst were unaware of participant group status.The study was approved by the Hamilton Integrated Research Ethics Board and was registered (ClinicalTrials.gov:NCT03654261).
## Participants
Participants could self-refer after seeing social media advertisements or be referred by staff at a partnering organization.They had to be ⩾18 years old, have an infant <12 months of age, live in a recruitment region, and have an Edinburgh Postnatal Depression Scale (EPDS) score ⩾10.This cut-off is commonly used in clinical and research settings to identify those who may have PPD [bib_ref] Perinatal depression: A systematic review of prevalence and incidence, Gavin [/bib_ref] [bib_ref] Prevalence and characteristics of postpartum depression symptomatology among Canadian women: A crosssectional..., Lanes [/bib_ref] and was chosen to maximize public health relevance [30% of mothers/birthing parents have these symptom levels [bib_ref] Perinatal maternal depressive symptoms as an issue for population health, Meaney [/bib_ref] ].Participants were also fluent in written and spoken English.
## Intervention
Workshops were delivered by one psychiatrist or one registered psychotherapist randomly assigned to workshop dates.Their training involved review of the participant manual and workshop script.Each delivered one workshop and received feedback from the other prior to delivering study workshops.Workshops were held in community settings such as libraries and community centers, and free childminding was provided.
Each interactive workshop was run from 0900 to 1600 and consisted of four modules containing didactic teaching, group exercises/discussion, and role plays.The first reviewed PPD etiology with an emphasis on cognitive factors, and the second focused on cognitive skills including cognitive restructuring.The third built behavioral skills such as problem solving, behavioral activation, and assertiveness, and the fourth provided an opportunity for action planning.Workshops were based on previous work by Brown and colleagues, but adapted for PPD [bib_ref] Large-scale health promotion stress workshops for the general public: A controlled evaluation, Brown [/bib_ref].Participants received a printed copy of the workshop manual.
## Psychological medicine
## Data collection and outcome measures
Data were collected from participants using REDCap [bib_ref] Research electronic data capture (REDCap) -A metadata-driven methodology and workflow process for..., Harris [/bib_ref] at baseline (T1) and 12 weeks later (T2).At enrollment, participants self-reported their sociodemographic characteristics (age, infant age, ethnicity, marital status, educational attainment, household income, antidepressant use, and past counseling).
We chose to assess our T2 outcomes at 12 weeks posttreatment because this corresponds to one of the most common post-treatment endpoints used in RCTs of gold-standard psychotherapies like interpersonal therapy (IPT) and CBT.This was intended to help determine if this very brief treatment could produce benefits over comparable periods of time of these well-established treatments.It was felt this could assist patients and clinicians determine if this briefer treatment could be effective and applicable to them.
The primary outcome (PPD) was assessed using the EPDS, a 10-item measure of symptoms of depression during the previous 7 days [bib_ref] Detection of postnatal depression. Development of the 10-item Edinburgh Postnatal Depression Scale, Cox [/bib_ref].Each item is scored on a 4-point scale (0-3) with higher scores indicating worse depressive symptoms.Based on Jacobson and Truax's reliable change index (1991), and consistent with the work of others [bib_ref] An international study exploring levels of postpartum depressive symptomatology, Affonso [/bib_ref] [bib_ref] Calculating clinically significant change in postnatal depression studies using the Edinburgh Postnatal..., Matthey [/bib_ref] , we defined an EPDS score change of at ⩾4 points as indicative of clinically significant change in PPD.
Secondary outcomes included anxiety, mother-infant bonding, infant temperament, behavior of older children, and costeffectiveness.The 7-item Generalized Anxiety Disorder Questionnaire (GAD-7) measured anxiety and contains seven items scored on a 0-3-point scale, with higher scores indicating worse anxiety [bib_ref] A brief measure for assessing generalized anxiety disorder: The GAD-7, Spitzer [/bib_ref].The GAD-7 has been validated for use in postpartum samples [bib_ref] Comparative efficacy of the generalized anxiety disorder 7-item scale and the Edinburgh..., Simpson [/bib_ref].In accordance with previous research, ⩾4 point change in GAD-7 score was defined as clinically significant [bib_ref] Sensitivity to change and minimal clinically important difference of the 7-item generalized..., Toussaint [/bib_ref].
The Postpartum Bonding Questionnaire (PBQ) assessed the mother-infant relationship.Its 25-items are scored on a 5-point scale (0-5), and contains four subscales: impaired bonding, rejection and pathological anger toward the infant, infant focused anxiety, and incipient abuse [bib_ref] The postpartum bonding questionnaire: A validation, Brockington [/bib_ref].The incipient abuse scale was not examined due to past performance issues [bib_ref] Mother-infant bonding disorders patients with postnatal depression: The postpartum bonding questionnaire in..., Klier [/bib_ref].
Infant temperament was rated by mothers with the Infant Behavior Questionnaire-Revised Very-Short Form (IBQ-R), a 37-item measure of infant temperament over the past week [bib_ref] Development and assessment of short and very short forms of the infant..., Putnam [/bib_ref].Items are scored on a 7-point scale and address three factors: positive affectivity/surgency, negative emotionality, and orienting/regulatory capacity.
Participants also rated the behavior of their 12-36-month-old children (when present) using the Early Childhood Behavior Questionnaire-Short Form (ECBQ), a 107-item measure of temperament comprised of surgency/extraversion, negative affectivity, and effortful control [bib_ref] Measurement of fine-grained aspects of toddler temperament: The early childhood behavior questionnaire, Putnam [/bib_ref].Children older than 36 months were rated using the Strength and Difficulties Questionnaire (SDQ), a 25-item measure of child behavior consisting of five scales (emotional, conduct, hyperactivity/inattention, peer relationship problems, and prosocial behavior).The total problems scale, consisting of the first four scales above, was used here [bib_ref] The strengths and difficulties questionnaire (SDQ): The factor structure and scale validation..., He [/bib_ref].
Participants reported their use of health resources on a scale created using the Canadian Community Health Survey and the Service Use and Resources Form, and adapted for the postpartum period.Data on service usage including hospital, physician, and other healthcare provider visits were collected, as were those pertaining to diagnostic tests and medication usage.The costs of workshop delivery were added to healthcare costs determined using a public payer perspective.Service costs were determined primarily by using the Province of Ontario's standardized Schedule of Benefits and its Schedule of Facility Fees.When those were not available, recommended hourly averages reported by the profession's official Ontario association were used (e.g. for physiotherapists, occupational therapists, etc.).The costs of public health programs were averaged based on provincial funding in 2020 (online Supplementary Table [fig_ref] Table 1: Baseline characteristics of experimental and control participants [/fig_ref].The costs of medications were calculated using the Ontario Drug Benefit Formulary.
The EuroQol Group's EQ-5D-3L is a preference-based health status questionnaire comprised of five domains: mobility, selfcare, usual activities, pain/discomfort, and anxiety/depression.Each domain is measured through three-level response options indicating no, some, and extreme problems [bib_ref] Assessing the impact of EQ-5D country-specific value sets on cost-utility outcomes, Van Dongen [/bib_ref].This study utilized a Canadian value set [bib_ref] Canadian valuation of EQ-5D health states: Preliminary value set and considerations for..., Bansback [/bib_ref].These index values were used to compute QALYs to compare the difference in effect between the intervention and control groups [bib_ref] Economic evaluation alongside randomised controlled trials: Design, conduct, analysis, and reporting, Petrou [/bib_ref].The EQ-5D-3L can also be scored as a 100-point Visual Analog Scale (VAS), providing a holistic summary of health status across the EQ-5D's five domains [bib_ref] Correspondence between EQ-5D health state classifications and EQ VAS scores, Whynes [/bib_ref].The VAS is reported as an outcome measure alongside the core results of this study.
## Statistical analyses
Demographic data were compared using t tests and χ 2 tests for continuous and dichotomous variables, respectively.
A priori sample size estimation suggested that a total of 476 participants would provide us with adequate statistical power to detect a medium effect size (d = 0.3) change in our primary outcome (EPDS) between groups (a group × time interaction).The power calculation used two-tailed statistical significance (α = 0.05) and 1 − β = 0.90.This estimation assumes a linear mixed model (LMM) applying a first-order autoregressive error covariance structure.This sample size estimate also presumed a 10% attrition rate between T1 and T2.Because of the COVID-19 pandemic, recruitment was stopped when 461 participants had been recruited.However, this sample size was still estimated to be capable of detecting significant effect sizes of similar magnitude (d ∼ 0.33).Sample size and power estimations were calculated using RMASS [bib_ref] Sample size determination for studies with repeated continuous outcomes, Bhaumik [/bib_ref] [bib_ref] Sample size estimation for longitudinal designs with attrition: Comparing time-related contrasts between..., Hedeker [/bib_ref] [bib_ref] Sample size determination for hierarchical longitudinal designs with differential attrition rates, Roy [/bib_ref].
To investigate the effectiveness of 1-day CBT workshops on continuous outcomes, we utilized LMMs with restricted maximum-likelihood estimation.A two-level hierarchy was employed in which outcomes at each timepoint (pre-and postworkshop) were nested within participant (split by group), allowing us to assess the effect of the intervention over time between experimental and control groups.A random-effects intercept was also included in the model controlling for unobserved heterogeneity at the level of the individual participant.These models also included workshop facilitator as a fixed effect covariate to control for individual differences in intervention effectiveness that may exist between workshop leaders.For outcome measures that revealed a significant group × time interaction, models stratified by group were employed to identify the magnitude of change in outcome measure score following workshop completion by the experimental group.Cohen's d defined the effect size of the change in mean scale score between baseline and follow-up.Finally, logistic regression was used to compare the odds of participants experiencing a ⩾4-point decrease in EPDS and GAD-7 scores (indicative of clinically significant improvement) between T1 and T2.Analyses were performed in SPSS 24 (IBM SPSS Statistics).
In conjunction with the QALY values calculated using the EQ-5D-3L Canadian index scores, healthcare cost data over the 12-week study period were used to calculate the ICER, comparing the cost-effectiveness of the workshop plus TAU v. TAU alone.In keeping with the intent-to-treat approach and given the limitations of imputing missing QALY and service use data, complete case analysis was employed.However, non-parametric bootstrapping was applied to the complete cases to estimate the uncertainty of the computed ICER across 1000 bootstrapped simulations.Finally, a cost-effectiveness acceptability curve (CEAC) was derived from the bootstrapped data, illustrating the probability of cost-effectiveness across a range of willingness-to-pay values per QALY gained.
# Results
A total of 1435 participants were assessed for eligibility and 461 were randomized to experimental (n = 229) or control (n = 232) groups.Sixty-nine participants declined to participate prior to T1 data collection, so 392 (experimental n = 189, control n = 203) completed baseline (T1) measures (Fig. [fig_ref] Figure 1: Fig [/fig_ref].
The majority of participants self-referred to the study after seeing information on social media (56%), on a partnering agency's website (5%), or after hearing about the study from family/friends (4%).Others (15%) were referred by healthcare providers including public health nurses, midwives, and/or family doctors.Remaining referral sources included community groups and early years centers.
In total, 33 workshops were run in regions across southern Ontario including Elgin county, Simcoe county, Prince Edward county, the city of Toronto, Huron county, Kitchener-Waterloo, and Halton regions.Workshops were delivered by a registered psychotherapist (n = 16) or a psychiatrist (n = 17).The baseline characteristics of experimental and control group participants are given in Table [fig_ref] Table 1: Baseline characteristics of experimental and control participants [/fig_ref].There were no statistically significant differences between groups.A total of 48 (12%) participants were lost to follow-up between T1 and T2, of which 31 were in the experimental group and 17 in the waitlist control group (χ 2 = 5.87, p = 0.015).Other than group assignment, no other baseline characteristics predicted dropout.At T1, 37 experimental and 44 control participants completed the ECBQ for their eligible toddlers, and 52 children of those in the experimental group and 55 control participants had children who were age-eligible for SDQ completion.
After adjusting for the fixed effect of workshop facilitator, statistically significant group × time interactions predicted EPDS, GAD-7, PBQ-IB, PBQ-RPA, PBQ-IFA, ECBQ-EC, and EQ-5D-3L scores (Table [fig_ref] Table 2: Group × time interaction predicting clinical outcomes [/fig_ref].After stratifying by group, experimental group participants reported statistically significant decreases in EPDS scores from T1 to T2, with scores decreasing from 15.77 pre-treatment to 11.22 post-treatment ( p < 0.01, Cohen's d = 0.62).Statistically significant decreases in GAD-7 scores 11.36 to 7.14 ( p < 0.01, d = 0.56), PBQ-IB (13.04 to 10.27; p < 0.01, d = 0.27), PBQ-RPA (7.05 to 4.89; p < 0.01, d = 0.32), ECBQ-EC (4.70 to 4.94, p = 0.04, d = 0.08), and EQ-5D-VAS (56.46 to 65.63; p < 0.01, d = 0.33) were also observed.While a statistically significant interaction between group and time predicted change in PBQ-IFA scores, no difference was detected between T1 and T2 among experimental group participants (3.52 v. 3.79, p = 0.25, d = 0.06) (Table [fig_ref] Table 3: Changes from T1 to T2 for primary and secondary outcome measures [/fig_ref].
Among experimental group participants, 58% reported clinically significant (⩾4-point) decreases in EPDS scores at T2, compared to 31% of control group participants [odds ratio (OR) 3.00, 95% confidence interval (CI) 1.93-4.67,number needed to treat (NNT) = 3.8].Likewise, 54% of experimental group participants reported a ⩾4-point decrease in GAD-7 scores at T2 v. 27% of control group participants (OR 3.20, 95% CI 2.03-5.04,NNT = 3.7).
The largest healthcare costs that accrued between T1 and T2 were attributable to general practitioner use (219 participants with at least one visit).Diagnostic imaging procedures (42 participants) were common but represented highly variable costs.While infrequent (eight participants), overnight hospital admissions also represented sizeable healthcare costs.Over the 12-week course of the study, experimental group participants accrued on average $2076.64 in healthcare costs, while control group participants accrued $4678.78.
QALYs were calculated for the 12-week time horizon between T1 and T2 for all participants who provided EQ-5D-3L index scores at T2 (experimental group n = 150, control group n = 179).The mean QALY increase was larger for experimental group participants [0.17 (S.D. = 0.02)] than for the control group [0.16 (S.D. = 0.03)] (the maximum of QALY over 12 weeks is 0.23).This difference was not statistically significant (t = −1.48,p = 0.14).However, the 1-day CBT workshop plus TAU achieved similar QALY at lower costs than TAU alone.The probability of the 1-day CBT workshop being cost-effective was 0.74 at a willingness-to-pay threshold of $0 and increased to 0.8 at $100 000 (Fig. [fig_ref] Figure 2: Fig [/fig_ref].
# Discussion
In this RCT of 461 mothers and birthing parents with PPD, a 1-day CBT-based workshop for PPD added to TAU led to statistically and clinically significant reductions in depression and anxiety relative to TAU alone.The workshops also resulted in improvements in the mother-infant relationship and the behavior of toddlers.In addition to its clinical effects, the workshops were cost-saving.
These findings replicate and extend those of an RCT of 1-day CBT-based workshops for PPD delivered online during the COVID-19 pandemic, and provide converging evidence supporting their effectiveness as a low-intensity intervention in steppedcare pathways .Indeed, medium effect size improvements were seen in PPD and anxiety in both in-person and online studies, and small effect size increases in the bonding subscale of the PBQ were seen in both interventions.The effectiveness of this 1-day workshop approach to depression treatment is also supported by studies that have examined 1-day interventions for general population samples of adults with depression [bib_ref] One-day cognitivebehavioural therapy self-confidence workshops for people with depression: Randomised controlled trial, Horrell [/bib_ref] and/or anxiety [bib_ref] Large-scale health promotion stress workshops for the general public: A controlled evaluation, Brown [/bib_ref] , and adolescents [bib_ref] School-based early intervention for anxiety and depression in older adolescents: A feasibility..., Brown [/bib_ref] , as well as other, briefer singlesession interventions [bib_ref] An online, singlesession intervention for adolescent self-injurious thoughts and behaviors: Results from..., Dobias [/bib_ref].The magnitude of the treatment effects seen are also similar to 1-day workshops in these other groups, as well as longer CBT treatment protocols previously utilized to treat depression in the perinatal period [bib_ref] A systematic review of the efficacy of cognitive behavioral therapy for treating..., Sockol [/bib_ref].
In addition to reductions in PPD, in-person and online workshops were both associated with meaningful improvements in anxiety.CBT is a first-line treatment for anxiety in adults [bib_ref] Royal Australian and New Zealand college of psychiatrists clinical practice guidelines for..., Andrews [/bib_ref] [bib_ref] & the Canadian Anxiety Guidelines Initiative Group on behalf of the Anxiety..., Katzman [/bib_ref] , and a growing body of evidence supports the effectiveness of CBT in those with postpartum anxiety (e.g. [bib_ref] Treatment of depression, anxiety, and trauma-related disorders during the perinatal period: A..., Nillni [/bib_ref].The size of the effect of this intervention on anxiety is comparable to our previous online workshop, as well as existing RCTs of CBT for anxiety [bib_ref] A randomized controlled trial of 'MUMentum pregnancy': Internet-delivered cognitive behavioral therapy program..., Loughnan [/bib_ref] [bib_ref] The efficacy of cognitive behavior therapy for the treatment of perinatal anxiety..., Maguire [/bib_ref] , and suggest that these workshops may be capable of reducing comorbid anxiety in those with PPD.
This intervention also led to some improvement in aspects of the mother-infant relationship including bonding, rejection, and pathological anger, and infant-focused anxiety.These findings replicate those of our previous online RCT (Van Lieshout et al., 2021), as well as other recent trials of CBT for PPD [bib_ref] Peer-delivered cognitive-behavioral therapy for postpartum depression: A randomized controlled trial, Amani [/bib_ref] [bib_ref] Mitigating the effect of persistent postnatal depression on child outcomes through an..., Stein [/bib_ref] [bib_ref] Public health nurse-delivered group cognitive behavioural therapy for postpartum depression: A randomized..., Van Lieshout [/bib_ref].Whether these changes are due to improvements in depression or anxiety, or the ways in which mothers interact with their infants are not known.However, while participants reported improvements in the mother-infant relationship, they did not note changes in infant temperament.This is at odds with the findings of the previous online 1-day trial where small but statistically significant increases in surgency were noted.Whether this is due to differences in our sampling frame, sample, or delivery modality (in-person v. online) is not known.
Despite the lack of statistically significant changes in infant temperament or the behavior of the participants' older children, improvements in the temperament (effortful control) of their toddlers were reported.Relatively few studies have examined the impact of interventions for PPD on non-infant offspring, even though it is well-known that PPD can have an adverse impact on their emotional and behavioral development.Pre-pandemic studies showed that children exposed to PPD manifest elevated levels of anxiety [bib_ref] Reported maternal postpartum depression and risk of childhood psychopathology, Walker [/bib_ref] , as well as hyperactivity and concentration difficulties at 2 years of age [bib_ref] Maternal postnatal depression and children's growth and behaviour during the early years..., Avan [/bib_ref].It is not clear why workshops may have had a positive effect on toddlers but not infants or older children.Perhaps changes in toddlers occur more quickly than in infants, or were more easily observable, and/or that the changes occur in temperament, but
## Psychological medicine
not child behavioral difficulties.However, replication in future samples is required in order to better understand the potential effects of workshops on offspring.We found that 1-day CBT-based workshops plus TAU were associated with similar QALYs to TAU alone, but were less expensive.This finding contrasts with a systematic review that found group CBT interventions to be associated with QALY gains but at a cost of £39 875 per QALY gained [bib_ref] Group cognitive behavioural therapy for postnatal depression: A systematic review of clinical..., Stevenson [/bib_ref] , well above the NICE-recommended cost-effectiveness threshold of less than £20 000-30 000, suggesting that these interventions may not be cost-effective.However, our costeffectiveness analyses suggested that 1-day CBT-based workshops for PPD added to TAU may be more effective and less expensive than TAU alone.In this study, the waitlist group also had worse overall health outcomes, though the incremental QALY difference was small.Regardless, these results support demonstrate the potential of the 1-day workshop model to be cost-effective, though further confirmation in future studies would be of value.
While promising, the findings of this study do need to be interpreted in light of its limitations.First, the intervention was delivered by only two expert therapists from a single center and occurred in-person prior to the pandemic.Facilitators of participation in in-person psychotherapies like implementing and clearly communicating safety protocols, conducting sessions outside of hospital settings, the provision of childcare, as well as flexible scheduling should be kept in mind in order to optimize participation in workshops delivered face-to-face [bib_ref] Barriers and facilitators to resuming in-person psychotherapy with perinatal patients amid the..., Andrejek [/bib_ref].While some prefer to attend interventions in-person, this modality may now be less popular than online delivery for some mothers and birthing parents.Online treatments have gained popularity since the COVID-19 pandemic for their increased accessibility [bib_ref] Videotherapy and therapeutic alliance in the age of COVID-19, Simpson [/bib_ref] , families and healthcare providers have expressed significant interest in using telemedicine for PPD [bib_ref] Implementing psychological interventions through nonspecialist providers and telemedicine in high-income countries: Qualitative..., Singla [/bib_ref] , and online therapeutic alliances have been rated as strongly as in in-person settings [bib_ref] Therapeutic alliance in videoconferencing psychotherapy: A review, Simpson [/bib_ref].
While full-day workshops are effective, we should acknowledge that some may prefer the intervention be delivered in two halfdays.However, in our online trial, just 10 of 161 participants in the experimental group (6%) expressed a preference that it be delivered differently (e.g. in half-days).Of these, 155 (96%) remained online for the entire session and 140 (87%) indicated that they were very satisfied with the full-day workshop [bib_ref] Effect of online 1-day cognitive behavioral therapy-based workshops plus usual care vs..., Van Lieshout [/bib_ref].
While facilitated interventions are often preferred by individuals with PPD over other self-guided options [bib_ref] Women's attitudes, preferences, and perceived barriers to treatment for perinatal depression, Goodman [/bib_ref] , future research should focus on exploring alternative delivery modalities, along with an examination of potential moderators (e.g.therapist type, participant PPD severity, delivery in a single-day v. two half-days) and mediators (intervention fidelity, cognitive, and/or behavioral skill use) of intervention effectiveness.In the interim, asking those with PPD what they feel might work best for them is likely the optimal approach.This study also took place in Canada where healthcare is universally available and so our findings may not generalize to all settings.Attrition was also higher in the experimental group in this study, but this may be due to the anticipation of those on the waitlist to receive the workshop.This is a consistently observed effect in studies of 1-day CBT-based workshops [bib_ref] One-day cognitivebehavioural therapy self-confidence workshops for people with depression: Randomised controlled trial, Horrell [/bib_ref] [bib_ref] Effect of online 1-day cognitive behavioral therapy-based workshops plus usual care vs..., Van Lieshout [/bib_ref].While the workshops did produce clinically significant improvements in PPD and anxiety, we did not conduct structured clinical interviews and so participants may not have had syndromal MDD or GAD, nor did they necessarily experience remission.However, since PPD is commonly seen as a continuous construct and the improvements observed in this and our previous study are clinically meaningful, the intervention may still have clinical utility.It is also important to note that all outcomes were self-reported by participants and so improvements in the mother-infant relationship and offspring outcomes may be contributed to by maternal symptom improvement.Moreover, it is possible that spontaneous remission could have contributed to our findings.Indeed, in previous trials of those with PPD, approximately 25% of participants were thought to have experienced spontaneous recovery [bib_ref] Effectiveness of cognitive behavioral therapy for perinatal maternal depression, anxiety and stress:..., Li [/bib_ref] [bib_ref] A systematic review of the efficacy of cognitive behavioral therapy for treating..., Sockol [/bib_ref] [bib_ref] A systematic review and meta-analysis of interpersonal psychotherapy for perinatal women, Sockol [/bib_ref].We feel that we should also emphasize that while we chose to assess our outcomes at a single timepoint (3 months postintervention) to try to optimize the comparability of our posttreatment findings to trials of full courses of structured psychotherapies, we did not assess participants immediately posttreatment (the day after the workshop).Because our study was powered to detect a moderate (d = 0.3) treatment effect in our primary outcome of interest (EPDS), it had less statistical power than might have been necessary to detect an equivalent effect in those outcomes that applied only to those with toddlers.Future studies may more comprehensively investigate the effect of CBT treatment on these outcomes with a larger sample size, specifically recruiting participants with children aged 2-3 years.Finally, we utilized a waitlist control design which may have inflated the size of the effects observed and prevented us from assessing the longer-term impact of the intervention.
Those focused on the mental health of mothers and birthing parents are in urgent need of safe, efficient, economical, evidencebased, and scalable means of treating PPD.One-day CBT-based workshops may be an effective treatment for PPD delivered in-person or online and has significant potential for scaling and task-shifting.It is also consistent with mothers' preferences and the proportionate universalism approach to public mental health utilized around the world, which attempts to address the whole
## Psychological medicine
[fig] Figure 1: Fig. 1.Flowchart of participants through the trial. [/fig]
[fig] Figure 2: Fig. 2. CEAC of treatment across increasing willingness to pay thresholds. [/fig]
[table] Table 1: Baseline characteristics of experimental and control participants [/table]
[table] Table 2: Group × time interaction predicting clinical outcomes [/table]
[table] Table 3: Changes from T1 to T2 for primary and secondary outcome measures [/table]
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10.1002/mbo3.732
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CCBY
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6528562
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30298571
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s2orc_pubmed_articles
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W361R mutation in GaaR, the regulator of D‐galacturonic acid‐responsive genes, leads to constitutive production of pectinases in Aspergillus niger
[bib_ref] A novel Zn 2 Cys 6 transcription factor BcGaaR regulates D-galacturonic acid..., Zhang [/bib_ref] [bib_ref] A novel Zn 2 Cys 6 transcription factor BcGaaR regulates D-galacturonic acid..., Zhang [/bib_ref] [bib_ref] The transcriptional activator GaaR of Aspergillus niger is required for release and..., Alazi [/bib_ref] [bib_ref] The transcriptional activator GaaR of Aspergillus niger is required for release and..., Alazi [/bib_ref] [bib_ref] The transcription factor PDR-1 is a multi-functional regulator and key component of..., Thieme [/bib_ref] [bib_ref] Aspergillus niger RhaR, a regulator involved in L-rhamnose release and catabolism, Gruben [/bib_ref] [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref] [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref] [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref] [bib_ref] Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic..., Alazi [/bib_ref] [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref] [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref] [bib_ref] Gene organization and regulation in the qa (quinic acid) gene cluster of..., Giles [/bib_ref] [bib_ref] Genetic regulation of the quinic acid utilization (QUT) gene cluster in Aspergillus..., Grant [/bib_ref] [bib_ref] The QUTA activator and QUTR repressor proteins of Aspergillus nidulans interact to..., Lamb [/bib_ref] [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref] [bib_ref] The QUTA activator and QUTR repressor proteins of Aspergillus nidulans interact to..., Lamb [/bib_ref] [bib_ref] Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic..., Alazi [/bib_ref] [bib_ref] Pectin-rich biomass as feedstock for fuel ethanol production, Edwards [/bib_ref] [bib_ref] Applications of pectinases in the commercial sector: A review, Kashyap [/bib_ref] [bib_ref] Potential application of pectinase in developing functional foods, Khan [/bib_ref] [bib_ref] Functional applications of lignocellulolytic enzymes in the fruit and vegetable processing industries, Toushik [/bib_ref]
## | material s and me thods
## | strains, media, and growth conditions
All strains used in this study are listed in . Media were prepared as described in [bib_ref] Using non-homologous end-joining-deficient strains for functional gene analyses in filamentous fungi, Arentshorst [/bib_ref].
Radial growth phenotype of the strains was analyzed on minimal medium (pH 5.8) containing 1.5% (w/v) agar (Scharlau, Barcelona, Spain) and various carbon sources: 50 mM D-glucose (VWR International, Amsterdam, The Netherlands), D-fructose (Sigma-Aldrich, Zwijndrecht, The Netherlands) or GA (Chemodex, St Gallen, Switzerland); or 1% (w/v) PGA (Sigma-Aldrich) or apple pectin (AP) (Sigma-Aldrich). Media of the uridine auxotrophic strains were supplemented with 10 mM uridine. Plates were inoculated with 5 μl 0.9% NaCl containing 1 × 10 4 or 5 × 10 4 freshly harvested spores, and cultivated at 30°C for 7 or 5 days, respectively. MM (pH 5.8) containing 1.5% (w/v) agar, 10 mM acetamide (Sigma-Aldrich, Steinheim, Germany) as the sole nitrogen source, and glucose, Dfructose, GA or 2-keto-3-deoxy-L-galactonate as the carbon source was prepared as described previously [bib_ref] Using non-homologous end-joining-deficient strains for functional gene analyses in filamentous fungi, Arentshorst [/bib_ref]. Plates were inoculated with 5 μl 0.9% NaCl containing 10 4 freshly harvested spores and cultivated at 30°C for 7 or 10 days.
Filter-sterilized carbon source solutions were added after autoclaving MM containing agar. PGA and AP were autoclaved together with the medium. All growth experiments were performed in duplicate.
For enzymatic analysis, 300 ml shake flasks that include 100 ml MM (pH 5.8) containing 50 mM D-fructose and 0.003% yeast extract were inoculated with 7.5 × 10 7 freshly harvested spores and cultivated for 36 hr in a rotary shaker at 30°C and 250 rpm. Experiments were performed in duplicate.
For microscopic analysis of the localization of the eGFP-tagged GaaR or GaaR W361R proteins, 4 × 10 5 freshly harvested spores were inoculated on cover slips in Petri dishes that include 4 ml MM containing 0.003% yeast extract and 10 mM D-fructose or 2-keto-3-deoxy-L-galactonate, and grown at 30°C for approximately 21 hr. For each condition, two biological replicates were performed.
## | sequencing gaax and gaar genes from mutants displaying constitutive production of enzymes involved in pga utilization
Sixty-four trans-acting mutants spontaneous or obtained after mild UV mutagenesis with constitutive production of enzymes involved in PGA utilization were obtained as previously described [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref] on solid MM containing 50 mM glucose as the carbon source and 10 mM acetamide as the sole nitrogen source. Genomic DNA of 11 spontaneous and 53 UV mutants was extracted as described by [bib_ref] Using non-homologous end-joining-deficient strains for functional gene analyses in filamentous fungi, Arentshorst [/bib_ref] , and gaaX and/or gaaR genes were PCR-amplified using the primers gaaX_P5f and gaaX_GSP9r, or gaaRP7f and gaaRP8r, respectively . The PCR fragments were sequenced in both directions using gaaX or gaaR sequencing primers, respectively .
## | construction of the promoter-reporter strains expressing gaar w361r
Protoplast-mediated transformation of A. niger and purification of the transformants were performed as described by [bib_ref] Using non-homologous end-joining-deficient strains for functional gene analyses in filamentous fungi, Arentshorst [/bib_ref].
The gaaR W361R gene together with its 964-bp promoter and 992-bp terminator regions was amplified by PCR using the primers gaaRP5f and gaaRP6r Transformants were selected on plates [bib_ref] Using non-homologous end-joining-deficient strains for functional gene analyses in filamentous fungi, Arentshorst [/bib_ref] containing 10 mM acetamide as the sole nitrogen source. Correct gene replacements in strains JN130.4 and JN129.1 were verified by Southern blot and sequencing analyses. For sequence analysis, the gaaR locus was amplified using the primers gaaRP7f and gaaRP8r and JN130.4 and JN129.1 genomic DNA as template, and ligated into pJET1.2/blunt cloning vector (Thermo Fisher Scientific, Carlsbad, CA). The resulting plasmids were amplified in Escherichia coli and sequenced using gaaR sequencing primers. Integration of the gaaR W361R into the endogenous gaaR locus was confirmed via Southern blot analysis. Genomic DNA was digested overnight with NcoI or HindIII restriction enzymes. A 501-bp fragment containing the gaaR gene was PCR-amplified using the primer pairs listed in with N402 genomic DNA as template and was used as a probe.
## | construction of strains expressing gaar-egfp or gaar-(ga) 4 -egfp
The plasmid pEA9 containing the PgaaR-gaaR-eGFP-TgaaR construct was created as follows: The eGFP gene and the 715-bp terminator of the gaaR gene were amplified by PCR using the primer pairs listed in with the plasmid pFG029 (unpublished vector, containing PgpdA-eGFP-TtrpC) and N402 genomic DNA as template, respectively. eGFP and TgaaR were combined by fusion PCR using primers GaaR_GFP1F and GaaR_GFP3R. The gaaR gene without the stop codon and together with the 731-bp promoter region was PCR amplified using the primer pairs listed in . PgaaR-gaaR and eGFP-TgaaR were combined by fusion PCR using primers eGFP-gaaR-For and eGFP-gaaR-Rev, ligated into pJET1.2/blunt cloning vector (Thermo Fisher Scientific) and amplified in E. coli. Sequencing of pEA9 showed that a PCR error (T to C) has occurred in TgaaR at a distance of 519 bp downstream of the stop codon of the eGFP gene.
The plasmid pEA10 containing the PgaaR-gaaR-(GA) 4 -eGFP-TgaaR construct was created in a similar way to pEA9, except that PgaaR-gaaR-(GA) 4 was amplified using the primer GaaR_GFP6R adding a linker containing four repeats of glycine and alanine residues between GaaR and eGFP. Sequencing of pEA10 showed that a PCR error (C to T) has occurred in TgaaR at a distance of 430 bp downstream of the stop codon of the eGFP gene.
To create the strain EA29.14, pEA9 was co-transformed into strain JN36.1 (ΔgaaR) together with the plasmid pMA357 containing the A. nidulans amdS gene behind the A. nidulans gpdA promoter [bib_ref] The transcriptional activator GaaR of Aspergillus niger is required for release and..., Alazi [/bib_ref]. EA30.6 was created by co-transformation of pEA10 into strain JN36.1 (ΔgaaR) together with the plasmid pMA357. Transformants were selected on transformation plates containing acetamide as the sole nitrogen source.
## | construction of the δgaar::aopyrg deletion strain
SO1.1 (ΔgaaR::AOpyrG) was created using the split marker approach . 5′ and 3′ flanks of gaaR gene were PCR-amplified using the primer pairs listed in genomic DNA as template. The Aspergillus oryzae pyrG gene was PCR-amplified as two fragments using the primer pairs listed in and the plasmid pAO4-13 (de Ruiter-Jacobs et al., 1989) as template. Split marker fragments with the AOpyrG selection marker were created by fusion PCR and used to transform the strain MA169.4 [bib_ref] Expanding the ku70 toolbox for filamentous fungi: Establishment of complementation vectors and..., Carvalho [/bib_ref] , resulting in the strain SO1.1. Proper deletion of gaaR was confirmed by diagnostic PCR (data not shown) and via Southern blot analysis.
## | construction of strains expressing gaar w361r , gaar w361r -egfp, and gaar-egfp at the endogenous gaar locus
To construct plasmid pSO1.2, the PgaaR-gaaR W361R -TgaaR allele was amplified by PCR using the primers gaaRP5f and gaaRP6r with JN103.1 genomic DNA as template and ligated into pJET1.2/ blunt cloning vector (Thermo Fisher Scientific). The resulting plasmid pSO1.2 was amplified in E. coli. pSO2.1 (containing the PgaaR-gaaR W361R -eGFP-TgaaR construct) was created by digesting the plasmids pSO1.2 and pEA9 (containing the PgaaR-gaaR-eGFP-TgaaR construct) with the restriction enzymes BcuI and BamHI, and by ligating the 2408-bp BcuI-BamHI fragment from pSO1.2 (containing the gaaR W361R mutation) with the BcuI and BamHI cut opened pEA9.
pSO1.2 and pSO2.1 were sequenced to ensure no PCR errors have occurred and proper ligation and orientation of the fragments.
Repair fragments for CRISPR-Cas9 mediated targeted integration (see below) were obtained as follows: The PgaaR-gaaR W361R -TgaaR construct containing the 964-bp PgaaR and 992-bp TgaaR was excised from pSO1.2 using the restriction enzymes NotI and XbaI.
The PgaaR-gaaR-eGFP-TgaaR and PgaaR-gaaR W361R -eGFP-TgaaR constructs containing the 731-bp PgaaR and 419-bp TgaaR were excised from pEA9 and pSO2.1, respectively, using the restriction enzyme
## Bglii.
The strains SO2.1 (gaaR W361R ), EA31.1 (gaaR-eGFP), and EA32.1 (gaaR W361R -eGFP) were created using the CRISPR-Cas9 technique [bib_ref] A CRISPR-Cas9 system for genetic engineering of filamentous fungi, Nødvig [/bib_ref]. A guide RNA protospacer sequence GTGGATACGTACTCCTTTTA targeting the AOpyrG gene was designed using E-CRISP [bib_ref] E-CRISP: Fast CRISPR target site identification, Heigwer [/bib_ref].
To create SO2.1, the DNA template for the in vitro synthesis of the guide RNA via the T7 promoter was amplified by PCR using the primers sgRNAP1 and OTL19 , and the plasmid p426- as template. The PCR consisted of the following reactions: Initial denaturation at 98°C for 30 s; 30 cycles of denaturation at 98°C for 5 s, annealing at 77°C for 5 s, extension at 72°C for 2 s; and final extension at 72°C for 30 s. The amplified DNA template was transcribed in vitro using the MEGAscript T7 Kit (Thermo Fisher Scientific). PgaaR-gaaR W361R -TgaaR repair fragment was co-transformed into strain SO1.1 (ΔgaaR::AOpyrG) together with the in vitro transcribed guide RNA and the plasmid pFC332 containing the cas9 gene and the hygromycin selection marker [bib_ref] A CRISPR-Cas9 system for genetic engineering of filamentous fungi, Nødvig [/bib_ref] , yielding to SO2.1.
[formula] SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, USA) (DiCarlo [/formula]
Transformants were selected on transformation plates containing hygromycin and uridine, and purified twice on MM containing GA, 5′-fluoroorotic acid, uridine, and hygromycin. pFC332 was cured by further purifying transformants twice without hygromycin selection on MM containing glucose and uridine. Transformants were not able to grow on MM containing glucose, uridine, and hygromycin after the first round of purification, indicating that pFC332 was successfully cured.
The DNA template for the in vivo synthesis of the guide RNA via the RNA polymerase III promoter was amplified in two parts by PCR using the primers Fw_LIC2 and Rev_P1, and Fw_P1 and Rev_LIC2 using the plasmid ANEp8_Cas9-gRNAalbA (manuscript in preparation [bib_ref] Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger, Song [/bib_ref] as template. The two PCR products were combined by fusion PCR using the primers Fw_LIC2 and Rev_ LIC2. The fusion PCR product was amplified by PCR using the primers for_pTE1 and rev_pTE1 to introduce PacI restriction sites at both ends, digested with PacI, and ligated into PacI cut opened pFC332, yielding to plasmid pTE1. pTE1 was co-transformed together with PgaaR-gaaR-eGFP-TgaaR or PgaaR-gaaR W361R -eGFP-TgaaR repair fragments into the strain SO1.1 (ΔgaaR::AOpyrG) to create EA31.1 or EA32.1, respectively. Transformants were selected on transformation plates containing hygromycin and uridine, and purified twice on MM containing GA, 5′-fluoroorotic acid and uridine. Transformants
were not able to grow on MM containing GA, 5′-fluoroorotic acid, uridine, and hygromycin after the first round of purification, indicating that pTE1 was successfully cured.
The gaaR locus in SO2.1 and EA31.1 was amplified using the primers gaaRP7f and gaaRP8r, and the one in EA32.1 using the primers PgaaR_seq_P2 and TgaaR_seq_P2, and ligated into pJET1.2/blunt cloning vector (Thermo Fisher Scientific). The resulting plasmids were amplified in E. coli and sequenced using gaaR sequencing primers to confirm the presence of the mutated gaaR allele in SO2.1 and EA32.1, and the wild-type gaaR allele in EA31.1. Integration of the repair fragments into the endogenous gaaR locus in SO2.1, EA31.1, and EA32.1 was confirmed via Southern blot analysis .
# | enzymatic analysis
Supernatants from shake flask cultures were obtained by filtration through glass microfiber filters (Whatman, Buckinghamshire, UK), and the filtrate was stored at −80°C. PGA plate assays were performed as described by [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref]. Twenty-five microliters of supernatant from each culture were spotted on plates containing 0.2% PGA, and plates were incubated at 37°C for 24 hr before staining with 0.1% Congo Red (Sigma-Aldrich, Zwijndrecht) solution.
## | microscopy
The cover slips with adherent germlings were placed upside down on glass slides. The GFP fluorescence was excited using 488 nm laser line in a Zeiss Observer confocal laser scanning microscope (Zeiss, Jena, Germany). The images were analyzed with the ImageJ software. On each image, the exact same brightness and contrast adjustments were applied.
## | re sults
## | genetic characterization of a. niger mutants showing constitutive production of pectinases
In a previous study, we isolated 64 mutants showing constitutive production of pectinases [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref]. Genome sequencing of five mutants showed that all five mutants harbor a mutation in a putative repressor protein that represses the expression of pectinases under non-inducing conditions [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref]. We sequenced the gaaX gene in the remaining 59 mutants showing constitutive production of pectinases (Table S3, . In total, 28 and nine mutants were found to carry nonsense and frameshift mutations, respectively. Twenty-three mutants were found to have missense mutations in the gaaX gene. These missense mutations were found throughout gaaX, indicating the importance of the different domains of GaaX for proper functioning. The nonsense or frameshift mutation closest to the C-terminus was the frameshift mutation V653G, indicating the importance of the last 45 amino acid residues for a functional GaaX, where the missense mutations L676P and V684K were also found . Four mutants did not carry any mutations in gaaX. Sequencing the gaaR gene in these mutants F I G U R E 1 Schematic representation of the domains present in GaaR (a) and conservation of GaaR W361 in Aspergillus species (b) and other Ascomycetes (c). Domains in GaaR (NRRL3_08195) were identified using NCBI's CDD [bib_ref] CDD: NCBI's conserved domain database, Marchler-Bauer [/bib_ref]. Protein sequences homologous to the Aspergillus niger GaaR were retrieved using the blastp algorithm from NCBI [bib_ref] Basic local alignment search tool, Altschul [/bib_ref] against the nonredundant protein sequences database and were aligned using Clustal Omega ( The UV mutant PpgaX-amdS ∆creA gaaR W361R-UV (JN103.1) carries a missense mutation (W 361 to R) caused by codon change from TGG to CGG at a distance of 1285 bp from the start codon of gaaR.
The tryptophan361 residue lies in the fungal-specific transcription factor domain (cd12148) that spans the residues 139-518 in the 740 amino acid-long GaaR , and is 100% conserved in the homologous GaaR sequences present in Aspergillus species and in other Ascomycetes analyzed belonging to the Pezizomycotina subdivision .
## | analyses of pectinase production in gaar w361r
To show that the W361R mutation in GaaR is responsible for the constitutive production of pectinases, the GaaR gene containing the W361R mutation was introduced via a CRISPR/Cas9 approach into the endogenous gaaR locus in SO1.1, a strain carrying The expression of pgaX was previously shown to be induced only in the presence of an inducing carbon source, such as GA . Radial growth assays on plates containing acetamide showed that PpgaX-amdS gaaR W361R (JN130.4) and PpgaX-amdS ∆creA gaaR W361R (JN129.1) can grow on glucose or D-fructose, while their parental strains cannot . This indicates that GaaR W361R can activate the expression of amdS via the pgaX promoter in an inducer-independent way, confirming the results obtained by PGA plate assays.
The expression of pgaX is known to be repressed strongly by glucose and mildly by D-fructose in a CreA-dependent way [bib_ref] Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic..., Alazi [/bib_ref] [bib_ref] The interaction of induction and repression mechanisms in the regulation of galacturonic..., Niu [/bib_ref]. These previous results were confirmed as shown in , where growth of the promoter-reporter strains on plates containing acetamide and GA decreased when glucose was added to the growth media, and a short delay in growth and sporulation was observed when fructose was added as a repressing sugar.
The repressing effect of glucose or fructose was lost upon creA deletion, indicating that CreA is required for the repressing effect of glucose and fructose. To examine whether the CreA-mediated car-
## | subcellular localization of gaar-egfp and gaar w361r -egfp
We previously showed that eGFP-gaaR (EA19.2), a strain expressing the N-terminally eGFP-tagged gaaR, exhibits slightly reduced growth on GA compared to the reference strain [bib_ref] Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic..., Alazi [/bib_ref] indicating that N-terminal eGFP-tagging might affect the function of GaaR. To check whether C-terminal eGFP-tagging affects GaaR functionality, strains expressing C-terminally eGFP-tagged GaaR, with or without a linker between GaaR and eGFP, were created in a ∆gaaR background. While ∆gaaR (JN35.1) could not grow on GA, growth on GA was restored in both gaaR-eGFP (EA29.14) and gaaR-(GA) 4 -eGFP (EA30.6) . This indicates that C-terminal eGFP-tagging does not affect the function of GaaR, independently of the presence of a linker between GaaR and eGFP.
To analyse the subcellular localization of GaaR-eGFP and GaaR W361 -eGFP, strains that express C-terminally eGFP-tagged gaaR or gaaR W361R from the endogenous gaaR locus were created, and integration of the PgaaR-gaaR-eGFP-TgaaR or PgaaR-gaaR W361R -eGFP-TgaaR constructs into the endogenous gaaR locus was verified by Southern blot analysis . Growth of gaaR-eGFP (EA31.1) and gaaR W361R -eGFP (EA32.1) was complemented on GA and GAcontaining carbon sources , confirming previous results that GaaR-eGFP is functional under inducing conditions. The ability of gaaR-eGFP (EA36.1) and gaaR W361R -eGFP (EA39.1) to constitutively produce pectinases was assessed via a PGA plate assay and compared to that of gaaR W361R (EA34.1) . No polygalacturonase activity was observed in the culture supernatant of the reference (MA234.1) strain or gaaR-eGFP (EA36.1) after growth in D-fructose, indicating that C-terminal eGFP-tagging does not affect the inactive state of GaaR under non-inducing conditions, similar to N-terminal eGFP-tagging [bib_ref] Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic..., Alazi [/bib_ref]. While the gaaR W361R (EA34.1) strain expressing the constitutively active gaaR W361R produced pectinases in D-fructose, the gaaR W361R -eGFP (EA39.1) strain expressing gaaR W361R -eGFP did not . This indicates that GFP-tagging of the C-terminus of GaaR W361R interferes with constitutive function of the protein.
The subcellular localization of the C-terminally eGFP-tagged GaaR and GaaR W361R in gaaR-eGFP (EA36.1) and gaaR W361R -eGFP (EA39.1) grown under inducing (i.e., on 2-keto-3-deoxy-L-galactonate) or noninducing (i.e., on D-fructose) conditions was analyzed using confocal laser scanning microscopy . Beforehand, the potency of 2-keto-3-deoxy-L-galactonate, the physiological inducer , to induce the expression of the GA-responsive genes was tested using the promoter-reporter strain PpgaX-amdS ∆creA (JN29.2) and was compared to that of GA . Addition of 100 nM 2-keto-3-deoxy-L-galactonate to the growth media resulted in a major increase in growth, whereas addition of a 100 nM GA was not sufficient to induce the growth of PpgaX-amdS ∆creA (JN29.2), indicating that 2-keto-3-deoxy-L-galactonate is able to induce pgaX expression and that 2-keto-3-deoxy-L-galactonate is more potent than GA in inducing the expression.
We previously showed that the partially functional N-terminally eGFP-tagged GaaR in eGFP-gaaR (EA19.2) is localized mainly in the nucleus under both inducing (i.e., on GA) and non-inducing (i.e., on D-fructose) conditions [bib_ref] Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic..., Alazi [/bib_ref]. Here, we repeated this experiment using 2-keto-3-deoxy-L-galactonate as an inducer instead of GA and confirmed a visual signal of eGFP-GaaR in the nucleus after induction by 2-keto-3-deoxy-L-galactonate or on the non-inducing carbon source D-fructose . C-terminally eGFP-tagged GaaR and GaaR W361R were also visualized mostly in the nucleus after growth in 2-keto-3-deoxy-L-galactonate. Surprisingly, this was not observed after growth in D-fructose. This result indicates that on D-fructose (under non-inducing and mildly repressing F I G U R E 3 Nuclear and cytoplasmic fluorescence intensities of the eGFP-tagged GaaR and GaaR W361R proteins. The eGFP-gaaR (EA19.2), gaaR-eGFP (EA36.1) and gaaR W361R -eGFP (EA39.1) strains were grown in MM containing 10 mM D-fructose or 2-keto-3-deoxy-L-galactonate for 21 hr. Example micrographs representing each condition are shown. Scale bar: 10 μm conditions) nuclear accumulation of C-terminally eGFP-tagged GaaR is restrained and suggests that the C-terminal tagging of GaaR with eGFP affects is localization under non-inducing conditions. The inability of the GaaR-eGFP protein to localize in the nucleus under non-inducing conditions is most likely also the reason why the GaaR W361R -eGFP protein does not exert its constitutive activity.
## | d iscuss i on
We previously isolated via a forward genetic screen A. niger strains displaying constitutive expression of pectinases [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref].
The majority of the mutants in the collection (60 out of 64 strains) was found to contain mutations in GaaX and are probably all loss-offunction mutations as deletion of gaaX leads to the same phenotype [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref]. From the four mutants that did not carry mutations in gaaX, one mutant was found to possess a missense mutation leading to an amino acid change from tryptophan to arginine at position 361 in GaaR. The nature of the mutations in the three UV mutants with no mutations in gaaX or gaaR remains to be discovered. Introducing the GaaR W361R mutation in the wild-type genetic background and subsequent phenotypic analyses allowed us to conclude that this mutation results in constitutive activation of GaaR and therefore constitutive production of pectinases under non-inducing conditions.
To our knowledge, XlnR/Xyr1 is the only transcription factor involved in plant biomass degradation in which missense mutations leading to constitutive expression of enzymes are described.
The two mutations are the XlnR V756F mutation in A. niger, and the Xyr1 A824V mutation in Trichoderma reesei and orthologous mutations in N. crassa and Penicilium oxalicum, resulting in constitutive production of xylanases [bib_ref] Mutation of the Xylanase regulator 1 causes a glucose blind hydrolase expressing..., Derntl [/bib_ref] [bib_ref] Combining manipulation of transcription factors and overexpression of the target genes to..., Gao [/bib_ref] [bib_ref] Functional analysis of the transcriptional activator XlnR from Aspergillus niger, Hasper [/bib_ref]. In protein sequence alignment, these two mutations are only three amino acids apart from each other . Whereas the GaaR W361R mutation is located roughly half way of the 740 amino acid-long GaaR protein, the XlnR V756F mutation is located closer to the C-terminal in the 875 amino acid-long XlnR protein. The GaaR W361R mutation is predicted to be in the fungal-specific transcription factor domain (cd12148) which comprises amino acids 139-518 in GaaR. The cd12148 domain comprises amino acids 341-653 in A. niger XlnR and 368-717 in T. reesei Xyr1 (NCBI's CDD [bib_ref] CDD: NCBI's conserved domain database, Marchler-Bauer [/bib_ref] , and the constitutive mutations in XlnR/Xyr1 are therefore outside of the predicted fungal-specific transcription factor domain in the Cterminal parts of XlnR and Xyr1, which has been suggested to be the activation domain. The XlnR/Xyr1 constitutive mutations are not located in a defined protein domain family, but the C-terminal parts of XlnR and Xyr1, including the amino acids V756 and the A824, respectively, have been suggested to be the activator domain [bib_ref] Mutation of the Xylanase regulator 1 causes a glucose blind hydrolase expressing..., Derntl [/bib_ref] [bib_ref] Functional analysis of the transcriptional activator XlnR from Aspergillus niger, Hasper [/bib_ref]. Alignment of protein sequences of XlnR and GaaR from A. niger, A nidulans, Aspergillus fumigatus and A. oryzae and Xyr1 of T. reesei using Clustal omega [bib_ref] Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega, Sievers [/bib_ref] showed low sequence similarity in the amino acid residues surrounding the GaaR W361 residue between GaaR and XlnR/Xyr1 . Whereas the W361 residue is fully conserved in GaaR in all four Aspergilli, only A. niger, A. oryzae, and T. reesei had a W residue positioned at the aligned protein sequence of XlnR/Xyr1 .
Of note is that sequence similarity surrounding this region of XlnR/ Xyr1 is weak, making it possible that this conservation has no biological relevance. To test its biological relevance, it will be of interest to examine whether mutations in XlnR of A. niger at position W639 would also result in a constitutive phenotype. Protein sequence alignment revealed that the V756 and A824 residues are fully conserved in the XlnR/Xyr1 proteins of the four Aspergilli and T. reesei, but not strictly conserved between XlnR/Xyr1 and GaaR .
Because of the low level of conservation between the regions and amino acids that result in constitutive activation of the respective transcription factors, it is reasonable to suggest that the mechanisms leading to the activation of GaaR and XlnR/Xyr1 are different. We identified in the A. niger genome four transcription factor/repressor modules [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref]. For two modules, we know that they are involved in either GA or quinic acid utilization [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref]. Analysis of the knockouts for the two remaining genes encoding the repressors, and subsequent phenotypic and transcriptomic analysis has shown that the two repressors are not involved in the expression of genes encoding xylanases . Therefore, the activity of XlnR is likely not controlled by a GaaX-like repressor protein.
The activity of GaaR is proposed to be inhibited by GaaX under non-inducing conditions via protein-protein interactions [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref]. Pectinase genes are expressed under inducing conditions (i.e., in the presence of inducer), or under non-inducing conditions (i.e., in the absence of inducer) when gaaX is deleted or gaaR is overexpressed [bib_ref] Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic..., Alazi [/bib_ref] [bib_ref] An evolutionary conserved D-galacturonic acid metabolic pathway operates across filamentous fungi capable..., Martens-Uzunova [/bib_ref] [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref]. These findings indicate that GA-responsive gene expression only requires the presence of active GaaR relieved/escaped from GaaX inhibition but does not require the presence of the inducer. This suggests that the inducer binds to GaaX under inducing conditions resulting in a GaaX-unbound and active form of GaaR.
Therefore, we propose that W361R in GaaR disrupts the interaction between GaaX and GaaR under non-inducing conditions. Both PpgaX-amdS gaaR W361R and PpgaX-amdS ∆gaaX mutants were found to produce pectinases under non-inducing conditions . The qualitative phenotypic similarity of these strains resulting from the W361R mutation in GaaR or the absence of GaaX, respectively, suggests that the GaaR-GaaX interaction is disturbed in the GaaR W361R mutant. In future experiments, the proposed proteinprotein interaction of GaaR and GaaX and mutant variants thereof will be studied further in detail. Initial attempts to express GaaR and GaaX in E. coli to perform interaction studies have been unsuccessful and will be subject for further research. In this respect, it would also be of interest to isolate and identify additional mutations in GaaR that lead to constitutive expression of pectinases to identify regions in GaaR that are necessary for the proposed interaction with GaaX.
GaaX of A. niger, QutR of A. nidulans, and qa-1S of N. crassa are all multidomain repressor proteins with sequence similarity to each other and to the three C-terminal domains of AROM, a large pentafunctional protein involved in the shikimate pathway. This suggests that these repressors share a common evolutionary origin. The last three domains of the AROM protein encode the shikimate kinase, 3-dehydroquinate dehydratase, and shikimate dehydrogenase enzymes [bib_ref] The QUTA activator and QUTR repressor proteins of Aspergillus nidulans interact to..., Lamb [/bib_ref]. The sequence similarity of the repressor domains to AROM is low (between 20% and 30% identity), suggesting that the enzymatic functions that are present in AROM are lost in the repressors. Loss-of-function mutations (nonsense and missense mutations) were found throughout all three domains of GaaX (shikimate kinase, 3-dehydroquinate dehydratase, and shikimate dehydrogenase domains) . The 22 missense mutations could especially be valuable for structure/function relationships to predict which domains exert different function as they might affect the proposed protein-protein interaction between GaaX and GaaR. It is however difficult to predict whether a missense mutation affects the overall secondary/tertiary structure of the protein or whether a mutation affects the interaction without affecting its structure.
We identified a hot spot for mutations in the region 190-194 in which four mutants were found carrying mutations in three amino acid positions. This region could either be important for GaaR-GaaX interaction or could be important for the structure of GaaX.
## Alignment of protein sequences homologous to gaax present in 18
Pezizomycetes species using Clustal omega [bib_ref] Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega, Sievers [/bib_ref] revealed that these four mutations were found in three residues that are highly conserved . Several other missense mutations were found in conserved residues and are indicated in and S7. Relatively more missense mutations were found in the shikimate dehydrogenase domain (one mutation per 24 amino acids) compared to the shikimate kinase (one mutation per 76 amino acids), or 3-dehydroquinate dehydratase domains (one mutation per 37 amino acids), indicating that the shikimate dehydrogenase domain might be more important in relation to binding to GaaR than the other domains.
In N. crassa, dehydroquinic acid is suggested to be the physiological inducer of the quinic acid catabolic pathway genes, as mutants that accumulate or cannot produce dehydroquinic acid showed induced or diminished expression of these genes, respectively [bib_ref] The occurrence of two dehydroquinases in Neurospora crassa, one constitutive and one..., Giles [/bib_ref] [bib_ref] The qa repressor gene of Neurospora crassa: Wild-type and mutant nucleotide sequences, Huiet [/bib_ref]. [bib_ref] The pre-chorismate (shikimate) and quinate pathways in filamentous fungi: Theoretical and practical..., Hawkins [/bib_ref] have proposed that the QutR domain homologous to the AROM 3-dehydroquinate dehydratase can recognize and bind to dehydroquinic acid produced from quinic acid, but does not react on it enzymatically in A. nidulans. For A. niger, accumulation of 2-keto-3-deoxy-L-galactonate in ΔgaaC results in hyper-induction of GA-responsive genes and the inducer 2-keto-3-deoxy-L-galactonate is likely to interact with the GaaX repressor protein . The GA catabolic pathway enzyme GaaC is an aldolase and converts 2-keto-3-deoxy-L-galactonate to pyruvate and L-glyceraldehyde.
Both the aldolase domain of GaaC and the 3-dehydroquinate dehydratase domain of GaaX belong to the same Aldolase Class I_superfamily (NCBI's CDD [bib_ref] CDD: NCBI's conserved domain database, Marchler-Bauer [/bib_ref]. These findings suggest that 2-keto-3-deoxy-L-galactonate could bind to the GaaX domain homologous to the AROM 3-dehydroquinate dehydratase. However, direct protein-metabolite interaction studies are required to determine which part (or parts) of the repressor protein interacts with the inducer.
N-terminally eGFP-tagged GaaR was previously shown to mainly localize in the nucleus under both inducing and noninducing conditions but was unable to fully complement the growth of ∆gaaR when grown on inducing carbon sources [bib_ref] Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic..., Alazi [/bib_ref]. C-terminally eGFP-tagged GaaR/GaaR W361R accumulated in the nucleus under inducing conditions and complemented the growth of ∆gaaR when grown on inducing carbon sources.
Based on these observations, we conclude that C-terminally eGFP-tagged GaaR/GaaR W361R is functional under inducing conditions. However, prominent nuclear eGFP-fluorescence signal was not observed in the strains producing the C-terminally eGFPtagged GaaR/GaaR W361R under non-inducing conditions. This result indicates that C-terminal eGFP-tagging restrained nuclear accumulation of GaaR/GaaR W361R under non-inducing conditions, explaining the inability of GaaR W361R -eGFP to constitutively activate the production of pectinases, as GaaR W361R can. In B. cinerea, expression of the C-terminally GFP-tagged BcgaaR via the strong oliC promoter resulted in higher nuclear GFP fluorescence intensity under both inducing and non-inducing conditions [bib_ref] A novel Zn 2 Cys 6 transcription factor BcGaaR regulates D-galacturonic acid..., Zhang [/bib_ref]. This indicates that nuclear accumulation of overexpressed and wild-type BcGaaR is not restrained by C-terminal GFP-tagging under non-inducing conditions, unlike the GaaR-eGFP/GaaR W361 -eGFP expressed from its endogenous promoter.
Constitutive production of plant biomass degrading enzymes like pectinases is interesting for the industrial production of these enzymes because it allows the production independently of the feedstock. By understanding the transcriptional regulatory mechanism of pectinases, several approaches have been conducted leading to inducer independent and abundant production of pectinases, such as deletion of GaaX [bib_ref] An evolutionarily conserved transcriptional activatorrepressor module controls expression of genes for D-galacturonic..., Niu [/bib_ref] , overexpression of GaaR from a constitutive promoter [bib_ref] Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic..., Alazi [/bib_ref] , or accumulation of the inducer . Here, we show that a mutation in GaaR can also lead to constitutive production of pectinases. Combination of these approaches can be considered to further boost pectinase production in A. niger. The conservation of GaaR and GaaX in other fungi also allows improved pectinase production in other industrially important filamentous fungi.
## Ack n owled g m ents
EA and JN were supported by grants from BE-Basic (Flagship 10) and the China Scholarship Council, respectively. This work was in part supported by the Natural Sciences and Engineering Research Council of Canada Strategic Industrial Biocatalysis Network.
[fig] F: I G U R E 2 Effect of the W361R mutation in gaaR on the expression of pectinase genes. (a-c) PGA plate assay. Strains were grown in MM containing 50 mM D-fructose for 36 hr, and culture supernatants were spotted PGA plates. Enzymatic activities in the supernatants from duplicate cultures are shown: (a) the reference (MA234.1), PpgaX-amdS ∆creA gaaR W361R-UV (JN103.1), gaaR W361R (EA34.1), PpgaX-amdS ∆gaaX (JN123.1); (b) PpgaX-amdS (JC1.5), PpgaX-amdS ∆creA (JN29.2), PpgaX-amdS gaaR W361R (JN130.4), PpgaX-amdS ∆creA gaaR W361R (JN129.1); and (c) reference (MA234.1), gaaR W361R (EA34.1), gaaR-eGP (EA36.1), and gaaR W361R -eGP (EA39.1). (d) Growth phenotype of the PpgaX-amdS (JC1.5), PpgaX-amdS ∆creA (JN29.2), PpgaX-amdS gaaR W361R (JN130.4), and PpgaX-amdS ∆creA gaaR W361R (JN129.1) strains after 7 days at 30°C on solid MM containing no carbon source (−); 50 mM glucose, D-fructose or GA as the sole carbon source; or 50 mM glucose or D-fructose together with 50 mM GA. All plates contain 10 mM acetamide as the sole nitrogen source ΔgaaR::AOpyrG. Southern blot analysis indicated that the PgaaR-gaaR W361R -TgaaR construct was successfully integrated into the endogenous gaaR locus in the genome of strain SO2.1 (igure S3), thereby replacing the A. oryzae pyrG marker which was used for deleting gaaR. Growth of SO2.1 (gaaR W361R ) was analyzed on different monomeric and polymeric carbon sources (igure S2). Strain SO1.1 (ΔgaaR) displayed strongly impaired growth on GA, PGA, and AP, as shown previously(Alazi et al., 2016). Introduction of gaaR W361R gene (strain SO2.1) resulted in a restoration of growth on GA-containing carbon sources, indicating the presence of a functional GaaR. The presence of the gaaR W361R allele did not affect growth on any other carbon sources tested. The pectinase production capacity of gaaR W361R (EA34.1), was assessed via PGA plate assay (igure 2a). Strains were grown in liquid medium containing D-fructose as a non-inducing carbon source, and the culture supernatants were spotted on PGA plates. Comparison of hydrolysis zones on PGA plates showed that gaaR W361R (EA34.1) constitutively produces pectinases similar to the UV mutant PpgaX-amdS ∆creA gaaR W361R-UV (JN103.1) (igure 2a). This indicates that the W361R mutation in gaaR, and no other additional mutation(s) induced by UV mutagenesis in PpgaX-amdS ∆creA gaaR W361R-UV (JN103.1), is responsible for the constitutive production of pectinases. The effect of the W361R mutation in gaaR on the expression of pectinase genes was also analyzed using the promoter-reporter strains expressing the amdS gene via the pgaX promoter. The gaaR W361R gene was transformed into the PpgaX-amdS (JC1.5) and PpgaX-amdS ∆creA (JN29.2) strains. Transformants were selected on medium containing a non-inducing carbon source and acetamide as the nitrogen source, and only transformants in which the gaaR W361R was integrated were expected to grow on the transformation plate because of constitutive activation of the PpgaX-amdS reporter. Southern blot analysis and sequencing of the gaaR locus showed that the endogenous gaaR gene was replaced with gaaR W361R in the resulting strains PpgaX-amdS gaaR W361R (JN130.4) and PpgaX-amdS ∆creA gaaR W361R (JN129.1), respectively (igure S3). Growth of PpgaX-amdS gaaR W361R (JN130.4) and PpgaX-amdS ∆creA gaaR W361R (JN129.1) was similar to the growth of their parental strains on MM containing glucose, D-fructose, GA, PGA, or AP (igure S2). The PGA plate assay showed that the parental strains PpgaX-amdS (JC1.5) and PpgaX-amdS ∆creA (JN29.2) did not produce any polygalacturonases in D-fructose, while PpgaX-amdS gaaR W361R (JN130.4) and PpgaX-amdS ∆creA gaaR W361R (JN129.1) showed polygalacturonase activity in their culture supernatants (igure 2b). The decreased polygalacturonase activity in the culture supernatant of PpgaX-amdS ∆creA gaaR W361R (JN129.1) compared to that of PpgaX-amdS gaaR W361R (JN130.4) might be caused by the growth defect due to creA deletion. [/fig]
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10.1038/s41598-021-03908-2
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CCBY
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8742006
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s2orc_pubmed_articles
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Tunable circular dichroism through absorption in coupled optical modes of twisted triskelia nanostructures
Supplementary Information Tunable Circular Dichroism through Absorption in Coupled Optical Modes of Twisted Triskelia NanostructuresSupplementary Information Tunable Circular Dichroism through Absorption in Coupled Optical Modes of Twisted Triskelia Nanostructures
Figure S1. Absorption, scattering and extinction cross-sections under LCP (blue solid line) and RCP (red solid line) light for a double triskelia system forming an anticlockwise in-plane angle of 0°, 10°, 60° and 90° and at an edge-to-edge distance = 30 nm (seeFig. 1(b)). . Near-field distributions of the square modulus of the electric field normalized to that of the incident light under RCP and LCP illumination for in-plane angles of 0° and 90°, at a wavelength of 1100 nm. . Near-field distributions of the square modulus of the electric field normalized to that of the incident light under RCP and LCP illumination for in-plane angles of 30° and 60°, at a wavelength of 1000 nm. . Near-field distributions of the square modulus of the electric field normalized to that of the incident light under RCP and LCP illumination for in-plane angles of 30° and 60°, at a wavelength of 760 nm. . Near-field distributions of the square modulus of the electric field normalized to that of the incident light under RCP and LCP illumination for in-plane angles of 30° and 60°, at a wavelength of 710 nm.
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10.3390/foods12112246
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10253062
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Textural and Consumer-Aided Characterisation and Acceptability of a Hybrid Meat and Plant-Based Burger Patty
The hamburger has been targeted for substitution by numerous plant-based alternatives. However, many consumers find the taste of these alternatives lacking, and thus we proposed a hybrid meat and plant-based burger as a more acceptable alternative for these consumers. The burger was made from 50% meat (beef and pork, 4:1) and 50% plant-based ingredients, including texturised legume protein. Texture and sensory properties were evaluated instrumentally and through a consumer survey (n = 381) using the check-all-that-apply (CATA) method. Expressible moisture measurements indicated a significantly juicier eating experience for the hybrid compared to a beef burger (33.5% vs. 22.3%), which was supported by the CATA survey where "juicy" was used more to describe the hybrid than the beef burger (53% vs. 12%). Texture profile analysis showed the hybrid burger was significantly softer (Young's modulus: 332 ± 34 vs. 679 ± 80 kPa) and less cohesive than a beef burger (Ratio 0.48 ± 0.02 vs. 0.58 ± 0.01). Despite having different textural and CATA profiles, overall liking of the hybrid burger and a beef burger were not significantly different. Penalty analysis indicated that "meat flavour", "juiciness", "spiciness" and "saltiness" were the most important attributes for a burger. In conclusion, the hybrid burger had different attributes and was described with different CATA terms than a beef burger but had the same overall acceptability.
# Introduction
World population has now surpassed eight billion, and at the same time global meat consumption has steadily increased to a global average of more than 42 kg per capita per year in 2020. In addition, even though the consumption of meat per capita is beginning to show signs of plateauing in high income countries, there is no indication of the increase in overall global consumption stagnating during the coming years. It is estimated that between a fourth and a third of global anthropogenic greenhouse gas (GHG) emissions are due to food systems. Furthermore, it is well established that intensive animal farming for meat production is associated with substantial GHG emissions [bib_ref] Reducing Food's Environmental Impacts through Producers and Consumers, Poore [/bib_ref] [bib_ref] Food Systems Are Responsible for a Third of Global Anthropogenic GHG Emissions, Crippa [/bib_ref] , with ruminants being the largest contributor, predominantly due to the production of methane from intestinal fermentation of ingested biomass. Therefore, due to climate concerns, policymakers in many parts of the world are pushing for a reduction in consumption of animal derived foods, and beef in particular. The aim is to incite consumers to move towards a more plantforward diet for a reduction of its carbon footprint. The recently updated Danish dietary guidelines were developed to also take into account the climate impact of food systems, an approach that is seen in several other European countries as well, i.e., sustainability is specifically mentioned in the dietary guidelines of countries including Italy, France, Germany, the Netherlands and the United Kingdom.
Reducing meat in the diet without replacing it with another protein-rich food will reduce the consumer's overall protein intake. Legumes are often highlighted as a suitable replacement for meat because most legume seeds have a comparatively high protein content. They do, however, have a less optimal amino acid profile than animal protein [bib_ref] Digestible Indispensable Amino Acid Score (DIAAS) Is Greater in Animal-Based Burgers than..., Fanelli [/bib_ref] and USA), tomato paste, milled extruded pea starch (Crispy Food, Gørlev, Denmark), dried porcini mushroom, salt, monosodium glutamate (Ajinomoto Foods Europe, Paris, France), yeast flakes (Nutty Vegan, Holstebro, Denmark), garlic powder and black pepper), and mixed thoroughly in a stand mixer until a visible gluten network had formed. Bacon (Danish Crown, Randers, Denmark) was added to the mixture which was then processed in a meat mincer fitted with a 6 mm hole plate. The resulting mince was mixed with minced beef and finely chopped frozen coconut oil-in-water emulsion, which was made as follows. Methylcellulose (Special Ingredients, Chesterfield, United Kingdom) (final concentration in emulsion 0.5% w/v) was mixed with one part water using an immersion blender, followed by slowly adding three parts melted coconut oil until a mayonnaise-like texture was achieved. The emulsion was then frozen and subsequently was finely chopped before use. The estimated nutritional composition of the hybrid burger patties is shown in Supplementary Table S1. Data for estimation of nutritional composition was acquired from ingredient and raw material specifications, as well as the official Danish food composition database. The final mixture was shaped into 100 g patties with a diameter of 8 cm and pan-fried to an internal temperature of 75 - C, as were reference patties of identical size and shape made from minced beef. Salt and ground black pepper were added to the reference patties at similar amounts as the hybrid burger patties immediately before frying. Afterwards, the burger patties were either analysed or frozen for later use in the consumer survey. Freezing of the patties for the survey was necessary for logistical reasons. Texture profile analysis: Circular samples were cut using a 30 mm diameter steel ring. These were then cut to a height of 19 mm. Texture profile analysis (TPA) was carried out on a TMS Pro texture analyser (Food Technology Corporation, Sterling, VA, USA), using a flat cylindrical probe with a diameter of 50 mm. The TPA settings were as follows: trigger force = 2 N, compression and return speed = 2 mm/s, strain = 60% of initial height, with no pause between compressions. The attributes calculated were as follows: Young's modulus = stress/strain (kPa); cohesiveness = ratio of work performed during second and first compression, respectively; springiness = ratio of sample height at second compression to initial height. Note, Young's modulus was chosen because it is independent of sample dimensions and therefore is more generalisable [bib_ref] Young's Modulus Estimation in Food Samples: Effect of Experimental Parameters, Sinha [/bib_ref].
Cooking loss: Hybrid and beef burger patties were pan-fried to 75 - C internal temperature. The cooking loss was calculated as the percentage of weight lost after cooking. Expressible moisture: Expressible moisture was assessed using the procedure described by Earl et al. [bib_ref] A Modification of a Method to Determine Expressible Moisture in Ground, Dark..., Earl [/bib_ref] with minor modifications. Briefly, approximately 3 g pieces of cooked burger patty were placed in a small cup fashioned from one inner layer of Whatman no. 50, 70 mm and three outer layers of Whatman no. 3, 50 mm filter papers. These were placed inside a 50 mL centrifuge tube and centrifuged at 15,000× g and room temperature for 15 min. Expressible moisture was taken as the percentage of weight lost after centrifugation of the cooked patty.
Consumer survey: We conducted a consumer survey at a foodservice expo held over three days in March 2022 in Herning, Denmark. The survey was structured as a CATA questionnaire including a nine-point hedonic liking scale for each product anchored at "dislike very much" and "like very much" and with five meaning "neither like nor dislike", as described by Ares and Jaeger [bib_ref] Check-All-That-Apply (CATA) Questions with Consumers in Practice: Experimental Considerations and Impact on..., Ares [/bib_ref]. The CATA question included 20 descriptive terms generated by a focus group of students from Business Academy Aarhus' Food Technology and Application programme (n = 35; M = 16; F = 19). The terms, which are shown in [fig_ref] Table 2: Consumer-generated sensory terms for check-all-that-apply survey of hybrid vs [/fig_ref] , were printed in randomised order on the survey questionnaire to minimise primacy bias [bib_ref] Randomization of CATA Attributes: Should Attribute Lists Be Allocated to Assessors or..., Meyners [/bib_ref]. The questionnaire also prompted participants to describe their perceived ideal burger patty using the same 20 CATA terms which permitted identification of important attributes through penalty analysis [bib_ref] Check-All-That-Apply (CATA) Questions with Consumers in Practice: Experimental Considerations and Impact on..., Ares [/bib_ref]. In addition to the CATA and liking questions, the participants were asked to state age and sex, and whether or not they were a regular consumer of plant-based meat alternatives. Questionnaires from 381 assessors were filled out correctly with regard to the CATA and liking questions and were therefore included in the analysis. The assessors were recruited among visitors to the expo and were asked to evaluate the hybrid and beef burgers and fill out the questionnaire at a table behind the expo booth under ambient conditions. The samples were served single-blinded labelled with a three-digit code and in randomised order on white paper plates after reheating to 75 - C over a water bath in a standard household oven. Assessors were instructed to taste and check terms for one burger patty at a time and were given tap water for palate rinsing between samples. The terms for the "ideal" burger patty were checked at the end. Climate impact (CO 2 -eq): The climate impact of the beef and hybrid burgers were estimated in terms of CO 2 -equivalents (CO 2 -eq) per burger in a use case exemplified here by a standard cheeseburger. The CO 2 -eq for the different ingredients were obtained from The Big Climate Database, which is the official Danish database for climate impact of food products. In the cases where a specific entry for an ingredient was not available in the database, a similar product was chosen instead, e.g., wheat flour substituted for gluten in the hybrid burger patty, and Danbo cheese 45+ substituted for burger cheese.
Data analysis: The results of the texture profile analysis, cooking loss and expressible moisture were analysed using two-tailed student's t-test. Hedonic liking of the test burgers was analysed using the paired Wilcoxon signed rank test, and differential use of CATA terms for hybrid, beef and ideal burgers was analysed using Cochran's Q test.
Correspondence analysis was carried out on the contingency table of CATA term use from the questionnaire, and a biplot was created to assess relationships between terms and samples.
Hierarchal clustering was performed using the CLUSCATA algorithm in XLSTAT-Sensory [bib_ref] A New Approach for the Analysis of Data and the Clustering of..., Llobell [/bib_ref]. After clustering, all assessors with incomplete demographic data were excluded. Differential liking of the burgers in the generated clusters was tested using two-way non-parametric ANOVA with interaction on Aligned Rank Transformed data according to Wobbrock et al. [bib_ref] The Aligned Rank Transform for Nonparametric Factorial Analyses Using Only Anova Procedures, Wobbrock [/bib_ref]. The factors were "cluster" and "type of burger" and assessor was included as random effect. Potential differences in cluster demographic composition were assessed using X 2 -test on counts.
Penalty analysis was carried on the CATA and liking data from the questionnaire to assess the importance of attributes for consumer acceptability of the burgers.
Differences were considered as significant at the 0.05 level, and all analyses were carried out in R version 4.3.1and XLSTAT Sensory 2022.2.1 (Addinsoft, Paris, France).
# Results
## Texture attributes
In addition to taste, the texture and mouthfeel of food has a profound impact on consumer acceptability. The texture of soft solid foods can be measured instrumentally using TPA, which involves a two-cycle compression of uniformly cut samples of the food under investigation. A number of attributes can be calculated from the force-time and stress-strain curves, each of which approximate different aspects of the sensory experience of eating the food [bib_ref] Texture Is a Sensory Property, Szczesniak [/bib_ref] [bib_ref] On the Texture Profile Analysis Test, Trinh [/bib_ref]. The results from the TPA are shown in [fig_ref] Table 3: Physical attributes related to texture and sensory perception [/fig_ref]. The Young's modulus of the beef burger was two-fold higher than that of the hybrid, indicating that the beef burger would possibly be experienced as somewhat firmer than the hybrid. The beef burger's cohesiveness was twenty percent higher than the hybrid's, suggesting that the hybrid burger will require less total energy input during mastication before swallowing. In contrast, the springiness was marginally higher for the hybrid burger; however, a difference of such limited magnitude will in all likelihood not be detectable by the consumer. Taken together, the hybrid burger will presumably be experienced as softer and easier to chew. Cooking loss measures the amount of moisture lost during frying. The hybrid burger lost markedly less moisture than the beef burger [fig_ref] Table 3: Physical attributes related to texture and sensory perception [/fig_ref] and, as expected, the subsequent amount of expressible moisture was correspondingly higher for the hybrid burger. This should translate to a juicier sensory experience when eating the hybrid burger.
## Climate impact
The amounts and estimated CO 2 -eq footprint for each ingredient in the regular cheeseburgers is shown in [fig_ref] Table 4: Climate impact of ingredients used in a regular cheeseburger [/fig_ref]. The CO 2 -eq footprint of the hybrid burger patty is less than half that of the beef patty. This is because not only is the amount of beef in the hybrid patty reduced by 50%, a further 8.5% is exchanged with bacon, which has a lower CO 2 -eq footprint than beef (4.8 vs. 33 kg CO 2 -eq/kg). The CO 2 -eq footprints for each cheeseburger sums to 1.80 and 3.60 kg CO 2 -eq for the hybrid and beef burger, respectively. Thus, re-placing the beef patty in a cheeseburger with the hybrid patty halves the overall GHG emissions associated with the burger.
## Consumer survey, evaluation of hybrid and beef burgers
The assessors in the survey were recruited at an expo for foodservice providers in Denmark. After exclusion of incomplete or incorrectly filled out forms, 381 assessors were included in the analysis. Assessor demographics are shown in [fig_ref] Figure 1: Age and gender distribution of assessors in the consumer survey [/fig_ref].
## Overall liking
On a discrete nine-point scale anchored at "dislike very much" and "like very much" the consumers' scores for the two burgers were not significantly different, as assessed by the Wilcoxon paired signed rank test (p = 0.32). The respective liking scores (±s.e.) were 5.4 (±0.1) for the beef and 5.3 (±0.1) for the hybrid.
## Cata term usage
The assessors were asked to check any of the 20 CATA terms they felt described the burger they were tasting at a given moment. The frequency of term usage is shown in [fig_ref] Table 5: Frequency [/fig_ref]. Cochran's Q test was employed to determine which terms' use was significantly different for the meat and hybrid burger, which was most of the terms, except "Metallic", "Pepper", "Meat colour", and "Brown surface". Thus, there is independence between rows and columns, indicating that the assessors experienced them as having different sensory characteristics. For six of the twenty attributes the difference in usage frequency between the meat and hybrid burger was more than 3.5-fold. These were "Soft", "Dry", "Juicy", "Tough", "Pink", and "Off-flavour". The hybrid burger was perceived as softer, less dry and juicier, more tender, and pinker than the beef burger but was also perceived by 29% of the assessors as having an off-flavour, in contrast with just 3% indicating this
## Overall liking
On a discrete nine-point scale anchored at "dislike very much" and "like very much" the consumers' scores for the two burgers were not significantly different, as assessed by the Wilcoxon paired signed rank test (p = 0.32). The respective liking scores (±s.e.) were 5.4 (±0.1) for the beef and 5.3 (±0.1) for the hybrid.
## Cata term usage
The assessors were asked to check any of the 20 CATA terms they felt described the burger they were tasting at a given moment. The frequency of term usage is shown in [fig_ref] Table 5: Frequency [/fig_ref]. Cochran's Q test was employed to determine which terms' use was significantly different for the meat and hybrid burger, which was most of the terms, except "Metallic", "Pepper", "Meat colour", and "Brown surface". Thus, there is independence between rows and columns, indicating that the assessors experienced them as having different sensory characteristics. For six of the twenty attributes the difference in usage frequency between the meat and hybrid burger was more than 3.5-fold. These were "Soft", "Dry", "Juicy", "Tough", "Pink", and "Off-flavour". The hybrid burger was perceived as softer, less dry and juicier, more tender, and pinker than the beef burger but was also perceived by 29% of the assessors as having an off-flavour, in contrast with just 3% indicating this for the beef burger. The relationship between the products being tested and the CATA terms was explored using correspondence analysis. A graphical representation is shown in [fig_ref] Figure 2: Biplot of rows [/fig_ref]. There is a clear separation of the two tested burgers as well as the ideal burger. Apparently, neither of the tested burgers represented an ideal burger to the assessors, which according to the biplot, is associated with a juicy, fat, spicy and salty taste and has a pink appearance with a crust. The hybrid burger was more associated with being soft and having off-flavour, whereas the beef burger was perceived as more firm, tough, well done and dry.
# Penalty analysis
Penalty analysis can be used to determine how much the overall liking of a product drops when the usage of a specific CATA term is different for a product relative to the "ideal". This provides a tool for research or for product development whereby it is possible to identify product attributes that are either detrimental to consumer acceptability, if present, or are required for optimal acceptability. An overview of the penalty analysis carried out for the hybrid and beef burgers tested in the present study is shown in [fig_ref] Figure 3: Mean drop in overall liking score for a burger when a CATA... [/fig_ref]. The graph shows the mean drop associated with certain terms and how large a proportion of assessors had checked this term differently for product vs. "ideal". The vertical line shows the cut-off at 20% of assessors, above which a given term is deemed relevant for further consideration. Thus, terms in the upper right corner of the graph are the most important for the product being tested. In this case "Meat flavour", "Spicy", "Juicy", "Salt", "Crust", and "Pink" can be considered must-have attributes, and conversely "Rubberlike" and "Dry" can be considered must-not-have attributes. The terms "Soft", "Dark surface", "Off-flavour", "Grainy", and "Tough" could be considered somewhat important for acceptability but should not be prioritised over those that are must-have and must-not-have. For the rest of the terms in [fig_ref] Figure 3: Mean drop in overall liking score for a burger when a CATA... [/fig_ref] either the proportion of assessors or the penalty is too small for them to be considered relevant.
Cochran's Q test for significance of difference between use of terms for each sample. Asterisks indicate statistically significant difference between samples: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, n.s. = p > 0.05.
The relationship between the products being tested and the CATA terms was explored using correspondence analysis. A graphical representation is shown in [fig_ref] Figure 2: Biplot of rows [/fig_ref]. There is a clear separation of the two tested burgers as well as the ideal burger. Apparently, neither of the tested burgers represented an ideal burger to the assessors, which according to the biplot, is associated with a juicy, fat, spicy and salty taste and has a pink appearance with a crust. The hybrid burger was more associated with being soft and having off-flavour, whereas the beef burger was perceived as more firm, tough, well done and dry. [fig_ref] Figure 2: Biplot of rows [/fig_ref]. Biplot of rows (terms) and columns (burgers, including ideal) from correspondence analysis of the CATA data. Only terms with significantly different usage are shown (p ≤ 0.05).
# Penalty analysis
Penalty analysis can be used to determine how much the overall liking of a product drops when the usage of a specific CATA term is different for a product relative to the "ideal". This provides a tool for research or for product development whereby it is possible to identify product attributes that are either detrimental to consumer acceptability, if present, or are required for optimal acceptability. An overview of the penalty analysis carried out for the hybrid and beef burgers tested in the present study is shown in [fig_ref] Figure 3: Mean drop in overall liking score for a burger when a CATA... [/fig_ref]. The graph shows the mean drop associated with certain terms and how large a proportion of assessors had checked this term differently for product vs. "ideal". The vertical line shows the cut-off at 20% of assessors, above which a given term is deemed relevant for further consideration. Thus, terms in the upper right corner of the graph are the most important for the product being tested. In this case "Meat flavour", "Spicy", "Juicy", "Salt", "Crust", and "Pink" can be considered must-have attributes, and conversely "Rubberlike" and "Dry" can be considered must-not-have attributes. The terms "Soft", "Dark surface", "Off-flavour", "Grainy", and "Tough" could be considered somewhat important for acceptability but should not be prioritised over those that are must-have and mustnot-have. For the rest of the terms in [fig_ref] Figure 3: Mean drop in overall liking score for a burger when a CATA... [/fig_ref] either the proportion of assessors or the penalty is too small for them to be considered relevant.
# Cluster analysis
Using the CLUSCATA hierarchal clustering algorithm, the assessors were assigned to clusters where within-cluster dissimilarity between assessors' CATA term usage was minimised. This resulted in two clusters and a K+1 cluster containing the assessors that did not fit either of the two obtained clusters. Of the original 381 assessors, after exclusion of assessors with incomplete demographic data, 92 were assigned to cluster 1 and 138 to cluster 2. The relationship between the burgers and the CATA term usage is visualised by correspondence analysis-generated biplots [fig_ref] Figure 4: Mean liking scores for the two burgers in two clusters [/fig_ref] ,C for clusters 1 and 2, respectively. Assessors in both clusters apparently associate the beef burger with "Well done", "Firm", "Tough", and "Dry", and an ideal burger with "Pepper", "Pink", "Salt", "Crust", and "Spicy". Conversely, the hybrid burger is more associated with "Rubberlike" and "Grainy" in cluster 1, where in cluster 2 the association with "Soft" and "Juicy" is strongest. Analysis of the liking scores [fig_ref] Figure 4: Mean liking scores for the two burgers in two clusters [/fig_ref] for the two clusters showed that assessors in cluster 2 scored both burgers higher, and preferred the hybrid over the beef burger. In contrast, the assessors in cluster 1 preferred the beef over the hybrid burger, although this difference was not significant. There was a non-significant trend (p = 0.08) for fewer women than men in cluster 1 and more women than men in cluster 2. The ratio of regular
# Cluster analysis
Using the CLUSCATA hierarchal clustering algorithm, the assessors were assigned to clusters where within-cluster dissimilarity between assessors' CATA term usage was minimised. This resulted in two clusters and a K+1 cluster containing the assessors that did not fit either of the two obtained clusters. Of the original 381 assessors, after exclusion of assessors with incomplete demographic data, 92 were assigned to cluster 1 and 138 to cluster 2. The relationship between the burgers and the CATA term usage is visualised by correspondence analysis-generated biplots [fig_ref] Figure 4: Mean liking scores for the two burgers in two clusters [/fig_ref] ,C for clusters 1 and 2, respectively. Assessors in both clusters apparently associate the beef burger with "Well done", "Firm", "Tough", and "Dry", and an ideal burger with "Pepper", "Pink", "Salt", "Crust", and "Spicy". Conversely, the hybrid burger is more associated with "Rubberlike" and "Grainy" in cluster 1, where in cluster 2 the association with "Soft" and "Juicy" is strongest. Analysis of the liking scores [fig_ref] Figure 4: Mean liking scores for the two burgers in two clusters [/fig_ref] for the two clusters showed that assessors in cluster 2 scored both burgers higher, and preferred the hybrid over the beef burger. In contrast, the assessors in cluster 1 preferred the beef over the hybrid burger, although this difference was not significant. There was a non-significant trend (p = 0.08) for fewer women than men in cluster 1 and more women than men in cluster 2. The ratio of regular plant-based eaters to non-eaters also tended to be higher in cluster 2 than in cluster 1; however, it was also not significant (p = 0.11). plant-based eaters to non-eaters also tended to be higher in cluster 2 than in cluster 1; however, it was also not significant (p = 0.11).
# Discussion
The current and growing demand for meat and other animal foods is unsustainable with regard to climate impact [bib_ref] Reducing Meat Consumption in Today's Consumer Society: Questioning the Citizen-Consumer Gap, De Bakker [/bib_ref] , with beef possibly being the largest single contributor, when factoring in the volumes consumed. This is also reflected in the EAT-Lancet commission recommendations of consuming just 14 g/day of beef, lamb or pork. Hamburgers are a popular beef-based food and are therefore an excellent target product category for introducing a reduction in beef consumption. Nonetheless, the beef in a burger patty has distinct technological and sensory characteristics that should be mimicked, such as texture, mouthfeel and meaty and fatty flavours. Replacing the burger patty with one that is plant-based and more climate friendly is not sensorially acceptable to many consumers [bib_ref] High-Moisture Extrusion: Meat Analogues, Osen [/bib_ref]. The use of legume protein can be associated with "grassy", "beany", bitter" and "astringent" off-flavours from volatile compounds [bib_ref] Nonmeat Protein Alternatives as Meat Extenders and Meat Analogs, Asgar [/bib_ref]. For example, a CATA-based study by Neville et al. [bib_ref] Consumer-orientated Development of Hybrid Beef Burger and Sausage Analogues, Neville [/bib_ref] compared hybrid burgers and sausages to fully plant-based and fully meat-based products and reported that "processed appearance" and "off-flavour" together with lack of "meat flavour" were important attributes for acceptability of burgers. In addition, some consumers may have an affective connection with meat, and therefore the idea of giving up meat in favour of a plant-based alternative is associated with negative emotions [bib_ref] Attached to Meat? (Un)Willingness and Intentions to Adopt a More Plant-Based Diet, Graça [/bib_ref]. Thus, they continue eating all-meat burgers. However, a discrete choice analysis survey with German and Belgian consumers indicated that meat-hybrid foods would be preferred over fully plant-based foods. The authors concluded that meat-hybrid foods could help facilitate the transition to a more plant forward diet.
The hybrid burger patty developed here had a nutritional composition comparable to a beef burger, albeit with a slightly lower fat but a higher carbohydrate content and some dietary fibre at 1 g per 100 g of burger patty. A study evaluated the effects of substituting TVP made from pea, sunflower, or pumpkin protein for 30% of the meat in pork meatballs on nutritional and climate impact characteristics. It was found that fibre and unsaturated fatty acid content was increased. The former was similar to the present study, and the latter was due to the use of canola oil. We used coconut oil as the source of plant-based fat. Coconut oil contains almost exclusively saturated fatty acids, and therefore the unsaturated fatty acid content in our hybrid burger is likely to be low, although it was not specifically analysed. Compared to preparation of a beef burger patty,
# Discussion
The current and growing demand for meat and other animal foods is unsustainable with regard to climate impact [bib_ref] Reducing Meat Consumption in Today's Consumer Society: Questioning the Citizen-Consumer Gap, De Bakker [/bib_ref] , with beef possibly being the largest single contributor, when factoring in the volumes consumed. This is also reflected in the EAT-Lancet commission recommendations of consuming just 14 g/day of beef, lamb or pork. Hamburgers are a popular beef-based food and are therefore an excellent target product category for introducing a reduction in beef consumption. Nonetheless, the beef in a burger patty has distinct technological and sensory characteristics that should be mimicked, such as texture, mouthfeel and meaty and fatty flavours. Replacing the burger patty with one that is plant-based and more climate friendly is not sensorially acceptable to many consumers [bib_ref] High-Moisture Extrusion: Meat Analogues, Osen [/bib_ref]. The use of legume protein can be associated with "grassy", "beany", bitter" and "astringent" off-flavours from volatile compounds [bib_ref] Nonmeat Protein Alternatives as Meat Extenders and Meat Analogs, Asgar [/bib_ref]. For example, a CATAbased study by Neville et al. [bib_ref] Consumer-orientated Development of Hybrid Beef Burger and Sausage Analogues, Neville [/bib_ref] compared hybrid burgers and sausages to fully plant-based and fully meat-based products and reported that "processed appearance" and "off-flavour" together with lack of "meat flavour" were important attributes for acceptability of burgers. In addition, some consumers may have an affective connection with meat, and therefore the idea of giving up meat in favour of a plant-based alternative is associated with negative emotions [bib_ref] Attached to Meat? (Un)Willingness and Intentions to Adopt a More Plant-Based Diet, Graça [/bib_ref]. Thus, they continue eating all-meat burgers. However, a discrete choice analysis survey with German and Belgian consumers indicated that meat-hybrid foods would be preferred over fully plant-based foods. The authors concluded that meat-hybrid foods could help facilitate the transition to a more plant forward diet.
The hybrid burger patty developed here had a nutritional composition comparable to a beef burger, albeit with a slightly lower fat but a higher carbohydrate content and some dietary fibre at 1 g per 100 g of burger patty. A study evaluated the effects of substituting TVP made from pea, sunflower, or pumpkin protein for 30% of the meat in pork meatballs on nutritional and climate impact characteristics. It was found that fibre and unsaturated fatty acid content was increased. The former was similar to the present study, and the latter was due to the use of canola oil. We used coconut oil as the source of plant-based fat. Coconut oil contains almost exclusively saturated fatty acids, and therefore the unsaturated fatty acid content in our hybrid burger is likely to be low, although it was not specifically analysed. Compared to preparation of a beef burger patty, preparation of the hybrid burger patty requires several additional steps, as described in Section 2, which will make scale-up challenging. In the present lab-scale form of the procedure the coconut oil emulsion is prepared separately and requires freezing, and ingredients are mixed and minced separately. This makes scale-up unlikely, and thus the procedure for this proof-of-concept hybrid burger patty should be simplified to be industrially relevant.
The hybrid burger was evaluated both instrumentally and in a consumer survey. In order to imitate the texture of beef, TVP was combined with gluten as a binder and secondary textural element and with extruded pea starch as a water and fat binder. Similar texture properties to a beef patty were not achieved, as revealed by the TPA. However, thirty percent of assessors in the survey prefer a soft burger, as indicated by the frequency of "Soft" checked for the ideal burger; however, this will need to be within an acceptable range, and not too soft, as 21% checked "Firm" as a preferred attribute for an ideal burger. The penalty analysis also indicated that softness is a desired attribute. The moisture-related attributes of the burgers showed that the hybrid burger would provide a much juicier mouthfeel than the beef burger, which was also supported by more than half of the assessors checking "Juicy" for the hybrid burger. Juiciness is the most desired attribute, with an 82% checked frequency for an ideal burger. The improved juiciness of the hybrid burger is a result of the lower observed cooking loss, which translated to more moisture released during mastication, in line with the higher expressible moisture measurement. The underlying molecular mechanism is likely the lower content of connective tissue in the form of collagen in the hybrid burger, and possibly the water-holding properties of the added extruded pea starch. Loss of moisture during cooking is largely a result of collagen contraction due to heat [bib_ref] Modelling the Effect of Sarcomere Length on Collagen Thermal Shortening in Cooked..., Lepetit [/bib_ref] , and since the hybrid burger contains less meat and therefore less collagen, less moisture is expelled.
The CATA questionnaire indicated that the burgers were perceived as dissimilar, presumably owing to the textural and moisture-related differences discussed above. That taste and flavour related terms differed was perhaps not entirely surprising, as it is wellknown that legume proteins and volatile compounds can result in various off-taste and offflavours [bib_ref] Flavor Challenges in Extruded Plant-Based Meat Alternatives: A Review, Wang [/bib_ref]. The CATA demonstrated that the hybrid burger was not an indistinguishable replacement for a beef burger; however, it could be an acceptable one, as judged by the similar overall liking score. In the study by Neville et al. [bib_ref] Consumer-orientated Development of Hybrid Beef Burger and Sausage Analogues, Neville [/bib_ref] the liking scores for two different hybrid burgers were statistically the same as for a beef burger, as in the present study. Conversely, in their correspondence analysis the hybrid burgers were clustered together with the meat burger, and thus were associated with the same CATA terms, which was quite different in our analysis. However, this was likely caused by the inclusion of two fully vegetarian burger variants in their study, and the dissimilarity between their hybrid and vegetarian burgers was more pronounced than between their hybrid and meat burgers. In our correspondence analysis the different use of the CATA terms is clearly illustrated. To assess this further we performed a penalty analysis to learn which attributes should be prioritised when developing a burger patty. The penalty analysis corroborated the previous results that pointed to "Spicy", "Juicy", and "Salt" as important attributes, but also "Meat flavour", which incurred the largest penalty when absent. Based on this, and since the hybrid burger was already favourably evaluated in juiciness, effort should be put into enhancing spiciness, saltiness, and meat flavour to accommodate consumer preferences.
An interesting question to ask could be whether it is a specific type of consumer that embraces a product like the hybrid burger, and if so, what separates them from other consumers. In an attempt at answering this question we conducted a cluster analysis using the CLUSCATA hierarchal algorithm resulting in two separate clusters of assessors. The clustering is carried out solely on the use of the CATA terms in the survey, and therefore also provides some insight to the question of whether there is a connection between term usage and liking. Cluster 2 assessors liked both burgers more than cluster 1 assessors and preferred the hybrid over the beef burger. Cluster 2, in contrast to cluster 1, trended towards being composed of more women than men. Historically, eating meat has been associated more with masculinity than femininity, which may help to explain this observation. Furthermore, Bush and Clayton recently reported that there is a significant gender difference in attitudes toward climate change [bib_ref] Facing Change: Gender and Climate Change Attitudes Worldwide, Bush [/bib_ref] , which is more pronounced in high income countries. They showed a clear correlation between a country's GDP and the gap between women's and men's perceived seriousness of climate change, with women being more concerned than men. Even though the burgers were presented in a blinded manner, only labelled with three-digit codes, it is likely that some assessors were able to guess which was the hybrid and which was the beef burger, which may then have influenced their attitude towards them. The correspondence analysis of the term usage shows a distinctly different pattern in the two clusters. In cluster 1 the hybrid burger is more associated with terms that have a negative impact on liking, whereas in cluster 2 the association with more positive or neutral terms was stronger. Along with the penalty analysis, this serves to underpin an apparent connection between term usage and overall liking of the burgers.
Potential weaknesses of the study are mainly associated with the consumer survey. Firstly, for logistical reasons it was not possible to prepare the burger patties on site, which meant they had to be cooked in advance, then frozen and subsequently thawed and reheated at the expo where the survey was carried out. Nevertheless, this method is also used in fast food restaurants in Denmark. In addition, in order to comply with food safety regulations, they were cooked to an internal temperature of no less than 75 - C, which may be higher than what some consumers would prefer regarding colouring, texture and mouthfeel. However, the two burgers received identical treatments, so whatever negative effect the above may have had would be the same for both.
# Conclusions
The trend towards a more plant-forward diet as a tool for climate change mitigation has shown signs of stagnation. This may reflect lacking sensory acceptability towards plant-based meat alternatives and an emotional attachment to meat among the consumers who have not embraced the trend. Therefore, we hypothesised that a hybrid burger might be a less challenging change of habitual diet for this consumer segment. We developed a hybrid burger patty that was softer and juicier but associated with an off taste as well as less meat flavour than a beef burger. However, consumers found the hybrid burger overall as acceptable as a reference beef burger. The climate footprint, expressed in CO 2 -eq, is also substantially reduced in the hybrid burger. Our observations point to specific critical sensory attributes to develop for possibly increasing acceptability of a hybrid plant/meat burger patty. Cluster analysis only indicated trends toward defining distinct consumer characteristics that could predict liking and CATA term usage for the hybrid burger. In conclusion, we describe here proof-of-concept that a 50%/50% plant/meat hybrid burger may be an acceptable substitution for a meat-only burger, however, this should be evaluated more directly through willingness to purchase and behavioural studies.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods12112246/s1, [fig_ref] Table 1: Ingredients used in hybrid [/fig_ref] : Estimated nutritional content per 100 g of a hybrid (50%/50% plant/meat) and a beef (100% meat) burger patty. Funding: This research did not receive any external funding, and was carried out entirely using Business Academy Aarhus' basic funding from the Ministry of Higher Education and Science in Denmark.
# Data availability statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the participants of this study not giving written consent for their data at an individual level to be shared publicly.
[fig] Foods 2023 ,Figure 1: 12Age and gender distribution of assessors in the consumer survey. Shaded part of each bar shows proportion of assessors who sometimes eat plant-based meat alternatives. [/fig]
[fig] Figure 1: Age and gender distribution of assessors in the consumer survey. Shaded part of each bar shows proportion of assessors who sometimes eat plant-based meat alternatives. [/fig]
[fig] Figure 2: Biplot of rows (terms) and columns (burgers, including ideal) from correspondence analysis of the CATA data. Only terms with significantly different usage are shown (p ≤ 0.05). [/fig]
[fig] Foods 2023 ,: 12, x FOR PEER REVIEW 9 of 14 [/fig]
[fig] Figure 3: Mean drop in overall liking score for a burger when a CATA term was either checked for ideal and not for tested burger (−P(No)|(Yes)), or not checked for ideal and checked for tested burger (+P(Yes)|(No)). [/fig]
[fig] Figure 4: Mean liking scores for the two burgers in two clusters (panel A) generated by the CLUS-CATA method. Different letters above bars indicate statistically significant difference (p < 0.05). (panels B and C) Biplot of rows (terms) and columns (burgers, including ideal) from correspondence analysis of cluster 1 and 2, respectively. [/fig]
[fig] Author: Contributions: Conceptualization, B.P.-M.; writing-original draft preparation, B.P.-M.; writing-review and editing, B.P.-M. and S.D.; visualization, B.P.-M. and S.D. All authors have read and agreed to the published version of the manuscript. [/fig]
[table] Table 1: Ingredients used in hybrid (50%/50% plant/meat) and beef burger patty. [/table]
[table] Table 2: Consumer-generated sensory terms for check-all-that-apply survey of hybrid vs. beef burger patty. [/table]
[table] Table 3: Physical attributes related to texture and sensory perception. Results are given as means ± s.d. (n = 6). [/table]
[table] Table 4: Climate impact of ingredients used in a regular cheeseburger. [/table]
[table] Table 5: Frequency (%) of assessors' (n = 381) use of CATA terms to describe burgers and their ideal burger. [/table]
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10.1186/s12934-023-02228-6
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CCBY
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10590003
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37865739
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s2orc_pubmed_articles
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Identification of a novel family B DNA polymerase from Enterococcus phage IME199 and its overproduction in Escherichia coli BL21(DE3)
Background Identification and characterization of novel, faithful and processive DNA polymerases is a driving force in the development of DNA amplification methods.Purification of proteins from natural phages is often time-consuming, cumbersome and low yielding.Escherichia coli is a host bacterium widely used for the production of recombinant proteins, is the cell factory of choice for in vitro studies of phage protein function.ResultsWe expressed the gene encoding Enterococcus faecium phage IME199 DNA polymerase (IME199 DNAP) in Escherichia coli BL21(DE3), and characterized protein function.IME199 DNAP has 3′-5′ exonuclease activity, but does not have 5′-3′ exonuclease activity.In addition, IME199 DNAP has dNTP-dependent 5′-3′ polymerase activity and can amplify DNA at 15-35 °C and a pH range of 5.5-9.5.The amino acid residues Asp30, Glu32, Asp112 and Asp251 are the 3′-5′ exonuclease active sites of IME199 DNAP, while residues Asp596 and Tyr639 are essential for DNA synthesis by IME199 DNAP.More importantly, the IME199 DNAP has strand displacement and processive synthesis capabilities, and can perform rolling circle amplification and multiple displacement amplification with very low error rates (approximately 3.67 × 10 -6 ).Conclusions A novel family B DNA polymerase was successfully overproduced in Escherichia coli BL21(DE3).Based on the characterized properties, IME199 DNAP is expected to be developed as a high-fidelity polymerase for DNA amplification at room temperature.
# Background
DNA polymerase is an important enzyme necessary for all living organisms and is responsible for DNA replication and repair.Arthur Kornberg discovered the first DNA polymerase (DNA polymerase I) in Escherichia coli in 1955 [bib_ref] Enzymic synthesis of deoxyribonucleic acid, Kornberg [/bib_ref] , and subsequently a variety of DNA polymerases have been found in other prokaryotes and eukaryotes [bib_ref] Eukaryotic DNA polymerases, Jain [/bib_ref] [bib_ref] Prokaryotic DNA replication, Marians [/bib_ref].The discovery of DNA polymerase largely contributed to our understanding of how DNA is replicated and repaired today, as well as to the creation and development of DNA amplification techniques.
Based on nucleotide sequence and structural similarity, DNA polymerases can be classified into seven families: A, B, C, D, X, Y and RT [bib_ref] Eukaryotic DNA polymerases, Hubscher [/bib_ref].Family B DNA polymerases are relatively well-discovered, present in all domains of life, and have been reported in bacteria, archaea, eukaryotes, and viruses [bib_ref] DNA polymerases drive DNA sequencing-by-synthesis technologies: both past and present, Chen [/bib_ref] [bib_ref] An updated structural classification of replicative DNA polymerases, Raia [/bib_ref].Most family B DNA polymerases have at least two common domains: DNA polymerase domain and 3′-5′ exonuclease domain [bib_ref] Diversity and evolution of B-family DNA polymerases, Kazlauskas [/bib_ref].The family B DNA polymerases that have been reported can participate in genome replication in organisms using three different ways: replication using pre-existing nucleic acids (DNA or RNA) primers [bib_ref] Human DNA polymerase α has a strong mutagenic potential at the initial..., Lisova [/bib_ref] , protein-primed replication [bib_ref] Protein-priming of DNA replication, Salas [/bib_ref] , and template-dependent de novo synthesis [bib_ref] Primer-independent DNA synthesis by a family B DNA polymerase from self-replicating mobile..., Redrejo-Rodríguez [/bib_ref].As the biochemical properties and functions of a large number of family B DNA polymerases have been investigated, their widespread application in molecular biology and biotechnology has been facilitated.
As the most abundant and widely distributed biological entities in the biosphere, bacteriophages (phages) have diverse genomes that also confer diversity in their DNA polymerase functions [bib_ref] Bacteriophage-encoded DNA polymerases-beyond the traditional view of polymerase activities, Morcinek-Orłowska [/bib_ref].Phages that can encode their own DNA polymerase are constantly being discovered, and the DNA polymerases of phage phi29, T4, Bam35 and RB69 that have been characterized belong to the family B DNA polymerases [bib_ref] Phi29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved..., Eisenbrandt [/bib_ref] [bib_ref] Analysis of inhibitors of bacteriophage T4 DNA polymerase, Khan [/bib_ref] [bib_ref] DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and..., Berjón-Otero [/bib_ref] [bib_ref] Protein determinants of RNA binding by DNA polymerase of the T4-related bacteriophage..., Petrov [/bib_ref].Among all phage-encoded DNA polymerases, phi29 DNA polymerase (phi29 DNAP) is a model replicative DNA polymerase that has been extensively studied at the genetic, physiological and biochemical levels, and has been used in a variety of applications [bib_ref] Rolling-circle amplification of viral DNA genomes using phi29 polymerase, Johne [/bib_ref] [bib_ref] DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication, Salas [/bib_ref].The phi29 DNAP uses a protein as the primer to initiate genome replication, which is a paradigm for protein-primed replication.The high processivity and the capacity to couple polymerization to strand displacement endow phi29 DNA polymerase with powerful replication efficiency, which led to the development of its protocol for isothermal amplification in circular and linear genomic DNA.The development of rolling circle amplification (RCA) and multiple displacement amplification (MDA) methods has made phi29 DNA polymerase widely used in single-cell whole-genome amplification [bib_ref] Single-cell whole-genome amplification and sequencing: methodology and applications, Huang [/bib_ref] , genotyping of single nucleotide polymorphisms [bib_ref] L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide..., Qi [/bib_ref] and miRNA detection [bib_ref] Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle..., Murakami [/bib_ref].However, phi29 DNAP still has some limitations in its application, such as incomplete and uneven genome amplification and poor amplification of high G+C templates [bib_ref] Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells..., Stepanauskas [/bib_ref] [bib_ref] Calibrating genomic and allelic coverage bias in single-cell sequencing, Zhang [/bib_ref].The phi29 DNAP is only the tip of the iceberg of many phage DNA polymerases, and there are countless other promising DNA polymerases that need to be studied and developed, which will certainly provide new impetus to the field of molecular biology.
Phages rely on host bacteria for rapid reproduction, and it is very difficult to obtain phage-encoded proteins from the host after infection.Recombinant DNA expression technology in Escherichia coli (E.coli) provides assistance in studying functional proteins in phages.E. coli BL21 (DE3) has many advantages to support heterologous gene expression, such as increasing the production of soluble proteins, enhancing the formation of disulfide bonds, and facilitating the proper folding of target proteins [bib_ref] Recombinant protein expression in Escherichia coli (E.coli): what we need to know, Hayat [/bib_ref] , making it the first cell factory for heterologous production of phage proteins.
In our previous study, we isolated an Enterococcus phage IME199 with a total length of 18,838 bp [bib_ref] Complete genome sequence of a novel, virulent Ahjdlikevirus bacteriophage that infects Enterococcus..., Xing [/bib_ref].Genome sequencing analysis revealed a 65 bp reverse repeat sequence at the end of the IME199 genome, which has the same properties as phi29-like phages [bib_ref] Phi29 family of phages, Meijer [/bib_ref].In addition, the IME199 DNAP shares weak amino acid sequence similarity with phi29 DNAP, suggesting that they may be functionally similar, although IME199 and phi29 are very different taxonomically.In this study, we investigated the function of IME199 DNAP in terms of evolutionary analysis, protein production, and activity identification, and found that it has properties of high processivity, strand displacement, and high fidelity, which enable IME199 DNAP to perform RCA and MDA.This discovery enriches our understanding of phage DNA polymerase and provides a reference for the application of IME199 DNAP in the field of molecular biology.
# Results
## Phylogenetic analysis of ime199 dnap
Analysis of the amino acid sequence of IME199 DNAP (GenBank accession no.ALO81005.1)using InterPro [bib_ref] InterPro: the integrative protein signature database, Hunter [/bib_ref] revealed that it has a DNA-directed DNA polymerase structural domain, which belongs to the family B DNA polymerases (Additional file 1: Fig. .To understand the evolutionary relationships of IME199 DNAP in homologous species, we constructed a phylogenetic tree based on amino acid sequence similarity.As shown in Fig. , we found that IME199 DNAP has some interrelationship with phage DNA polymerases of Enterococcus, Streptococcus, Staphylococcus, Lactococcus, Clostridium and Bacillus.Intriguingly, these different phage genomes have terminal inverted repeat sequences ranging from 6 to 378 bp (Additional file 1: Table .In the extensively studied phi29 like phages (during infection of Bacillus), terminal inverted repeat sequences are a prerequisite for protein-initiated DNA replication, allowing DNA polymerase to employ a sliding-back mechanism to recover information of the terminal nucleotides in genome replication [bib_ref] Phi29 family of phages, Meijer [/bib_ref] [bib_ref] Functional characterization of the genes coding for the terminal protein and DNA..., Illana [/bib_ref] [bib_ref] Initiation of phi 29 DNA replication occurs at the second 3′ nucleotide..., Méndez [/bib_ref].Despite the lack of sequence similarity between the genomes of these phages which infect different genera of bacteria, their DNA polymerases may have important common functional and structural features.Amino acid sequence alignment of IME199 DNAP with other family B DNA polymerases showed that their 3′-5′ exonuclease active site is evolutionarily conserved which consists of three motifs (Exo I, Exo II, and Exo III), and that they also have conserved motifs associated with polymerase activity (Additional file 1: Fig. [fig_ref] Figure 2: Exonuclease activity test of IME199 DNAP [/fig_ref].The conserved motifs of family B DNA polymerases are essential for understanding the evolution of species [bib_ref] Diversity and evolution of B-family DNA polymerases, Kazlauskas [/bib_ref] , and are required for the biological functions of these enzymes.The evolutionary similarity of DNA polymerases and the features of inverted repeats at the end of the genome suggest that these phages may have the same replication mechanism and a common ancestor.
## Ime199 dnap possesses 3′-5′exonuclease activity
To study the biochemical function of IME199 DNAP, its gene was cloned in pET-28a(+) and overexpressed in E. coli BL21(DE3), and the recombinant protein was purified after Ni-column affinity and gel filtration chromatography.Successfully produced and purified recombinant IME199 DNAP protein showed a band of approximately 95 kDa on SDS-PAGE stained with Coomassie brilliant blue (Additional file 1: Fig. [fig_ref] Figure 3: Polymerase activity test of IME199 DNAP [/fig_ref] , which is consistent with the theoretical molecular mass of the amino acid sequence.We first examined the exonuclease activity of IME199 DNAP using modified ssDNA, and the results showed that IME199 DNAP has 3′-5′ exonuclease Fig. Phylogenetic analysis of IME199 DNAP.The phylogenetic tree was constructed based on the amino acid sequences of DNA polymerases using the Neighbor-joining method with 1000 bootstrap replicates in MEGA 7. All the amino acid sequences were downloaded from NCBI.IME199 DNAP is marked with a red square activity (Fig. [fig_ref] Figure 2: Exonuclease activity test of IME199 DNAP [/fig_ref] but not 5′-3′exonuclease activity (Fig. [fig_ref] Figure 2: Exonuclease activity test of IME199 DNAP [/fig_ref] , which is typical of family B DNA polymerases.
DNA polymerase with 3′-5′ exonuclease activity plays an important role in promoting the accuracy of DNA replication and DNA proofreading and repair [bib_ref] Genetic and biochemical studies of bacteriophage T4 DNA polymerase 3′->5′-exonuclease activity, Reha-Krantz [/bib_ref].We found that IME199 DNAP with 3′-5′ exonuclease activity could remove the incorrect bases at the 3′ end of the primer that did not match the template, thus amplifying the correct product (Additional file 1: Fig. [fig_ref] Figure 4: Biochemical characteristics of IME199 DNAP [/fig_ref].This indicates that 3′-5′ exonuclease activity makes IME199 DNAP highly specific for inserting the correct base when performing replication and ensures high fidelity of DNA replication.
## Dna polymerase activity of ime199 dnap
To evaluate the polymerase activity of IME199 DNAP, we analyzed the extension of labeled primer/template substrates (Fig. [fig_ref] Figure 3: Polymerase activity test of IME199 DNAP [/fig_ref].As shown in Fig. [fig_ref] Figure 3: Polymerase activity test of IME199 DNAP [/fig_ref] , when dNTPs were sufficient, the yield of full-length products gradually increased with increasing IME199 DNAP concentration, and the products no longer increased after the IME199 DNAP concentration was greater than 50 nM.The 50 nM IME199 DNAP was incubated with the substrate in the presence of different concentrations of dNTPs, and the results showed that significant degradation products could be observed without total products in the absence or presence of low concentrations of dNTPs (< 100 nM) (Fig. [fig_ref] Figure 3: Polymerase activity test of IME199 DNAP [/fig_ref] , lanes 3-5).In addition, the yield of full-length products gradually increased with increasing dNTPs concentration, and no degradation products were observed in the presence of 800 nM dNTPs (Fig. [fig_ref] Figure 3: Polymerase activity test of IME199 DNAP [/fig_ref] , lanes 6-10).
## Biochemical characterization of ime199 dnap
To determine the biochemical properties of DNA synthesized by IME199 DNAP, we examined the ability of IME199 DNAP to amplify labeled primer/template substrates in different metal cation buffers, different pH buffers and different temperatures.It is well known that replicative DNA polymerases require bivalent metal cations for polymerization.In this study, we found that IME199 DNAP could amplify full-length products in the presence of Mg 2+ , Mn 2+ , Ca 2+ , K + or Na + (Fig. [fig_ref] Figure 4: Biochemical characteristics of IME199 DNAP [/fig_ref] , lanes 2-6), whereas Zn 2+ could not assist IME199 DNAP in the polymerization reaction.Studies on phage phi29 and Bam35 DNA polymerases have demonstrated that Mg 2+ and Mn 2+ can act as activators of B family DNA polymerases to promote the persistence of DNA replication [bib_ref] Modulation of DNA polymerase noncovalent kinetic transitions by divalent cations, Dahl [/bib_ref].Unlike phi29 DNAP [bib_ref] Metal activation of synthetic and degradative activities of phi 29 DNA polymerase,..., Esteban [/bib_ref] , Ca 2+ , K + and Na + can also act as metal activators of IME199 DNAP replication, but they are not as efficient as Mg 2+ and Mn 2+ in promoting polymerase activity.
Thermal stability tests showed that IME199 DNAP has polymerase activity in the range of 15-35 ℃, and 30 °C is the optimum reaction temperature (Fig. [fig_ref] Figure 4: Biochemical characteristics of IME199 DNAP [/fig_ref] , lanes 2-6).IME199 DNAP is sensitive to high temperatures and loses polymerase activity at 40 °C and above (Fig. [fig_ref] Figure 4: Biochemical characteristics of IME199 DNAP [/fig_ref] , lanes 7-9).The tolerance temperature range of IME199 DNAP is essentially close to room temperature, facilitating its development as a polymerase that does not require heating assistance for DNA amplification in the future.
Next, we investigated the effect of pH on the polymerase activity of IME199 DNAP.As shown in Fig. [fig_ref] Figure 4: Biochemical characteristics of IME199 DNAP [/fig_ref] , IME199 DNAP had good polymerase activity in the pH range of 5.5-8.5, and the polymerase activity decreased significantly when the pH was higher than 9.5.
## Mutational studies of ime199 dnap
The tertiary structure of IME199 DNAP was predicted using SWISS-MODEL [bib_ref] SWISS-MODEL: homology modelling of protein structures and complexes, Waterhouse [/bib_ref].Although the similarity is less than 30%, the best reference model is the phi29 DNA polymerase orthorhombic crystal form (results not shown), indicating that IME199 DNAP and phi29 DNAP have structural similarities.Since the structure and active site of phi29 DNAP have been well studied, we compared the amino acid sequences of IME199 DNAP with those of phi29 DNAP and found that amino acid residues Asp30, Glu32, Asp112 and Asp251 of IME199 DNAP are similar to Asp12, Glu14, Asp66 and Asp169 of phi29 DNAP (Additional file 1: Fig. .These conserved amino acid residues are a part of the catalytic site of phi29 correspond to 3′-5′ exonuclease activity, and their mutation led to a 100-fold reduction in exonuclease activity [bib_ref] A conserved 3′--5′ exonuclease active site in prokaryotic and eukaryotic DNA polymerases, Bernad [/bib_ref] [bib_ref] 40 years with bacteriophage ø29, Salas [/bib_ref].In addition, amino acid residues Tyr333, Gly335, Ser360, Thr575, Asp596 and Tyr639 of IME199 DNAP are also similar to Tyr226, Gly228, Ser252, Thr434, Asp458 and Tyr500 of phi29 DNAP (Additional file 1: Fig. , and these amino acid residues of phi29 DNAP have been shown to be essential for polymerase activity.Mutations of Tyr226 and Gly228 in phi29 DNAP can affect polymerase/exonuclease balance [bib_ref] A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases, Truniger [/bib_ref] , and mutations of Ser252, Thr434, Asp458 and Tyr500 can affect in template-primer binding and cause loss of polymerase activity [bib_ref] Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage, Hendrix [/bib_ref] [bib_ref] Phi 29 DNA polymerase active site. Residue ASP249 of conserved amino acid..., Blasco [/bib_ref] [bib_ref] Primer-terminus stabilization at the psi 29 DNA polymerase active site. Mutational analysis..., Méndez [/bib_ref] [bib_ref] The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases..., Bernad [/bib_ref] [bib_ref] Primer terminus stabilization at the phi 29 DNA polymerase active site. Mutational..., Blasco [/bib_ref].
To confirm whether these similar amino acid residues are also essential for IME199 DNAP, we mutated 10 amino acids (Asp30, Glu32, Asp112, Asp251, Tyr333, Gly335, Ser360, Thr575, Asp596, Tyr639) of IME199 DNAP to alanine by targeted mutagenesis and tested the 3′-5′ exonuclease and polymerase activities of the mutant proteins.The results showed that the Asp30A, Glu32A, Asp112A and Asp251A mutant proteins lost 3′-5′ exonuclease activity (Fig. , lanes 2-5) but still had polymerase activity (Fig. , lanes 2-5), indicating that residues Asp30, Glu32, Asp112 and Asp251 are essential for IME199 DNAP 3′-5′ exonuclease activity.Meanwhile, the Tyr333A, Gly335A, Ser360A, Thr575A, Asp596A and Tyr639A mutant proteins still had 3′-5′ exonuclease activity (Fig. , lanes 3-8), but only the Asp596A and Tyr639A mutant proteins almost lost polymerase activity (Fig. , lanes 7 and 8), indicating that residues Asp596 and Tyr639 are essential for DNA synthesis by IME199 DNAP.
Amino acid residues mutational analysis of IME199 DNAP showed that the exonuclease structural domains of IME199 DNAP and phi29 DNAP are similar, but there are differences in their polymerase active structural domains, which also suggests that they are evolutionarily related.In addition, mutational analysis has also improved our understanding of the functional role of IME199 DNAP amino acid residues.
## Strand displacement and processive synthesis capability of ime199 dnap for rolling circle amplification
Strand displacement and processive synthesis are the outstanding characteristics of phi29 DNAP, making it widely used in the field of genetic engineering and biotechnology.Given the evolutionary and structural similarities between IME199 DNAP and phi29 DNAP, we hypothesized that IME199 DNAP also has strand displacement and processive synthesis capabilities.To verify this, we performed replication assay using the single-stranded circular DNA M13mp18 as a substrate (Fig. [fig_ref] Figure 6: Rolling circle amplification of single-stranded circular M13mp18 by IME199 DNAP [/fig_ref].The results showed that IME199 DNAP can continuously amplify on the circular DNA template and produce a replication product larger than the full-length M13mp18 genome (7.25 kb), which is consistent with the product amplified by phi29DNAP (Fig. [fig_ref] Figure 6: Rolling circle amplification of single-stranded circular M13mp18 by IME199 DNAP [/fig_ref] , lanes 5-6).Some amplification products of IME199 DNAP and phi29 DNAP were larger compared to the amplification products of T4 DNA polymerase (T4 DNAP), which caused them to remain in the upper sample void.These results suggest that IME199 DNAP is similar to phi29 DNAP in its ability to couple strand displacement and processive polymerization during rolling circle DNA replication.Subsequently, we investigated the difference in different length primers for rolling loop amplification, and the results showed that IME199 DNAP amplified the most products using 28 bp primers (Fig. [fig_ref] Figure 6: Rolling circle amplification of single-stranded circular M13mp18 by IME199 DNAP [/fig_ref] , which was the same in different regions of the M13mp18 genome (Additional file 1: Fig. [fig_ref] Figure 6: Rolling circle amplification of single-stranded circular M13mp18 by IME199 DNAP [/fig_ref].Under the same conditions, phi29 DNAP also had the highest yield using 28 bp primers (Additional file 1: Fig. [fig_ref] Figure 6: Rolling circle amplification of single-stranded circular M13mp18 by IME199 DNAP [/fig_ref].However, we found that IME199 DNAP has a preference for high G+C primers when primer lengths are consistent.Significantly more products were amplified using high G+C (75%) primers than those using low G+C (≤ 54%) primers (Fig. [fig_ref] Figure 6: Rolling circle amplification of single-stranded circular M13mp18 by IME199 DNAP [/fig_ref] , while phi29DNAP did not have this preference under the same conditions (Additional file 1: Fig. .
## Fidelity of ime199 dnap for multiple displacement amplification
Phi29 DNAP can amplify DNA without sequence information using random hexamer primers, which is called multiple displacement amplification [bib_ref] Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase..., Dean [/bib_ref] , and coupled with the high fidelity of amplification, phi29 DNAP has been widely used for whole genome amplification.We examined the amplification of the pUC19 plasmid by IME199 DNAP using random hexamer primers.The results showed that IME199 DNAP could amplify a product of the same size as phi29 DNAP using random hexamer primers (Fig. , lanes 6 and 7).Simultaneous digestion of the amplified product and the pUC19 plasmid with endonuclease SalI followed by agarose gel electrophoresis analysis, the results showed that almost all of the amplified DNA was linearized after digestion, showing bands of the identical size as the digested pUC19 plasmid (approximately 2.7 kbp) (Fig. , lanes 1-7), which was also the same as that of phi29 DNAP (Fig. , lane 8).These results indicate that IME199 DNAP can be subjected to multiple displacement amplification and that the amplification products are tandem repeats of the template DNA.
Accurate DNA synthesis during replication is essential to maintain the stability of the genome over multiple generations [bib_ref] DNA replication fidelity, Kunkel [/bib_ref].The error rate of DNA replication is an important reflection of the fidelity of DNA polymerase, and a lower error rate represents higher fidelity.We determined the fidelity of IME199 DNAP for multiple displacement amplification using the blue-white screening method [bib_ref] An improved method for simple and efficient hepatitis B virus genome cloning, Tu [/bib_ref] by exploiting the properties of the lacZα gene in the pUC19 plasmid.The average error rate of IME199 DNAP amplification was calculated to be 3.67 ± 0.55 × 10 −6 by counting blue/white clones and detecting the yield of amplified products (Table , similar to that of phi29 DNAP and Bam35 [fig_ref] Figure 3: Polymerase activity test of IME199 DNAP [/fig_ref] DNAP, which was 5 × 10 −6 [46] and 5.1 ± 1.7 × 10 -6 [bib_ref] DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and..., Berjón-Otero [/bib_ref] , respectively.This result indicates that IME199 DNAP is also a high-fidelity DNA polymerase.Moreover, we sequenced the lacZα gene on the pUC19 plasmid in the mutant (white) clones and found that the mutation type of IME199 DNAP amplification was substitution and deletion, with the highest substitution rate and all C:G → T:A transitions (Additional file 1: Fig. .
# Discussion
Over the past 60 years, the field of biotechnology has greatly benefited from the application of a large number of DNA polymerases with different properties.In order to fully develop and apply the functionality of novel DNA polymerases, systematic expression, purification and characterization processes are required.In this study, we constructed a heterologous expression system in E. coli for the production and purification of Enterococcus Fig. Study on the activity of IME199 DNAP mutant proteins.A Exonuclease activity test of IME199 DNAP and its mutant proteins (199P-D30A, 199P-E32A, 199P-D112A and 199P-D251A).The 5′-FAM-labeled 24 nt oligonucleotide (5′FAM-TCC TAA CGA GAT TAG TTT TGC TGT -3′) was incubated at 400 nM with 50 nM IME199 DNAP or its mutant proteins (199P-D30A, 199P-E32A, 199P-D112A and 199P-D251A) at 30 °C for 20 min and then analyzed on a 20% denaturing PAGE gel.B Polymerase activity test of IME199 DNAP and its mutant proteins (199P-D30A, 199P-E32A, 199P-D112A and 199P-D251A).The primed-template (24/50 nt) substrate was incubated at 400 nM with 50 nM IME199 DNAP or its mutant proteins (199P-D30A, 199P-E32A, 199P-D112A and 199P-D251A) and 125 μM dNTPs at 30 °C for 20 min, and then analyzed on a 20% denaturing PAGE gel.C Exonuclease activity test of IME199 DNAP and its mutant proteins (199P-Y333A, 199P-G335A, 199P-S360A, 199P-T575A, 199P-D596A and 199P-Y639A).The 5′-FAM-labeled 24 nt oligonucleotide (5′FAM-TCC TAA CGA GAT TAG TTT TGC TGT -3′) was incubated at 400 nM with 50 nM IME199 DNAP or its mutant proteins (199P-Y333A, 199P-G335A, 199P-S360A, 199P-T575A, 199P-D596A and 199P-Y639A) at 30 °C for 20 min and then analyzed on a 20% denaturing PAGE gel.(D) Polymerase activity test of IME199 DNAP and its mutant proteins (199P-Y333A, 199P-G335A, 199P-S360A, 199P-T575A, 199P-D596A and 199P-Y639A).The primed-template (24/50 nt) substrate was incubated at 400 nM with 50 nM IME199 DNAP or its mutant proteins (199P-Y333A, 199P-G335A, 199P-S360A, 199P-T575A, 199P-D596A and 199P-Y639A) and 125 μM dNTPs at 30 °C for 20 min, and then analyzed on a 20% denaturing PAGE gel.199P represents IME199 DNAP in the figure phage IME199 DNAP, and characterized the function of IME199 DNAP by biochemical methods.Evolutionary and structural domain analyses indicate that IME199 DNAP is a family B DNA polymerase, and as expected, IME199 DNAP possesses the basic activity characteristics of family B DNA polymerases: polymerase and 3′-5′ exonuclease activity.The 3′-5′ exonuclease activity of IME199DNAP allows it to remove mismatch bases from extended primers, thereby improving fidelity.The importance of proofreading activity for family B DNA polymerases has been demonstrated by the fact that their error rate increases more than tenfold when 3′-5′ exonuclease activity is inactivated [bib_ref] Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase-an extremely heat..., Mattila [/bib_ref] [bib_ref] PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases, Cline [/bib_ref].
As a substrate for DNA polymerase, dNTPs are essential for DNA biosynthesis and repair, and they can regulate the polymerase activity and 3′-5′ exonuclease activity of DNA polymerase [bib_ref] The 3′-5′ exonuclease of human DNA polymerase delta (pol delta) is regulated..., Lee [/bib_ref].In this study, IME199 DNAP mainly exerted 3′-5′ exonuclease activity when the concentration of dNTPs was insufficient, while the 3′-5′ exonuclease activity of IME199 DNAP was .D Yield of M13mp18 amplified by IME199 DNAP using primers with different G+C content.25 ng of single-stranded M13mp18 DNA was amplified by 100 nM IME199 DNAP at 30 °C for 1 h in the presence of primers with different G+C content.The primer sequences were listed in Additional file 1: Table .Data are shown as the mean ± SD.Statistical analysis was performed by one-way analysis of variance following a Dunnett's multiple comparisons test.**P < 0.01, ****P < 0.0001 replaced by polymerase activity when the concentration of dNTPs increased (Fig. [fig_ref] Figure 3: Polymerase activity test of IME199 DNAP [/fig_ref].IME199 DNAP was able to amplify substrates in the presence of low concentrations (100 nM) of dNTPs, which was generally consistent with the DNA polymerase of temperate phage Bam35 (phi29like phage) [bib_ref] DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and..., Berjón-Otero [/bib_ref].However, different family B DNA polymerases exhibit different sensitivities to dNTPs.DNA polymerase from Thermococcus gammatolerans (Tga PolB) requires at least 2 μM dNTPs to amplify the substrate [bib_ref] Characterization and application of a family B DNA polymerase from the hyperthermophilic..., Zhang [/bib_ref].
Metal ions are cofactors necessary for DNA polymerase to replicate DNA.Mg 2+ and Mn 2+ have been shown to be effective activators of family B DNA polymerase [bib_ref] Characterization and application of a family B DNA polymerase from the hyperthermophilic..., Zhang [/bib_ref] [bib_ref] Effect of manganese on in vitro replication of damaged DNA catalyzed by..., Villani [/bib_ref].Our results also indicate that Mg 2+ and Mn 2+ promote IME199DNAP replication capacity significantly better than other metal ions (Fig. [fig_ref] Figure 4: Biochemical characteristics of IME199 DNAP [/fig_ref].DNA polymerase is sensitive to metal ion-induced changes in DNA conformation, which makes the ability of different metal ions to activate DNA polymerase to amplify DNA very different.Although the amino acid sequences of IME199 DNAP and phi29 DNAP share many similar conserved motifs, their metal ion catalytic sites are different.Ca 2+ activates DNA replication by IME199 DNAP, but is not an activator of phi29 DNAP.To date, only PabPolB (Pyrococcus abyssi GE5 DNA polymerase) in the family B DNA polymerases has been described to utilize Ca 2+ as a cofactor for DNA polymerization [bib_ref] Calcium-driven DNA synthesis by a high-fidelity DNA polymerase, Ralec [/bib_ref].However, the rate of DNA synthesis using Ca 2+ is low compared to Mg 2+ , so Mg 2+ remains the preferred metal cofactor for DNA polymerase replication.
When DNA polymerase synthesizes deoxynucleoside triphosphate into nascent DNA, pyrophosphate and hydrogen ions are released, and the pH of the reaction system changes as the concentration of hydrogen ions Fig. Multiple displacement amplification of pUC19 plasmid by IME199 DNAP.A The primed pUC19 DNA in which the heat-denatured pUC19 plasmid hybridized with random hexamer primers was incubated with 100 nM IME199 DNAP or 10 U phi29 DNAP (NEB, USA) at 30 °C for 2 h, and then analyzed by non-denaturing 1% agarose electrophoresis.B The pUC19 plasmid DNA and multiple displacement amplification of product by IME199 DNAP or phi29 DNAP was digested with SalI-HF for 2 h at 37 °C, and then analyzed by non-denaturing 1% agarose electrophoresis.Experimental details are described in Experimental Procedures
## Table 1 determination of error rate values for ime199 dnap during mda
The error rate was calculated using the equation ER = mf/(bp×d), where mf is the mutation frequency that was calculated using the equation mf = white colonies/ total colonies, and bp is the number of detectable sites (324) in the lacZ gene of plasmid pUC19, and d is the number of template doublings that were calculated using the equation d = (ng product)/(ng input) increases.This change has made it possible to detect DNA amplification by using pH-sensitive indicator dyes, which has been used in loop mediated isothermal amplification (LAMP) and rolling circle amplification (RCA) [bib_ref] Simple rolling circle amplification colorimetric assay based on pH for target DNA..., Hamidi [/bib_ref] [bib_ref] Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes, Tanner [/bib_ref].In this study, IME199 DNAP has been shown to be effective for rolling circle amplification while exhibiting stable polymerase activity at pH 5.5-9.5.Based on this property, the amplification reaction of IME199 DNAP can be visually detected using weakly alkaline pH indicators (results not shown), which provides a possibility for the application of IME199 DNAP in isothermal amplification detection in the future.DNA polymerases with strand displacement activity, such as phi29 DNAP, Bst DNAP and Bsu DNAP, are core to isothermal amplification technology, and these enzymes are able to displace downstream DNA during replication, thus eliminating the need for thermal denaturation [bib_ref] Isothermal multiple displacement amplification of DNA templates in minimally buffered conditions using..., Tenaglia [/bib_ref] [bib_ref] An enhanced activity and thermostability of chimeric Bst DNA polymerase for isothermal..., Li [/bib_ref] [bib_ref] Zeptomolar-level one-pot simultaneous detection of multiple colorectal cancer microRNAs by cascade isothermal..., Chen [/bib_ref].Our study confirmed that IME199 DNAP has similar strand replacement and processive synthesis capabilities as phi29 DNAP.Unlike phi29 DNAP, IME199 DNAP has better amplification effect using high G+C primers, and the yield of amplifying high G+C sequences is also higher than that of amplifying low G + C sequences under the same conditions (Additional file 1: Fig. .This property could enable IME199 DNAP to be used to amplify high G+C content DNA templates, such as soil microorganisms with high G+C genomes [bib_ref] Environments shape the nucleotide composition of genomes, Foerstner [/bib_ref].In addition, based on the characteristics of RCA, IME199 DNAP could be used for point-of-care testing of circular DNA viruses (such as Transfusion Transmitted viruses and Norovirus).
# Conclusion
In summary, we overproduced a DNA polymerase from Enterococcus faecium phage IME199 in E. coli BL21 (DE3), and described its biological properties.Evolutionary analysis and mutation studies demonstrated that IME199 DNAP has great similarity to the extensively studied phi29 DNAP.IME199 DNAP has both 3′-5′ exonuclease activity and polymerase activity, which are essential properties of family B DNA polymerases.IME199 DNAP has the ability for strand displacement and processive synthesis, and can perform rolling circle amplification and multiple displacement amplification at 30 °C similar to phi29 DNAP.However, unlike phi29 DNAP, Ca 2+ ions can act as metal activators to assist IME199 DNAP in DNA replication.Meanwhile, IME199 DNAP can amplify more DNA products using high G+C primers or on high G+C templates.Our study provides an important reference for the further development of IME199 DNAP and its application in isothermal amplification.
## Experimental procedures
## Phylogenetic analyses of ime199 dnap
The amino acid sequences of IME199 DNAP and other phage polymerases were obtained from the National Center for Biotechnology Information (NCBI), and the phylogenetic tree was constructed with MEGA7 using the Neighbour-Joining algorithm [bib_ref] MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets, Kumar [/bib_ref].
## Protein production and purification
The sequences of IME199 DNAP were amplified by PCR from bacteriophage IME199 genomic DNA.The target sequence was constructed into the pET-28a ( +) vector using BamHI/XhoI enzyme cutting sites, and the protein was expressed in E. coli BL21 (DE3) as described elsewhere [bib_ref] Mammalian commensal streptococci utilize a rare family of class VI lanthipeptide synthetases..., He [/bib_ref] with some modifications.In brief, E. coli BL21(DE3) cells harboring the recombinant plasmids were cultured in LB medium containing 50 μg/mL kanamycin at 37 °C until they reached an OD600 of 0.6.Then, target proteins were induced by adding 0.1 mM isopropyl β-D-thiogalactoside (IPTG), and the cells continued to be cultured for 20 h at 16 °C.Subsequently, cells were collected by centrifuging at 10,000 × g for 10 min, and resuspended in buffer A (20 mM Tris-HCl, 500 mM NaCl, 10% glycerol, pH 7.4).Further protein purification steps were carried out using a His-tag Protein Purification Kit (Beyotime, China) according to the manufacturer's instructions.The target proteins were further purified by size exclusion chromatography via a Superdex 200 gel filtration column (GE Healthcare Life Sciences China) with buffer B (10 mM HEPES, 150 mM NaCl, 5% glycerol, pH 7.4).The effluent containing target proteins was pooled and concentrated using an Amicon Ultra-50 centrifugal filter unit.After examining its purity by 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the IME199 DNAP was maintained in the presence of 50% glycerol at − 20 °C for storage.
In addition, protein variants of IME199 DNAP (D30A, E32A, D112A, D251A, Y333A, G335A, S360A, T575A, D596A, Y639A) were constructed using the Fast Mutagenesis System (Cat# FM111-01, TransGen Biotech, Beijing, China) according to the manufacturer's instructions, and these proteins were expressed and purified using the same method as described above.All primers used in this study are listed in Additional file 1: Table .
## Exonuclease and polymerase activity assay
The exonuclease and polymerase activity assay were performed by using oligonucleotides as previously described [bib_ref] Crystal structure and biochemical studies of the bifunctional DNA primase-polymerase from phage..., Guo [/bib_ref] with minor modifications.The 5′-FAMlabeled 24 nt oligonucleotides ENC-1 (5′-TCC TAA CGA GAT TAG TTT TGC TGT -3′) and the 3′-FAMlabeled 24 nt oligonucleotides ENC-2 (5′-TCC TAA CGA GAT TAG TTT TGC TGT -3′) were used as substrates for the exonuclease activity test.Different concentrations of IME199 DNAP or mutant proteins were incubated with 400 nM oligonucleotides ENC-1 or ENC-2, 50 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT and 10 mM MgCl 2 in a final volume of 20 μL (pH 7.5) at 30 °C for 20 min.
Oligonucleotides ENC-1 were hybridized to PLM-R (5′-CCC ATA CAA ATA AAC CAA AAA ACA ATA CAG CAA AAC TAA TCT CGT TAG GA -3′) forming a primer/ template structure for the polymerase activity test.Different concentrations of IME199 DNAP or mutant proteins were incubated with 400 nM primer/template, 50 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM MgCl 2 , 4 mM DTT and different concentrations of dNTPs in a final volume of 20 μL (pH 7.5) at 30 °C for 20 min.
Finally, a 20% denaturing PAGE gel was prepared using 20% Urea-PAGE electrophoresis kit (Coolaber, Beijing, China) and used to separate the reaction products under electrophoretic conditions at 30 mA for 50 min.Then, the gel was visualized using fluorescence imaging with the Tanon Imaging System (Tanon 5200 Multi, Biotanon, China).All oligonucleotides were synthesized from Beijing Rui Biotech Co., Ltd and listed in Additional file 1: Table .
## Optimization of the polymerase activity of ime199 dnap
To investigate the effects of different metal ions on IME199 DNAP polymerase activity, 10 mM MgCl 2 , MnCl 2 , CaCl 2 , KCl, NaCl, ZnCl 2 or FeSO 4 was added to 20 μL of reaction mixture (50 nM IME199 DNAP, 400 nM primer/template, 50 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 125 μM dNTPs, 4 mM DTT, pH 7.5) individually and incubated at 30 °C for 20 min.The results were visualized as described above.
For the optimal temperature assay, the reaction mixture (20 μL, pH 7.5) contained 50 nM IME199 DNAP, 400 nM primer/template, 50 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM MgCl 2 , 125 μM dNTPs, 4 mM DTT were incubated at 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C and 45 °C for 20 min, respectively.The results were visualized as described above.
To determine the optimal pH, 50 nM IME199 DNAP, 400 nM primer/template, 10 mM (NH 4 ) 2 SO 4 , 10 mM MgCl 2 , 125 μM dNTPs and 4 mM DTT were added into the Tris buffers with different pH values ranging from 5.5 to 10.5, and the reaction mixture (20 μL) were incubated at 30 °C for 20 min.The results were visualized as described above.
## Strand displacement and rolling circle amplification analysis
Strand displacement assay using single-stranded circular M13mp18 (NEB, USA) as the template.The assay was carried out in the presence of 100 nM IME199 DNAP or 10 U phi29 DNAP (NEB, USA) or 10U T4 DNAP (NEB, USA), 10 mM MgCl 2 , 50 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 250 μM dNTPs, 4 mM DTT, 25 ng M13mp18 (NEB, USA) and 1 μM primer Site1-P4 (5′-TCG TAA TCA TGG TCA TAG CTG TTT CCTG -3′) in a 25 μL reaction.After incubation for the indicated times at 30 °C, the reaction was stopped by adding 25 mM EDTA and 0.5% SDS.DNA replication products were analyzed by electrophoresis in 1.0% agarose contained Genecolour II nucleic acid dyes under electrophoretic conditions at 120 V for 50 min, and the gel was analyzed using fluorescence imaging with the Tanon Imaging System (Tanon 5200 Multi, Biotanon, China).
In addition, the characteristics of IME 199DNAP were investigated in RCA using different primers with M13mp18 (NEB, USA) as the substrate according to the above method.The concentration of DNA was measured using the DNA HS Assay Kit (Cat# FS-T1002, Beijing Foreverstar Biotech Co., Ltd, China) according to the manufacturer's instructions.The yield of DNA was calculated using the equation Yield = v × c, where v is the reaction volume and c is the concentration of detected DNA.
## Multiple displacement amplification and fidelity analysis
Multiple displacement amplification assay was performed using the plasmid pUC19 (NEB, USA) as substrate.In brief, the pUC19 plasmid (NEB, USA) was mixed with random hexamer primers (NEB, USA) and incubated at 95 °C for 3 min and cooled to room temperature.The formed primed pUC19 DNA was added to the reaction mixture (25 μL, pH 7.5) contained 100 nM IME199 DNAP or 10 U phi29 DNAP (NEB, USA), 50 mM Tris -HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM MgCl 2 , 500 μM dNTPs, 4 mM DTT, and incubated at 30 °C for 2 h.Electrophoresis and analysis were performed as described above.
The fidelity of IME199 DNAP was measured as previously described [bib_ref] DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and..., Berjón-Otero [/bib_ref] with some modifications.In brief, IME199 DNAP or phi29 DNAP was incubated with 1 ng pUC19 (NEB, USA) at 30 °C for 8 h according to the above system.Amplification products were digested with restriction endonuclease SalI-HF (NEB, USA), heat inactivated, ligated, transformed into XL-10 competent cells (Vazyme, China), and plated onto Luria-Bertani solid plates containing ampicillin (50 μg/mL), X-Gal (0.8 mg) and IPTG (0.8 mg).Plates were incubated at 37 °C for 16 h and then the blue and white colonies were counted.In addition, the plasmids extracted from the white clones were sent to Ruibiotech (Beijing, China) for sequencing to identify the mutation site.
[fig] Figure 2: Exonuclease activity test of IME199 DNAP.Different concentrations of IME199 DNAP were incubated with 400 nM 5′-FAM-labeled 24 nt oligonucleotides (5′FAM-TCC TAA CGA GAT TAG TTT TGC TGT -3′) (A) or 3′-FAM-labeled 24 nt oligonucleotides (5′-TCC TAA CGA GAT TAG TTT TGC TGT -FAM3′) (B) at 30 °C for 20 min.The control is the FAM-labeled 24 nt oligonucleotide without IME199 DNAP.No deoxyribonucleoside triphosphates (dNTPs) were added in any of the reactions.All results are presented on 20% denaturing PAGE [/fig]
[fig] Figure 3: Polymerase activity test of IME199 DNAP.A Schematic representation of polymerase activity research.A primed-template (24/50 nt) substrate in which the primer was 5′-FAM-labeled was extended by IME199 DNAP into a complete double strand (50/50 nt) in the presence of dNTPs.B Extension of a primed-template (24/50 nt) substrate by IME199 DNAP.400 nM substrate was incubated with different concentrations of IME199 DNAP and 125 μM dNTPs at 30 °C for 20 min, and then analyzed on a 20% denaturing PAGE gel.C Effect of dNTPs on IME199 DNAP polymerase activity.A primed-template (24/50 nt) substrate was incubated at 400 nM with 50 nM IME199 DNAP and different concentrations of dNTPs at 30 °C for 20 min, and then analyzed on a 20% denaturing PAGE gel.The primed-template (24/50 nt) substrate was made by hybridizing two oligonucleotide strands (24 nt oligonucleotide sequence: 5′FAM-TCC TAA CGA GAT TAG TTT TGC TGT -3′, 50 nt oligonucleotide sequence 5′-CCC ATA CAA ATA AAC CAA AAA ACA ATA CAG CAA AAC TAA TCT CGT TAG GA-3′) [/fig]
[fig] Figure 4: Biochemical characteristics of IME199 DNAP.A Effect of different metal ions on the polymerase activity of IME199 DNAP.400 nM primed-template (24/50 nt) substrate was incubated with 50 nM IME199 DNAP and 125 μM dNTPs at 30 °C for 20 min in reaction buffers containing different metal ions (final concentration of 10 mM), and then analyzed on a 20% denaturing PAGE gel.B Effect of temperature on the polymerase activity of IME199 DNAP.400 nM primed-template (24/50 nt) substrate was incubated with 50 nM IME199 DNAP and 125 μM dNTPs at different temperatures for 20 min in reaction buffers containing Mg 2+ , and then analyzed on a 20% denaturing PAGE gel.C Effect of different pH values on the polymerase activity of IME199 DNAP.400 nM primed-template (24/50 nt) substrate was incubated with 50 nM IME199 DNAP and 125 μM dNTPs at 30 °C for 20 min in different pH reaction buffers, and then analyzed on a 20% denaturing PAGE gel.The primed-template (24/50 nt) substrate as same as Fig. 3 [/fig]
[fig] Figure 6: Rolling circle amplification of single-stranded circular M13mp18 by IME199 DNAP.A Schematic diagram of rolling circle amplification.DNA polymerase with strand displacement and processive synthesis capability amplifies primed M13mp18 DNA into a large single-stranded DNA product.B Agarose gel electrophoresis of IME199 DNAP, phi29 DNAP and T4 DNAP amplification primed M13mp18 products.The primed (primer sequence: 5′-TCG TAA TCA TGG TCA TAG CTG TTT CCTG -3′) M13mp18 DNA was incubated at 25 ng with 10 U T4 DNAP, 10 U phi29 DNAP or 100 nM IME199 DNAP at 30 °C for 40 min, and then analyzed by non-denaturing 1% agarose electrophoresis.Lane 1, DNA marker; lane 2, M13mp18 single-stranded DNA; lane 3, M13mp18 double-stranded DNA; lane 4, T4 DNA polymerase; lane 5, phi29 DNA polymerase; lane 6, IME199 DNA polymerase.C Yield of M13mp18 amplified by IME199 DNAP using primers of different lengths.The assay was performed using 25 ng of single-stranded M13mp18 DNA and 100 nM IME199 DNAP.The primer sequences were listed in Additional file 1: TableS2.D Yield of M13mp18 amplified by IME199 DNAP using primers with different G+C content.25 ng of single-stranded M13mp18 DNA was amplified by 100 nM IME199 DNAP at 30 °C for 1 h in the presence of primers with different G+C content.The primer sequences were listed in Additional file 1: TableS2.Data are shown as the mean ± SD.Statistical analysis was performed by one-way analysis of variance following a Dunnett's multiple comparisons test.**P < 0.01, ****P < 0.0001 [/fig]
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Retraction: Down-regulation of HOXB5 inhibits TGF-β-induced migration and invasion in hepatocellular carcinoma cells via inactivation of the PI3K/Akt pathway
Retraction: Down-regulation of HOXB5 inhibits TGF-b-induced migration and invasion in hepatocellular carcinoma cells via inactivation of the PI3K/Akt pathwayRetraction: Down-regulation of HOXB5 inhibits TGF-b-induced migration and invasion in hepatocellular carcinoma cells via inactivation of the PI3K/Akt pathway
Retraction of 'Down-regulation of HOXB5 inhibits TGF-b-induced migration and invasion in hepatocellular carcinoma cells via inactivation of the PI3K/Akt pathway' by Jin-Ping Sun et al., RSC Adv., 2018, 8, 41415-41421, https://doi.The Royal Society of Chemistry hereby wholly retracts this RSC Advances article due to concerns with the reliability of the data.The western blots inFig. 1B, 1D, 2A, 2B and 4A have been over-contrasted to the point where the background has almost been erased.InFig. 2C, the 'shControl/À' and 'shHOXB5/+' panels are rotated versions of the same image. Some additional features can be observed in the 'shHOXB5' image, although the majority of the features overlap between the two images.InFig. 2D, the 'shControl/À' and 'shHOXB5/À' panels contain many duplicating features. Some additional features can be observed in the 'shControl' image, although the majority of the features overlap between the two images.Two of the images in the 'shHOXB5 + TGF-b' panel inFig. 3Aare identical. InFig. 4B, the 'shControl/À/À' and 'shHOXB5/+/À' panels are rotated versions of the same image. Some additional features can be observed in the 'shHOXB5/+/À' image, although the majority of the features overlap between the two images.The authors were asked to provide the raw data for this article, but did not respond. Given the signicance of the concerns about the validity of the data, and the lack of raw data, the ndings presented in this article are not reliable.The authors were informed but have not responded to any correspondence regarding the retraction. Signed: Laura Fisher, Executive Editor, RSC Advances Date: 18 th August 2022
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Full mitochondrial genome sequences of two endemic Philippine hornbill species (Aves: Bucerotidae) provide evidence for pervasive mitochondrial DNA recombination
Background: Although nowaday it is broadly accepted that mitochondrial DNA (mtDNA) may undergo recombination, the frequency of such recombination remains controversial. Its estimation is not straightforward, as recombination under homoplasmy (i.e., among identical mt genomes) is likely to be overlooked. In species with tandem duplications of large mtDNA fragments the detection of recombination can be facilitated, as it can lead to gene conversion among duplicates. Although the mechanisms for concerted evolution in mtDNA are not fully understood yet, recombination rates have been estimated from "one per speciation event" down to 850 years or even "during every replication cycle". Results: Here we present the first complete mt genome of the avian family Bucerotidae, i.e., that of two Philippine hornbills, Aceros waldeni and Penelopides panini. The mt genomes are characterized by a tandemly duplicated region encompassing part of cytochrome b, 3 tRNAs, NADH6, and the control region. The duplicated fragments are identical to each other except for a short section in domain I and for the length of repeat motifs in domain III of the control region. Due to the heteroplasmy with regard to the number of these repeat motifs, there is some size variation in both genomes; with around 21,657 bp (A. waldeni) and 22,737 bp (P. panini), they significantly exceed the hitherto longest known avian mt genomes, that of the albatrosses. We discovered concerted evolution between the duplicated fragments within individuals. The existence of differences between individuals in coding genes as well as in the control region, which are maintained between duplicates, indicates that recombination apparently occurs frequently, i.e., in every generation. Conclusions: The homogenised duplicates are interspersed by a short fragment which shows no sign of recombination. We hypothesize that this region corresponds to the so-called Replication Fork Barrier (RFB), which has been described from the chicken mitochondrial genome. As this RFB is supposed to halt replication, it offers a potential mechanistic explanation for frequent recombination in mitochondrial genomes.
# Background
Since Desjardins and Morais [bib_ref] Sequence and gene organisation of the chicken mitochondrial genome. A novel gene..., Desjardins [/bib_ref] have presented the mt gene organization of the domestic chicken (Gallus gallus), it is known that birds possess a different gene order compared to other vertebrates. While the chicken gene order was found in many other avian taxa as well, Mindell et al. [bib_ref] Multiple independent origins of mitochondrial gene order in birds, Mindell [/bib_ref] , Eberhard et al. [bib_ref] Duplication and concerted evolution of the mitochondrial control region in the parrot..., Eberhard [/bib_ref] , Abbott et al. [bib_ref] An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in..., Abbott [/bib_ref] , and Verkuil et al. [bib_ref] A novel mitochondrial gene order in shorebirds (Scolopacidae, Charadriiformes), Verkuil [/bib_ref] subsequently presented alternate avian mt gene orders and discussed their potential origin. Gibb et al. [bib_ref] Mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations, Gibb [/bib_ref] suggested a conversion scenario for avian species according to the tandem duplication and random loss [TDRL] model [bib_ref] Tandem duplications in animal mitochondrial DNAs: variation in incidence and gene content..., Moritz [/bib_ref] [bib_ref] The duplication/random loss model for gene rearrangement exemplified by mitochondrial genomes of..., Boore [/bib_ref]. Specifically, they assume the derived avian gene order to have originated from an initial tandem duplication of the Cytb/tRNA T/tRNA P/NADH6/tRNA E/CR region, followed by several gene losses or reductions. Up to now, the completely conserved tandem duplicate is only reported for albatrosses [bib_ref] An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in..., Abbott [/bib_ref] [bib_ref] Mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations, Gibb [/bib_ref] , spoonbills [bib_ref] Tandem duplication of mitochondrial DNA in the black-faced spoonbill, Platalea minor, Cho [/bib_ref] , and boobies [bib_ref] Concerted evolution of duplicated mitochondrial control regions in three related seabird species, Morris-Pocock [/bib_ref]. Although further intermediate forms with two apparently functional gene or control region (CR) duplicates are rarely found (but see [bib_ref] Duplication and concerted evolution of the mitochondrial control region in the parrot..., Eberhard [/bib_ref] [bib_ref] A novel mitochondrial gene order in shorebirds (Scolopacidae, Charadriiformes), Verkuil [/bib_ref] [bib_ref] Mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations, Gibb [/bib_ref] [bib_ref] Bird mitochondrial gene order: insight from 3 warbler mitochondrial genomes, Singh [/bib_ref] [bib_ref] Repeated sequence homogenization between the control and pseudo-control regions in the mitochondrial..., Cadahía [/bib_ref] , it is generally assumed that the derived gene order has evolved independently more than once [bib_ref] Multiple independent origins of mitochondrial gene order in birds, Mindell [/bib_ref].
Studying mantellid frogs from Madagascar, Kurabayashi et al. [bib_ref] Phylogeny, recombination, and mechanisms of stepwise mitochondrial genome reorganization in mantellid frogs..., Kurabayashi [/bib_ref] infer two other possible mechanisms of mt genome reorganization than the TDRL model, both duplication modes mediated by recombination. One mechanism is the "illegitimate recombination via minicircle" (e.g., [bib_ref] Animal mitochondrial DNA recombination, Lunt [/bib_ref] [bib_ref] Intramitochondrial recombination: is it why some mitochondrial genes sleep around?, Dowton [/bib_ref] , where one part of the mt gene region is excised from one mt genome, forming a separate minicircle molecule. This molecule is then inserted into another genome, resulting in nontandem-duplicated regions within the mtDNA molecules. Another mechanism is the "general (homologous) recombination" (e.g., [bib_ref] Mammalian mitochondria possess homologous DNA recombination activity, Thyagarajan [/bib_ref] , where DNA strands of two genomic portions with identical or similar nucleotide sequences between chromosomes or within a DNA molecule are exchanged. When the exchanged DNA strands contain the same set of genes or regions, this recombination process does not cause gene duplication but can homogenize the sequences (gene conversion). On the contrary, when the exchanged DNA strands carry unequal sets of genes, one of the resultant molecules or genomic portions will have an extracopied gene region (unequal crossing over).
Recombination of the putative clonally maternally inherited mitochondrial DNA has been detected in several animal species, including birds and mammals [bib_ref] A broad survey of recombination in animal mitochondria, Piganeau [/bib_ref]. The frequency of such recombination, however, remains controversial; its estimation is not straightforward, as recombination under homoplasmy (i.e., among identical mt genomes) is likely to be overlooked [bib_ref] Animal mitochondrial DNA recombination revisited, Rokas [/bib_ref]. In species with tandem duplications of large mtDNA fragments such as in albatrosses [bib_ref] An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in..., Abbott [/bib_ref] , spoonbills [bib_ref] Tandem duplication of mitochondrial DNA in the black-faced spoonbill, Platalea minor, Cho [/bib_ref] and boobies [bib_ref] Concerted evolution of duplicated mitochondrial control regions in three related seabird species, Morris-Pocock [/bib_ref] , the detection of recombination is possible, as it can lead to gene conversion among duplicates. Although the mechanisms for concerted evolution in mtDNA are not fully understood yet, recombination rates have been estimated from "one per speciation event" among albatross species [bib_ref] An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in..., Abbott [/bib_ref] , down to 850 years among populations of killifish [bib_ref] Rapid concerted evolution in animal mitochondrial DNA, Tatarenkov [/bib_ref]. The study of Ogoh and Ohmiya [bib_ref] Concerted evolution of duplicated control regions within an ostracod mitochondrial genome, Ogoh [/bib_ref] even shows that gene conversion in the mt genome of ostracods occurs during every replication cycle. To explain their results, Ogoh and Ohmiya [bib_ref] Concerted evolution of duplicated control regions within an ostracod mitochondrial genome, Ogoh [/bib_ref] suggest a different mechanism. According to them, an exact replication mechanism, not recombination, controls the concerted evolution: the duplicated fragment is deleted and duplicated afresh in every replication cycle.
Besides studies on recombination and on rearrangements of mt genes, mtDNA as such is considered as a valuable tool in population genetic, phylogeographic, and phylogenetic studies [bib_ref] Numts: a challenge for avian systematics and population biology, Sorenson [/bib_ref]. Because useful information can be detected from many of the mt genes and due to primers being functional for a wide range of avian taxa [bib_ref] Primers for a PCRbased approach to mitochondrial genome sequencing in birds and..., Sorenson [/bib_ref] , the number of completely sequenced avian mt genomes is steadily increasing (e.g., [bib_ref] Mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations, Gibb [/bib_ref] [bib_ref] The mtDNA sequence of the ostrich and the divergence between paleognathous and..., Härlid [/bib_ref] [bib_ref] The complete mitochondrial genome of Rhea americana and early avian divergences, Härlid [/bib_ref] [bib_ref] Interordinal relationships of birds and other reptiles based on whole mitochondrial genomes, Mindell [/bib_ref] [bib_ref] Determination of the complete nucleotide sequence and haplotypes in the D-loop region..., Yamamoto [/bib_ref] [bib_ref] Complete mitochondrial genome sequences of two extinct moas clarify ratite evolution, Cooper [/bib_ref] [bib_ref] Complete mitochondrial DNA genome sequences of extinct birds: ratite phylogenetics and the..., Haddrath [/bib_ref] [bib_ref] The complete sequence of the mitochondrial genome of Buteo buteo (Aves, Accipitridae)..., Haring [/bib_ref] [bib_ref] Complete sequence of the Japanese quail (Coturnix japonica) mitochondrial genome and its..., Nishibori [/bib_ref] [bib_ref] Complete mitochondrial DNA genome sequences show that modern birds are not descended..., Paton [/bib_ref] [bib_ref] Two new mitochondrial genomes (penguin and goose) and a summary of birds..., Slack [/bib_ref] [bib_ref] Four new avian mitochondrial genomes help get to basic evolutionary questions in..., Harrison [/bib_ref] [bib_ref] Resolving the root of the avian mitogenomic tree by breaking up long..., Slack [/bib_ref] [bib_ref] Bird evolution: testing the Metaves clade with six new mitochondrial genomes, Morgan-Richards [/bib_ref] [bib_ref] Toward resolving deep neoaves phylogeny: data, signal enhancement, and priors, Pratt [/bib_ref]. Nevertheless, no mt genome from the family Bucerotidae is known so far and hornbills are missing from many phylogenetic analyses.
Here we present the complete mt genomes of two Philippine hornbills, endemic to the West Visayas, the Rufous-headed Hornbill Aceros waldeni and the Visayan Tarictic Hornbill Penelopides panini, and compare their characteristic mt genome features to each other and to those of other birds. We specifically test the hypothesis that recombination of mtDNA occurs regularly, i.e., within individuals of local animal populations.
# Results and discussion
## Genome organization
The two new mt genome sequences of the Philippine hornbills have been deposited in NCBI GenBank under the accession numbers HQ834450 (A. waldeni) and HQ834451 (P. panini). As expected and known from other birds, NADH6 and 8 tRNAs are transcribed from the light strand. All other 12 protein coding genes, 14 tRNAs and the two rRNAs 12S and 16S are located on the heavy strand (see Additional file 1: . Sequence annotation of the mt genome of A. waldeni/P. panini (as in deposited sequence)). The mt genomes of the two hornbills are longer than any other avian mt genome reported so far. The deposited sequences are 21,657 bp in A. waldeni and 22,737 bp in P. panini, both substantially exceeding the hitherto longest known avian mt genome of Diomedea melanophris (18,967 bp) [bib_ref] Mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations, Gibb [/bib_ref]. The gene order is characterized by a tandemly duplicated region beginning with the last 526 bp of Cytb, continuing over tRNA T/tRNA P/NADH6/tRNA E, and ending after CRII. The final length of the mt genome is the result of this duplication event, a repeat motif with remarkably long units found in both CRs, and another tandem repeat at the end of CRII. As there was some intraindividual variation (heteroplasmy) in the number of these repeats, total mt genomic lengths of both hornbill species were variable (see below).
## Structure of the duplicated region
Within individuals, the duplicated fragments are identical for the first 1,368bp (A. waldeni) or 1,388 bp (P. panini), except for a single substitution in two specimens (position 565 (tRNA T) in Pp-1 and position 1384 (CR) in Pp-6; cf., C). This region contains the duplicated part of Cytb, tRNA T, tRNA P, NADH6, and tRNA E, and the first part of domain I of the control regions (CR,. In the following 159 bp of A. waldeni, the duplicated fragments greatly differ within each individual at a total of 48 polymorphic sites, including three indels such that CRII is 2-3 bp shorter than CRI. In P. panini, the duplicates differ within individuals at 28 polymorphic sites and 1 indel position over a length of 59 bp. In the remaining part of domain I and in the complete domain II (403 bp in A. waldeni, 483 bp in P. panini; defined from the start of the conserved F box to the start of the conserved sequence block (CSB) 1 according to the sequence of the chicken [bib_ref] Sequence and gene organisation of the chicken mitochondrial genome. A novel gene..., Desjardins [/bib_ref] , the duplicates are again identical within all analyzed specimens of A. waldeni, but exhibit a few variable sites in P. panini, C). Domain III is characterized by a tandem repeat occuring in variable copy numbers, starting 37 bp (A. waldeni) or 27 bp (P. panini) after the end of the corresponding CSB1 of the domestic chicken. In the hornbills' CRI, the number of repeat units (determined by cloning and subsequent sequencing) varied between 1 and 10 (A. waldeni) or 1 and 17 (P. panini). The most common number was 9 units in A. waldeni and 12 units in P. panini [fig_ref] Figure 2: Nested PCR-amplificates spanning over the repetitive units in domain III of the... [/fig_ref] , B). In the total of 33 sequenced clones, 23 (A. waldeni) or 16 (P. panini) different repeat unit types between 111 bp and 123 bp in length were found.. The different repeat units did not occur in random order. Instead, repeat unit types were either (i) only found at the beginning and/or the end of the repeat region, or (ii) never occurred in that position, but only in between other repeats. Within each species, the first repeat unit always started with an identical motif of 19 bp (A. waldeni) or 98 bp (P. panini). Likewise, the last repeat unit always ended with an identical motif of 18 bp (A. waldeni) or 49 bp (P. panini). In CRII, the same types of repeat units were found. On average, CRII contains fewer repeat units than CRI. The number varies between 1 and 8 in A. waldeni (dominant 6, [fig_ref] Figure 2: Nested PCR-amplificates spanning over the repetitive units in domain III of the... [/fig_ref] and between 1 and 10 in P. panini (dominant 10, [fig_ref] Figure 2: Nested PCR-amplificates spanning over the repetitive units in domain III of the... [/fig_ref].
The following sequence of domain III (around 130 bp in A. waldeni and around 160 bp in P. panini) is identical among CRI and CRII within each of the species and exhibits at its beginning some similarity to the repeat motifs (data not shown; cf. to full mt genome sequences in Genbank accession numbers HQ834450 and HQ834451). In A. waldeni, CRI ends directly at the duplicated part of Cytb, whereas CRII continues after a spacer of 15 bp with another tandem repeat region. In P. panini, CRII passes directly into this second repeat region, whereas a spacer of 12 bp is situated between CRI and the duplicated part of Cytb. Concerning the second repeat region, the fully sequenced mt genome copies contained a truncated unit and between 1 and 27 (A. waldeni) or 33 (P. panini) complete units of 34 bp length. Even longer repeat regions (with presumably higher repeat numbers) existed, but sequence analysis did not reach through the entire repeat region of these very long mt genome variants, such that the exact number of repeats could not unambiguously be determined. The tandem repeat units exhibit a high A-content (47%) on the heavy strand. While A. waldeni possesses only one type of these tandem repeat units, different units with transitions at two sites are found in P. panini.
The organization of the mitochondrial genome found in the two hornbill species is most similar to that reported for albatrosses (Thalassarche spp. [bib_ref] An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in..., Abbott [/bib_ref] , Diomedea melanophris [bib_ref] Mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations, Gibb [/bib_ref] , for the black-faced spoonbill [bib_ref] Tandem duplication of mitochondrial DNA in the black-faced spoonbill, Platalea minor, Cho [/bib_ref] , and for boobies [bib_ref] Concerted evolution of duplicated mitochondrial control regions in three related seabird species, Morris-Pocock [/bib_ref]. It might constitute a general pattern at least in these two related taxonomic groups of seabirds (i.e., Procellariiformes and Pelecaniformes [bib_ref] Mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations, Gibb [/bib_ref] [bib_ref] Concerted evolution of duplicated mitochondrial control regions in three related seabird species, Morris-Pocock [/bib_ref].
Despite the general similarity in mitochondrial genome organization among albatrosses, the black-faced spoonbill, boobies, and hornbills, there are some fundamental differences among them: The last part of Cytb, with which the duplicated part begins, is significantly longer in hornbills, the spoonbill, and boobies, whereas in Thalassarche albatrosses this short Cytb part is preceded by a further part of Cytb considered to be degenerated [bib_ref] An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in..., Abbott [/bib_ref].
Concerning CR domain III, the albatrosses and the black-faced spoonbill possess repeat motifs only in CRII, whereas in both hornbill species and in the three booby species studied by Morris-Pocock et al. [bib_ref] Concerted evolution of duplicated mitochondrial control regions in three related seabird species, Morris-Pocock [/bib_ref] , the first repeat motif is also found in CRI. Equally to the spoonbill study [bib_ref] Tandem duplication of mitochondrial DNA in the black-faced spoonbill, Platalea minor, Cho [/bib_ref] , neither Abbott et al. [bib_ref] An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in..., Abbott [/bib_ref] nor Gibb et al. [bib_ref] Mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations, Gibb [/bib_ref] mention length heteroplasmy in this motif of the albatross in their publications; however, in an updated version of the respective sequence of Diomedea melanophris albatross [GenBank: AY158677], heteroplasmy is assumed, as well as for the boobies [bib_ref] Concerted evolution of duplicated mitochondrial control regions in three related seabird species, Morris-Pocock [/bib_ref]. Furthermore, confirmed length heteroplasmy in these motifs is described for other bird species, e.g., for the loggerhead shrike Lanius ludovicianus [bib_ref] Tandem repeats and heteroplasmy in the mitochondrial DNA control region of the..., Mundy [/bib_ref] , for the little blue penguin Eudyptula minor [bib_ref] Two new mitochondrial genomes (penguin and goose) and a summary of birds..., Slack [/bib_ref] , and for the ivorybilled aracari Pteroglossus azara [bib_ref] Mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations, Gibb [/bib_ref]. Two sets of repeat motifs, as found in the studied hornbills in CRII, are also described for the control region of penguins (Adélie penguin, Pygoscelis adeliae [bib_ref] A repeat complex in the mitochondrial control region of Adelie penguins from..., Ritchie [/bib_ref] and little blue penguin [bib_ref] Two new mitochondrial genomes (penguin and goose) and a summary of birds..., Slack [/bib_ref]. The little blue penguin was found to be heteroplasmic for both of these motifs [bib_ref] Two new mitochondrial genomes (penguin and goose) and a summary of birds..., Slack [/bib_ref].
## Evidence for frequent recombination among mt genomes
The analysis of the tandemly duplicated region in 6 individuals of each hornbill species enabled us to evaluate sequence evolution patterns across orthologous and paralogous duplicates over a total length of 1,930 bp, encompassing the duplicated parts of Cytb, tRNA T/ tRNA P/NADH6/tRNA E and domains I and the first parts of domains II of the control regions, accession numbers HQ834450-HQ834471). This analysis revealed a remarkable shift in similarity pattern: In a central section (159 bp in A. waldeni and 59 bp in P. panini, grey shaded in, C), orthologous copies (duplicate I of all individuals and duplicate II of all individuals, respectively) are more closely related to one another across individuals than to paralogous copies (duplicate I and duplicate II) within individuals (A. waldeni: πInd = 0.020, πDupl = 0.255; P. panini: πInd = 0.043, πDupl = 0.429). This section is situated in domain I of the control regions. It is surrounded by sections with a reversed diversity pattern (1,368 bp and 403 bp in A. waldeni, 1,388 bp and 483 bp in P. panini), i.e., where paralogous copies within individuals are more closely related (and in fact fully identical for most specimens; πDupl between 0.000 and 0.003) than orthologous copies across specimens (πInd up to 0.032;, C). This striking shift in sequence similarity from similarity among orthologues across specimens (grey) to similarity/identity among paralogues within specimens (white) is also reflected in our sequence section-specific phylogenetic analyses using ML [fig_ref] Figure 4: C [/fig_ref]. These analyses suggest a homogenization among the duplicated fragments within individuals [fig_ref] Figure 4: C [/fig_ref] , from which a distinct central sequence stretch (the grey shaded sections in(B) A. waldeni, section with high similarity between orthologues across individuals (putative Replication Fork Barrier (RFB) region, grey in(C) P. panini, region of inferred recombination (white in(D) P. panini, putative RFB region (grey in. Roman letters (I, II) indicate CRI vs. CRII copies of single individuals. Aw, Aceros waldeni; Pp, Penelopides panini. waldeni and 1,871 bp in P. panini). In this entire region, both duplicates within individuals are identical (except for one or two single base pair differences in a few specimens of P. panini (one synonymous Single Nucleotide Polymorphism (SNP) in tRNA T, the others in the CR); cf.. At the same time, there is sequence variation at orthologues among individuals, both in coding genes (one synonymous and one non-synonymous SNP in Cytb and one synonymous SNP in NADH6 of A. waldeni; one synonymous SNP in tRNA T and one synonymous SNP in NADH6 of P. panini) and in the control region of both species. If we compare any pair of specimens, their sequences in this region differ from one another at exactly the same nucleotide sites in both duplicates. This pattern indicates that the homogenization process must occur frequently, as it appears to have occurred in every single mitochondrial lineage within both species. Kurabayashi et al. [bib_ref] Phylogeny, recombination, and mechanisms of stepwise mitochondrial genome reorganization in mantellid frogs..., Kurabayashi [/bib_ref] postulated a novel scheme for vertebrate mtDNA replication, which can explain high frequencies of recombination. Applying this model to birds, replication of avian mtDNA is initiated throughout the mt genome, excluding the Replication Fork Barrier (RFB) [bib_ref] Bidirectional replication initiates at sites throughout the mitochondrial genome of birds, Reyes [/bib_ref]. During each replication cycle, the 3' end of the nascent L-strand is suspected to remain free at the RFB region until replication restarts. During the relatively long time of exposure, the free strands can easily be exchanged, leading to a high rate of recombination. This exchange can take place between two mtDNA molecules, but also within a single molecule (intragenomic gene conversion) if two independent replication forks occur.
For both hornbill species, the homogenization of the duplicates within individuals may be well explained with this recombination model. The part of the control region without intra-individual homogenization across duplicates (grey-shaded in, C; see above) putatively represents the RFB. Kurabayashi et al. [bib_ref] Phylogeny, recombination, and mechanisms of stepwise mitochondrial genome reorganization in mantellid frogs..., Kurabayashi [/bib_ref] found recombination only on one side of the RFB. Our observation of homogenized sections on both sides of the putative RFB in hornbills (as also described for albatrosses [bib_ref] An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in..., Abbott [/bib_ref] , the black-faced spoonbill [bib_ref] Tandem duplication of mitochondrial DNA in the black-faced spoonbill, Platalea minor, Cho [/bib_ref] , boobies [bib_ref] Concerted evolution of duplicated mitochondrial control regions in three related seabird species, Morris-Pocock [/bib_ref] , and for the ruff [bib_ref] A novel mitochondrial gene order in shorebirds (Scolopacidae, Charadriiformes), Verkuil [/bib_ref] may be explained by the fact, that replication occurs in both directions around the circular mtDNA in birds [bib_ref] Bidirectional replication initiates at sites throughout the mitochondrial genome of birds, Reyes [/bib_ref] , with a prominent initiation zone between Cytb and 12S, i.e., exactly in the genomic region duplicated in hornbills, albatrosses, the blackfaced spoonbill, boobies, and the ruff.
For the CR of mammals, two types of so-called Extended Termination Associated Sequences (ETAS) have been described [bib_ref] Mammalian mitochondrial D-loop region structural analysis: identification of new conserved sequences and..., Sbisà [/bib_ref]. It is suggested that ETAS1 could contain recognition signals (primary and secondary structural elements) for the termination of the nascent DNA or RNA chain, while ETAS2 could contain the binding sites for termination factors [bib_ref] Mammalian mitochondrial D-loop region structural analysis: identification of new conserved sequences and..., Sbisà [/bib_ref]. Subsequently, ETAS were also found in birds (e.g., [bib_ref] Organization and evolution of the mitochondrial DNA control region in the avian..., Randi [/bib_ref]. If we align the consensus sequence of mammalian ETAS1 and ETAS2 to our mt genome data of hornbills, they best match within the CR of the hornbills exactly in front of the putative RFB (i.e., exactly adjacent upstream to the grey-shaded region in. Putatively inferring this to be an ETAS region would further explain the exclusion of the inferred RFB from the recombination process.
## Organization of the mt genome in hornbills compared to other birds
Our complete sequences of the mitochondrial DNA of two hornbill species reveal many peculiar features in these mt genomes. Each of these features has been occasionally detected in a few avian taxa, but hornbills exhibit a unique combination in one single mt genome, i.e., (1) a tandem duplication of a region spanning over three tRNAs, one partial and one complete proteincoding gene, and the control region, (2) the existence of two sets of CR repeat motifs, of which one is duplicated as well, and (3) the remarkably long single units of these motifs, altogether making hornbills' mt genome with 21,657 bp (A. waldeni) and 22,737 bp (P. panini) the longest mt genome known from birds so far.
The hornbills' gene order differs from the gene order of most other avian taxa, except for two related groups of seabirds (Procellariiformes, Pelecaniformes [bib_ref] An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in..., Abbott [/bib_ref] [bib_ref] Mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations, Gibb [/bib_ref] [bib_ref] Concerted evolution of duplicated mitochondrial control regions in three related seabird species, Morris-Pocock [/bib_ref] and for the black-faced spoonbill (Ciconiiformes [bib_ref] Tandem duplication of mitochondrial DNA in the black-faced spoonbill, Platalea minor, Cho [/bib_ref] , assumed to be related to Procellariiformes and Pelecaniformes. It has been named "duplicate tThr-CR" [bib_ref] Mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations, Gibb [/bib_ref]. Because Procellariiformes/Pelecaniformes/Ciconiiformes and hornbills are generally not assumed to be sister taxa, our study indicates the independent evolution of the rare "duplicate tThr-CR" gene order. This gene order has been assumed to constitute an intermediate form between the "ancestral avian" and the "remnant CR(2)" gene orders, which is characterized by two apparently functional control region duplicates [bib_ref] Mitochondrial genomes and avian phylogeny: complex characters and resolvability without explosive radiations, Gibb [/bib_ref] [bib_ref] Bird mitochondrial gene order: insight from 3 warbler mitochondrial genomes, Singh [/bib_ref].
None of the peculiar features found in both Philippine hornbills was found in the control region of African hornbills -the closest relatives for which the CR has been examined [bib_ref] Characterization and evolution of the mitochondrial DNA control region in hornbills (Bucerotiformes), Delport [/bib_ref]. Instead, the authors assumed the typical avian gene order ("ancestral type") for Bucorvus leadbeateri and several species of the genus Tockus. However, in the light of our results this assumption has to be re-examined. Delport et al. [bib_ref] Characterization and evolution of the mitochondrial DNA control region in hornbills (Bucerotiformes), Delport [/bib_ref] sequenced only part of the mt genome (between tRNAs T and F), thus a duplication event might have been overlooked. Concerning further studies on the CR, we suggest for all avian taxa to sequence at least once the complete fragment between the end of NADH5 and the beginning of 12S because all duplication events known so far have been located in this section.
Whereas repeat motifs are found in domain III of the Philippine hornbills, Delport [bib_ref] Characterization and evolution of the mitochondrial DNA control region in hornbills (Bucerotiformes), Delport [/bib_ref] discovered such a motif in domain I of all African hornbills examined. Thus, an alignment of the CRs of the Philippine with those of the African hornbills was only possible for the conserved central domain II. In a phylogenetic analysis of this central domain, the two Philippine hornbills cluster together and are sister to the African species (see Additional file 2:. ML-analysis of the central domain of the control region of Bucerotidae). In this data set, however, the authenticity of the published sequence of Bucorvus leadbeateri is rendered questionable, as it is indistinguishable from Tockus erythrorhynchus, despite the fact that Bucorvus and Tockus species are morphologically clearly apart. Furthermore, the assumption that the repeat motif in domain I of the African hornbills had arisen before the adaptive radiation of all hornbill species, but after the divergence of hornbills from other avian taxa [bib_ref] Characterization and evolution of the mitochondrial DNA control region in hornbills (Bucerotiformes), Delport [/bib_ref] clearly has to be rejected, as (1) Philippine hornbills do not show this sequence pattern and (2) our re-analysis of the data on African hornbills strongly suggest that Delport et al. [bib_ref] Characterization and evolution of the mitochondrial DNA control region in hornbills (Bucerotiformes), Delport [/bib_ref] studied only members of the genus Tockus.
# Conclusion
Our full mt genome analysis of hornbill species revealed a large tandem duplication. Sequences within individuals are homogenized, except for the central putative Replication Fork Barrier. This sequence pattern suggests very frequent recombination of the mitochondrial genome. The studies of Eberhard et al. [bib_ref] Duplication and concerted evolution of the mitochondrial control region in the parrot..., Eberhard [/bib_ref] and Ogoh and Ohmiya [bib_ref] Concerted evolution of duplicated control regions within an ostracod mitochondrial genome, Ogoh [/bib_ref] were the first on concerted evolution in mitochondrial genomes in single species. In the ostracod Vargula hilgendorfii, Ogoh and Ohmiya [bib_ref] Concerted evolution of duplicated control regions within an ostracod mitochondrial genome, Ogoh [/bib_ref] detected frequent gene conversion and suggested repeated deletion and exact duplication in every replication cycle as the underlying mechanism. For hornbills, this mechanism is very unlikely, as it would imply that deletion and exact duplication occur synchronously on both sides of the RFB in every replication cycle. In a study on mtDNA of mangrove killifishes, Tatarenkov and Avise [bib_ref] Rapid concerted evolution in animal mitochondrial DNA, Tatarenkov [/bib_ref] found indications for frequent recombination, but did not present any underlying molecular mechanism. In addition, they -as others -detected recombination specifically in the control region. For the case of Philippine hornbills, we present evidence for frequent gene conversion by recombination of a large section of the mitochondrial genome, encompassing several coding genes. Morris-Pocock et al. [bib_ref] Concerted evolution of duplicated mitochondrial control regions in three related seabird species, Morris-Pocock [/bib_ref] sequenced only one individual of each booby species, but they also assume that more than the CR, namely part of Cytb/tRNA T/tRNA P/NADH6/ tRNA E, evolve in concert. A possible mechanism is the existence of a Replication Fork Barrier, where mt genome replication is halted such that the 3' end of the replicated strand remains free and might hence easily recombine [bib_ref] Phylogeny, recombination, and mechanisms of stepwise mitochondrial genome reorganization in mantellid frogs..., Kurabayashi [/bib_ref]. While there is no reason to assume that this mechanism is restricted to those species with a duplication in their mt genome, we argue that such duplication greatly facilitates our ability to unravel recombination: Without a duplication, recombination might affect orthologues and might hence go undetected. The duplication creates the additional possibility of intraindividual recombination among paralogues sequence parts. Such recombination causes a homogenization among these paralogues, clearly differing from the expectation of independent evolution after gene duplication.
# Methods
## Sampling and dna extraction
One drop of blood per sample was taken from captive hornbills kept by the Philippine Endemic Species Conservation Project (PESCP) on Panay, Philippines, and stored in 1 ml Queen's Lysis Buffer [bib_ref] Preservation of avian blood and tissue samples for DNA analyses, Seutin [/bib_ref]. DNA extraction was performed using the DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions for blood samples. The two genomes presented here were sequenced using DNA from two single individuals. To test the hypothesis that recombination occurs within individuals, five further unrelated individuals of each species were sampled.
The research followed internationally recognized guidelines and applicable national legislation. We received ethical approval from the deputy of animal welfare of the University of Potsdam.
## Amplification, sequencing and cloning
To minimize the possibility of obtaining nuclear copies of mt genes, we amplified longer fragments starting by using the primers Pen_Cyt1065-for and Buce_12S240rev (~4.5 kb) designed from published sequences of Penelopides spp. and other Bucerotidae and related taxa from NCBI databank (see Additional file 3: . PCR primers used to amplify and sequence mt gene fragments). The PCRs were performed using a longrange polymerase (LA Taq™, TaKaRa Bio Inc, Shiga, Japan). 15 μl-reaction volumes were set up as follows: 7.5 μl sterilized distilled water, 1.5 μl 10 x LA PCR™ Buffer II (Mg 2+ free), 1.5 μl 25 mM MgCl 2 -solution, 2.4 μl dNTP Mixture (2.5 mM each), 0.5 μl of each primer (2 mM), 1 μl DNA template (~20 ng/μl), 0.08 μl TaKaRa LA Taq™ (5 u/μl). The reaction was performed under the following conditions: denaturing at 94°C for 1 min, followed by 30 cycles of denaturing at 98°C for 10 s and elongating at 70°C for 4 min (without additional primer annealing step), and finally, an extended elongation period of 10 min at 72°C. The EXO-AP purified product was sequenced directly with PCR primers and additionally with internally primers designed by primer walking (see fragment 5 in Additional file 3: . PCR primers used to amplify and sequence mt gene fragments). The sequencing reactions were performed using BigDye Terminator Cycle Sequencing reagents version 3.1 and run on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.
To test for the existence of a duplicated control region we amplified a product with primers AcePen_KRII-for and AcePenGlu-rev (conditions as above, but 56°C annealing temperature for 20 s and 68°C elongating temperature for 3 min). This product was sequenced with PCR primers and internal primers (seeand fragment 4 in Additional file 3: . PCR primers used to amplify and sequence mt gene fragments).
Due to the detected duplication event, we amplified the complete mt genomes in five overlapping fragments, using the following primer combinations:
1.~14 kb: AcePen_12S_68-for and AcePen_Cytb250rev/AcePen_Cyt1018-rev (annealing and elongation both at 68°C for 14 min together); 2.~1.6 kb: AcePen_Cytb253-for and AcePenGlu-rev (annealing at 60°C, elongation at 68°C for 1.5 min); 3.~3.2 kb (A. waldeni),~3,6 kb (P. panini): Ace-Pen_Glu-for and AcePen_Cyt1018-rev (annealing at 60°C, elongation at 68°C for 3 min); 4.~2.8 kb (A. waldeni),~3,1 kb (P. panini): Ace-Pen_KRII-for and AcePen_Glu-rev (annealing at 56°C , elongation at 68°C for 3 min); 5.~3.8 kb (A. waldeni),~4,5 kb (P. panini): Pen_-Cyt1065-for and Buce_12S240-rev (annealing and elongation both at 70°C for 4 min together).
PCR products were subsequently sequenced by using PCR primers and internal primers (see Additional file 3: . PCR primers used to amplify and sequence mt gene fragments). Because of apparent length heteroplasmy at the end of the control regions, we performed nested PCRs on fragment 4 with the primers Ace-Pen_KR_Rep-for and AcePen_Cyt638-rev and on fragment 5 with the primers AcePen_KR_Rep-for and Acewal_KR_Z-rev for A. waldeni and Penpan_KR_Z-rev for P. panini, respectively (annealing at 56.2°C, elongation at 70°C for 2 min (for sequences of primers see Additional file 3: . PCR primers used to amplify and sequence mt gene fragments). Another nested PCR was performed on fragment 5 using primers AcePen_K-RII-for and AvesDiv_Phe-rev. All these nested PCR products were cloned using the TOPO TA Cloning Kit for Sequencing (Invitrogen) according to manufacturer's instructions. At least 30 clones of each PCR were sequenced.
1,930 bp encompassing the duplicated parts of Cytb, tRNA T/rRNA P/NADH6/tRNA E and domains I and the first parts of domains II of the CRs of 10 additional individuals were determined by PCR amplification and sequencing the fragments 2, 3, 4, and 5 (cf..
## Alignments and gene annotation
Transfer RNA genes (tRNA) were identified by their potential secondary structure and anticodon sequence using the tRNAscan-SE Server [bib_ref] tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic..., Lowe [/bib_ref]. tRNA S2 was not found by the server. Thus, we constructed the secondary structure manually using tRNA S2 alignments from other birds. The boundaries of ribosomal RNA genes (rRNA) and the control region were inferred from boundaries of flanking genes under the assumption that there are neither intergenic spacers nor overlaps. Start positions of protein-coding genes preceded by tRNAs were defined by the first potential start codon after the end of the flanking tRNA. Start positions of proteincoding genes preceded by other protein-coding genes were determined by aligning them to other avian mt genomes using the BioEdit Sequence Alignment Editor [bib_ref] BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows..., Hall [/bib_ref]. This method was also used to verify the boundaries of all other genes. Stop codons of all protein-coding genes were determined according to Slack et al. [bib_ref] Two new mitochondrial genomes (penguin and goose) and a summary of birds..., Slack [/bib_ref].
## Analyses of sequence data
To infer the pattern of evolution of the duplicated region, we conducted a phylogenetic analysis for 6 individuals per species. The two copies were included separately in each analyses and are designated by an abbreviation of the species name, a unique number for the individual, and a roman letter to distinguish the copies (e.g., Pp-1-I and Pp1-1-II for the two duplicates of individual number 1 of Penelopides panini). We conducted separate analyses for the region without the putative Replication Fork Barrier (RFB) and for the putative RFB fragment only (for determination of the RFB region seeand text below).
All four datasets (Pp_without_RFB, Pp_RFB_only, Aw_without_RFB, Aw_RFB_only) were aligned in BioEdit [bib_ref] BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows..., Hall [/bib_ref]. Maximum likelihood (ML) analysis of the dataset was conducted using RAxML version 7.0.3 [bib_ref] Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models, Stamatakis [/bib_ref] , using GTR+GAMMA+P-Invar model parameters (4 gamma categories). GTR is the only available nucleotide substitution model in RAxML.
To test for gene conversion, we compared (a) the mean p-distance between duplicates within any individual (πdupl) and (b) the mean p-distance between individuals separately for each of the two duplicates (πind). This
[fig] Figure 1: C....A............................A...T....C...C....A..C Aw-3 AT.......-................................T.........C.T.A... Aw-4 AT.......-................................T.........C.T.A... Aw-5 .........-...................A............T...........T.A... Aw-6 ..T......-.........................G......T..C....T..G.TAGG. Aw-1 .....AACT-ACATC-CCACAAA-.CTGCAGCAACTCGAACCTGT.A.GGT......... Aw-2 ...C.AACT-ACATC-CCACAAA-.CTGCAGCAACTCGAACCTGT.A.GGTC....A..C Aw-3 AT.C.AACT-ACAT.-CCACAAA-.CTG.AGCAACTCGAA.CTGT.A.GGT.C.T.A... Aw-4 AT.C.AACT-A.AT.-CCACAAA-.CTG.AGCAACTCGAA.CTGT.A.GGT.C.T.A... Aw-5 .....AACT-ACAT.-CCACAAA-CCTG.AGCAACTCGAACCTGT.A..GT...T.A... Aw-6 ..T..AACT-ACATC-CCACAAA-.CTGCAGCAACTCGAACCTGT.A.GGT..G.TAGG. ..........C..............T....ACG.C.A..T.T.C.GTGT.TG.T.TAAG.C Pp-3 .GTC..........C.........T....T....ACG.C.A..T...C.GTGT.TG.TTTAAG.C Pp-4 ..T..........C.....................C.CCT..A.C.A.....T..G.....A.AC Pp-5 ..T..........C........................CT.C....A.....T.TG........C Pp-6 ..T...........C.......................CT......A.A...TC.GTT......C Pp-1 .....CCTTA.CTCACGTAAC.CT.CACATA-.C............................... Pp-2 .GTC.CCTTAACTCACGTAAC.CT.CACA.A-.CA...C.A..T.T.C.GTGT.TG.T.TAAG.C Pp-3 .GTC.CCTTAACTCACGTAAC.C..CACA.A-.CA...C.A..T...C.GTGT.TG.TTTAAG.C Pp-4 ..T..CCTTA.CTCACGTAACCC..CACATA-.C....CT..A.C.A.....T..G.....A.AC Pp-5 ..T..CCTTA.CTCACGTAACCC..C.CATA-.C....CT.C....A.A...T.TG........C Pp-6 ..T.ACCTTA.CTCACGTAACCC..CACATA-CC...CCT......A.A...TC.GTT.....mtDNA genome organization in Philippine Hornbills (only tandemly duplicated part) (A) Gene order and position of five overlapping PCR-amplificates. (B, C) Variable sites of 1,930 bp alignments of the duplicated fragments of A. waldeni (B) and P. panini (C). The grey shaded sections represent the putative Replication Fork Barrier (RFB) regions. πDupl is the average diversity among duplicates within any individual. πInd is diversity among individuals for the same duplicate (I or II). [/fig]
[fig] Figure 2: Nested PCR-amplificates spanning over the repetitive units in domain III of the control region. Number of repeat units is indicated by the scale superimposed over the amplificates. (A) CRI of A. waldeni (dominant: 9 repeats). (B) CRI of P. panini (dominant: 12 repeats). (C) CRII of A. waldeni (dominant: 6 repeats). (D) CRII of P. panini (dominant: 10 repeats). [/fig]
[fig] Figure 3: |....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. Aw 1 (B) -TTTCTAACAACACTAGGAACATTTAACTAAAAATTTTACTAAATTTTTGTCATTT-ATTGCTTACACG--TTTATTTCACATACTATTTCCGCTGAAATTG-------CATTAATAAGCCC---------Aw 2 (B) -................................TA..C.............T....-..........TA--C.............................A-------........TTA.ATTAACAAA-Aw 3 (B) -.......................................................-............--..............................A-------........TTA.ATTAACAAA-Aw 4 (B) -.......................................................C...........ATGC..............................-------.............---------Aw 5 (B) -................................TA..C..................C...........ATGC..............................-------.............---------Aw 6 (B) -...................T............TA..C......A......T....-..........TA--C.............................A-------........TTA.ATTAACAAA-Aw 7 (M) -...T...............T............TA..C......A......T....-..........TA--C..............................-------.............---------Aw 8 (M) -...T...............T............TA..C..................C...........ATGC..............................-------.............---------Aw 9 (M) -...T...............T............TA..C..................C...........ATGC.............................A-------........TTA.ATTAACAAA-Aw10 (M) --..T...............T............TA..C......A......T....-..........TA--C.............................A-------........TTA.ATTAACAAA-Aw11 (M) -...T...............T............TA..C.............T....-..........TA--C.............................A-------........TTA.ATTAACAAA-Aw12 (M) CCA.................T............TA..C.............T....-..........TA--C.............................A-------........TTA.ATTAACAAA-Aw13 (M) CCA.................T............TA..C......A......T....-..........TA--C.............................A-------........TTA.ATTAACAAA-Aw14 (E) CCA.................T............TA..C.............T....-..........TA--C.............................A-------.......--TA.ATCAATAG--Aw15 (E) CCA.................T............TA..C......A......T....-..........TA--C.............................A-------.......--TA.ATCAATAG--Aw16 (BE) -.......................................................-............--...............................-------.......--TA.ATCAATAG--Aw17 (BE) -.......................................................-............--..............................A-------.......--TA.ATCAATAG--Aw18 (BE) -.......................................................-..........TA--C.............................A-------.......--TA.ATCAATAG--Aw19 (BE) -...................T............TA..C..........-..T....-..........TA--C.............................A-------.......--TA.ATCAATAG--Aw20 (BE) -................................TA..C.............T....-..........TA--C.............................A-------.......--TA.ATCAATAG--Aw21 (E) -...T...............T............TA..C......A......T....-..........TA--C.............................A-------.......--TA.ATCAATAG--Aw22 (E) -...T...............T............TA..C.............T....-..........TA--C.............................A-------.......--TA.ATCAATAG--Aw23 (E) -...T...............T............TA..C..................C...........ATGC.............................A-------.......--TA.ATCAATAG--Pp 1 (B) -.................................A..CGT................-....A.--T..ATA..............................A-------............T---------Pp 2 (B) -.................................A..CGT................-....A.--T..ATA..............................A-------.........T...CCCAACAAC Pp 3 (M) -...................T............TA..C.............T....-......--T..ATAC.............................A-------.........T...CCCAACAAC Pp 4 (M) -...T...............T............TA..C...........A..G...C...A..C....ATGC.............................A-------............T---------Pp 5 (M) -...T...............T............TA..C...........A..G...C...A..C....ATGC.............................A-------.........T...CCCAACAAC Pp 6 (M) -...T...............T............TA..C...........A..G...T......--T..ATAC.............................A-------.........T...CCCAACAAC Pp 7 (M) -...T...............T............TA..C.............T....-......--T..ATAC.............................A-------............T---------Pp 8 (M) -...T...............T............TA..C.............T....-......--T..ATAC.............................A-------.........T...CCCAACAAC Pp 9 (M) -CC.T...............T............TA..C.............T....-......--T..ATAC.............................A-------.........T...CCCAACAAC Pp10 (M) -CC.T...............T............TA..C.............T....-....----T..ATAC.............................A-------.........T...CCCAACAAC Pp11 (BE) -.................................A..CGT................-....A.--T..ATA..............................ATTAATTG..C......C..----------Pp12 (E) -...T...............T............TA..C...........A..G...C...A..C....ATGC.............................ATTAATTG..C......C..----------Pp13 (E) -...T...............T............TA..C.............T....-......--T..ATAC.............................ATTAATTG..C......C..----------Pp14 (E) -...T...............T............TA..C.............T....-....----T..ATAC.............................ATTAATTG..C......C..----------Pp15 (E) -CC.T...............T............TA..C.............T....-......--T..ATAC.............................ATTAATTG..C......C..----------Pp16 (E) -CC.T...............T............TA..C.............T....-....----T..ATAC.............................ATTAATTG..C......C..----------Repeat units found in CRI und CRII of A. waldeni (Aw) and P. panini (Pp). B: found only at the beginning. M: found only between B and E. E: found only at the end. BE: repeat unit of clones with only one unit. [/fig]
[fig] Figure 4: C) are exempted (B, D). This homogenization encompasses the whole duplicated part of Cytb, tRNA T/tRNA P/NADH6/tRNA E, the first and Phylogenetic analyses for sections of the duplicated mt genome region. ML, GTR+Γ+I-model of sequence evolution. Datasets are: (A) A. waldeni, section with high similarity between paralogues within individuals (=region of inferred recombination, white in [/fig]
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10.7555/JBR.29.20150068
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CCBY
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4585439
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26442749
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s2orc_pubmed_articles
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A simple practical balloon anchoring technique within the guide catheter for chronic total occlusion (CTO) of the coronary artery
[fig_ref] Figure 1: Detailed steps of the new technique for withdrawing the microcatheter [/fig_ref] [fig_ref] Figure 1: Detailed steps of the new technique for withdrawing the microcatheter [/fig_ref]
[fig] Figure 1: Detailed steps of the new technique for withdrawing the microcatheter. A: The microcatheter is withdrawn to the end of the ascending aorta. The predilation balloon is advanced into the distal microcatheter alongside the guide catheter. B: Release the predilation balloon at 8-10atm. The guidewire is anchored at the wall of the guide catheter to fix the guidewire and then the microcatheter is withdrawn. [/fig]
[fig] Figure 2: Operation process of the patient performed with the new technique. A: CAG showed completely obstruction in the proximal RCA.B: The microcatheter advanced into the distal part of the RCA through the CTO lesion. C: The microcatheter was withdrew within the guide catheter (at the end of the the ascending aorta). The balloon was dilated with 8-10atm when the predilation balloon was inserted 2-3cm distal the microcatheter to fix the guidewire at the inside wall of the guide catheter. The microcatheter was withdrawn slowly with no shifting of the guidewire. D: CAG after the operation showed no stenosis in the distal vessels of RCA and TIMI 3 flow. [/fig]
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10.5811/cpcem.2020.6.46961
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CCBY
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7434238
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32926716
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s2orc_pubmed_articles
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Chest Wall Pain after Minor Trauma
## Case presentation
While deployed during Hurricane Harvey with a Federal Emergency Management Agency Task Force, a 30-year-old male presented to the medical team for left-sided chest pain. He had been leaning over the rail of a military truck during search and rescue operations and developed pain and a "pulling sensation" when moving his left upper extremity. He had no significant past medical history and was well appearing with normal vital signs. Examination of the chest revealed a tender palpable cord along the left anterolateral chest wall without overlying erythema or warmth (Image).
# Discussion
This case highlights a presentation of Mondor's disease secondary to blunt trauma in a unique, austere environment. Mondor's disease is a superficial thrombophlebitis first described by Charles Fagge in 1870 and later described by French surgeon Henri Mondor in 1939. [bib_ref] Trauma on a recently augmented breast as a trigger for Mondor's disease, Yordanov [/bib_ref] [bib_ref] Mondor's disease: a review of the literature, Amano [/bib_ref] [bib_ref] Bodybuilding-induced Mondor's disease of the chest wall, Tröbinger [/bib_ref] Initially, the diagnosis referred specifically to superficial thrombophlebitis of the lateral thoracic, thoracoepigastric, or superior epigastric veins of the thoracoabominal wall. Currently, the diagnosis has expanded to include thrombosis of the dorsal penile vein. [bib_ref] Mondor's disease: a review of the literature, Amano [/bib_ref] [bib_ref] Mondor's disease of the penis, Hamilton [/bib_ref] California Task Force 6 -Urban Search and Rescue, Riverside, California Arrowhead Regional Medical Center, Department of Emergency Medicine, Colton, California California Task Force 1 -Urban Search and Rescue, Los Angeles City Fire Department, Los Angeles, California
[formula] * † ‡ [/formula]
The underlying etiology of Mondor's disease is varied and in many cases unknown. It can be related to trauma, physical activity, breast surgery, and rarely breast carcinoma. [bib_ref] Mondor's disease, Vijayalakshmi [/bib_ref] thought that an initial injury to the vein leads to inflammation, thrombosis, and fibrosis, although some cases are believed to be related to lymphangitis. 1,2 Symptoms typically last 4-8 weeks with spontaneous resolution. Treatment consists of nonsteroidal anti-inflammatory drugs and warm compresses. It is imperative that possible underlying causes are considered such as disease processes that result in a hypercoagulable state, vasculitis/vascular diseases, carcinoma and, in the case of penile Mondor's disease, sexually transmitted diseases. [bib_ref] Mondor's disease: a review of the literature, Amano [/bib_ref] Diagnosis is generally based on clinical presentation; however, it can be confirmed with ultrasound.
Institutional Review Board approval has been obtained and filed for publication of this image in emergency medicine.
[table] It is: Image. Subcutaneous cord along the anterolateral thoracoabdominal wall. Clinical Practice and Cases in Emergency Medicine [/table]
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10.1186/s12871-015-0104-y
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CCBY
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4566491
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26357836
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s2orc_pubmed_articles
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Spinal anaesthesia at low and moderately high altitudes: a comparison of anaesthetic parameters and hemodynamic changes
Background: Hypoxemia caused high altitude leads to an increase and variability in CSF volume. The purpose of this prospective study was to detect the differences, if any, between moderately highlanders and lowlanders in terms of anaesthetic parameters under neuroaxial anaesthesia. Methods: Consecutive patients living at moderately high altitude (Erzurum, 1890 m above the sea level) and sea level (Sakarya, 31 m above the sea level) scheduled for elective lower extremity surgery with spinal anaesthesia were enrolled in this study (n = 70, for each group). Same anaesthesia protocol was applied for all patients. Spinal anaesthesia was provided with hyperbaric bupivacaine 0.5 %, 9 mg (1.8 mL) in all patients. Anaesthetic characteristics and hemodynamic parameters of patients were recorded. The findings obtained in two different altitudes were compared using appropriate statistical tests. If data was not normally distributed, comparisons were determined using the Mann-Whitney U-test. Comparisons were determined using the Independent T test when data was normally distributed and Fisher's exact test was used to compare the percentage values. Results: Duration of the block procedure (minutes) was significantly shorter at the sea level (14.34 ± 0.88) than at moderate altitude (20.38 ± 1.46) (P < 0.001). Motor block duration (minutes) was higher at the sea level compared to the moderate altitude (310.2 ± 104.2, 200.4 ± 103.2; respectively; P < 0.05). Also, the sensory block time (minutes) was higher at the sea level compared to moderate altitude (200.2 ± 50. minutes vs. 155.2 ± 60.7 min; respectively; P < 0.05). Moderate altitude group had significantly higher MABP values at baseline, during surgery and at postoperative 1 st and 2nd hours than in the sea level group (P < 0.05, for all). Moderately high altitude group had lower heart rate values at baseline, during surgery and postoperative 1 st and 2 nd hours compared with the sea level group (P < 0.05). PDPH was seen more frequently (7.14 vs. 2.85 %; P < 0.05) at moderate altitude.Conclusions: Hemodynamic variations and more anaesthetic requirements following the spinal anaesthesia may be observed at moderately high altitudes compared to the sea level.
# Background
Individuals living at high and moderate altitudes have respiratory, cardiovascular and haematological changes in relation to the oxygen uptake and transport [bib_ref] Physiology and pathophysiology at high altitude: considerations for the anesthesiologist, Leissner [/bib_ref] [bib_ref] The heart and pulmonary circulation at high altitudes: healthy highlanders and chronic..., Penaloza [/bib_ref]. An increase in blood viscosity, a decrease in carbon monoxide diffusion capacity, an increase in cerebral arterial blood flow, a reduction in blood volume and a decline in cardiac output occur in response to hypoxemia at high altitude [bib_ref] The heart and pulmonary circulation at high altitudes: healthy highlanders and chronic..., Penaloza [/bib_ref]. It has been reported that the amount of cerebrospinal fluid (CSF) increases [bib_ref] Cerebral artery dilatation maintains cerebral oxygenation at extreme altitude and in acute..., Wilson [/bib_ref] and acid-base balance of CSF changes because of hypoxemia at high altitude [bib_ref] Ccomparison of cisternal and lumbar cerebrospinal fluid pH in high altitude natives, Blayo [/bib_ref]. On the other hand, Sorensen et al. [bib_ref] Cerebrospinal fluid acid-base composition at high altitude, Sorensen [/bib_ref] showed lower pH values of CSF in high-landers than in low-landers. They suggested that these differences in pH values of CSF may be associated with hyperventilation in high-altitude natives.
Spinal anaesthesia provides nerve blockade in a large part of the body during surgery with a smaller dose of local anaesthetic and shorter surgery onset time. However, it is difficult to keep the spread of local anaesthetic under control through the CSF after adequate block for surgery was provided [bib_ref] Intrathecal drug spread, Hocking [/bib_ref]. So, there is an increased risk of complications due to extensive spread of local anaesthetic through the CSF. Many factors such as age, height, weight and the lumbosacral cerebrospinal fluid volume affect the intrathecal spread of the injected local anaesthetics [bib_ref] Intrathecal drug spread, Hocking [/bib_ref]. It was shown that hypoxemia caused high altitude leads to an increase and variability in CSF volume [bib_ref] Cerebral artery dilatation maintains cerebral oxygenation at extreme altitude and in acute..., Wilson [/bib_ref] and these changes are the most important factor that contributes to the variability in the spread of the spinal sensory anaesthesia [bib_ref] Lumbosacral cerebrospinal fluid volume is the primary determinant of sensory block extent..., Carpenter [/bib_ref].
The high altitude anaesthesia literature is limited [bib_ref] Propofol-fentanyl anaesthesia at high altitude: anaesthetic requirements and haemodynamic variations when compared..., Puri [/bib_ref] [bib_ref] Characteristics of mepivacaine axillary brachial plexus block performed at 2800 m of..., Fuzier [/bib_ref] and the effect of moderately high altitude on central neuraxial block has not been studied. Because there is clinical evidence demonstrating the changes in CSF volume and contents under hypoxic conditions [bib_ref] Cerebral artery dilatation maintains cerebral oxygenation at extreme altitude and in acute..., Wilson [/bib_ref] [bib_ref] Ccomparison of cisternal and lumbar cerebrospinal fluid pH in high altitude natives, Blayo [/bib_ref] [bib_ref] Cerebrospinal fluid acid-base composition at high altitude, Sorensen [/bib_ref] , we hypothesized that duration of sensory and motor blockade in lowlanders would be equal or greater than that in moderately highlanders. Therefore, the purpose of this prospective study was to detect the differences, if any, between moderately highlanders and lowlanders in terms of anaesthetic parameters under neuroaxial anaesthesia.
# Methods
This prospective study was approved by the Ethics Committee of Ataturk University, Medical Faculty, Erzurum, Turkey (the date of approval by the ethics committee: 26.12.2013, the protocol number: 12). A total of 155 consecutive male subjects who were admitted to 2 study institutions at Ataturk University, Medical Faculty, Erzurum, Turkey (1890 m above the sea level) and Sakarya University, Medical Faculty, Sakarya, Turkey (31 m above the sea level) between January 1, 2014 and June 30, 2014 and who were scheduled elective lower extremity surgery with spinal anaesthesia were enrolled in this study. Patients with the age between 25 and 40 years, a body mass index between 20 and 25 kg/m2, ASA (the classification of the American Society of Anesthesiologists) physical status I or II were included [bib_ref] Propofol-fentanyl anaesthesia at high altitude: anaesthetic requirements and haemodynamic variations when compared..., Puri [/bib_ref]. Smokers, alcohol consumers, patients with psychiatric or neurological disorders, chronic diseases such as diabetes, a body mass index over 25, ASA physical status III or IV and contraindications to spinal anaesthesia such as coagulation disorder and infection at the puncture site were excluded from the study. All participants were permanently resident at moderately high altitude, as well as the sea level. Written informed consent was obtained from all participating patients. Demographic characteristics (age, weight and height) and indications for surgery of the patients were recorded. Before transferred to the operating room, every patient received an infusion of 500 mL ringer's lactate solution in 30 min via 18-gauge cannula in a forearm peripheral vein. Standard monitoring included non-invasive arterial pressure, electrocardiography and pulse oximetry were established for all patients in the operating room and post-anaesthesia care unit (PACU). Same anaesthesia protocol was applied for the patients living at moderately high altitude and the sea level. Each patient was premedicated with intravenous (iv) fentanyl (0.1 μg/kg) and midazolam (2 mg). After the skin infiltration with 2 % lidocaine, 26-gauge Quincke's needle was inserted through the L 2-3 / L 3-4 intervertebral space of patient in sitting position. Once free flow of the cerebrospinal fluid was obtained, hyperbaric bupivacaine 0.5 %, 9 mg (1.8 mL) was injected intrathecally. Then, the patient was enrolled in the supine position. Sensory block level was tested using pinprick tests and motor block level was evaluated with Modified Bromage scale (scale 0 = full flexion of foot, knee and hip, ie, no motor block; scale 1 = full flexion of foot and knee, unable to hip flexion; scale 2 = full flexion of foot, unable to knee and hip flexion; scale 3 = total motor block; unable to foot, knee, and hip flexion). When the sensory block reached the T 12 dermatome, surgery was initiated. If no signs of analgesia were observed within the first 10 min after the intrathecal injection, technique was considered as failed and general anaesthesia was administered for these patients. Oxygen was delivered with a face mask during surgery; iv midazolam (1 mg) for the complaint of discomfort was administered to each patient if necessary. During the operation, patients' mean arterial blood pressure (MABP), heart rate (HR) were monitored and recorded every 5 min. Ephedrine (iv, 2.5 mg) was administered in case of hypotension (a 30 % decrease in systolic blood pressure compared to preoperative values) and atropine (iv, 0.5 mg) was applied when bradycardia (the heart rate < 45 beats/min) was observed. The application time of the spinal anaesthesia, duration of the block procedure (the time from the start of the anaesthetic procedure to the development of full motor block), duration of surgery (the time from the start of the surgical incision to the completion of surgery), highest sensory block level, anaesthetic complications and the number of patients required midazolam during the surgical procedure were recorded. After the surgery, patients were transferred to the PACU. In PACU, postoperative analgesia was provided with iv 50 mg tramadol and an anaesthetist assessed the sensory and motor block levels every half-hour from the end of surgery until there is a complete recovery of the motor block and recovery of the sensation of the S 2 dermatome. Sensory block time (from the local anaesthetic injection to the recovery of S 2 dermatome), motor block duration (the time from the local anaesthetic injection to the complete motor function recovery) were recorded. Following a complete recovery of the motor and sensory blocks, patients were transferred to the orthopaedic ward. On the first and seventh days after the operation, patients were questioned in terms of post-dural puncture headache (PDPH, increased pain intensity upon standing up from a supine position) by an investigator via telephone interview. Crystalloid infusions (500 ml, 8-h intervals) and a non-steroidal anti-inflammatory drug applied to the patients diagnosed with PDPH.
Sample size was calculated as minimum 63 patients, based on our preliminary results to detect a minimum difference of 25 % in the duration of sensory block between the two groups with a power of 80 %, α of 0.05 and β of 0.20. Data were analysed using SPSS software 12.0 (SPSS Inc., Chicago, IL, USA) and calculated as mean ± standard deviation, P < 0.05 was considered significant. The findings obtained in two different altitudes were compared using appropriate statistical tests. The Kolmogorov-Smirnov test was used to assess the normal distribution of data. If data was not normally distributed, comparisons were determined using the Mann-Whitney U-test. Comparisons were determined using the Independent T test when data was normally distributed and Fisher's exact test was used to compare the percentage values.
# Results
Eligible patients for this study were analysed for the primary outcomes and are shown in the CONSORT flow diagram [fig_ref] Figure 1: CONSORT flow diagram [/fig_ref] [bib_ref] CONSORT 2010 vstatement: updated guidelines for reporting parallel group randomised trials, Schulz [/bib_ref]. During study period, 200 patients were eligible for this study in two institutes and 160 patients had inclusion criteria. One hundred and forty five patients agreed to participate in the study. Five patients were excluded from the study due to general anaesthesia requirement caused by prolonged surgery. Study population was consisted of 140 patients divided as 70 patients in each group. Clinical characteristics of the patients in both groups were similar. However, moderate altitude group had higher baseline MABP and lower baseline heart rate values than in the sea level group (P < 0.05) [fig_ref] Table 1: Baseline characteristics of the patients living at the sea level and moderately... [/fig_ref]. The spinal anaesthesia was performed at the L 3-4 (n = 120), L 2-3 (n = 20, nine in moderately high altitude group and eleven in the sea level group) interspace at the midline. Adequate surgical anaesthesia was provided within 15 min in all patients. The application time of the anaesthetic technique (5.85 ± 0.75 min vs. 5.90 ± 1.65 min) and duration of the surgery (52.15 ± 20.25 min vs. 51.68 ± 15.19 min) were similar between the groups. Duration of the block procedure was significantly shorter at the sea level (14.34 ± 0.88 min) than at moderate altitude (20.38 ± 1.46 min; P < 0.001). No patient in both groups required midazolam during surgery. Eight patients in the sea level group and 3 patients in the moderately high altitude group needed ephedrine 5-30 mg for treatment of hypotension during surgery (P < 0.05). PDPH was seen more frequently (7.14 vs. 2.85 %; P < 0.05) at moderate altitude [fig_ref] Table 2: Anaesthetic characteristics in groups [/fig_ref]. Motor block duration (minutes) was higher at the sea level compared to moderate altitude (310.2 ± 104.2, 200.4 ± 103.2; respectively; P < 0.05). Also, the sensory block time (minutes) was higher at the sea level compared to moderate altitude [fig_ref] Table 2: Anaesthetic characteristics in groups [/fig_ref]. The upper limit of the sensory blockade was significantly higher at the sea level compared to moderately high altitude (T 8 vs. T 10 ) (P < 0.001) [fig_ref] Table 3: Mean spread of the sensory block of the operative side at timed... [/fig_ref]. The period from the local anaesthetic injection to the complete motor function recovery was shorter in moderately high altitude group than in the sea level group [fig_ref] Table 4: Mean bilateral motor block intensity [/fig_ref].
Moderate altitude group had significantly higher MABP values at baseline, during surgery and in postoperative 1 st and 2 nd hours than in the sea level group (P < 0.05, for all). Patients in both groups had significantly lower MABP values during surgery and in postoperative 1 st hour compared to the baseline values (P < 0.005, for all) . Arterial hypotension was observed in 2 patients in the sea level group and 3 patients in moderate altitude group (P > 0.05). Moderately high altitude group had lower heart rate values at baseline, at any time points during surgery and postoperative 1 st and 2 nd hours compared to the sea level group (P < 0.05). Patients in both groups had significantly lower heart rate values during surgery compared to baseline values (P < 0.05) . Bradycardia was observed in two patients in each group. No patients in both groups had respiratory complications during surgery and post-operative period.
# Discussion
According to our literature review, this is the first study to compare onset and duration of central neuraxial block in patients living in moderately highland areas (1890 m above the sea level) with those in lowland areas (close to the sea level). Onset time of complete sensory and motor blocks was shorter in the sea level group compared to the moderately high altitude group. Also, higher upper limit of the sensory blockade and higher motor and sensorial block times were found at the sea level compared to moderately high altitude.
It has been reported that an anaesthetic technique that has the least impact on ventilation should be chosen for the patients at high altitude [bib_ref] Physiology and pathophysiology at high altitude: considerations for the anesthesiologist, Leissner [/bib_ref]. Also aspiration risk during anaesthesia induction increases in patients at high altitude due to the significantly delay in gastric emptying [bib_ref] Gastric emptying effects of dietary fiber during 8 hours at two simulated..., Hinninghofen [/bib_ref]. In this instance, the use of neuroaxial anaesthesia techniques at high altitude is more appropriate [bib_ref] Physiology and pathophysiology at high altitude: considerations for the anesthesiologist, Leissner [/bib_ref]. Spinal anaesthesia, a neuroaxial anaesthesia technique provides adequate anaesthesia for surgery with a shorter surgery onset time. Many factors affecting the intrathecal spread of injected local anaesthetics were identified such as sex, weight, height, intra-abdominal pressure and spinal anatomy [bib_ref] Intrathecal drug spread, Hocking [/bib_ref]. Also, variability in CSF volume was reported as the most important factor that contributes to the variability in the spread of the spinal sensory anaesthesia [bib_ref] Lumbosacral cerebrospinal fluid volume is the primary determinant of sensory block extent..., Carpenter [/bib_ref]. Increasing altitude leads to the changes in cardiac, cerebral and pulmonary systems such as an increase in cerebral arterial blood flow, a decline in cardiac output and an increase in CSF volume [bib_ref] Physiology and pathophysiology at high altitude: considerations for the anesthesiologist, Leissner [/bib_ref] [bib_ref] The heart and pulmonary circulation at high altitudes: healthy highlanders and chronic..., Penaloza [/bib_ref] [bib_ref] Cerebral artery dilatation maintains cerebral oxygenation at extreme altitude and in acute..., Wilson [/bib_ref].
We chose median effective dose of hyperbaric bupivacaine as 9 mg based upon a pilot study assessing clinical impression of motor blockade and analgesia duration in two different altitudes which this study was carried out. We observed that the sensory block spread to the T 10 Data was expressed as mean ± SD or n. *P <0.05, compared with the sea level group Results were expressed as mean ± SD dermatome was faster in the sea level group compared to moderately high altitude group. Also, sensory block spread was significantly more extensive at the sea level than in moderately high altitude. The duration of the sensory and motor blocks were significantly shorter in moderately high altitude group than in the sea level group. These significant differences between the groups may be a result of the greater CSF volume in the moderately high altitude group as increased cerebral arterial blood flow and CSF volume has been shown to be one of the compensatory mechanisms at high altitude in response to hypoxemia [bib_ref] The heart and pulmonary circulation at high altitudes: healthy highlanders and chronic..., Penaloza [/bib_ref] [bib_ref] Cerebral artery dilatation maintains cerebral oxygenation at extreme altitude and in acute..., Wilson [/bib_ref]. Although it was reported that CSF volume may have a crucial effect on intrathecal drug spread [bib_ref] Lumbosacral cerebrospinal fluid volume is the primary determinant of sensory block extent..., Carpenter [/bib_ref] , there are no detailed study on this issue in the literature due to the difficulties of measuring CSF volume accurately [bib_ref] Intrathecal drug spread, Hocking [/bib_ref]. On the other hand, cerebral blood flow alterations occur in high altitude [bib_ref] The heart and pulmonary circulation at high altitudes: healthy highlanders and chronic..., Penaloza [/bib_ref] and there is an inverse correlation between the intracranial blood volume and CSF volume [bib_ref] The Monro-Kellie hypothesis: applications in CSF volume depletion, Mokri [/bib_ref]. Also, it was reported that highlanders have lower CSF pH values than in lowlanders depending on the hyperventilation caused by hypoxia [bib_ref] Cerebrospinal fluid acid-base composition at high altitude, Sorensen [/bib_ref]. Higuchi et al. [bib_ref] Influence of lumbosacral cerebrospinal fluid density, velocity, and volume on extent and..., Higuchi [/bib_ref] reported a significant correlation between the CSF density and peak sensory block level, a positive correlation between the lumbosacral CSF volume and onset time of the complete motor block and a significant inverse correlation between the peak diastolic CSF velocity and duration of motor blockade. They concluded that CSF density and volume influence the spread of the spinal anaesthesia and that CSF volume also influences the duration of the spinal anaesthesia. It is unclear, however, as to which physiological mechanisms explain these differences in anaesthetic characteristics following spinal anaesthesia in moderately highlanders.
Heart rate values at baseline and during surgery were found to be lower in moderately highlanders compared to lowlanders in this present study. Similar to our results, Puri et al. [bib_ref] Propofol-fentanyl anaesthesia at high altitude: anaesthetic requirements and haemodynamic variations when compared..., Puri [/bib_ref] reported lower heart rate values at the baseline and during surgery in patients living at high altitude compared to patients living at low altitude. The cause of these low heart rates in moderately highlanders may be the increased parasympathetic neural activity caused chronic hypoxia at high altitude [bib_ref] Parasympathetic neural activity accounts for the lowering of exercise heart rate at..., Boushel [/bib_ref]. On the other hand, we observed higher MAP values at baseline and during surgery in patients living at moderately high altitude compared to the patients living at the sea level. Consistent with our results, Calbet [bib_ref] Chronic hypoxia increases blood pressure and noradrenaline spillover in healthy humans, Calbet [/bib_ref] reported that chronic hypoxia causes an increase in the systemic arterial pressure in healthy humans due to the marked activation of the sympathetic nervous system and reduced tissue hypoxia associated with acclimatization.
PDPH was observed more frequently at moderate altitude in this current study. Although the exact cause of the increased incidence of PDPH at moderately high altitude remains unknown, the changes in CSF volume and pressure due to increased cerebral blood flow caused by hypoxia may be a cause for this increased incidence of PDPH at high altitude. Also, a case of headache aggravated at altitude was reported by Batsis et al. [bib_ref] Intracranial hypotension: aggravation of headache at high altitude, Batsis [/bib_ref] They suggested that hyperventilation caused by hypoxia leads to an intracranial dehydration, cerebral vasodilation and brain engorgement. On the other hand, Wilson et al. [bib_ref] Cerebral venous system and anatomical predisposition to high-altitude headache, Wilson [/bib_ref] concluded that restriction in cerebral venous outflow is associated with the increased cerebral venous engorgement and with greater headache burden in response to hypoxia.
Puri et al. [bib_ref] Propofol-fentanyl anaesthesia at high altitude: anaesthetic requirements and haemodynamic variations when compared..., Puri [/bib_ref] investigated the effectiveness of the general anaesthetic agents on the anaesthetic requirements and hemodynamic variations at high and low altitudes. They concluded that high-altitude dwellers require significantly larger amounts of intravenous anaesthetic propofol. In another study, Fuzier et al. [bib_ref] Characteristics of mepivacaine axillary brachial plexus block performed at 2800 m of..., Fuzier [/bib_ref] evaluated the feasibility and pharmacodynamic profile of axillary brachial plexus nerve blocks performed in high altitude. They found no difference between the patients at high altitude (2877 m) and low altitude (150 m) in terms of onset times for blocks, duration of the sensory and motor blocks. However, there is no study in the literature comparing the effects of spinal 30th min T 10 (T 4 -T 12 )* T 8 (T 5 -T 12 )
45th min T 11 (T 4 -T 12 )* T 9 (T 6 -T 12 )
60th min T 12 (T 6 -L 4 )* T 11 (T 6 -L 2 ) 75th min L 1 (T 7 -S 2 )* T 12 (T 6 -L 3 )
90th min L 2 (T 7 -S 4 )* T 12 (T 6 -L 5 )
120th min L 4 (T 10 -S 4 )* L 2 (T 7 -S 2 )
150th min S 1 (T 10 -S 4 )** L 5 (T 2 -S 4 ) *P < 0.001, **P = 0.026, compared to the sea level group. Data were expressed as median (range) anaesthesia on hemodynamic and anaesthetic parameters in different altitudes.
The limitation of this present study is that a noninvasive hemodynamic evaluation was used. The present study was focused on the anaesthetic parameters. Also, our study population was consisted of young and good health patients. So, an advanced hemodynamic monitoring was not needed. Another limittion was the relatively small patient population. On the other hand, observer bias may be a factor influencing our results.
# Conclusions
Hemodynamic variations and further anaesthetic requirements following spinal anaesthesia may be observed at moderately high altitudes compared to the sea level. Shorter onset time of complete sensory and motor blocks and higher sensory level were found in the sea level group compared to the moderately high altitude. Also, the duration of the sensory and motor blocks was longer in the sea level group than in the moderately high altitude group. These differences may be associated with a physiological Mean arterial blood pressure values of patients in groups. *P <0.05, compared to moderately high altitude group. Patients in both groups had significantly lower MABP values during the surgery compared to baseline values (P < 0.05, for all) The comparison of heart rate values in groups. *P < 0.05, compared to the sea level group. Patients in both groups had significantly lower heart rate values during surgery and postoperative 1 st hour compared to baseline values (P < 0.05, for all) adaptation to chronic hypoxia in individuals living at moderately high altitude. Further studies including a greater number of patients and reporting data on cardiac output or stroke volume variations are needed to support our findings.
[fig] Figure 1: CONSORT flow diagram. The course of patients through this study was shown (200.2 ± 50. minutes vs. 155.2 ± 60.7 min; respectively; P < 0.05) ( [/fig]
[table] Table 1: Baseline characteristics of the patients living at the sea level and moderately high altitude [/table]
[table] Table 2: Anaesthetic characteristics in groups [/table]
[table] Table 3: Mean spread of the sensory block of the operative side at timed intervals following local anaesthetic injection in groups [/table]
[table] Table 4: Mean bilateral motor block intensity (Bromage scale: 0-3) at timed intervals following local anaesthetic injection in groups *P < 0.001, **P = 0.001, compared to the sea level group [/table]
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10.1155/2020/9125752
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CCBY
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7421792
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32832008
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s2orc_pubmed_articles
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Gentiopicroside, a Secoiridoid Glycoside from Gentiana rigescens Franch, Extends the Lifespan of Yeast via Inducing Mitophagy and Antioxidative Stress
Gentiopicroside (GPS), an antiaging secoiridoid glycoside, was isolated from Gentiana rigescens Franch, a traditional Chinese medicine. It prolonged the replicative and chronological lifespans of yeast. Autophagy, especially mitophagy, and antioxidative stress were examined to clarify the mechanism of action of this compound. The free green fluorescent protein (GFP) signal from the cleavage of GFP-Atg8 and the colocation signal of MitoTracker Red CMXRos and GFP were increased upon the treatment of GPS. The free GFP in the cytoplasm and free GFP and ubiquitin of mitochondria were significantly increased at the protein levels in the GPS-treated group. GPS increased the expression of an essential autophagy gene, ATG32 gene, but failed to extend the replicative and chronological lifespans of ATG32 yeast mutants. GPS increased the survival rate of yeast under oxidative stress condition; enhanced the activities of catalase, superoxide dismutase, and glutathione peroxidase; and decreased the levels of reactive oxygen species and malondialdehyde. The replicative lifespans of Δsod1, Δsod2, Δuth1, and Δskn7 were not affected by GPS. These results indicated that autophagy, especially mitophagy, and antioxidative stress are involved in the antiaging effect of GPS.
# Introduction
Aging is a time-dependent functional decline that is the primary risk factor for many diseases [bib_ref] The hallmarks of aging, López-Otín [/bib_ref] [bib_ref] Structure characterization and action mechanism of an antiaging new compound from Gastrodia..., Farooq [/bib_ref] , such as Alzheimer's disease, Parkinson's disease, and diabetes. With the intensive increase in the aging population worldwide, enormous personal, social, and economic challenges have been encountered by the modern society [bib_ref] Geroscience and the challenges of aging societies, Sierra [/bib_ref]. Therefore, studies on prolonging the lifespan, especially improving the healthspan to increase the quality of life and retard suffering of inevitability of elders, have been intensively conducted worldwide [bib_ref] The road ahead for health and lifespan interventions, Gonzalez-Freire [/bib_ref].
Gentiana rigescens (G. rigescens) grows in the southwest region of China. The roots of this Chinese herb medicine have been used to treat inflammation, hepatitis, and functional dyspepsia [bib_ref] Review on "Long-Dan", one of the traditional Chinese medicinal herbs recorded in..., Wang [/bib_ref]. Sheng Nong's Herbal Classic, a classic book of traditional Chinese medicine on Materia Medica, described that G. rigescens has effects on cognition improve-ment and antiaging. In our previous study, 11 novel neuritogenic benzoate-type molecules (named gentisides A-K) were isolated from G. rigescens [bib_ref] Gentisides A and B, two new neuritogenic compounds from the traditional Chinese..., Gao [/bib_ref] [bib_ref] Gentisides C-K: Nine new neuritogenic compounds from the traditional Chinese medicine Gentiana..., Gao [/bib_ref]. The mixture of gentisides (n-GS) was confirmed to have alleviation effects on the impaired memory of scopolamine-induced mouse model by inhibiting acetylcholinesterase activities and antioxidative stress and regulating the insulin-like growth factor 1 receptor/extracellular signal-regulated kinase (IGF-1R/ERK) signal pathway [bib_ref] Benzoate fraction from Gentiana rigescens Franch alleviates scopolamine-induced impaired memory in mice..., Li [/bib_ref]. These results possibly proved the cognition-improving effect of G. rigescens on the molecular basis, which was mentioned in Sheng Nong's Herbal Classic.
In the present study, we investigated the antiaging molecular base of G. rigescens. Budding yeast Saccharomyces cerevisiae is a common aging model that has replicative and chronological lifespans. The K6001 strain, a yeast mutant strain derived from W303, has a characteristic that only mother cells can produce daughter cells and daughter cells do not continue to mitosis in glucose medium [bib_ref] Structure characterization and action mechanism of an antiaging new compound from Gastrodia..., Farooq [/bib_ref] [bib_ref] A novel assay for replicative lifespan in Saccharomyces cerevisiae, Jarolim [/bib_ref]. The use of K6001 as a model to study replicative lifespan possesses many merits, such as low cost, short generation time, and easy operation. Therefore, the replicative lifespan assay of K6001 was utilised as a bioassay system to screen antiaging substances from food extracts and traditional Chinese medicines.
Autophagy is a highly conservative process used to degrade large structures, such as long-lived or damaged organelles and protein aggregates [bib_ref] Autophagy and metabolism, Rabinowitz [/bib_ref]. Accordingly, autophagy serves an adaptive role to protect organisms against diverse pathologies, including infections, cancer, neurodegeneration, aging, and heart disease [bib_ref] Autophagy in the pathogenesis of disease, Levine [/bib_ref]. The regulation of autophagy is extremely important for the maintenance of normal physiological functions [bib_ref] Decreased ovarian function and autophagy gene methylation in aging rats, Li [/bib_ref]. Reports show that enhancing autophagy can regulate aging and aging-related diseases and prolong lifespan [bib_ref] Autophagy and the cell biology of age-related disease, Leidal [/bib_ref]. For example, rapamycin, a well-known molecule, as an antiaging candidate, is reported to extend the lifespan of Caenorhabditis elegans (C. elegans), fruit fly, and mice, but the effects are abolished when the autophagy-related genes are knocked out or knocked down, suggesting that autophagy plays an important role in the life-prolonging effect of rapamycin [bib_ref] Rapamycin fed late in life extends lifespan in genetically heterogeneous mice, Harrison [/bib_ref] [bib_ref] Autophagy is required for extension of yeast chronological life span by rapamycin, Alvers [/bib_ref]. Mitophagy, a selective type of autophagy, which targets long-lived or damaged mitochondria to degradation, is regarded as a major mechanism responsible for mitochondrial quality control. The accumulation of damaged mitochondria is a common marker of aging and is related to diseases. However, the level of mitophagy markedly declines in mammalian tissues during normal aging, and the decline in mitophagy might fuel the vicious circle of induced oxidative stress and age-related tissue damage [bib_ref] The role of mitochondria in aging, Jang [/bib_ref]. Many studies have shown that stimulating mitophagy might have widespread beneficial effects in antiaging or age-related functional decline. For example, urolithin A, a metabolite of natural products, extends the lifespan of C. elegans, depending on the expression levels of the mitophagy genes pink-1, dct-1, and skn-1 and improves the muscle function in old mice with a high expression of mitophagy (Park2) transcripts [bib_ref] Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases..., Ryu [/bib_ref].
Oxidative stress is a disturbance in the balance between the production of free radicals and antioxidant defences, thereby causing cell aging. Free radicals, known as reactive oxygen species (ROS) and reactive nitrogen species [bib_ref] Role of ROS and RNS sources in physiological and pathological conditions, Meo [/bib_ref] , will be excessively produced when cells are under oxidative stress condition. The superfluous free radical attacks cell components and produces harmful substances, such as malondialdehyde (MDA), the product of lipid oxidation that can cause cross-linking polymerisation of proteins, nucleic acids, and other biological macromolecules [bib_ref] A review of recent studies on malondialdehyde as toxic molecule and biological..., Rio [/bib_ref]. However, these types of oxidative damage can be alleviated by antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). In our previous published paper, SOD1, SOD2, SUN family protein Uth1 (UTH1), and SKN7 genes were confirmed to be involved in oxidative stress and played an important role in the antiaging effects [bib_ref] Parishin from Gastrodia elata extends the lifespan of yeast via regulation of..., Lin [/bib_ref].
In our previous study, many antiaging substances were isolated from natural products, such as parishin, phloridzin, cucurbitacin B, cucurbitane glycosides, and a new compound named bis(4-hydroxybenzyl)ether mono-β-L-galactopyranoside from Gastrodia elata, apple branches, Pedicellus melo, fruits of Momordica charantia L and G. elata Blume, under the guidance of the K6001 yeast bioassay system [bib_ref] Structure characterization and action mechanism of an antiaging new compound from Gastrodia..., Farooq [/bib_ref] [bib_ref] Parishin from Gastrodia elata extends the lifespan of yeast via regulation of..., Lin [/bib_ref] [bib_ref] Anti-aging effects of phloridzin, an apple polyphenol, on yeast via the SOD..., Xiang [/bib_ref] [bib_ref] Cucurbitacin B exerts antiaging effects in yeast by regulating autophagy and oxidative..., Lin [/bib_ref] [bib_ref] Antiaging of cucurbitane glycosides from fruits of Momordica charantia L, Cao [/bib_ref]. In the present study, GPS was isolated from G. rigescens using the same bioassay system. Here, the antiaging activity and the mechanism of action of GPS were reported.
# Materials and methods
2.1. General. G. rigescens was purchased from Huqingyutang Chinese Pharmacy Hangzhou, Zhejiang Province. The identity of this plant was confirmed by Associate Professor Liurong Chen (College of Pharmaceutical Sciences, Zhejiang University), and a voucher specimen (no. 20190620) was preserved in Zhejiang University, Institute of Materia Medica. Preparative high-performance liquid chromatography (HPLC) analysis was performed using a HPLC system equipped with Elite P-230 pumps (Dalian Elite Inc., China). An Agilent Technologies 6224A Accurate-Mass time-of-flight liquid chromatography-mass spectrometry (TOF LC/MS) system was used to measure the mass spectra (Agilent Technologies Inc., Beijing, China). A Bruker AV III-500 spectrometer was used to record the nuclear magnetic resonance (NMR) spectra (Bruker, Karlsruhe, Germany). NMR chemical shifts in δ (ppm) were referenced to solvent peaks of δ C 49.0 for CD 3 OD (Cambridge Isotope Laboratories, Inc., MA, USA). Octadecyl-silane (Cosmosil 75 C18-OPN, Nacalai Tesque, Japan) and RP-18 plates (0.25 mm) (Merck KGaA, Darmstadt, Germany) were used for column chromatography and thin-layer chromatography analysis, respectively [bib_ref] Structure characterization and action mechanism of an antiaging new compound from Gastrodia..., Farooq [/bib_ref]. Resveratrol (Res) was purchased from J&K Scientific Ltd. (Beijing, China) and was used as a positive control.
## Preparation and
Determination of GPS. GPS was isolated from G. rigescens. Dried and powdered G. rigescens (100 g) was extracted in methanol (material/methanol, 1/10, w/v) at room temperature for 24 h with shaking. The methanol extract was filtrated, and the filtrate was concentrated, suspended in water, and successively partitioned with n-hexane, ethyl acetate, and n-butanol to obtain the active nbutanol fraction (7.5 g). The n-butanol fraction was separated by ODS open column and eluted with MeOH/H 2 O (20 : 80, 30 : 70, 40 : 60, 50 : 50, 70 : 30, 90 : 10, and 100 : 0) to give seven fractions. A part of the third fraction (30 mg) was purified through HPLC (Cosmosil 5C30-UG-5 (10 mm × 250 mm), flow rate: 3 mL/min, 30% aq. MeOH) to yield a pure active substance (23 mg, t R = 44 min). The chemical structure of the active molecule was identified to be gentiopicroside (GPS) by comparing HR ESI-MS and 13 C NMR data with those reported in literature [bib_ref] Iridoids. An updated review, part II, Boros [/bib_ref]. [bib_ref] Autophagy and the cell biology of age-related disease, Leidal [/bib_ref] Oxidative Medicine and Cellular Longevity K6001 background, YOM36, Δatg32 of YOM36, YOM38-Atg8-GFP, and BY4741 yeast strains were used in the present study. K6001 yeast was provided by Professor Breitenbach (Salzburg University, Austria), and other mutant yeast strains were constructed by Professor Matsuura (Chiba University, Japan). The replicative lifespan assay was performed in accordance with the protocol described in our previous study [bib_ref] Structure characterization and action mechanism of an antiaging new compound from Gastrodia..., Farooq [/bib_ref] [bib_ref] Parishin from Gastrodia elata extends the lifespan of yeast via regulation of..., Lin [/bib_ref]. Briefly, the K6001 yeast was inoculated in galactose medium (3% galactose, 2% hipolypeptone, and 1% yeast extract) for 24 h in a shaking incubator at 180 rpm and 28°C. The harvested yeast cells were then washed with PBS for three times, and approximately 4000 cells were spread on glucose agar plates (2% glucose, 2% hipolypeptone, 1% yeast extract, and 2% agar) containing GPS at concentrations of 0, 0.3, 1, 3, and 10 μM. The well-known antiaging molecule, Res, at a concentration of 10 μM was used as positive control. The agar plates were then incubated at 28°C, and 40 microcolonies formed on agar plate were randomly selected and counted daughters produced by each mother cell under microscopy. The replicative lifespan assay methods of mutants with K6001 background (Δuth1, Δskn7, Δsod1, Δsod2, and Δatg32 of K6001) were the same as that of the K6001 strain. The chronological lifespan assay was conducted as described in our previous report [bib_ref] Structure characterization and action mechanism of an antiaging new compound from Gastrodia..., Farooq [/bib_ref] [bib_ref] Cucurbitacin B exerts antiaging effects in yeast by regulating autophagy and oxidative..., Lin [/bib_ref]. YOM36 yeast were cultured in synthetic complete (SC) medium (2% glucose, 2% peptone, and 1% yeast extract) or synthetic defined (SD) medium (0.17% yeast nitrogen base without amino acids and ammonium sulphate (BD Difco), 0.5% ammonium sulphate, and 0.2% glucose) in a shaking incubator at 180 rpm and 28°C. After 24 h, the cells were inoculated into the SC or SD medium containing GPS at concentrations of 0, 1, and 3 μM with the initial OD600 value of 0.01 and incubated in a shaking incubator at 180 rpm and 28°C. On the third day, 200 cells from each treat or untreated groups were spread on glucose agar plates and cultured in an incubator at 28°C for 2 days. The colony-forming units (CFUs) of each plate were counted, and the CFUs were denoted as 100% survival at day 3. On the fifth day, the steps of the third day were repeated, and the survival rate of each group was calculated (CFUs of each plate/CFUs of the same plate on the third day × 100%). The steps were repeated every two days until the survival rate decline to less than 10%. The chronological lifespan assay methods of YOM36 mutants (Δatg32 of YOM36) were the same as those of YOM36 strain.
## Visualisation of autophagy and mitophagy of yeast
through Confocal Fluorescence Microscopy. The experiment was performed following a previously published literature [bib_ref] Cucurbitacin B exerts antiaging effects in yeast by regulating autophagy and oxidative..., Lin [/bib_ref]. Briefly, YOM38 yeast cells containing pR316-GFP-Atg8 plasmid were cultured in liquid glucose medium in a shaking incubator at 180 rpm and 28°C under dark conditions. After 24 h, the cells were collected and washed with SD medium and divided into several different groups with OD600 value of 0.1. The cells were then treated with GPS at concentrations of 0, 1, and 3 μM or Res at 300 μM and cultured for 22 h in the dark. Subsequently, the cells were stained with 4 ′ ,6-diamidino-2-phenylindole (DAPI) 20 μg/mL) for 10 min in the dark and then washed thrice with PBS. Yeast cells were observed and photographed with a two-photon confocal fluorescence microscope (Olympus FV1000BX-51, Tokyo, Japan). The experimental procedure for mitophagy was similar to that of autophagy. The difference is that the cells were stained with 250 nM MitoTracker Red CMXRos (Beyotime, Shanghai, P. R. China) at 37°C in the dark for 1 h before staining with DAPI. The percentage of cells with green fluorescence and the colocation of red and green fluorescence were calculated, and the data obtained were analysed on software. (c) Effect of GPS on the chronological lifespan of YOM36 yeast. * , * * , and * * * represent significant difference compared with the control group at p < 0:05, p < 0:01, and p < 0:001, respectively. The chronological lifespan assay is performed in the SC medium, and the survival rate less than 5% of each group is defaulted to 100% death.
3 Oxidative Medicine and Cellular Longevity dark conditions. After 24 h, the cells were collected and washed with SD medium and divided into several different groups with OD600 value of 0.1. The cells were then treated with GPS at concentrations of 0, 1, and 3 μM or Res at 300 μM and cultured for 22 h in the dark. Subsequently, yeast cells of different groups were collected and sonicated for 5 min. The cell lysates were centrifuged at 12,000 g for 15 min, and the supernatant was obtained for western blot analysis. The mitochondria were isolated from yeasts to obtain the protein, as described in references [bib_ref] The extraction of Schizosaccharomyces pombe mitochondria and mitochondrial RNA, Yang [/bib_ref] [bib_ref] Isolation of highly purified mitochondria from Saccharomyces cerevisiae, Glick [/bib_ref]. Briefly, YOM38 yeast cells containing pR316-GFP-ATG8 plasmid were treated GPS similar to the above description. Subsequently, the cell lysate was centrifuged twice for 5 min at 5000 g. The supernatants were removed to new tubes and centrifuged for 15 min at 12,000 g to obtain the mitochondrial pellet. The mitochondrial pellet was dissolved in a RAPI lysis buffer and incubated in ice for 20 min. The supernatants were obtained as protein sample after centrifugation at 12,000 g for 15 min. The protein concentrations were measured with a BCA Protein Assay Kit (CoWin Biotech, Beijing, China). Western blot analysis was performed as described in our previous study [bib_ref] Cucurbitacin B exerts antiaging effects in yeast by regulating autophagy and oxidative..., Lin [/bib_ref]. Approximately 20 μg protein was separated with SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies followed by secondary antibodies. The primary antibodies used are as follows: anti-GFP (Medical & Biological Laboratories, Nagoya, Japan), anti-β-actin (CoWin Biotech, Beijing, China), antiubiquitin (Cell Signalling Technology, Massachusetts, USA), and anti-VDAC1/Porin (Abcam Trading (Shanghai) Company Ltd., Shanghai, China) antibodies. The secondary antibodies used are as follows: horseradish peroxidase-linked antirabbit and antimouse IgGs (CoWin Biotech, Beijing, China). Antigens were visualised using an ECL Western Blot Kit (CoWin Biotech, Beijing, China) and digitised with ImageJ software.
## Real-time polymerase chain reaction (rt-pcr)
Analysis. The wild-type BY4741 were incubated with GPS at concentrations of 0 and 1 μM in glucose medium for 12 h at 28°C with shaking at 180 rpm. Total RNA was extracted using a hot phenol method. A reverse transcription method was utilised to synthesise cDNA using a HiFi-MMLV cDNA Kit (CoWin Biotech, Beijing, China) and 5 μg of RNA. Quantitative RT-PCR was performed by using CFX96 Touch (Bio-Rad, Hercules, USA) and SYBR Premix EX Taq (Takara, Otsu, Japan), as described in our previous study [bib_ref] Structure characterization and action mechanism of an antiaging new compound from Gastrodia..., Farooq [/bib_ref] [bib_ref] Parishin from Gastrodia elata extends the lifespan of yeast via regulation of..., Lin [/bib_ref]. The thermal cycling parameters are as follows: 40 cycles, 94°C for 15 s, 51.6°C for 15 s, and 68°C for 20 s. The sequences of the primers for RT-PCR are as follows: for ATG32, sense 5′-ACC GTC TCA TCC CTT TAA AC-3′ and antisense 5 ′ -CTT CCT CAA AAG CCT CAT CT-3 ′ and for TUB1, sense 5 ′ -CCA AGG GCT ATT TAC GTG GA-3 ′ and antisense 5 ′ -GGT GTA ATG GCC TCT TGC AT-3 ′ . The relative gene expression data were analysed using the 2 −ΔΔCt method. The levels of ATG32 mRNA were normalised to those of TUB1. 2.7. Antioxidative Assay. BY4741 yeast cells in glucose medium with initial OD600 value of 0.1 were treated with GPS at doses of 0, 1, and 3 μM or Res as positive control at 10 μM for 24 h. Subsequently, the yeast cells from each group were diluted to 1.5 OD600 value. Approximately 5 μL of yeast culture from each group was dropped on glucose agar plates containing H 2 O 2 at a dose of 10.5 mM and incubated at 28°C for 3 days. The growth of yeast was observed and photographed.
To quantify the antioxidative activity of GPS, BY4741 yeast cells were cultured with GPS at concentrations of 0, 1 and 3 μM or 10 μM Res at 28°C for 24 h. Then, 200 yeast cells from each treated group were painted on glucose agar plates with or without 5 mM of H 2 O 2 . After 2 days, the growth of yeast was observed, and the number of microcolonies in each plate was counted to evaluate the antioxidative activity. The survival rate was calculated as the ratio of the number of microcolonies with H 2 O 2 at 5 mM divided by the number of microcolonies in the absence of H 2 O 2 .
2.8. CAT, GPx, and SOD Enzyme Activity Assays. BY4741 yeast cells were cultured in liquid glucose medium to reach the logarithmic growth phase. The cells were divided into five groups with OD600 value of 0.1 and treated with GPS at 0, 1, 3, and 10 μM or Res at 10 μM and cultured in a glucose medium for 24 h. Subsequently, the yeast cells from different groups were collected and sonicated for 5 min. The cell lysates were centrifuged at 12,000 g for 15 min, and the supernatant was obtained to test the activity of enzymes. The activities of CAT, GPx, and SOD were determined with CAT and GPx assay kits (Beyotime Biotechnology Limited Company, Shanghai, China) and SOD assay kits (Nanjing Jiancheng Bioengineering Institute (Nanjing, China) in accordance with the manufacturer's instructions. All experimental procedures were conducted in strict accordance with the protocol instructions provided by the manufacturers.
2.9. Measurement of ROS and MDA Level in Yeast. ROS and MDA assays were performed by following the same methods as reported in literature [bib_ref] Parishin from Gastrodia elata extends the lifespan of yeast via regulation of..., Lin [/bib_ref]. Briefly, BY4741 yeast cells were treated with GPS at 0, 1, 3, and 10 μM or Res at 10 μM and cultured for 24 h. Subsequently, DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) was added to the culture to obtain a final concentration of 40 μM. The cells were then incubated in a shaker under the dark condition at 28°C for 1 h. The cells were then collected and washed with PBS thrice. The DCF fluorescence intensity of 1 × 10 7 cells were detected with a fluorescence plate reader using 488 nm as excitation and 525 nm as emission wavelengths.
To detect the MDA level in yeast, BY4741 yeast cells were cultured as described in the ROS assay for 24 h. These cells were then washed with PBS thrice, suspended in 250 μL PBS, and ultrasonicated on ice for 5 min. Subsequently, these cells were centrifuged at 4°C for 10 min at 12,000 g to obtain the supernatant, and the MDA level was measured with a MDA assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the manufacturer's instruction.
2.10. Statistical Analysis. Experimental data were expressed as mean ± SEM value of two or three independent experiments. Significant differences among the groups in all experiments were analysed through one-way ANOVA, followed by two-tailed multiple t-tests with Bonferroni's correction on GraphPad Prism 5 (GraphPad Software Inc.). Survival analysis was used for chronological lifespan assay. A p value of less than 0.05 was considered statistically significant.
# Results
## Gps extends the replicative and chorological lifespans of
Yeast. Yeast as an aging model is simple and amenable to genetic and molecular manipulations [bib_ref] Recent developments in yeast aging, Kaeberlein [/bib_ref]. It is widely used to study aging mechanisms and screen potential drug candidates for attenuating aging. In this study, a K6001 yeast replicative lifespan bioassay system was used as a bioassay system to screen antiaging samples. Res was used as a positive control to evaluate the reliability of the yeast bioassay system and bioassay results. The antiaging potential of GPS at concentrations of 0, 0.3, 1, 3, 10, and 30 μM was evaluated using the K6001 replicative lifespan assay and S1). The average replicative lifespan of each treatment or negative control group is as follows: 8:05 ± 0:60 generations for the negative control group, 11:13 ± 0:67 generations for the positive control group treated with Res at 10 μM, 8:08 ± 0:51 generations for treatment with GPS at 0.3 μM, 11:35 ± 0:64 generations for treatment with GPS at 1 μM, 10:25 ± 0:63 generations for treatment with GPS at 3 μM, 10:48 ± 0:73 generations for treatment with GPS at 10 μM, and 9:73 ± 0:59 generations for treatment with GPS at 30 μM. These results indicated that GPS can significantly prolong the replicative lifespan of K6001 yeast cells at concentrations of 1, 3, and 10 μM (p < 0:01, p < 0:05, and p < 0:05), respectively. The chronological lifespan assay of YOM36 yeast was performed to evaluate the antiaging activity of GPS. GPS significantly increased the survival rate of yeast at concentrations of 1 and 3 μM (p < 0:001, p < 0:01) . These results revealed that GPS extends the replicative and chronological lifespans of yeast.
## Effects of gps on autophagy and mitophagy in yeast.
Autophagy, particularly mitophagy, is a degenerative process that degrades cellular components, such as the mitochondria, for recycling into amino acids and other metabolites. Autophagy and aging is closely related [bib_ref] Autophagy and the cell biology of age-related disease, Leidal [/bib_ref] [bib_ref] Mitophagy, mitochondrial dynamics, and homeostasis in cardiovascular aging, Wu [/bib_ref]. Therefore, the effects of GPS on autophagy and mitophagy were examined. We used the YOM38-GFP-Atg8 yeast strain, which expresses GFP-Atg8 at a physiological level, to monitor the level of free GFP upon the treatment with GPS through fluorescent microscopy. The fluorescent images are displayed in , and the digital result is shown in . GPS significantly enhanced the percentage of cells with free GFP from 21:4 ± 1:6% to 31:6 ± 1:5% and 26:23 ± 0:8% at the concentrations of 1 and 3 μM (p < 0:001 and p < 0:05), respectively. The positive control Res at 300 μM increased the percentage from 21:4 ± 1:6% to 31:7 ± 0:5% (p < 0:001).
To confirm the effect of GPS on the regulation of autophagy, western blot analysis was performed to test the generation of free GFP that is released into the vacuole during the autoph-agy flux. The results of western blot analysis are displayed in and . GPS significantly increased the expression levels of Atg8-GFP and free GFP protein at the concentration of 1 μM (p < 0:001). These results implied that GPS can increase the level of autophagy in yeast.
Mitochondria are the sites of oxidative phosphorylation and ROS production, such as H 2 O 2 and superoxide anion (O 2 ⋅− ) in eukaryotic cells [bib_ref] Selective degradation of mitochondria by mitophagy, Kim [/bib_ref]. Hence, mitophagy, a selective type of autophagy, is extremely important in degrading the damaged mitochondria. MitoTracker Red CMXRos to localise the mitochondria of YOM38-GFP-Atg8 cells [fig_ref] 2. 3: Yeast Strains and Lifespan Assay [/fig_ref] was used to monitor mitophagy. GPS increased the percentage of cells with the colocation of green and red fluorescence from 10:2 ± 0:1% to 23:3 ± 1:5% at the concentrations of 1 μM [fig_ref] 2. 3: Yeast Strains and Lifespan Assay [/fig_ref] ) (p < 0:001). GPS increased the Atg8-GFP and free GFP protein levels in the mitochondrial fraction of yeast [fig_ref] 2. 3: Yeast Strains and Lifespan Assay [/fig_ref] and , p < 0:001). The ubiquitinproteasome system is a major intracellular pathway for the degradation of proteins that govern important life processes of the cell [bib_ref] Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases..., Ryu [/bib_ref]. Therefore, we tested the expression of ubiquitin in the mitochondria of yeast at the protein level and found that GPS can enhance the expression of ubiquitin at the protein level [fig_ref] 2. 3: Yeast Strains and Lifespan Assay [/fig_ref] and . These results revealed that GPS can significantly induce authophagy and mitophagy in yeast.
## Atg32 involved in the antiaging effect of gps.
In yeast, the Atg32 protein localises on the mitochondria, interacts with Atg8 and Atg11, and plays an important role on mitophagy [bib_ref] Mitochondria-anchored receptor Atg32 mediates degradation of mitochondria via selective autophagy, Okamoto [/bib_ref]. Therefore, we explored the effects of GPS on the ATG32 gene expression in yeast [fig_ref] Figure 4: G [/fig_ref]. The PCR analysis result showed that the expression of ATG32 was increased by GPS at concentrations of 1 and 3 μM [fig_ref] Figure 4: G [/fig_ref] , p < 0:01 and p < 0:05), respectively. We examined the effect of GPS on the replicative lifespan of the atg32 mutant with K6001 background and chronological lifespan of the atg32 mutant with YOM36 background. The replicative lifespan of the atg32 mutant with K6001 background is shown in [fig_ref] Figure 4: G [/fig_ref]. The average replicative lifespan of the K6001 strain was 7:65 ± 0:59 generations in the negative control, and the generations increased to 9:95 ± 0:51 (p < 0:001) and 9:95 ± 0:59 (p < 0:001) after treatment of Res at 10 μM and GPS at 1 μM, respectively. The replicative lifespan of the atg32 mutant of K6001 was 7:18 ± 0:53 generations in the control, and the generations were 7:45 ± 0:49 and 6:30 ± 0:37 after treatment with Res at 10 μM and GPS at 1 μM, respectively, suggesting that Res and GPS cannot extend the replicative lifespan of the atg32 mutant of K6001. These results implied that ATG32 was involved in the effect of extending the replicative lifespan of GPS. The chronological lifespan assay was performed using YOM36 wild type and atg32 mutant with YOM36 background yeast. The absence of ATG32 gene shortened the chronological lifespan of yeast [fig_ref] Figure 4: G [/fig_ref]. A significant change was found on the survival rate of the atg32 mutant with YOM36 background after treatment with GPS at 1 μM [fig_ref] Figure 4: G [/fig_ref] , suggesting that ATG32 was not involved in the effect of extending the chronological lifespan of GPS. These results indicated that GPS extended the replicative 5 Oxidative Medicine and Cellular Longevity lifespan through the regulation of mitophagy that requires ATG32.
## Gps increases the survival rate of yeast under oxidative
Stress Condition. Oxidative stress is a major risk factor of aging and age-related diseases, and strong oxidative stress results in the damage of DNA, lipid peroxidation, and dysfunction of protein. Qualitative and quantitative experiments were performed to evaluate the antioxidative activity of GPS, as shown in [fig_ref] Figure 5: Effect of GPS on the survival rate of yeast under oxidative stress... [/fig_ref]. The survival rates of yeast in oxidative stress were 48:8% ± 0:8 in the control group, 57:7% ± 2:6 in the positive control with Res-treated group (p < 0:05), 66:4% ± 1:1 in the GPS at 1 μM treated group (p < 0:01), 66:0% ± 3:2 in the GPS at 3 μM group (p < 0:01) and 58:1% ± 0:9 in the treatment of GPS at 10 μM (p < 0:05), respectively. These results indicated that GPS has antioxidative stress and plays an important role in the antiaging activity of GPS in yeast. Oxidative Medicine and Cellular Longevity 107:7 ± 7:2 (p < 0:05, p < 0:05), respectively [fig_ref] Figure 5: Effect of GPS on the survival rate of yeast under oxidative stress... [/fig_ref]. These results confirmed that GPS exhibited an antioxidative activity in yeast.
## Gps decreases the ros and mda levels in yeast.
Increased production of ROS can cause mitochondrial deterioration and global cellular damage, thereby supporting the role of ROS in aging [bib_ref] Cucurbitacin B exerts antiaging effects in yeast by regulating autophagy and oxidative..., Lin [/bib_ref]. MDA is a product of lipid peroxidation of polyunsaturated fatty acids and is a popular indicator used to determine oxidative stress [bib_ref] A review of recent studies on malondialdehyde as toxic molecule and biological..., Rio [/bib_ref]. Thus, we evaluated the levels of ROS and MDA after treatment with Res at 10 μM and GPS at 1, 3, and 10 μM for 24 h in yeast. As shown in Figures 5(g) and 5(h), the level of ROS decreased from 2:47 ± 0:09 to 1:62 ± 0:07, 1:32 ± 0:10, 1:38 ± 0:08, and 1:88 ± 0:31 (p < 0:01, p < 0:001, p < 0:001, and p < 0:05), respectively. The level of MDA decreased from 0:34 ± 0:02 to 0:23 ± 0:01, 0:27 ± 0:01, 0:27 ± 0:01, and 0:29 ± 0:01 (p < 0:001, p < 0:001, p < 0:01, and p < 0:05), respectively. These results suggested that GPS can significantly reduce the ROS level and production of MDA.
3.7. SOD1, SOD2, UTH1, and SKN7 Involved in the Antiaging Effect of GPS. SOD1, SOD2, UTH1, and SKN7 are antioxidative or oxidative-related genes. Replicative lifespan of K6001 strain and sod1, sod2, uth1, and skn7 mutants with K6001 background were performed to evaluate the effects of GPS on these genes. The results are demonstrated in Figures 6(a)-6(d). The average replicative lifespan of the K6001 strain was 7:80 ± 0:49 generations in the negative control, and the generations increased to 10:38 ± 0:64 (p < 0:01) and 9:88 ± 0:60 (p < 0:01) after the treatment of positive control (Res at 10 μM) and GPS at 1 μM, respectively . The average replicative lifespan of the sod1 mutant of K6001 was 6:35 ± 0:31 generations in the negative control, and the generations were 6:33 ± 0:33 and 6:90 ± 0:49 after treatment of Res at 10 μM and GPS at 1 μM, respectively , suggesting that Res and GPS did not extend the replicative lifespan of the sod1 mutant of K6001. In the case of the negative control, the average replicative lifespan of the sod2 mutant of K6001 was 7:60 ± 0:63 generations, and the generations were 7:95 ± 0:54 and 6:68 ± 0:51 after treatment with Res at 10 μM and GPS at 1 μM, respectively , suggesting that Res and GPS did not prolong the replicative lifespan of the sod2 mutant of K6001. The average replicative lifespan of the K6001 strain was 7:65 ± 0:59 generations in the negative control, and the generations increased to 9:95 ± 0:51 (p < 0:01) and 9:95 ± 0:59 (p < 0:01) after treatment of Res at 10 μM and GPS at 1 μM, respectively (Figures 6(c) and 6(d)). The replicative lifespan of the uth1 mutant of K6001 was 10:75 ± 0:65 generations in the negative control, and the generations were 11:50 ± 0:68 and 10:83 ± 0:58 after treatment with Res at 10 μM and GPS at 1 μM, respectively ), suggesting that Res and GPS did not extend the replicative lifespan of the uth1 mutant of K6001. The replicative lifespan of the skn7 mutant of K6001 was 7:98 ± 0:52 generations in the negative control, and the generations were 7:33 ± 0:43 and 7:58 ± 0:49 after treatment with Res at 10 μM and GPS at 1 μM, respectively , suggesting that Res and GPS did not extend the replicative lifespan of the skn7 mutant with the background of K6001. These results indicated that SOD1, SOD2, UTH1, and SKN7 are involved in the antiaging effect of GPS. Effect of GPS on the chronological lifespan of atg32 mutants with YOM36 background. * , * * , and * * * indicate significant differences from the corresponding control at p < 0:05, p < 0:01, and p < 0:001, respectively. Each experiment is repeated twice. [bib_ref] Benzoate fraction from Gentiana rigescens Franch alleviates scopolamine-induced impaired memory in mice..., Li [/bib_ref] Oxidative Medicine and Cellular Longevity biological studies on active natural products and their derivatives from G. rigescens for drug discovery against Alzheimer's disease and antiaging. In our previous studies, many novel molecules were isolated from G. rigescens and hundreds of their derivatives were synthesised. We discovered the lead compound, tetradecyl 2,3-dihydroxybenzoate through a structure-activity relationship study. This compound demonstrated significant neuritogenic effects in cell Each experiment is repeated thrice. * , * * , and * * * indicate significant differences from the corresponding control (p < 0:05, p < 0:01, and p < 0:001), respectively. 9 Oxidative Medicine and Cellular Longevity culture and animal models [bib_ref] Pro-neurogenesis and anti-dementia properties of tetradecyl 2,3-dihydroxybenzoate through TrkA receptor-mediated signalling pathways, Zhou [/bib_ref]. These results implied that we may have discovered substances that have cognition improvement from G. rigescens, as described in Sheng Nong's Herbal Classic. However, the active molecules on antiaging effect described in Sheng Nong's Herbal Classic are still under discovery. In the present study, GPS, which is a major component in G. rigescens, was isolated as the antiaging molecule under the guidance of the yeast K6001 replicative lifespan bioassay system. A previous literature reported that GPS has antioxidant, hepatoprotect, anti-inflammation, analgesic, and antibacterial effects [bib_ref] Research progress of natural product gentiopicroside -a secoiridoid compound, Wu [/bib_ref]. However, intensive studies on the antiaging activity of GPS and its mechanism of action are lacking. The extension of replicative and chronological lifespans of yeast after treatment of GPS and 1(c)) suggested that GPS had an antiaging activity on yeast.
# Discussion
Subsequently, two types of classical antiaging mechanisms, namely, autophagy and antioxidative stress, were investigated to understand the antiaging mechanism of GPS. Atg8 is an important component for autophagic machinery, participates in the entire process of autophagy, and is a biomarker of autophagy in yeast [bib_ref] Two newly identified sites in the ubiquitin-like protein Atg8 are essential for..., Amar [/bib_ref]. In our previous study, we found that autophagy was involved in the antiaging activity of cucurbitacin B (CuB), a natural product [bib_ref] Cucurbitacin B exerts antiaging effects in yeast by regulating autophagy and oxidative..., Lin [/bib_ref]. Therefore, we firstly examined whether autophagy was involved in the antiaging effect of GPS. The increase in free GFP in YOM38-GFP-Atg8 yeast after treatment of GSP confirmed that autophagy played an important role in the antiaging activity of GPS. Considering that autophagy, including mitochondrial, peroxisome, and nonselective autophagies, is categorised in selective autophagy, understanding which type of autophagy plays an important role in the antiaging effect of GPS is crucial. Therefore, we focused on mitophagy, especially Atg8 and Atg32, which are essential for mitophagy [bib_ref] Atg32 is a mitochondrial protein that confers selectivity during mitophagy, Kanki [/bib_ref] , to perform investigation using Mito-Tracker Red CMXRos stain method, western blot analysis, lifespan assay of the atg32 mutant yeast with K6001 or YOM36 background, and RT-PCR. The increase in free GFP and ubiquitin in the mitochondria and gene expression of ATG32 and no changes in the replicative lifespan of the atg32 mutant with K6001 background after treatment of GPS [fig_ref] 2. 3: Yeast Strains and Lifespan Assay [/fig_ref] clarified that mitophagy was involved in the antiaging effect of GPS. GPS, a secoiridoid glycoside, : Effect of GPS on the replicative lifespans of Δ sod1 (a), Δsod2 (b), Δ uth1 (c), and Δskn7 (d) yeast with K6001 background. The procedure for the replicative lifespan assay is the same as that for the K6001 lifespan assay. Each experiment is conducted thrice. * * indicates significant difference between the control group of K6001 and GPS-treated group at p < 0:01. and CuB, a highly oxidised triterpenoid, belonged to completely different types of chemical molecules. They exhibited significant effect on inducing autophagy. Moreover, a phenolic type molecule, bis(4-hydroxybenzyl)ether mono-β-L-galactopyranoside from the Gastrodia elata [bib_ref] Structure characterization and action mechanism of an antiaging new compound from Gastrodia..., Farooq [/bib_ref] , as well as GPS and CuB, showed a significant antiaging activity on yeasts. All of these three different structural types of molecules exhibited antiaging activities through antioxidative stress. The decrease of ROS and MDA levels significantly were common features of these three different structural types of compounds. Therefore, studying the similarities and differences among the three different types of molecules in inducing autophagy and antioxidative stress in the future is extremely valuable.
We investigated the effects of antioxidative stress on the antiaging effect of GPS. The increase in the survival rate under oxidative stress; enhancement of CAT, SOD, and GPx activities; and the decrease in ROS and MDA levels on yeast [fig_ref] Figure 5: Effect of GPS on the survival rate of yeast under oxidative stress... [/fig_ref] suggested that GPS exhibited antiaging effect on yeast with the increase in antioxidant enzyme activity and antioxidative stress. We performed the replicative lifespan assay of sod1, sod2, uth1, and skn7 yeast mutants with a K6001 background to provide many evidence for supporting the conclusion. The replicative lifespan assay results of yeast mutants in showed that SOD1, SOD2, UTH1, and SKN7 were required in the antiaging effect of GPS.
In the present study, we found that SD and SC mediums had different effects on the chronological lifespan of YOM36 yeast. The survival rate of yeast in the SC medium was better than that in the SD medium and S1(b)). The culture medium for chronological lifespan assay will be optimised in the future study.
# Conclusion
The findings revealed that GPS isolated from G. rigescens, a traditional Chinese medicine, has a significant antiaging activity on yeast. GPS prolonged the replicative and chronological lifespans through the regulation of mitophagy and antioxidative stress. GPS is an extremely high-content molecule in G. rigescens and is a representative component of the plant. This condition indicates that large quantities of the compound can be readily obtained for subsequent chemical and biological studies. This study provides an important foundation for future studies on the mechanism of GPS and evaluation of the antiaging effect of GPS in different antiaging animal models for novel drug development.
## Data availability
The data used to support the findings of this study are included within the article.
## Conflicts of interest
The authors declare no financial or commercial conflict of interest.
[fig] 2. 3: Yeast Strains and Lifespan Assay. The K6001 yeast, Δuth1, Δskn7, Δsod1, Δsod2, and Δatg32 yeast mutants with 2 [/fig]
[fig] 2. 5, Figure 1: Western Blot Analysis. YOM38 yeast cells containing pR316-GFP-Atg8 plasmid were cultured in liquid glucose medium in a shaking incubator at 180 rpm and 28°C under Chemical structure and effect of GPS on the lifespan of yeast. (a) Chemical structure of GPS. (b) The changes in the replicative lifespan of K6001 yeast after treatment of GPS. RES at 10 μM is used as the positive control. [/fig]
[fig] Figure 2, Figure 3: Effect of GPS on autophagy in yeast. (a) Fluorescent images of YOM38 yeast containing plasmid pR316-GFP-Atg8 after treatment of RES or GPS observed with a two-photon confocal fluorescent microscope. (b) The percentage of YOM38 cells containing plasmid pR316-GFP-Atg8 with autophagosome (green). Three pictures containing more than 60 cells in each group are used for statistical analysis. (c) Western blot analysis of GFP-Atg8 and free GFP in yeast after treatment with RES or GPS for 22 h in the SD medium. * and * * * indicate significant differences from the corresponding control at p < 0:05 and p < 0:001, respectively. Each experiment is repeated twice. . GPS Improves the CAT, SOD, and GPx Activities in Yeast. CAT, SOD, and GPx play important roles in antioxidative stress. The enzyme activities of CAT, total-SOD, CuZn-SOD and GPx in yeast were measured after treatment with Res at 10 μM and GPS at 1, 3, and 10 μM to confirm the antioxidative effect of GPS. The enzyme activity (units/mg) of CAT significantly increased from 58:3 ± 3:4 to 72:2 ± 2:8, 72:0 ± 2:0, 68:0 ± 3:4, and 66:7 ± 3:7 (p < 0:05, p < 0:05, p < 0:05, and p < 0:05), respectively(Figures 5(c)). As shown inFigures 5(d)and 5(e), the enzyme activity (units/mg) of total-SOD significantly increased from 84:p < 0:01, and p < 0:05), respectively. The enzyme activity (units/mg) of CuZn-SOD significantly increased from 46:1 ± 4:8 to 56:4 ± 1:0, 59:1 ± 0:7, 58:9 ± 1:2, and 44:8 ± 2:1 (p < 0:05 and p < 0:01 and p < 0:01, respectively. The enzyme activity of GPx (mU/mg) significantly increased from 102:Effect of GPS on mitophagy in yeast. (a) Fluorescent images of YOM38 yeast containing plasmid pR316-GFP-Atg8 after treatment of GPS through MitoTracker Red CMXRos staining. (b) The percentage of YOM38 cells containing plasmid pR316-GFP-Atg8 with the colocation of autophagosome (green) and MitoTracker Red CMXRos (red). (c, d) The changes in ATG8-GFP, free GFP, and ubiquitin at the protein level in the mitochondria after treatment of GPS. Three pictures containing more than 60 cells in each group are used for statistical analysis. [/fig]
[fig] Figure 4: G. rigescens Franch is a traditional Chinese medicine produced in Yunnan and Guizhou Provinces, China. Our research group has long been engaged in the chemical and Effect of GPS on ATG32 gene expression and lifespan of Δatg32 mutant yeast with K6001 and YOM36 backgrounds. (a) Effect of GPS on ATG32 gene expression after 12 h treatment. (b) Effect of GPS on the replicative lifespan of atg32 mutants with K6001 background. (c) [/fig]
[fig] Figure 5: Effect of GPS on the survival rate of yeast under oxidative stress condition and antioxidative enzyme activity in yeast. (a) Photograph of yeast growth after treatment of GPS under oxidative stress condition induced by H 2 O 2 at 10.5 mM. (b) The survival rate changes in yeast under oxidative conditions at 5 mM H 2 O 2 . (c-f) The changes in CAT, T-SOD, SOD1, and GPx enzyme activities in yeast after treatment of GPS for 48 h. (g, h) Effect of GPS on ROS and MDA levels. [/fig]
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10.3389/fpubh.2021.611652
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CCBY
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8591105
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34790639
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s2orc_pubmed_articles
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Prevalence and Incidence Rate of Diabetes, Pre-diabetes, Uncontrolled Diabetes, and Their Predictors in the Adult Population in Southeastern Iran: Findings From KERCADR Study
Background: Diabetes mellitus is among the most serious health challenges worldwide. We assessed the prevalence of pre-diabetes (pre-DM) and diabetes (DM), the effectiveness of diabetes management, the 5-year incidence rate, and associated variables in the adult population in southeastern Iran.Methods: In a random cluster household survey (2014-2018), 9,959 adult individuals aged 15-80 years were assessed for coronary artery disease risk factors, including diabetes mellitus in Kerman (KERCADRS, phase 2). Among these people, 2,820 persons had also participated in phase 1 of the study 5 years earlier (2009)(2010)(2011). Univariable and multivariable survey logistic regression models were used to identify the potential predictors of diabetes and pre-diabetes.Results: The prevalence of pre-DM was 12% (males 13.2% vs. females 11.1%), steadily increasing from 7.1% in the 15-24 years group to 18.4% in the 55-64 years group. The prevalence of DM was 10.2% (male and female, 7.9 and 10.8%, respectively), of which 1.9% were undiagnosed. DM was diagnosed in 10.6% of educated and 15.1% of illiterate people. The prevalence of diagnosed DM was lower in smokers (5.2 vs. 8.7%) and dependent opium users (5.4 vs. 8.8%). The prevalence of uncontrolled DM (HbA1c > 7%) was 48.8%, increasing with age. The frequency of uncontrolled DM among people without and with treatment was 32 and 55.9%, respectively. Illiterate people had worse uncontrolled DM (55.6 vs. 39.6%). The 5-year incidence rate (persons/100 person-years) was 1.5 for pre-DM and 1.2 for DM, respectively. The lowest and the highest incidence rate of DM belonged to the 15-34 years old group (0.5) and dependent opium users (2.4). The incidence rate was found to have a direct relationship with BMI and a reverse relationship with physical activity.Conclusion: Pre-DM and DM affected 22.2% of the population. One-third of patients with diabetes had undiagnosed DM, and in 55.9% of people with diagnosed DM, Najafipour et al.Diabetes Epidemiology in Southeastern Iran treatment had been ineffective. Appropriate health interventions are needed to reduce the prevalence and health consequences of diabetes in the region.
# Introduction
Type 2 diabetes mellitus (T2DM) is widely associated with an increased prevalence of cardiovascular disease (CVD) [bib_ref] Prevalence of cardiovascular disease in type 2 diabetes: a systematic literature review..., Einarson [/bib_ref]. In fact, T2DM patients have a 2-to 4-fold higher risk for CVD morbidity and mortality than healthy non-diabetic patients [bib_ref] Cardiovascular risk assessment in patients with diabetes, Bertoluci [/bib_ref]. In addition, accounting for almost 80% of deaths among T2DM patients, CVD is the leading cause of mortality in people suffering from DM [bib_ref] Complications of diabetes 2017, Papatheodorou [/bib_ref]. The association between T2DM and CVD is not only supported by meta-analyses and observational data [bib_ref] Excess risk of fatal coronary heart disease associated with diabetes in men..., Huxley [/bib_ref] but is also based on the pathophysiological background characterizing T2DM on the CV continuum [bib_ref] The pathologic continuum of diabetic vascular disease, Orasanu [/bib_ref]. T2DM involves a chronic state of vascular inflammation and endothelial and platelet dysfunction induced by hyperglycemia and insulin resistance, which predisposes the patient to macro-vascular complications even before T2DM is diagnosed [bib_ref] ESC guidelines on diabetes, pre-diabetes, and cardiovascular diseases developed in collaboration with..., Rydén [/bib_ref]. It has been reported that coronary plaques with larger necrotic cores and increased inflammation (with more T lymphocytes and macrophages) in addition to a higher rate of plaque ruptures and positive remodeling are generally observed in T2DM patients with non-diabetic controls, suggesting a more active atherosclerotic process [bib_ref] Pathology of human coronary and carotid artery atherosclerosis and vascular calcification in..., Yahagi [/bib_ref] [bib_ref] C-reactive protein is associated with prevalence of the metabolic syndrome, hypertension, and..., Mazidi [/bib_ref]. The prevalence of diabetes for all age groups worldwide was estimated to be 2.8% in 2000 and 4.4% in 2030 [bib_ref] Global prevalence of diabetes: estimates for the year 2000 and projections for..., Wild [/bib_ref]. According to the latest data published in the International Federation of Diabetes Atlas, 463 million adults live with diabetes (14). This is an important contributor to disease burden, particularly in developing countries (15).
According to the national CAD risk factors surveillance report, the overall prevalence of diabetes in Iran were estimated to be 8.7% in people aged 15-64 years, about half (4.1%) of whom were newly diagnosed cases [bib_ref] Third national surveillance of risk factors of non-communicable diseases (SuRFNCD-2007) in Iran:..., Esteghamati [/bib_ref]. Based on a systematic review, the prevalence of T2DM in Iran was estimated as one out of four among adults aged ≥ 40 years [bib_ref] Prevalence of type 2 diabetes in the Islamic Republic of Iran: systematic..., Haghdoost [/bib_ref]. However, it is not clear how many are in the pre-diabetic stage, are prone to developing diabetes, and need timely interventions to avoid developing it.
In addition to late diagnosis, diabetes management is a challenging issue in Iran, as only 39.2% of individuals with diagnosed diabetes receive treatment [bib_ref] Effectiveness of diabetes and hypertension management by rural primary healthcare workers (Behvarz..., Farzadfar [/bib_ref]. Taking fasting plasma glucose ≥ 130 mg/dl as the criterion for poor management of diabetes, it was found that about 57% of individuals with diagnosed diabetes had a high level of plasma glucose [bib_ref] Related factors to disparity of diabetes care in Iran, Mirzazadeh [/bib_ref].
In a population-based research named the Kerman Coronary Artery Diseases Risk Factors study (KERCADRS) from 2009 to 2011 on 5,900 adults aged 15-75 in Kerman, the prevalence was found to be 18.7% (23.4% men and 13.7% women) for pre-diabetes and 9.0% (7.7% men 10.3% women) for diabetes. The present study is the second phase of the KERCADRS performed on a larger sample size of 9,959 to determine the prevalence and predictors of pre-diabetes and diabetes in the adult population aged 15-80 living in an urban setting in southeastern Iran. The results of this study are compared with the findings of the first phase to explore the trend of changes in prevalence and the 5-year incidence rate of pre-diabetes and diabetes. This will provide a better insight into the severity and growth rate of these important CVD risk factors in this region in the past 5 years. We also assessed the effectiveness of diabetes management (using Hb1C as the indicator) in people with diagnosed diabetes. The prevalence of the main CADrelated comorbidities is also reported in normal, pre-diabetic, and diagnosed, and undiagnosed diabetic subpopulations.
# Methods
## Kercadrs, the kerman coronary artery disease risk factors
Study, is a population-based cohort study with multiple surveys. The study was conducted according to the Declaration of Helsinki, and the study protocols were approved by the Ethics Committee of Kerman University of Medical Sciences (ethics code: IR.KMU.REC.1392.405). Between 2014 and 2018, 9,959 15-to 80-year-old individuals were recruited through a nonproportional-to-size one-stage cluster sampling into the second stage of the study. The methodology of the KERCADR study (phase 1) has been explained in detail elsewhere [bib_ref] Coronary artery disease risk factors in an urban and peri-urban setting, Kerman,..., Najafipour [/bib_ref]. In brief, 420 zip codes were randomly selected in phase 2, each representing a house (called a seed). The seed household and other households in the neighborhood from the right direction of the seed household were then systematically approached by social mobilizers, and all eligible people (15-to 80-year-olds) in the household were invited to participate in the study. The recruitment continued until 24 persons (12 men and 12 women who provided written informed consent) in each cluster were recruited, reaching a total target sample size of 10,000. For participants under 18, informed consent was acquired from both themselves and their parents, and they usually attended the interview site accompanied by their parents.
## Interview and measurements
Details on what was measured and how this was achieved are presented elsewhere [bib_ref] Coronary artery disease risk factors in an urban and peri-urban setting, Kerman,..., Najafipour [/bib_ref]. In brief, trained interviewers assessed the study subjects for different CAD risk factors using a structured questionnaire including questions on demographic information, cigarette smoking (yes/no), opium addiction (no/occasional/dependent), level of physical activity (low/moderate/high), and level of depression and anxiety (BECK questionnaire). The subjects were also asked about their medical and familial history of DM, and whether they were under insulin or non-insulin treatment.
Physical activity was determined by the Global Physical Activity Questionnaire, and the intensity of physical activities was expressed using metabolic equivalents of task (MET). The total MET in time (min) was computed for the status of the activity in work-, transport-, and recreation-related physical activity and was then categorized into three levels of low, moderate, and intense. MET is defined as the rate of energy use in a person in a sitting position (equivalent to 3.5 ml oxygen consumption/kg body weight per minute). Moderate physical activity is the consumption of four times, and intense physical activity is the consumption of eight or more times the energy consumed while sitting [bib_ref] The prevalence of low physical activity in an urban population and its..., Najafipour [/bib_ref]. Hypertension was defined as systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg, or taking any antihypertensive drug. Body mass index (BMI) between 25 and 29.9 kg/m 2 and BMI ≥ 30 kg/m 2 were interpreted as overweight and obese, respectively.
Diabetes was diagnosed according to the ADA recommendation. Every individual who had previously been diagnosed with diabetes (by a physician) or was taking insulin or oral anti-diabetic medications or had FPG ≥ 126 mg/dl at the time of recruitment (provided that the HbA1c was more than 6.5%) was considered diabetic. Those with FPG between 100 and 125 mg/dl were considered pre-diabetic (pre-DM). Participants with FPG ≥ 126 mg/dl for the first time were called back, and HbA1c was checked, and if HbA1c was more than 6.5%, diabetes was confirmed. None of the patients had GLP-1 agonists as a treatment option.
Those with FPG ≥ 126 and HbA1c lower than 6.5% were not included as diabetics (these were 46 subjects vs. 1,020 individuals with diabetes, see study limitations). Subjects who had no previous history of diabetes or anti-diabetic medication but were found to have FPG ≥ 126 mg/dl and HbA1c higher than 6.5% on recruitment were considered undiagnosed (new) diabetic cases. Every diabetic case was tested for HbA1c to determine the glycemic control status of diagnosed patients with diabetes.
Based on ADA recommendation, "Older adults who are otherwise healthy with few coexisting chronic illnesses and intact cognitive function and functional status should have lower glycemic goals [such as HbA1C 7.5% (58 mmol/mol)], while those with multiple coexisting chronic illnesses, cognitive impairment, or functional dependence should have less stringent glycemic goals [such as HbA1c 8.0-8.5% (64-69 mmol/mol)]".
Uncontrolled diabetes was specified as HbA1c > 7%, and in people with a progression of microvascular and chronic complications and those aged more than 70, HbA1c > 8% was considered the cut-off for poor glycemic control (uncontrolled DM).
## Laboratory measurements
All participants were asked to fast for 12 h before coming to the clinic. Venous blood samples were obtained from the antecubital vein between 7:00 and 9:00 a.m., and fasting plasma glucose (FPG) was measured in serum (Kimia Kit, Code 890410, Iran). Subjects with diagnosed diabetes and new cases with FPG higher than 100 mg/dl were called back for another FPG and HbA1c test (NycoCard Kit, Code 1042184, Austria). Serum lipids for all participants were measured using KIMIA Kit; total cholesterol was assessed using KIMIA Kit, Code 890303, Iran, while triglycerides were assessed using KIMIA Kit, Code 890201, Iran.
## Incidence rate of diabetes/pre-diabetes
We used the same method to calculate the incidence rate for prediabetes and diabetes. Therefore, we only present the method for measuring the latter here. To calculate the incidence rate of diabetes, we used the data from those who had participated in both phases, had normal FBS with no anti-diabetes treatment in phase 1, and, therefore, were at risk of becoming diabetic during the follow-up [fig_ref] FIGURE 1 |: The flow chart of people participated in both phases of the study [/fig_ref]. Therefore, 27.7% of the 5,900 participants (1,634 cases) in phase 1 who were already prediabetic/diabetic were excluded from the incidence calculation. Out of the remaining participants (4,265 cases), 1,445 persons (24.5% of total participants) were lost to follow-up (did not take part in Phase 2 or had died). The number of new diabetic cases (among 4,265 cases) identified during the followup period was considered the numerator. The time difference (in years) between the visit in phase 1 and the visit in phase 2 was calculated as person-year at risk for those who had normal FPG in the phase 1 visit. Therefore, the denominator is the sum of the time each person was followed (person-year), totaled for all 4,265 persons at risk of becoming diabetic. For those lost to follow-up, on average, 2.5 years (half of overall follow time) was taken as years at risk. Then incidence rate (expressed as person per 100 person-years) was calculated by the formula (24)
# Statistical analysis
All statistical analyses were conducted under survey data analysis using Stata v. 15 (Stata Corp. 2015 College Station, Texas USA). Data are presented as absolute and relative frequencies and 95% confidence intervals (95% CI). To account for the clustering effect, we used the survey data package analysis, in which we set clusters to be the primary sampling units. Then, because of the non-proportionate-to-size sampling method, the total estimates were standardized based on the real age distribution of the target population (national census of Kerman population size in 2016). We reported weighted prevalencefor pre-DM and DM. We ran the bivariate analysis to assess the association between all covariates and the study outcome [pre-DM and DM (both diagnosed and undiagnosed) binary outcomes] one at a time. Then, we included all covariates with P-values < 0.05.n (based on likelihood test) in the multivariable logistic regression. Outputs from univariable and multivariable survey logistic regressions were reported as crude and AOR. Data from the second phase of the study were used in the logistic regression. Z-test and the Chi-square test were used to compare the prevalence between phases 1 and 2 .
# Results
## Demographic characteristics
The 9,959 people recruited in this study had a median age of 47 years with an interquartile range (IQR) of 24 years, and 59.4% were female. There was no significant difference between females (median 46, IQR 23 years) and males (median 47, IQR 26 years) in this regard. According to the self-report, 5.2% had never been to school, and one-third (33.5%) had not completed secondary education (had not finished high school).
## Pre-dm and dm prevalence
Overall, the age-and sex-standardized prevalence of prediabetes (pre-DM) was 12.0% (men 13.2% vs. women 11.1%) [fig_ref] TABLE 1 |: The standardized prevalence % [/fig_ref]. The pre-DM prevalence showed an increasing trend from 7.1% in young adults (age group 15-24 years) to 18.4% in middle-aged people (age group 55-64 years). Almost 19% of illiterate people were pre-DM, while the prevalence was lower in people with above-highschool education (11.5%). 8.9% of people with normal BMI, 13.1% of those with overweight, and 15.8% of obese individuals had pre-DM (P < 0.01). Smoking, opium use, the level of physical activity, and having a familial history of DM had no significant effect on the prevalence of pre-DM. The age-and sex-standardized prevalence of pre-DM was 18.9% (men 23.8% vs. women 15.1%; P < 0.001) in phase 1 and 12% (men 13.2% vs. women 11.1%; P < 0.01) in phase 2. Overall, pre-DM in both genders was significantly lower in phase 2 than in phase 1 (P < 0.01 between two phases). The prevalence of pre-DM increased with age in both phases and it was lower in phase 2 in all age groups . 2 | The prevalence of pre-diabetes (A,B) and diabetes (C,D) in KERCADRS by sex and age groups. Total participants = 9,959 in phase 2 and 5,900 in phase 1. The data of phase 1 were used here for comparison and are extracted from our paper published previously. *P < 0.05, **P < 0.01, ***P < 0.001 compared to phase 1.
The prevalence of DM was 10.8% in phase 1 (men 9.7% vs. women 11.7%) and 10.3% in phase 2 (men 8% vs. women 11.8%) . Only males had a significantly lower prevalence of DM in phase 2 compared to phase 1. There was no difference in age group trends of DM between the two phases.
## Predictors of diabetes
In the multivariable model, after controlling for confounders, it was shown that odds of diabetes (diagnosed and undiagnosed combined) had an increasing trend by age group (AOR up to 30.2) and had a positive association with familial history of DM (AOR 2.5), overweight and obesity (AOR 1.37 and 1.77), and low physical activity (AOR 1.3) [fig_ref] TABLE 1 |: The standardized prevalence % [/fig_ref]. A significant negative association was found between smoking and odds of diabetes (AOR 0.7). Depression, anxiety, and opium use had no statistically significant association with diabetes.
## Undiagnosed and diagnosed diabetes
In total, the standardized prevalence of diabetes was 10.2% (men 7.9% and women 10.8%), of which 1.9% had undiagnosed diabetes (equal for men and women), and 8.3% had had their disease already diagnosed (men 6.0% vs. women 9.9%) [fig_ref] TABLE 1 |: The standardized prevalence % [/fig_ref].
Undiagnosed DM was more prevalent in illiterates (6.6%) compared to higher-educated people, in older individuals (5.1% among people over 75) compared to youngers, in obese people (2.6%) compared to those with normal weight, and in those with positive family history of DM (2.3%). People with anxiety symptoms (1.7%), high physical activity (1.6%), and depression (1.1%) had a lower prevalence of undiagnosed DM compared to those with low physical activity and normal mental status. There was an equal prevalence of undiagnosed DM in males and females [fig_ref] TABLE 1 |: The standardized prevalence % [/fig_ref].
The diagnosed-DM showed an increasing trend in the prevalence by age from 1% in young adults (age group 15-24) to 30.7% in older adults (aged 65-74 years). DM was diagnosed in 10.6% of educated (above high school) people compared to 15.1% illiterate participants. Compared to non-cigarette-smokers, the prevalence of diagnosed DM was lower among smokers (5.2 vs. 8.7%). The prevalence of diagnosed DM was 8.8% in nonopium-users, 6.6% in occasional opium users, and 5.4% among dependent users. Eight percent of overweight and 11.5% of FIGURE 3 | The prevalence of undiagnosed DM (A,B) and uncontrolled DM in the diagnosed diabetic participants (C,D) in the study (KERCADRS) by sex and age groups. Total participants were 9,957 in phase 2 and 5,900 in phase 1. The data of phase 1 were used here for comparison and are extracted from our paper published previously. **P < 0.01 compared to phase 1.
obese people had diagnosed DM. In people with high physical activity, 5.9% had diagnosed DM, compared with 8.8% in people with low physical activity. Compared to subjects with negative family history of DM, people with positive history had a higher prevalence of diagnosed DM (12.8 vs. 5.7%) [fig_ref] TABLE 1 |: The standardized prevalence % [/fig_ref].
## Diabetes mismanagement
Regarding the HbA1c > 7 (old definition) in people with diagnosed DM, the prevalence of uncontrolled DM was 50.2% (men 49.8% vs. women 50.4%). Uncontrolled DM showed an increasing trend in prevalence by age from 11.1% in young adults to 52.7% in older people (15-24 years old and 65-74 years old, respectively). The frequency of uncontrolled DM among people without treatment was 32%, but it was 55.9% in those under treatment (both insulin and oral). Less educated people had more uncontrolled DM than highly educated individuals (58.3 vs. 40.8%). Uncontrolled DM among non-smokers and no-opium users (50.3 and 50.3%) was higher than in smokers and dependent opium users (48.2 and 46.6%). Regarding obesity, uncontrolled DM ranged from 53.1% (normal BMI) to 49.3% (obese subgroup). More than fifty-two percent of patients with diabetes who had a positive history of familial DM were uncontrolled.
Given age and coexisting complications (details are mentioned above in the method), overall, uncontrolled DM (HbA1c > 8%, patient centered) was observed in 48.8% of diagnosed DM cases (men 48.4% vs. women 49%). The prevalence of uncontrolled DM ranged from 0.3 to 43% in different subpopulations. The greatest difference was observed among age groups (43.5%), in those receiving insulin therapy (30%), and in illiterates (16%). Familial history of DM (3.9%), opium use (3.4%), and the level of physical activity (0.4%) have minor association with the status of diabetes control. The frequencies of drug-naivety, use of oral agents, insulin monotherapy, and insulin combination therapy were 32.0% (n = 219), 60% (n = 435) 7.7% (n = 56), and 2.1% (n = 15) in phase 1 and 15.6% (n = 212), 68.7% (n = 933), 8.9% (n = 121), and 6.8% (n = 15) in phase 2, respectively.
The age-and sex-standardized prevalence of undiagnosed DM was 3% (men 3.2% vs. women 2.9%) in phase 1 and 1.9% (men 2% vs. women 1.9%) in phase 2. Both overall and age-dependent prevalence of undiagnosed DM were significantly lower in phase 2 compared to phase 1 of the study (P < 0.01 between the two phases, . The prevalence of uncontrolled-DM in the diagnosed DM patients was 48.8% (men 48.4% vs. women 49%) in phase 2 and 60.8% (men 59.7% vs. women 61.5%) in phase 1. Uncontrolled DM overall and in both genders was also significantly lower in phase 2 than in phase 1 (P < 0.01 between two phases, .
# Diabetes-related comorbidities
The maximum prevalence of comorbidities among patients with diagnosed DM included overweight/obesity (77.3%), hypertriglyceridemia (70.3%), hypertension (61.0%), and
## Incidence rate of pre-diabetes and diabetes
The incidence rate of pre-DM and DM during the 5 years between the two phases of the study is presented in.
Overall, the incidence rate per 100-person years was 1.5 for pre-DM and 1.2 for DM, with a higher incidence rate for men in pre-DM and for women in DM. The lowest incidence rate of pre-DM was among those who had high physical activity (1.2 persons/100 person-years) and in middle-aged (35-44 years old) adult participants (0.7 persons/100 person-years) while the highest incidence rate was observed among those in the age group of 65-74 years old (2.5 persons/100 person-years) and occasional opium users (2.2 persons/100 person-years). Similarly, the lowest incidence rate of DM was seen in young (15-34 years old) adults and cigarette smokers (0.5 and 0.8 persons/100 personyears, respectively) while the highest incidence rate was observed among dependent opium users (2.4 persons/100 person-years and those in the age group of 65-74 years (2.1 persons/100 person-years). There was a reverse relationship between the incidence rate of DM and the level of physical activity and a direct relationship between the incidence rate of DM and BMI.
# Discussion
Our analysis revealed that one out of four individuals living in the urban area in southeast Iran either had impaired glucose levels (pre-diabetes) or was diabetic. Close to 2% of studied individuals had undiagnosed diabetes, and in about 50% of diagnosed patients with diabetes, the treatment was not effective. Both the number of cases and the prevalence of diabetes have been steadily increasing over the past few decades, particularly in low-and middle-income countries. We observed an almost equal prevalence of DM in our study population (10.2%) with that reported by WHO in 2016 on the prevalence of DM in Iran (10.3%). However, we found that 13.2% of men and 11.1% of women are pre-diabetic. This should be taken as an opportunity by health authorities to reduce the burden of the disease by preventing pre-diabetic cases from developing the full-blown disease. It has been shown that by losing weight and increasing physical activity, individuals can prevent or delay pre-diabetes from progressing to diabetes [bib_ref] The role of physical activity in type 2 diabetes prevention: physiological and..., Burr [/bib_ref] [bib_ref] Van Dam RM. Physical activity of moderate intensity and risk of type..., Jeon [/bib_ref] [bib_ref] Evidence-based risk assessment and recommendations for physical activity clearance: diabetes mellitus and..., Riddell [/bib_ref] [bib_ref] Sex-based differences in diabetes prevalence and risk factors: a population-based cross-sectional study..., Zhang [/bib_ref].
Fortunately, the prevalence of pre-diabetes decreased significantly during the 5 years between the two phases of the study in all age groups although overall, this reduction was more prominent in males. Also, the prevalence of diabetes has stopped rising. Previously, in urban areas, patients' compliance with medication and regular visits to physicians depended on the patients themselves. About 7 years ago, the family physician program was piloted in a few cities and was then expanded to many urban areas in Iran to cover this gap in primary healthcare in urban settings [bib_ref] Family physicians' satisfaction in Iran: a long path ahead, Shalileh [/bib_ref] [bib_ref] Assessment of urban family physician program in pilot centers covered by Ahvaz..., Shahrokh [/bib_ref]. We think this study provides evidence of the positive effects of such interventions and the effect of the health reform plan, which was carried out mostly in urban areas of the country during the 5 years between the two phases of the study. The significant reduction in the frequency of drug-naivety and increase in the use of oral agents or insulin therapy in phase 2 compared to phase 1 (see results section under "Diabetes Mismanagement") is a validation of this hypothesis. Also, the prevalence of undiagnosed DM was significantly reduced in phase 2 compared to phase 1 , verifying the increase in patients' willingness to refer to physicians in the health system.
Unlike pre-diabetes, the prevalence of diabetes showed a slight reduction, only in males, during the 5 years between the two phases of the study . We hypothesize that the wave of current reduction in the prevalence of pre-diabetes will affect diabetes soon. This should be monitored over time by the next phase of the KERCADRs study or other populationbased surveys.
In both phases of the study, the prevalence of diabetes was significantly higher in women than in men, which agrees with the findings of a recent study, which found that females were more affected by diabetes [bib_ref] Prevalence and risk factors associated with type 2 diabetes in elderly patients..., Asiimwe [/bib_ref]. The frequencies of cardiometabolic risk factors were also significantly different in men and women, with diabetes and obesity the more predominant traits among women. The higher prevalence among the women can be partly explained by a higher prevalence of obesity [bib_ref] Housewives' obesity determinant factors in iran; national survey-stepwise approach to surveillance, Navadeh [/bib_ref] [bib_ref] Trends in overweight, obesity and central fat accumulation among Tehranian adults between, Azizi [/bib_ref] [bib_ref] First nationwide survey of prevalence of overweight, underweight, and abdominal obesity in..., Janghorbani [/bib_ref] and lower physical activity reported in several studies [bib_ref] A province-based surveillance system for the risk factors of non-communicable diseases: a..., Alikhani [/bib_ref] [bib_ref] Physical activity, sex, and socioeconomic status: a population based study, Talaei [/bib_ref] [bib_ref] Leisure time physical activity and its determinants among adults in Tehran: Tehran..., Momenan [/bib_ref] in them. These findings were different from a recent study in China, where there was no significant gender difference in the prevalence of DM (men, 14.1% and women, 14.5%) [bib_ref] Sex-based differences in diabetes prevalence and risk factors: a population-based cross-sectional study..., Zhang [/bib_ref]. There is a slight sex difference in the global numbers of people with diabetes, with an estimated 17 million more diabetic men than women in 2017. The prevalence increases sharply with age in both sexes [bib_ref] Epidemiology of diabetes, Forouhi [/bib_ref].
It has been reported in several studies that the prevalence of diabetes (diagnosed and undiagnosed), IGT, and IFG increases by age [bib_ref] Epidemiology of diabetes, Forouhi [/bib_ref] [bib_ref] High prevalence of undiagnosed diabetes and abnormal glucose tolerance in the Iranian..., Hadaegh [/bib_ref]. This is a physiologic phenomenon in which the prevalence of metabolic disorders increases with age, especially in women after menopause, when the level of sex hormones decreases sharply. The age dependency of diabetes was found in the present study up to the age of 64. The reduction in the prevalence of diabetes among individuals aged 65 years or more found in the present study could be due to higher mortality in patients with diabetes in comparison to the rest of the population; diabetes has been reported as a trigger for other cardiovascular diseases such as hypertension, stroke, and acute myocardial infarction [bib_ref] Established risk factors account for most of the racial differences in cardiovascular..., Henderson [/bib_ref].
In our study, the lowest incidence rate of pre-DM was observed among the middle-aged (35-44 years old) adult participants, while the highest incidence rate was seen among those in the 65-74 years age group. Fiorentino et al. showed that young IGT and adult IGT subjects exhibited a progressively greater degree of hepatic insulin resistance and reduced insulin clearance compared with older IGT subjects [bib_ref] Individuals with pre-diabetes display different age-related pathophysiological characteristics, Fiorentino [/bib_ref]. The incidence rate was lower in smokers, which seems unexpected [bib_ref] Genetic variation at CHRNA5-CHRNA3-CHRNB4 interacts with smoking status to influence body mass..., Freathy [/bib_ref]. However, it has been shown that smoking causes lower BMI and evidence from a systematic review and meta-analysis shows that the risk of type 2 diabetes is raised in new quitters compared with those who have never smoked [bib_ref] Relation of active, passive, and quitting smoking with incident type 2 diabetes:..., Pan [/bib_ref]. Also, smoking cessation is associated with deterioration in glycaemia control in smokers with type 2 diabetes [bib_ref] The association between smoking cessation and glycaemic control in patients with type..., Lycett [/bib_ref].
The observed trend of diabetes during the 5-year interval between the study's two phases indicated that both overall and age-dependent pre-diabetes decreased significantly. This, along with the overall stability in the prevalence of diabetes, is promising. Iran has a well-developed primary healthcare system in rural areas, with the "Behvarz" health workers responsible for population-based prevention and control services. The effectiveness of Behvarz health workers in rural areas on better diabetes management (both diagnosis and treatment) has been mentioned [bib_ref] Effectiveness of diabetes and hypertension management by rural primary healthcare workers (Behvarz..., Farzadfar [/bib_ref]. Likewise, in recent years, the government has made significant improvements in the primary health care system of urban areas. Unfortunately, based on the results of KERCADRS phase 2, physical activity has decreased, especially in young individuals, during the 5-year interval between the two phases of the study [bib_ref] The prevalence and 5-year incidence rate of low physical activity in an..., Najafipour [/bib_ref] , and overweight and obesity have increased in the area. These may decelerate the positive effect of improvements in other health factors.
The other important finding of the study was that the highest incidence rate of pre-DM and DM was related to occasional and dependent opium users. There is a belief among most opium users that this substance will reduce blood glucose [bib_ref] The impact of opium consumption on blood glucose, serum lipids and blood..., Najafipour [/bib_ref] and that it is beneficial to patients with diabetes. This study did not verify this belief. On the other hand, the lowest incidence rate of pre-DM and DM was in those with high physical activity, while low physical activity is quite prevalent among opium users [bib_ref] The prevalence and 5-year incidence rate of low physical activity in an..., Najafipour [/bib_ref].
# Strengths and weaknesses
In addition to the broad age range of the participants and the beneficial and definitive epidemiological information related to CAD risk factors in the population, which the study presented to health authorities, we should acknowledge the limitations of our survey. Firstly, KERCADRS was not an interventional study. Secondly, in the present study, those with FPG ≥ 126 mg/dl were examined for the first time, and HbA1c < 6.5% in recruitment were not included as people with diabetes. This happened because we had used HbA1c as the second test for confirming the diagnosis of diabetes. Assessing HbA1c as the second test confirmed the diagnosis and also showed us how diabetes is controlled. As there were 46 patients with the mentioned conditions, assuming that half of these were diabetic, the prevalence of reported diabetes in the present study would increase from 10.2 to 10.4%. We are going to correct the protocol in the third phase of KERCADRS to recheck FBS and HbA1c in the second blood sampling (recruitment). Thirdly, we could not distinguish type I from II diabetes as we did not review the individuals' medical records. Fourthly, although we tried to track all people who participated in phase 1, unfortunately, a significant number of them had changed their location between the two phases, and we were unable to contact them because mobile phones were not so popular in 2009-2011 or because some of them had died between the two phases. This may affect the incidence calculation in the study. However, an increase in the sample size to 10,000 in phase 2 strengthens the prevalence calculated in the study.
# Conclusion
There were promising signs of considerable reduction in the prevalence of pre-DM and undiagnosed DM and stability in the prevalence of DM in the 5-year interval between the two phases of the study. However, the finding that treatment is still ineffective in more than 55% of diagnosed DM individuals is a warning that the health care management system should take more effective measures in primary healthcare in urban areas. Early diagnosis and better management of diabetic cases are necessary to prevent further diabetes-caused morbidity and mortality in this area.
# Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.
# Ethics statement
The studies involving human participants were reviewed and approved by Ethics Committee of Kerman University of Medical Sciences (Ethical code: IR.KMU.REC.1392.405). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.
# Author contributions
HN, AM, and MSa designed the study. AM developed the analytic approach. HN, MSa, MF, and MSh participated in data collection. RA did the analysis with input from other authors. HN, MF, and MSh wrote the first draft of the paper. All authors contributed to finalizing of the paper.
# Funding
The KERCADR phase 2 study was a population-based cohort study implemented and funded by Kerman University of Medical Sciences (Grant No. KMU.93/310).
[fig] FIGURE 1 |: The flow chart of people participated in both phases of the study. [/fig]
[table] TABLE 1 |: The standardized prevalence % (95% confidence interval CI) of pre-diabetes, undiagnosed and diagnosed diabetes, and adjusted odds ratio for different predictors of diabetes mellitus; community-based cohort study (KERCADRS-2nd Phase-N = 9,959), Kerman, Iran 2014-2018. [/table]
[table] TABLE 3 |: The [/table]
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10.3389/fphys.2014.00464
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s2orc_pubmed_articles
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Utilizing past and present mouse systems to engineer more relevant pancreatic cancer models
The study of pancreatic cancer has prompted the development of numerous mouse models that aim to recapitulate the phenotypic and mechanistic features of this deadly malignancy. This review accomplishes two tasks. First, it provides an overview of the models that have been used as representations of both the neoplastic and carcinoma phenotypes. Second, it presents new modeling schemes that ultimately will serve to more faithfully capture the temporal and spatial progression of the human disease, providing platforms for improved understanding of the role of non-epithelial compartments in disease etiology as well as evaluating therapeutic approaches.
# Introduction
There has been a noticeable increase (near doubling) in the 5-year survival of pancreatic cancer patients, though the number remains quite low at about 6-7% (SEER Stat Fact Sheets: Pancreas Cancer-NCI). Much of this stems from a few more potent clinical therapies [folfirinox [bib_ref] FOLFIRINOX -a new paradigm in the treatment of pancreatic cancer, Papadatos-Pastos [/bib_ref] , nab-paclitaxol [bib_ref] Nab-paclitaxel and gemcitabine for the treatment of patients with metastatic pancreatic cancer, Borazanci [/bib_ref] , and various combinations with gemcitabine [bib_ref] Efficacy and safety profile of combining agents against epidermal growth factor receptor..., Tian [/bib_ref] ] that improve on previous survival rates. Thus, begins a new drive to employ relevant preclinical models with which to test novel drugs that can further improve patient survival. Indeed, there are already a few mouse models that can be used, with KPC mice (as described below) being one model that currently boasts a strong recapitulation of the paradigm observed in human pancreatic adenocarcinoma. Yet, further advances on mouse models will not only generate additional preclinical models but, perhaps more importantly, demonstrate the utility of newer diagnostic and/or therapeutic targets. The main objective of this review is to highlight past and present mouse models of pancreatic cancer [see [bib_ref] Genetically engineered mouse models of pancreatic adenocarcinoma, Guerra [/bib_ref] for a more thorough review of current models] in order to propose continued engineering of more relevant mouse systems. These future models could then be employed to better understand the role of non-parenchymal compartments during the development of disease as well as build inducible systems that allow multiple allelic changes at various intervals.
## Transgenic models
Initially, development of cancer in mouse pancreas was demonstrated by targeting Myc and TGFα to mouse pancreatic acinar cells (EL-Myc and EL-TGFα), which demonstrated acinar-to-ductal metaplasia leading to exocrine carcinoma with focally distinct ductal-like lesions [bib_ref] Overexpression of TGF alpha in transgenic mice: induction of epithelial hyperplasia, pancreatic..., Sandgren [/bib_ref] [bib_ref] Pancreatic tumor pathogenesis reflects the causative genetic lesion, Sandgren [/bib_ref] [bib_ref] Transforming growth factor alpha dramatically enhances oncogene-induced carcinogenesis in transgenic mouse pancreas..., Sandgren [/bib_ref] [bib_ref] Acinar-to-ductal metaplasia accompanies c-myc-induced exocrine pancreatic cancer progression in transgenic rodents, Grippo [/bib_ref]. Previous targeting of oncogene expression via the elastase (EL) promoter proved effective at inducing exocrine pancreatic neoplasms in transgenic mice, including EL-SV40 TAg and EL-Hras [bib_ref] Elastase I promoter directs expression of human growth hormone and SV40 T..., Ornitz [/bib_ref]. These two models developed acinar hyperplasia and carcinomawhile EL-TGFα mice produced severe fibrosis, tubular complexes, and aberrant cell morphology [bib_ref] Transforming growth factor alpha dramatically enhances oncogene-induced carcinogenesis in transgenic mouse pancreas..., Sandgren [/bib_ref]. Older EL-TGFα mice eventually develop carcinoma, and tumor development was enhanced in a p53 null background and concomitant with partial or whole loss of INK4a or SMAD4 . The metaplasia in EL-TGFα/p53 +/− mice was characterized along with its genomic signature [bib_ref] Pattern of secondary genomic changes in pancreatic tumors of Tgf alpha/Trp53 +/−..., Schreiner [/bib_ref] and increased expression of Pdx1, a gene necessary for pancreas development and often expressed in pancreatic cancer, was observed in mice with overexpression of TGFα [bib_ref] Expansion of Pdx1-expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpressing..., Song [/bib_ref]. Additionally, the EL-KRAS model, which directs human mutant KRAS transgene expression to pancreatic acinar cells via a rat elastase driver, demonstrates a common pancreatic cancer histotype by inducing neoplastic, ductal lesions [bib_ref] Preinvasive pancreatic neoplasia of ductal phenotype induced by acinar cell targeting of..., Grippo [/bib_ref] , often referred to as cystic papillary neoplasms (CPNs) similar to human cystic neoplasms including IPMN and MCN [bib_ref] Pathology of genetically engineered mouse models of pancreatic exocrine cancer: consensus report..., Hruban [/bib_ref].
## Conditional models
Conditional systems have become an asset to the mousemodeling field as they provide tissue specific targeting of genes. One prominent targeting strategy included Pdx1 and Ptf1a or p48-driven expression of Cre recombinase in mice with flanking Lox elements (floxed) that, upon Cre-mediated recombination, generated a mutant Kras in the endogenous mouse allele. These mice developed ductal lesions and mPanINs that occasionally progressed to invasive cancer [bib_ref] Preinvasive and invasive ductal pancreatic cancer and its early detection in the..., Hingorani [/bib_ref]. This model laid the foundation for the generation of the LSL-Kras G12D/+ ;LSL-Trp53 R172H/+ ;Pdx1-Cre (KPC) model which demonstrates a highly metastatic carcinoma that resembles human disease [bib_ref] Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic..., Hingorani [/bib_ref]. Models such as this one have allowed for the characterization of biomarkers in pancreatic cancer from disease initiation to metastasis [bib_ref] Spatiotemporal proteomic analyses during pancreas cancer progression identifies STK4 as a novel..., Mirus [/bib_ref]. It is important to note that these floxed alleles can be targeted to other cell types in the pancreas as demonstrated by expression of the LSL−Kras G12D/+ allele in Nestin positive cells leading to mPanINsand caerulein-induced PDAC.
Following the use of these models, other conditional targets were generated utilizing similar technology. Since Transforming Growth Factor β (TGFβ) signaling is commonly disrupted in cancer [bib_ref] TGF-beta: duality of function between tumor prevention and carcinogenesis, Principe [/bib_ref] and highly so in pancreatic cancer [bib_ref] Core signaling pathways in human pancreatic cancers revealed by global genomic analyses, Jones [/bib_ref] , LSL-Kras G12D/+ ;Tgfbr2 flox/flox ;Ptf 1a Cre/+ mice were generated to simultaneously express mutant Kras G12D and loss of the type 2 TGFβ receptor (Tgfbr2) in pancreatic epithelium. This model demonstrated an aggressive form of pancreatic ductal adenocarcinoma (PDAC) and explored the role of TGFβ signaling in the development of the disease [bib_ref] Aggressive pancreatic ductal adenocarcinoma in mice caused by pancreas-specific blockade of transforming..., Ijichi [/bib_ref]. As loss of downstream TGFβ target SMAD4 is common in pancreatic cancer [bib_ref] DPC4, a candidate tumor suppressor gene at human chromosome 18q21, Hahn [/bib_ref] , LSL-Kras G12D/+ ;Dpc4 flox/+ ;Pdx1-Cre and LSL-Kras G12D/+ ;Dpc4 flox/+ ;Ptf1a Cre/+ were generated to conditionally express Kras G12D in concert with Smad4/Dpc4 haploinsufficiency in the pancreas, thereby inducing MCNs and subsequent PDAC [bib_ref] Kras(G12D) and Smad4/Dpc4 haploinsufficiency cooperate to induce mucinous cystic neoplasms and invasive..., Izeradjene [/bib_ref]. Additionally, IPMN-like lesions accompanied by PDAC and metastatic disease were shown with the LSL-Kras G12D/+ ;Smad4 flox/flox ;Pdx1-Cre model [bib_ref] Inactivation of Smad4 accelerates Kras(G12D)-mediated pancreatic neoplasia, Kojima [/bib_ref].
Considering the implications for loss/inactivation of p16 Ink4a and p19 Arf in cellular transformation, a variety of models have pursued this target in concert with pancreas-specific mutations. An MT-TGFα;Ink4a/Arf −/− model was generated, ultimately demonstrating a serous cystadenoma (SCA) phenotype that resembled human disease [bib_ref] Obligate roles for p16(Ink4a) and p19(Arf)-p53 in the suppression of murine pancreatic..., Bardeesy [/bib_ref]. Following the creation of this model, pancreasspecific Kras targeting was coupled with a floxed Ink4a/Arf locus. These LSL-Kras G12D ;Ink4a/Arf flox/flox ;Pdx1-Cre mice presented with invasive, metastatic disease consistent with human disease [bib_ref] Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma, Aguirre [/bib_ref]. In addition, the LSL-Kras G12D/+ ;p16 flox/flox ;Pdx1-Cre model directed the knockout of the p16 Ink4a tumor suppressor gene in pancreatic epithelium. These mice developed mPanINs, PDAC, and metastases [bib_ref] Disruption of p16 and activation of Kras in pancreas increase ductal adenocarcinoma..., Qiu [/bib_ref]. Characterization of this tumor suppressive axis also prompted the generation of LSL-Kras G12D/+ ;Rb flox/flox ;Pdx1-Cre mice to assess the role of Rb inactivation and PDAC progression. These mice exhibited accelerated mPanIN progression and rapid PDAC development [bib_ref] Deletion of Rb accelerates pancreatic carcinogenesis by oncogenic Kras and impairs senescence..., Carriere [/bib_ref].
The activation of mutant Kras and heparin-binding epidermal growth factor-like growth factor (HB-EGF) by the Means group also demonstrated conditional targeting of two oncogenic events.
These mice featured rapid progression into the early stages of pancreatic cancer [bib_ref] Heparin-binding epidermal growth factor-like growth factor eliminates constraints on activated Kras to..., Ray [/bib_ref].
The tumor stroma's control of tumor growth was explored by utilizing two conditional models of pancreatic cancer. Shh flox/flox ;Pdx1-Cre;LSL-Kras G12D/+ ;p53 flox/+ ;Rosa26 LSL−YFP (Sh hPKCY) mice were generated to delete Sonic Hedgehog (SHH) in the context on PDAC. Due to lack of SHH, these mice presented with less tumor stroma yet more aggressive, proliferative tumors. This phenotype was also shown utilizing a Smoothened inhibitor in KPC mice. Additionally, VEGFR inhibition promoted SHHdeficient tumor survival, demonstrating that SHH-formed stroma limits tumor growth by restricting tumor angiogenesis. [bib_ref] Stromal elements act to restrain, rather than support, pancreatic ductal adenocarcinoma, Rhim [/bib_ref].
Additional study of the tumor stroma's contribution to cancer growth was explored via the generation of a mouse model that crosses LSL-Kras G12D/+ ;Tgfbr2 flox/flox ;Ptf1a Cre/+ mice to αSMAtk transgenic mice. Depletion of αSMA + myofibroblasts in the context of mPanINs or PDAC resulted in reduced survival characterized by hypoxia, EMT, and cancer stem cells. In addition, this model was characterized by the increase in regulatory T cells infiltrating myofibroblast-depleted tumors. Similar results were shown when the KPC model was used in cross with the αSMA-tk transgenic [bib_ref] Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer..., Ozdemir [/bib_ref].
Both of these studies hold implications for the future of stromal-directed therapies for the treatment of PDAC. Although mouse models have been successful for such therapies [bib_ref] Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model..., Olive [/bib_ref] , the recapitulation of these results in clinical trials has largely failed. Rhim and Ozdemir demonstrated that tumor stroma provided a protective effect for the host. Therefore, targeting the stroma may create a more aggressive form of PDAC. As noted by Gore and Korc, the stroma's capacity for both benefit and damage must be further explored in mouse models before potential therapies are reapplied in human trials [bib_ref] Stromal biology and therapy in pancreatic cancer, Neesse [/bib_ref] [bib_ref] Pancreatic cancer stroma: friend or foe?, Gore [/bib_ref]. However, ablation of a subpopulation of stromal cells (FAP+ cells) permitted immune control of tumor growth and uncovered the efficacy of immunotherapeutic antibodies (anti-CTLA-4 or anti-PD-L1), which resulted in acute tumor regression [bib_ref] Suppression of antitumor immunity by stromal cells expressing fibroblast activation protein-alpha, Kraman [/bib_ref] [bib_ref] Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic..., Feig [/bib_ref]. More recently it has been shown that VDR acts as a master transcriptional regulator of PSCs to reprise the quiescent state, resulting in induced stromal remodeling, increased intratumoral gemcitabine, reduced tumor volume, and a 57% increase in survival compared to chemotherapy alone [bib_ref] Vitamin d receptor-mediated stromal reprogramming suppresses pancreatitis and enhances pancreatic cancer therapy, Sherman [/bib_ref]. The distinct outcome of these studies underscores the need to better understand the role of desmoplastic stroma in pancreatic cancer.
## Inducible/conditional mouse modeling systems of pancreatic cancer
While the described conditional modeling systems have provided invaluable insight into disease incidence and progression, they do not fully capture the temporal component of human mutations observed in the clinic. For instance, in systems relying on Pdx or Ptf1 driven Cre, recombination occurs at E8.5 [bib_ref] IPF1, a homeodomain-containing transactivator of the insulin gene, Ohlsson [/bib_ref] or E9.5 [bib_ref] p48 subunit of mouse PTF1 binds to RBP-Jkappa/CBF-1, the intracellular mediator of..., Obata [/bib_ref] , respectively. While embryonic recombination often shortens the time to a cancer or neoplastic phenotype, the effects of these mutations on pancreatic development are not fully understood, and do not faithfully mimic the spontaneous mutations that occur in the fully formed gland of an adult human patient.
In recent years, conditional and inducible systems have prompted the unique ability to control when and where genes are expressed. In particular, the development of CreERT technology [bib_ref] Ligand-activated site-specific recombination in mice, Feil [/bib_ref] [bib_ref] Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains, Feil [/bib_ref] has prompted an array of tissue specific, temporally-controlled targeting models. Both the ElastaseCreERT2 [bib_ref] Preexisting pancreatic acinar cells contribute to acinar cell, but not islet beta..., Desai [/bib_ref] and the Ptf1a Cre-ERTM [bib_ref] Ongoing Notch signaling maintains phenotypic fidelity in the adult exocrine pancreas, Kopinke [/bib_ref] systems have advanced the field of pancreatic cancer modeling by providing a means for inducibly targeting pancreatic epithelium. Both of these systems feature a Cre recombinase cassette fused to a Tamoxifen-responsive mutant estrogen-receptor element that is driven by an acinar cell specific promoter region. The Cre recombinase in each of these systems is then able to activate gene expression in a loxP-mediated system. The utility of the CreERT system was further demonstrated in the Kras G12D ;Rosa26 NIC ;Pdx1-CreERT model, which temporally controlled the expression of Notch and Kras and showed synergistic effects between the two proteins with respect to mPanIN progression [bib_ref] Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia, De La [/bib_ref].
## Ikras * models
The Pasca di Magliano group has also generated several models that represent the full utilization of both spatial and temporal control of gene expression. The iKras * model functions through the transgenesis of three different types of mice. In these mice, the Ptf1a allele drives Cre expression [bib_ref] The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic..., Kawaguchi [/bib_ref] , which, in turn, excises a stop cassette bound by two loxP sites. This stop cassette functions to inhibit the reverse tetracycline transactivator (rtTa) for an IRES-EGFP cassette at the R26 locus [bib_ref] Conditional and inducible transgene expression in mice through the combinatorial use of..., Belteki [/bib_ref]. Since Ptf1a Cre/+ is mostly pancreas specific, the excision of the stop cassette allows for the expression of both rtTa and EGFP in the pancreatic epithelium beginning during embryogenesis [bib_ref] Oncogenic Kras is required for both the initiation and maintenance of pancreatic..., Collins [/bib_ref].
Administering doxycycline to these animals leads to activation of rtTa and subsequent Kras * expression through a TetO-Kras G12D transgene using rat mutant Kras [bib_ref] Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras..., Fisher [/bib_ref]. This inducible system provides a strong platform to explore several relevant issues. First, the mutation of Kras can be expressed in adult tissues, which is far more relevant to PanIN progression to cancer observed in humans. In addition, it allows for the abrogation of oncogenic Kras expression at various stages of cancer development and thus the study of the dependence of developing lesions and cancer on mutant Kras. Also, this system can be employed to investigate carcinogenesis in the context of tumor suppressor inactivation or additional oncogene activation. iKras * -p53 +/− mice were also generated to illustrate the development of PDAC when mutant Kras is paired with the concurrent inactivation of this tumor suppressor gene [bib_ref] Oncogenic Kras is required for both the initiation and maintenance of pancreatic..., Collins [/bib_ref]. This model provides a framework examining various features of oncogenic Kras in PDAC development. Inhibition of mutant Kras expression through doxycycline removal and subsequent reversion to a more normal phenotype supports continued efforts to target mutant Kras as a therapeutic option and eventual translation to the clinic. Furthermore, the Pasca di Magliano group generated a model that inducibly and conditionally activated Kras and a mutant p53 allele [bib_ref] Metastatic pancreatic cancer is dependent on oncogenic Kras in mice, Collins [/bib_ref]. These mice featured the same iKras * system described above with an additional mutant p53 allele preceded by a loxP-bound STOP cassette. Therefore, the same Ptf1a-driven Cre-recombinase that activates the rtTa for iKras * expression will also activate the mutant p53 R172H (p53 * ) allele [bib_ref] Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome, Olive [/bib_ref] by excising the preceding STOP cassette. However, in these iKras * p53 * mice, oncogenic Kras is not activated until doxycycline administration. This model demonstrated a dual functionality by allowing the simultaneous, pancreas-specific targeting of two alleles (iKras * and p53 * ) and the inducible/reversible expression of oncogenic Kras [bib_ref] Metastatic pancreatic cancer is dependent on oncogenic Kras in mice, Collins [/bib_ref]. Although the conditional LSL-Kras G12D/+ ;LSL-Trp53 R172H/+ ;Pdx1-Cre (KPC) model [bib_ref] Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic..., Hingorani [/bib_ref] of PDAC demonstrated a close mimicking of the human disease, it lacked inducible control of Kras. This type of control over mutant Kras expression allowed for the study of its role in primary and metastatic tumor maintenance when expressed concurrently with mutant p53 [bib_ref] Metastatic pancreatic cancer is dependent on oncogenic Kras in mice, Collins [/bib_ref] and the demonstration of mutant Kras-dependence on more aggressive and metastatic pancreatic cancer.
## Lsl-kras +/g12vgeo ;el-tta/teto-cre models
Additionally, the Barbacid group generated a model that accomplishes both temporal and spatial targeting of oncogenic Kras using a different mutant variant (G12V vs. G12D). By crossing a LSL-Kras +/G12Vgeo knockin strain [bib_ref] Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular..., Guerra [/bib_ref] to EL-tTA/tetO-Cre mice, their group was able to obtain an inducible system of endogenous Kras G12V mediated by doxycycline control of Cre recombinase activity [bib_ref] Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras..., Guerra [/bib_ref]. Essentially, removing doxycycline in this tet-off system permits an elastase-driven Cre specific to acinar and centroacinar cells of the pancreas. The Cre changes LSL-Kras G12Vgeo into the active, oncogenic Kras G12Vgeo by excising the loxP sites that contain a stop cassette. The utility of this system is further advanced by the detection of cells that ultimately end up with Kras G12V expression. A knockin of IRES-geo into the 3 untranslated sequences of the Kras allele allows for LacZ expression when the LSL cassette is removed [bib_ref] Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular..., Guerra [/bib_ref]. LacZ encodes β-galactosidase, which is then detectable via histochemical staining. Initially, this system was used to induce expression of oncogenic Kras at E16.5, leading to the production of mPanIN lesions that could advance in severity following caerulein administration [bib_ref] Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras..., Guerra [/bib_ref]. Surprisingly, doxycycline removal in adult stages resulted in widespread expression of Kras G12V in adult acinar cells with no phenotypic consequences. Interestingly, adult mice that express Kras G12V in the acinar cell compartment develop mPanINs and PDAC in the context of pancreatitis.
To explore the resistance of postnatal acinar cells to transformation via the expression of Kras, the Barbacid group also characterized the role of several tumor suppressors. These acinar cells were resistant to transformation even in the absence of tumor suppressors. Kras +/G12V ;p16Ink4a/p19Arf flox/flox ;EL-tTA/tetO-Cre and Kras +/G12V ;Trp53 flox/flox ;EL-tTA/tetO-Cre mice were generated and given doxycycline from birth until P60 [bib_ref] Pancreatitis-induced inflammation contributes to pancreatic cancer by inhibiting oncogene-induced senescence, Guerra [/bib_ref]. Acting under the same tet-off system as described above, these mice, when taken off doxycycline, were subject to expression of Cre recombinase in acinar and centroacinar cells of the pancreas. However, instead of just activating Kras, the Cre simultaneously excised the floxed p16Ink4a/p19Arf or Trp53 alleles. These models, when combined with caeruleininduced pancreatitis, presented an invasive, metastatic PDAC phenotype [bib_ref] Pancreatitis-induced inflammation contributes to pancreatic cancer by inhibiting oncogene-induced senescence, Guerra [/bib_ref].
## Tva-rcas models
Another mouse model system that features viral delivery for eventual induction of gene expression or loss of cell targets demonstrates the versatility of this field and another avenue for creating complex inducible/conditional schemes. Varmus and colleagues generated a model that introduced a replication-competent avian leukosis sarcoma virus long-terminal repeat with splice acceptor (ALSV-A-based RCAS) vector to mice that expressed the ALSV-A receptor, TVA, [bib_ref] An RCAS-TVA-based approach to designer mouse models, Orsulic [/bib_ref] under the control of the elastase promoter [bib_ref] The c-myc and PyMT oncogenes induce different tumor types in a somatic..., Lewis [/bib_ref]. This elastase-tva model allowed somatic acinar cells of the pancreas to incorporate RCASdelivered genes, such as polyoma virus middle T antigen (PyMT) [bib_ref] Natural biology of polyomavirus middle T antigen. Microbiol, Gottlieb [/bib_ref] or c-Myc, into the host cell genome. These elastase-tva mice were crossed to Ink4a/Arf null mice to create models characterizing the phenotype resulting from these initiating oncogenic events [bib_ref] The c-myc and PyMT oncogenes induce different tumor types in a somatic..., Lewis [/bib_ref]. They found that PyMT and c-Myc induced different types of pancreatic tumors, illustrating the impact of the initiating lesion on resulting tumor pathology.
The development of this TVA-RCAS model was further expanded with the coupling of the elastase-tva mice with Trp53 flox;Ptf1a-Cre [bib_ref] Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse..., Jonkers [/bib_ref] [bib_ref] The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic..., Kawaguchi [/bib_ref] mice [bib_ref] Trp53 deletion stimulates the formation of metastatic pancreatic tumors, Morton [/bib_ref]. In this model, delivery of the PyMT oncogene is accompanied by the pancreas-specific deletion of the tumor suppressor, Trp53. Results of this model showed metastatic disease to the liver. In addition, the elastasetva;Trp53 flox/flox ;Ptf1a Cre/+ mice were crossed to Ink4a/Arf flox/+ [bib_ref] Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice, Krimpenfort [/bib_ref] mice to assess tumor development in the context of a simultaneous p53 deficiency and Ink4a/Arf single allele deletion. Results of this model elucidated a much more aggressive tumor model after PyMT activation via virus administration [bib_ref] Trp53 deletion stimulates the formation of metastatic pancreatic tumors, Morton [/bib_ref]. This model succeeds as an example of both conditional and temporal control of gene expression by combining both pancreas-specific deletion of Trp53 via Crerecombinase activity and acinar cell-directed, inducible PyMT expression via elastase-tva targeting.
Lewis and his group expounded upon these findings by crossing the elastase-tva model with LSL-Kras G12D ;Ptf1a Cre/+ mice [bib_ref] Preinvasive and invasive ductal pancreatic cancer and its early detection in the..., Hingorani [/bib_ref] to assess the impact of activated Wnt signaling in the context of KRAS-induced pancreatic tumorigenesis [bib_ref] Activated wnt signaling in stroma contributes to development of pancreatic mucinous cystic..., Sano [/bib_ref]. These mice were injected with chick fibroblasts that produced ALSV-A-based RCAS vectors encoding Wnt1 or a GFP control, ultimately resulting in host genome uptake of these genes in pancreatic acinar cells and their progenitors. Thus, this model allowed for the targeting of Wnt1 to the pancreatic epithelium and subsequent characterization of its signaling activity when introduced in concert with Kras activation. They found that in this context, activated Wnt signaling induced the formation of mucinous cystic neoplasms (MCN). Interestingly, these mice displayed higher Wnt signaling in the stroma of the MCNs, rather than in the cyst epithelium, which is consistent with MCN patient data [bib_ref] Activated wnt signaling in stroma contributes to development of pancreatic mucinous cystic..., Sano [/bib_ref]. These results suggest that Wnt ligands may act in a paracrine fashion to stimulate MCN development.
## Exploring inducible/conditional systems coupled with epigenetic events
The significance of factors external to genomic changes in these models must not be overlooked. Multiple mutant Kras-expressing models have demonstrated the contribution of inflammation and dietary aspects to pancreatic cancer pathogenesis, improving our understanding of pancreatic cancer and pancreatitis as well as the interplay between the two. It was shown that high levels of Ras activity in cLGL-Kras G12V ;EL-CreERT generated high levels of fibrosis and inflammation that mimicked chronic pancreatitis. Since elevated Ras activity is also found in PDAC, this finding provided a mechanistic link between pancreatic cancer and chronic pancreatitis [bib_ref] Ras activity in acinar cells links chronic pancreatitis and pancreatic cancer, Logsdon [/bib_ref].
Other mechanisms have been explored with respect to inflammatory insult and subsequent neoplastic and cancerous phenotypes. Utilizing a breadth of models, Jack's group established that chronic pancreatitis may provide enough insult for insulin-expressing endocrine cells to become susceptible to KRAS-induced transformation [bib_ref] Context-dependent transformation of adult pancreatic cells by oncogenic K-Ras, Gidekel Friedlander [/bib_ref]. Logsdon and colleagues also demonstrated that with caerulein induction of acute pancreatitis in the presence of inducible mutant Kras (LSL-Kras G12V ;EL-CreERT) there was NF-κB mediated amplification of Ras activity. These mice presented with chronic inflammation and mPanIN lesions that subsided with the inhibition of Cox-2 or deletion of IKK2. This effect was also demonstrated in KC mice with loss of Cox-2 despite the additional loss of pTEN, highlighting the potential role of AKT activation in chemoresistance [bib_ref] Cell intrinsic role of COX-2 in pancreatic cancer development, Hill [/bib_ref]. Likewise, the LSL-Kras G12V ;EL-CreERT model was used in a cross with Cox-2 conditional knockout mice to study the effects of high fat diets on PDAC. LSL-Kras G12V ;EL-CreERT mice fed high fat diet presented with increased fibrosis, mPanINs, and PDAC compared to no increased mPanIN lesions or PDAC in COX flox/flox ;LSL-Kras G12V ;EL-CreERT mice fed the same diet [bib_ref] A high-fat diet activates oncogenic Kras and COX2 to induce development of..., Philip [/bib_ref]. Similarly, KC mice were shown to generate mPanIN lesions at an earlier onset following a high fat, high calorie diet with a subsequent increase in infiltration of macrophages and T cells in an expanded stromal bed [bib_ref] High-fat, high-calorie diet promotes early pancreatic neoplasia in the conditional KrasG12D mouse..., Dawson [/bib_ref].
Progression of mPanINs and PDAC has also been explored in the context of inhibitors to the Ras signaling pathway. Gefitinib, an EGFR inhibitor, was given to LSL-Kras G12D/+ ;Ptf1a Cre/+ mice, demonstrating a prevention of mPanIN and PDAC development [bib_ref] The epidermal growth factor receptor inhibitor gefitinib prevents the progression of pancreatic..., Mohammed [/bib_ref]. Similarly, it was shown that inhibition of EGFR does not allow for RAS levels sufficient for the transformation seen in PDAC [bib_ref] EGF receptor is required for KRAS-induced pancreatic tumorigenesis, Ardito [/bib_ref] [bib_ref] EGF receptor signaling is essential for k-ras oncogenedriven pancreatic ductal adenocarcinoma, Navas [/bib_ref].
## Future applications of inducible/conditional modeling systems
The mouse-modeling field has capitalized on conditional and/or inducible Cre-lox technology to target gene expression in numerous cell types. However, the overwhelming majority of pancreatic cancer models rely on Cre-lox to drive oncogenic Kras in the pancreatic epithelium, excluding the use of non-epithelial Cre systems and limiting the ability to target other cells types involved in carcinogenesis. Therefore, utilizing non-Cre-lox driven systems to target mutant Kras to pancreatic epithelium will allow compatibility with a vast array of preexisting Cre-lox systems that target genetic changes to additional cell types including the stroma and hematopoietic cell compartments. The use of single transgenic or knockin systems in combination with Cre-lox models that target non-parenchymal cells in the pancreas can circumvent some of the limitations that arise when using Cre-lox to drive an initiating event like mutant Kras.
## Figure 1 | mimicking human tumorigenesis through temporal modeling of pancreatic cancer.
A key difference between human pancreatic cancer and commonly used mouse models is in the timing of mutations. In human patients, Kras mutations are often considered an initiating event, occurring in adult cells, soon followed by mutations to p16, and later p53 and/or SMAD4. Yet in most models, Kras and altered tumor suppressor genes are induced simultaneously in the developing embryo. Despite a human-like histotype, these models have yet to be accurate predictors of outcomes observed in clinical trials. Therefore, we propose that using combinations of several systems to drive sequential Kras, p16, and SMAD4/p53 mutations may lead to more human-like disease that responds to therapy more like that observed in the clinic.
## Figure 2 | temporal modeling via two inducible systems.
In order to address the issue of successive induction of mutations as they occur in human, several modeling systems can be employed. In this example, as designed by the Pasca di Magliano group, expression of Cre-recombinase is driven by the Ptf1a promoter. This is combined with a LSL cassette followed by an rtTA sequence. In the presence of Cre, the stop codon is excised, and rtTA is transcribed. This allows for interaction with a third transgene, a TRE-Kras. When doxycycline is administered, oncogenic Kras expression is induced. By activating this system at 1 month, it would allow a simulated Kras mutation in near-adult tissues. Once lesions manifest, this can be followed by the induction of a second transgene, a mutant p53 driven by a Sox9-FLP ERT 2 recombinase. This will excise a stop codon in front of a mutant p53 sequence in the presence of tamoxifen, and drive mutant p53 expression. The p16 allele could also be engineered in the same manner. Timing of these events will likely have to be determined empirically, as mutant Kras expression in adult pancreas may not lead to the development of neoplastic lesions without an external stimulus (like caerulein). Indeed, a third allelic alteration may be necessary to drive a more aggressive metastatic phenotype (see .
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The EL-KRAS model may be a prime candidate for combined Cre-lox targeting of other cell types, as these mice develop acinarto-ductal metaplasia and cystic papillary neoplasms (CPN) that resemble human cystic disease in the pancreas. These lesions did progress to PDAC in a p16 null background or acinar carcinoma when in a p53 null background (personal communication with Dr. Eric Sandgren). EL-TGFα [bib_ref] Overexpression of TGF alpha in transgenic mice: induction of epithelial hyperplasia, pancreatic..., Sandgren [/bib_ref] and Mist1 KrasG12D/+ [bib_ref] Mist1-KrasG12D knock-in mice develop mixed differentiation metastatic exocrine pancreatic carcinoma and hepatocellular..., Tuveson [/bib_ref] models can serve as potential neoplastic drivers used in concert with Cre-lox targeting. EL-TGFα mice have been employed in combination with p53 loss [bib_ref] Pattern of secondary genomic changes in pancreatic tumors of Tgf alpha/Trp53 +/−..., Schreiner [/bib_ref] to generate a model of advanced pancreatic cancer with hallmark genetic features (loss of p16, inactivation of Cdkn2a) reminiscent in human disease and, in combination with mutant Kras, development of CPN that resembles human IPMN [bib_ref] Concomitant pancreatic activation of Kras(G12D) and Tgfa results in cystic papillary neoplasms..., Siveke [/bib_ref]. EL-TGFα does lead to proliferation of acinar cells and fibroblasts and focally generated metaplastic lesions derived from acini [bib_ref] Overexpression of TGF alpha in transgenic mice: induction of epithelial hyperplasia, pancreatic..., Sandgren [/bib_ref]. Yet, there was no reported observation of neoplasia or more advanced lesions in this model. Mist1 KrasG12D/+ mice developed a predictable lethal pancreatic cancer phenotype characterized by acinar metaplasia and dysplasia in its early stages [bib_ref] Mist1-KrasG12D knock-in mice develop mixed differentiation metastatic exocrine pancreatic carcinoma and hepatocellular..., Tuveson [/bib_ref]. Despite being a strong model of the pancreatic neoplasia to cancer paradigm as an ectopic model of mutant Kras expression, Mist1 KrasG12D/+ mice did, rather unexpectedly, develop hepatocellular carcinoma [bib_ref] Mist1-KrasG12D knock-in mice develop mixed differentiation metastatic exocrine pancreatic carcinoma and hepatocellular..., Tuveson [/bib_ref]. This feature of the model may be of potential concern when attempting to evaluate the phenotypes of genetically engineered mice that employ this particular initiating event. However, an inducible targeting of LSL-Kras G12D/+ with Mist1 CreERT2/+ produced mPanIN lesions, indicating the relevance of the Mist1-expressing compartment in the origins of PDAC [bib_ref] Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting..., Habbe [/bib_ref]. Although EL-KRAS FIGURE 3 | Temporal modeling via three inducible systems. As human malignancies often involve several mutations, a compound inducible system may be employed to target three successive transgenes to the same cell type. For example, mtKras may be first induced through a TVA/RCAS virus system. In this system, expression of a TVA receptor is targeted to the pancreas via the elastase promoter. Upon reaching adulthood, animals can be administered a RCAS virus coding for the mtKras gene. This will interact only with cells expressing the TVA receptor, allowing for targeted and inducible expression of KRAS in the pancreas. A second mutation, such as loss of p16, can then be induced in the same cells via an elastase driven tTA that, in the presence of doxycycline, will induce expression of Cre through TRE-Cre. Combining this with a p16 flox/flox gene will allow for doxycycline-induced loss of the p16 gene, and the second genetic hit. Finally, a tamoxifen-responsive Sox9-FLP ERT2 can target cells expressing ductal markers (including those having undergone acinar-ductal metaplasia), allowing for inducible expression of mtP53 via an FSF cassette, providing the third genetic hit as it often occurs in humans. It is important at each induction point that promoter/gene regulatory elements employed to run the next step be evaluated in the previous model. Hence, acinar-specific markers (eg., Amylase) should be assessed in pancreas following mutant Kras expression (TVA/RCAS delivery) and Sox9 antibodies should be used to demonstrate Sox9 expression in mtKras expressing pancreas with loss of p16. This would need to be done at the empirically derived time points (times provided in this figure are merely considerations) when the next induction is scheduled to begin.
## Frontiers in physiology | gastrointestinal sciences
December 2014 | Volume 5 | Article 464 | 6 mice do, on occasion, develop PanIN-like lesions, these are not the predominant histotype in the pancreas, as PanIN lesions are more frequently observed in human disease. Nonetheless, these transgenic approaches are compatible with non-mutant Kras driving Cre-lox systems and may prove useful in understanding disease etiology in combination with genetic manipulations in other cell compartments. These models do have utility with future approaches, though they lack recapitulation of the predominant clinical histotype (PanIN to PDAC). Therefore, a FLP/FRT KRAS model poses the most promise for inducing Kras mutations that result in a PanIN-like phenotype while allowing the use of Cre-lox to target different genetic events in other cell types. In a manner similar to the Cre-lox system, FLP/FRT utilizes a recombinase called flippase to target FLP recombinase targets that flank an endogenous gene [bib_ref] Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic..., Dymecki [/bib_ref]. Unlike Cre, which is derived from P1 bacteriophage, the FLP recombinase is derived from Saccharomyces cerevisiae [bib_ref] The Flp recombinase of the 2-microns plasmid of Saccharomyces cerevisiae, Sadowski [/bib_ref]. Ideally, a desirable model would involve the generation of a pancreas-specific FLP directed toward a FRT target sequence that flanks a stop codon upstream of oncogenic Kras. At this point, a pancreas-specific FLP may be possible with the intraductal injection of an adenovirus FLP or the generation of an EL-tTA;TetO-FLP;FSF-Kras G12D/+ mouse. Ideally, this mechanism would drive mutant Kras in a near identical fashion as EL-Cre;LSL-Kras G12D/+ while still allowing for the targeting of non-epithelial cell types with Cre-lox.
While this type of model would increase our understanding of the contributions of stromal, hematopoietic, and other cell types to pancreatic carcinogenesis, the ultimate goal of such a system would be the design of a layered model that is simultaneously and/or sequentially inducible. Mimicking a temporal progression of gene mutations in specific cellular compartments requires the use of multiple systems employing different modes of induction. As described, the CreERT system has been well established for many gene targets but alone can only deliver multiple mutations simultaneously [bib_ref] Maximizing mouse cancer models, Frese [/bib_ref]. Young and colleagues demonstrated the potential of the FLP/FRT system when coupled with Cre-lox in lung tissue. They generated mice with an Flp inducible allele of Kras G12D and Cre driven mutation of the tumor suppressor, p53 [bib_ref] Uncoupling cancer mutations reveals critical timing of p53 loss in sarcomagenesis, Young [/bib_ref]. The FLP-FRT system, FSF-Kras G12D , was induced through an adenovirus or lentivirus expressing Flpo, a version of Flp optimized FIGURE 4 | Spatial modeling of pancreatic cancer to explore cross compartmental interactions. Cre-loxP is the most widely used conditional targeting system. This is also true in models of pancreatic cancer, where it is primarily used to drive mtKRAS via a loxP-stop-loxP (LSL) cassette. However, reliance on Cre-loxP to induce a Kras mutation limits our ability to target other pertinent cell types in the tumor microenvironment. Should mtKras be induced by another system, for example a Ptf1a-FLP-driven Frt-stop-Frt (FSF) cassette, which would allow compatibility with one of the several hundred possible Cre-loxP combinations. For instance, an αSMA-Cre to explore the contributions of pancreas stellate cells to tumorigenesis, CD11b-Cre to target myeloid cells, Lck-Cre to target lymphoid cells, or Cdh5-Cre to target mature adipocytes (See [fig_ref] Table 1 |: Tissue Specific Cre-lox Targeting Systems [/fig_ref].
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## Compartment cell/tissue type targeting model reference
Epithelium Pancreatic epithelium, antral stomach, and duodenum in neonates. Pancreatic beta islet cells in adults. [bib_ref] Preinvasive and invasive ductal pancreatic cancer and its early detection in the..., Hingorani [/bib_ref] Pancreatic acinar cells ElastaseCreERT2 [bib_ref] Preexisting pancreatic acinar cells contribute to acinar cell, but not islet beta..., Desai [/bib_ref] Pancreatic acinar cells p48-Cre Ptf1a Cre/+ Ptf1a Cre-ERTM [bib_ref] Preinvasive and invasive ductal pancreatic cancer and its early detection in the..., Hingorani [/bib_ref] [bib_ref] Ongoing Notch signaling maintains phenotypic fidelity in the adult exocrine pancreas, Kopinke [/bib_ref] Pancreatic acinar cells Mist1 Cre-ERT 2/+ [bib_ref] Mist1-KrasG12D knock-in mice develop mixed differentiation metastatic exocrine pancreatic carcinoma and hepatocellular..., Tuveson [/bib_ref] Mesenchyme Myofibroblast αSMA-Cre [bib_ref] Detection of epithelial to mesenchymal transition in airways of a bleomycin induced..., Wu [/bib_ref] Smooth muscle SMA-CreERT2 [bib_ref] Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse, Wendling [/bib_ref] Interstitial stroma of mature tissues-prostate, forestomach, skin Fsp1-Cre [bib_ref] TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia, Bhowmick [/bib_ref] [bib_ref] Deletion of Smad4 in fibroblasts leads to defective chondrocyte maturation and cartilage..., Teng [/bib_ref] Bone, cartilage Dermo1-Cre Twist2-Cre [bib_ref] Conditional inactivation of FGF receptor 2 reveals an essential role for FGF..., Yu [/bib_ref] [bib_ref] TGF-beta receptor II in epithelia versus mesenchyme plays distinct roles in the..., Chen [/bib_ref] [bib_ref] Isolation of murine bone marrow derived mesenchymal stem cells using Twist2 Cre..., Liu [/bib_ref] Pancreatic exocrine lineages Nestin-Cre [bib_ref] Nestin expression in pancreatic exocrine cell lineages, Delacour [/bib_ref] Dermis, lung, pericardial connective tissue, blood vessel wall, splenic capsule, mesangial cells of glomerulus [bib_ref] Ligand-dependent genetic recombination in fibroblasts: a potentially powerful technique for investigating gene..., Zheng [/bib_ref] [bib_ref] Beta1 integrinextracellular matrix interactions are essential for maintaining exocrine pancreas architecture and..., Riopel [/bib_ref] Nestin-negative mesenchymal progenitors Prx1-Cre [bib_ref] CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance, Greenbaum [/bib_ref] Hematopoietic CD4+ for mammalian use. Utilization of this mammalian version of Flp, as opposed to Flpe, was utilized due to its higher recombination efficiency [bib_ref] Widespread recombinase expression using FLPeR (flipper) mice, Farley [/bib_ref]. Injection of the adenovirus/lentivirus activates mutant Kras and results in numerous lung tumors, ultimately confirming that FSF-Kras G12D results in a phenotype similar to LSL-Kras G12D/+ allele. This virus-driven FLP-FRT was coupled with a tamoxifen-driven p53 mutation via Cre recombinase activity [bib_ref] Uncoupling cancer mutations reveals critical timing of p53 loss in sarcomagenesis, Young [/bib_ref]. The TVA-RCAS targeting of epithelial tissue and subsequent stromal phenotype indicates further opportunity for the utilization of this system to target other cell types simultaneously. For example, the conditional nature of this model would allow for the targeting of genes to the stroma via a TVA-RCAS system utilizing a driver such as αSMA or Vimentin. Taking this further, the possibility arises for generation of a trigenic model. Utilizing Cre-lox, FLP/FRT, and TVA-RCAS targeting methods in the same mouse would provide a novel way to target several different cell types in both a conditional and inducible manner.
## Pdx1-cre
## Col1a2-creert
## Advancing the utility of inducible/conditional modeling
While the aforementioned models are undoubtedly technological achievements, their ability to faithfully recapitulate human disease is still limited. Clinically, at least two gene mutations occur to produce PDAC. Kras is believed to be the first mutation in a series of transformation events that lead to PDAC in adults. Subsequent major mutations include those to p53, SMAD4, or p16 INK4a , among several others [bib_ref] Genetics and biology of pancreatic ductal adenocarcinoma, Hezel [/bib_ref]. With current mouse models, recombination events affecting Kras and these other genes occur either during embryonic development or concomitantly sometime after pancreas formation, in the case of inducible systems. However, they fail to capture the step-wise mutation process that occurs in the adult pancreata of human patients.
Layering multiple inducible systems to target the same cell type and cause multiple mutations in a step-wise manner would assist in capturing a more faithful representation of human disease progression . For example, targeting Kras with an EL-tTA or EL-TVA system would provide a mechanism for issuing the first hit of genetic instability in both a temporal and tissue-specific manner. However, it should be noted that elastase targeting in these systems may be dramatically inefficient after pancreas cells advance to a ductal and/or abnormal phenotype. Ablation of a second gene such as p53, SMAD4, or p16 INK4a could then be controlled by a Cre-ERT2 system directed toward the same cells expressing mutant KRAS . Finally, a third system, the FLP/FRT, could be utilized to mutate a third gene in an effort to drive metastatic phenotypes. This trigenic model, which is just one example of many possible inducible/conditional mutation schemes, would better serve to mimic the progressive nature of PDAC . However, generation of such models inherently results in very complex breeding patterns. Additionally, once these trigenic mice are established the induction of different mutations requires a labor-intensive injection scheme and administration of doxycycline over extended periods of time.
From a functional standpoint, the utilization of inducible/conditional drivers other than Cre recombinase for the activation of mutant KRAS allows for subsequent Crelox targeting of cell types outside the epithelial compartment . Strategically, withholding Cre-lox targeting of Kras encourages the use of abundant, pre-existing Cre-lox systems [fig_ref] Table 1 |: Tissue Specific Cre-lox Targeting Systems [/fig_ref] that can target stromal, hematopoietic, and adipose compartments. However, this type of modeling is not necessarily relevant from a clinical standpoint, due lack of evidence that these non-epithelial mutations are common in human PDAC. Nevertheless, this approach allows for more rigorous evaluation of the contributions that different components of the tumor microenvironment (TME) have on carcinogenesis. Insight into the mechanism behind TME involvement in tumor progression and metastatic phenotypes may provide strategies and the rationale for targeting these compartments with certain therapeutic agents. These inducible/conditional systems will be highly relevant in studying the therapeutic value of a genetic target in mature tumors and not at the initiation stages. For instance, a model with expression of oncogenic Kras G12V and deletion of p53 with an EL-tTA FLP system used in conjunction with ablation of a target gene, such as EGFR, by an ubiquitous Cre-ERT2 system is under development in the Barbacid laboratory.
The goal of such systems is to recapitulate the human condition, which can only be done in part. Indeed, mouse models are simply that-models that will never completely recapitulate human PDAC. It is critical to generate these models in a clean background strain to eliminate the potential causative role that genetic variability among chimerics may play when comparing test and control animals, particularly as the complexity of these models increases. The layering of multiple schemes lends itself to amplifying the anomalies produced by one model and potentially augmenting those in another system as they are combined. Despite these caveats, current and future inducible and/or conditional models will lead to a more faithful representation of human disease, which is essential to teasing out the phenotypic and mechanistic aspects of pancreatic cancer that will ultimately improve outcomes in the clinic.
[table] Table 1 |: Tissue Specific Cre-lox Targeting Systems. [/table]
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Experimental Investigation of Additive Manufacturing Using a Hot-Wire Plasma Welding Process on Titanium Parts
# Introductions
Additive manufacturing (AM) describes technologies that are used to fabricate 3D objects by adding a cross-sectional layer upon a layer of liquid, powder, wire, or sheet material, whether the material is metal, polymer, or others. AM is currently being used to develop and customize end-use products in several industries. The relatively low cost for the low-volume development of near-net-shape components, lower net processing expenses, shorter lead times, and the construction of more complicated geometries are some advantages of using AM technologies over conventional tooling-based methods such as casting or machining. the basic parameters of hot-wire plasma welding to obtain good mechanical properties in a titanium welding workpiece. The definitions of good mechanical properties of titanium alloy grade 2 provided an excellent balance of medium strength and reasonably good ductility.
## Materials and experimental procedure
## Experimental material and equipment
Titanium alloy plates of grade 2 in the additive manufacture with the dimensions of 135 mm (width) × 350 mm (length) × 10 mm (thickness) were welded in this study. The substrate materials used a titanium alloy grade 2, the dimensions of 135 mm × 350 mm (width × length), and a thickness of 10 mm. These have a chemical composition measured using an energy-dispersive X-ray spectroscopy (EDS) machine (Shimadsu, Kyoto, Japan) with a standard chemical composition and mechanical physicalvalues as in . The filler wire was a titanium AMS 4951F (Grade 2) welding wire with a diameter of 1.6 mm in the welding process, which is a filler metal process by the American Welding Society (AWS, Miami, FL, USA). The workpiece was securely clamped by jigs and fixtures to prevent it from moving or from deflection during the hot-wire plasma welding process. The plasma arc welding torch was attached to a 6-axis ABB robot of freedom, which was used to generate the movement of the welding torch relative to the reference point on the substrate. The articulated robots, feature six axes, also called six degrees of freedom. The 6-axis robots allow for greater flexibility than robots with fewer axes. It also allows freedom of movement in three-dimensional space. The robot is the handle of the torch head only. Thus, we do not have to build up the Z-axis to facilitate the movement and maintain the arc distance of the robot. The movement in the additive build direction is performed by the robot and the writing codes to control the direction of the robot. Robot studio is software developed by ABB robot that writes a working sequence for robots. The power source was used to begin the process of welding and was operated with the robot in tandem. [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] shows the setup of the hot-wire plasma welding system, which includes a robot control cabinet, a plasma arc welding torch, a hot-wire machine, a monitoring system consisting of a CCD camera, and an ABB limited robot. Materials 2021, [bib_ref] High-Efficiency and Low-Heat-Input CO2 Arc-Welding Technology for Butt Joint of Thick Steel..., Wonthaisong [/bib_ref] 4 of 20 [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] shows the setup of the hot-wire plasma welding system, which includes a robot control cabinet, a plasma arc welding torch, a hot-wire machine, a monitoring system consisting of a CCD camera, and an ABB limited robot. Plasma welding also uses hot wires in the depositing process. The plasma welding controller from the Cebora machine brand (Bologna, Italy) was used for controlling various parameters for plasma welding. The hot-wire unit used in this research is from MAC brand, Power assist IV-642 model (Osaka, Japan) [bib_ref] Real-time temperature measurement using infrared thermography camera and effects on tensile strength..., Naksuk [/bib_ref] , with a wire feed system to the welding torch, which is attached to the robotic arm used for controlling the hot-wire plasma welding path. The plasma torch was at the front, while the hot-wire was fed behind the plasma welding torch. The experimental setup of the hot-wire unit depends on plasma welding parameters, where the arc current and welding speed of the robot are constant. We then adjusted the relationship of the wire current and the wire feed speed so that the added filler wire can move continuously in the melting pool. The hot-wire PAW process was carried out under the inter gas (argon) shield.
# Methodology
The designs of the full factorial experiments used in this study were based on three factors, (1) the welding speed [mm/s], (2) the wire current [A], and (3) the wire feeding speed [m/min] using a significant level, α = 0.05, by strictly controlling the other factors at the same value and condition for hot-wire plasma welding.
Thus, in this experiment, there were a total of 2 3 = 8 trials. An example of an image obtained from the results of the designed experiment is shown in [fig_ref] Table 3: The welding experimental conditions of eight welding specimens for hot-wire plasma welding [/fig_ref] and [fig_ref] Figure 2: Deposited single beads for a designed experiment for the hot-wire plasma welding... [/fig_ref]. Plasma welding also uses hot wires in the depositing process. The plasma welding controller from the Cebora machine brand (Bologna, Italy) was used for controlling various parameters for plasma welding. The hot-wire unit used in this research is from MAC brand, Power assist IV-642 model (Osaka, Japan) [bib_ref] Real-time temperature measurement using infrared thermography camera and effects on tensile strength..., Naksuk [/bib_ref] , with a wire feed system to the welding torch, which is attached to the robotic arm used for controlling the hot-wire plasma welding path. The plasma torch was at the front, while the hot-wire was fed behind the plasma welding torch. The experimental setup of the hot-wire unit depends on plasma welding parameters, where the arc current and welding speed of the robot are constant. We then adjusted the relationship of the wire current and the wire feed speed so that the added filler wire can move continuously in the melting pool. The hot-wire PAW process was carried out under the inter gas (argon) shield.
# Methodology
The designs of the full factorial experiments used in this study were based on three factors, (1) the welding speed [mm/s], (2) the wire current [A], and (3) the wire feeding speed [m/min] using a significant level, α = 0.05, by strictly controlling the other factors at the same value and condition for hot-wire plasma welding.
Thus, in this experiment, there were a total of 2 3 = 8 trials. An example of an image obtained from the results of the designed experiment is shown in [fig_ref] Table 3: The welding experimental conditions of eight welding specimens for hot-wire plasma welding [/fig_ref] and [fig_ref] Figure 2: Deposited single beads for a designed experiment for the hot-wire plasma welding... [/fig_ref]. The single bead number 2.1 had a better appearance and shape than the other weld beads and met the requirements. In addition to the above criteria, we found that the filling wire was not a problem, with no interruption, no splashes, and a smooth movement. The wire tip did not stick to the ceramic tip while the wire was pulled back (ceramic rod: wire support). Thus, the welding condition of single bead number 2.1 was applied for the deposited wall of the hot-wire PAW process. The hot-wire plasma welding parameters used in this research are shown in [fig_ref] Table 4: The parameters used for the hot-wire plasma welding process [/fig_ref].
Titanium is a metal that is sensitive to high-temperature oxidation. Titanium welding is required under the suitable shielding gas or inside a covered gas box. A shielded environment is required during the hot-wire plasma welding process for the wire + arc additive manufacture of titanium alloys to prevent oxidation and cracks. If air interacts with the weld, an oxide is formed with a color that depends on the thickness of the oxide layer. The acceptability level of the color depends on the application. The workpiece is found to have oxidation during the build of the first weld. Applying the shielding can increase the flexibility of the process. An example of a flow-purge welding chamber was used in the hot-wire PAW process shown in [fig_ref] Figure 3: The flow-purge welding chamber [/fig_ref]. The positions of the weld bead height and bead width measurement are shown in [fig_ref] Figure 4: Positions of the weld bead height and bead width measurement for the... [/fig_ref]. The single bead number 2.1 had a better appearance and shape than the other weld beads and met the requirements. In addition to the above criteria, we found that the filling wire was not a problem, with no interruption, no splashes, and a smooth movement. The wire tip did not stick to the ceramic tip while the wire was pulled back (ceramic rod: wire support). Thus, the welding condition of single bead number 2.1 was applied for the deposited wall of the hot-wire PAW process. The hot-wire plasma welding parameters used in this research are shown in [fig_ref] Table 4: The parameters used for the hot-wire plasma welding process [/fig_ref]. Titanium is a metal that is sensitive to high-temperature oxidation. Titanium welding is required under the suitable shielding gas or inside a covered gas box. A shielded environment is required during the hot-wire plasma welding process for the wire + arc additive manufacture of titanium alloys to prevent oxidation and cracks. If air interacts with the weld, an oxide is formed with a color that depends on the thickness of the oxide layer. The acceptability level of the color depends on the application. The workpiece is found to have oxidation during the build of the first weld. Applying the shielding can increase the flexibility of the process. An example of a flow-purge welding chamber was used in the hot-wire PAW process shown in [fig_ref] Figure 3: The flow-purge welding chamber [/fig_ref]. The positions of the weld bead height and bead width measurement are shown in [fig_ref] Figure 4: Positions of the weld bead height and bead width measurement for the... [/fig_ref]. The welding process started from the right-hand side of the workpiece and ended at the left-hand side, as shown in [fig_ref] Figure 5: Photographs of the deposited walls [/fig_ref]. The dimensions of the welded wall structures were 13.25 mm (width), 300 mm (length), and 63.2 mm (height). The welding process started from the right-hand side of the workpiece and ended at the left-hand side, as shown in [fig_ref] Figure 5: Photographs of the deposited walls [/fig_ref]. The dimensions of the welded wall structures were 13.25 mm (width), 300 mm (length), and 63.2 mm (height). The welding process started from the right-hand side of the workpiece and ended at the left-hand side, as shown in [fig_ref] Figure 5: Photographs of the deposited walls [/fig_ref]. The dimensions of the welded wall structures were 13.25 mm (width), 300 mm (length), and 63.2 mm (height).
# Porosity analysis
The current detection of the inside pore and other defects are frequently per-formed using the X-ray method after welding. This method is an X-ray application with computed tomography (XCT), which is a high-resolution system for 2D X-ray inspection, 3D computed tomography, and the measurement of various metal materials from GE Phoenix, system: V|tome|X S (Boston, MA, USA) using a high-power nano focus X-ray tube.
## Vickers microhardness
# Porosity analysis
The current detection of the inside pore and other defects are frequently per-formed using the X-ray method after welding. This method is an X-ray application with computed tomography (XCT), which is a high-resolution system for 2D X-ray inspection, 3D com-puted tomography, and the measurement of various metal materials from GE Phoenix, system: V|tome|X S (Boston, MA, USA) using a high-power nano focus X-ray tube.
## Vickers microhardness
Vickers microhardness tests for specimens were carried out with a diamond-shaped pyramid head. The pyramid's top angle was 136 degrees. This test can measure the hardness of a very soft metal, about 5 Kgf/mm 2 , or that of a very stiff metal, about 1500 Kgf/mm 2 , without changing the indenter. It only changes the pressure. The values between 1 and 120 Kgf depend on the hardness of the metal [bib_ref] Porosity measurements and analysis for metal additive manufacturing process control, Slotwinski [/bib_ref].
The wall was cut into three sections. A schematic illustration of micro-hardness measurement points on the profile of the wall, with the microhardness of the layer bands (top, middle, and bottom region) of the wall, is shown in [fig_ref] Figure 6: Schematic illustration of microhardness measurement on the profile of the microhardness of... [/fig_ref]. Each position was measured thrice; an average of these measurements was then calculated and is graphically displayed. The cut samples of all three specimens underwent grinding and polishing and became the cross-section area of the workpiece. During grinding, the workpiece was lubricated with water.
# Tensile strength
The tensile test is one of the most common tests used for material evaluation. The tensile test is performed in its easiest way by gripping the other ends of a test part (specimen) within the testing machine loading frame. In tensile tests, the test specimen will be pulled slowly. After that, the specimen will lengthily stretch, and the tensile may be increased steadily until the test specimens are fracture. The forces-extension data are normally monitored and recorded during the process, which is a quantitative measure of how the test part deforms under the applied tensile force. The stress and strain values are recorded, and the curves are plotted. The tensile testing according to ASTM is E8for metals, etc. ASTM E8 describes tensile testing of metals and is the actively used standard for the testing of metals. The sample used for testing will have different characteristics for metal, and may be made of sheets or bars. The dimensions of the tensile specimens in mm are shown in [fig_ref] Figure 7: Dimensions of the tensile specimens [/fig_ref]. The instrument used was a Microhardness Tester Anton-Paar, MHT-10, S/N 240826 (Anton Paar, Graz, Austria) The test conditions of the hardness test were performed under the ASTM specs standard test method E92, with a US specs standard test method AMS 4951 welding wire. The wall was cut into sections at three positions. In [bib_ref] Mechanical properties of Ti-6Al-4V specimens produced by shaped metal deposition, Baufeld [/bib_ref] , square samples, such as the top, the middle, and the bottom region of the wall, were taken in the direction of the horizontal construction at the center of layer bands.
# Tensile strength
The tensile test is one of the most common tests used for material evaluation. The tensile test is performed in its easiest way by gripping the other ends of a test part (specimen) within the testing machine loading frame. In tensile tests, the test specimen will be pulled slowly. After that, the specimen will lengthily stretch, and the tensile may be increased steadily until the test specimens are fracture. The forces-extension data are normally monitored and recorded during the process, which is a quantitative measure of how the test part deforms under the applied tensile force. The stress and strain values are recorded, and the curves are plotted. The tensile testing according to ASTM is E8for metals, etc. ASTM E8 describes tensile testing of metals and is the actively used standard for the testing of metals. The sample used for testing will have different characteristics for metal, and may be made of sheets or bars. The dimensions of the tensile specimens in mm are shown in [fig_ref] Figure 7: Dimensions of the tensile specimens [/fig_ref]. mally monitored and recorded during the process, which is a quantitative measure of how the test part deforms under the applied tensile force. The stress and strain values are recorded, and the curves are plotted. The tensile testing according to ASTM is E8for metals, etc. ASTM E8 describes tensile testing of metals and is the actively used standard for the testing of metals. The sample used for testing will have different characteristics for metal, and may be made of sheets or bars. The dimensions of the tensile specimens in mm are shown in [fig_ref] Figure 7: Dimensions of the tensile specimens [/fig_ref].
## Microstructure
We prepared the workpiece to observe the microstructure and microstructure picture of the hot-wire plasma welding process with a LEXT 3D measuring microscope OLS4100 (Olympus, Waltham, MA, USA). The microstructure analysis samples were polished with the sandpaper grades 120, 400, 600, 800, 1000, 1200, and 2500 grit by machine grinding and polishing. At the micro-level, we used the 50×, 200×, and 500× magnification to perform a complete visual analysis and observation.
## Analysis of the physical and mechanical properties
## Microstructure
We prepared the workpiece to observe the microstructure and microstructure picture of the hot-wire plasma welding process with a LEXT 3D measuring microscope OLS4100 (Olympus, Waltham, MA, USA). The microstructure analysis samples were polished with the sandpaper grades 120, 400, 600, 800, 1000, 1200, and 2500 grit by machine grinding and polishing. At the micro-level, we used the 50×, 200×, and 500× magnification to perform a complete visual analysis and observation.
## Analysis of the physical and mechanical properties
The physical and mechanical properties study in this research were performed in four steps (porosity, Vickers hardness, tensile strength, and microstructure), and the workpiece was divided into parts as shown in [fig_ref] Figure 8: Each part of the workpiece [/fig_ref]. The workpiece was cut with a wire cut EDM (electrical discharge machine, model: AQ325L, Sodick, Schaumburg, IL, USA) with a band saw and using a slow cutting speed. The physical and mechanical properties study in this research were performed in four steps (porosity, Vickers hardness, tensile strength, and microstructure), and the workpiece was divided into parts as shown in [fig_ref] Figure 8: Each part of the workpiece [/fig_ref]. The workpiece was cut with a wire cut EDM (electrical discharge machine, model: AQ325L, Sodick, Schaumburg, IL, USA) with a band saw and using a slow cutting speed.
# Results
## Parameters and welding process
The welding found that sample number 2 [fig_ref] Figure 2: Deposited single beads for a designed experiment for the hot-wire plasma welding... [/fig_ref] had a better appearance and shape than the other weld bead and met the requirements. The deposition of the hot-wire PAW process from sample number 2 is shown in [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref]. These parameters were constant: a welding speed of 1.83 mm/s, an arc current of 120 A, a wire feeding speed of 0.85 m/min, and a wire current of 35 A.
# Results
## Parameters and welding process
The welding found that sample number 2 [fig_ref] Figure 2: Deposited single beads for a designed experiment for the hot-wire plasma welding... [/fig_ref] had a better appearance and shape than the other weld bead and met the requirements. The deposition of the hot-wire PAW process from sample number 2 is shown in [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref]. These parameters were constant: a welding speed of 1.83 mm/s, an arc current of 120 A, a wire feeding speed of 0.85 m/min, and a wire current of 35 A.
## Parameters and welding process
The welding found that sample number 2 [fig_ref] Figure 2: Deposited single beads for a designed experiment for the hot-wire plasma welding... [/fig_ref] had a better appearance and shape than the other weld bead and met the requirements. The deposition of the hot-wire PAW process from sample number 2 is shown in [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref]. These parameters were constant: a welding speed of 1.83 mm/s, an arc current of 120 A, a wire feeding speed of 0.85 m/min, and a wire current of 35 A. when the current flow is maintained in the form of an arc. In addition, arc voltage depends on the shielding gas, current, electrode angle, workpiece composition, arc blow, and wire feeding, and it must be set for the particular conditions being used. The arc voltage rises and falls per layer because the arc voltage values are measured from the electrode tip to the melting pool, which leads to the results from the machine. In the experiment, it was found that the arc voltage was not clearly controlled, the last weld sagging (falling) causing the arc voltage to rise and instability. Arc voltage values will be measured during the arcing, and the resulting values change with the arc distance. These arc voltage values are the final arc voltage that occurs after the welding of each layer. [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] shows the temperature values of the deposition of the hot-wire PAW process per layer (all 36 layers). There were temperature measurements for the weld bead: infrared thermometer (IR) measures the temperature of the thermal radiation of the object, and can typically measure temperature in a range of −25 °C to +380 °C (14 °F to 716 °F). The temperature was measured on the surface of each deposited layer at the mid-point of each welded bead layer. After finishing each deposited layer, the deposition process resumed after five-minute break. The interpass temperature was not constant because the heat generated by the arc voltage was not constant. The fluctuation of the arc voltage during the deposition process was due to the fluctuation of the arc gap distance.
The values of the bead height titanium alloy walls built by the hot-wire PAW process from sample number 2 are shown in [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref]. [fig_ref] Figure 9: Arc voltage [/fig_ref] showed the final arc voltage value from 1 layer to 36 layers of the deposit process. The arc voltage gives a clear indication of arc length at a given current. These systems directly measure arc voltage and control the torch height to correct the error. It may be defined as the arc voltages that appear across the contact during the arcing period when the current flow is maintained in the form of an arc. In addition, arc voltage depends on the shielding gas, current, electrode angle, workpiece composition, arc blow, and wire feeding, and it must be set for the particular conditions being used. The arc voltage rises and falls per layer because the arc voltage values are measured from the electrode tip to the melting pool, which leads to the results from the machine. In the experiment, it was found that the arc voltage was not clearly controlled, the last weld sagging (falling) causing the arc voltage to rise and instability. Arc voltage values will be measured during the arcing, and the resulting values change with the arc distance. These arc voltage values are the final arc voltage that occurs after the welding of each layer. [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] shows the temperature values of the deposition of the hot-wire PAW process per layer (all 36 layers). There were temperature measurements for the weld bead: infrared thermometer (IR) measures the temperature of the thermal radiation of the object, and can typically measure temperature in a range of −25 - C to +380 - C (14 - F to 716 - F). The temperature was measured on the surface of each deposited layer at the mid-point of each welded bead layer. After finishing each deposited layer, the deposition process resumed after five-minute break. The interpass temperature was not constant because the heat generated by the arc voltage was not constant. The fluctuation of the arc voltage during the deposition process was due to the fluctuation of the arc gap distance.
The values of the bead height titanium alloy walls built by the hot-wire PAW process from sample number 2 are shown in [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref]. 1) The vernier caliper is a precise tool that can be used to accurately measure outside diameter, inside diameter, and depth. (2) The welding gauge is the main scale, height gauge, and undercut depth gauge. It is a welding inspection, for a variety of bevel angles detecting weldments, height, width, gap, and undercut depth.
Moreover, the arc distance is the layer-by-layer movement of the robot in the build direction. From the first layer (start), the arc distance was the distance between the torch and the baseplate at a height of 0 mm. After that, we moved the robot according to the average height of the welded bead in each layer. Therefore, the arc distance increases linearly.
# Porosity analysis
In the case of titanium alloy, there is always a certain proportion of porosity, and the content significantly depends on the use conditions [bib_ref] Microstructural evolution and mechanical property of Ti-6Al-4V wall deposited by continuous plasma..., Lin [/bib_ref] [bib_ref] XCT analysis of the influence of melt strategies on defect population in..., Tammas-Williams [/bib_ref]. [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] shows porosity in a sample specimen in the AM part: (a) front area; (b) back area. The porosity formed in the titanium alloy samples produced over a range of melt scan speeds, from 100 to 1000 mm/s, were investigated in [bib_ref] Influence of manufacturing parameters on microstructure and hydrogen sorption behavior of electron..., Pushilina [/bib_ref]. Meanwhile, the orange line and red line showed the height of arc distance and an average of bead height, respectively. There were two main measurements for weld bead height: (1) The vernier caliper is a precise tool that can be used to accurately measure outside diameter, inside diameter, and depth. (2) The welding gauge is the main scale, height gauge, and undercut depth gauge. It is a welding inspection, for a variety of bevel angles detecting weldments, height, width, gap, and undercut depth.
Moreover, the arc distance is the layer-by-layer movement of the robot in the build direction. From the first layer (start), the arc distance was the distance between the torch and the baseplate at a height of 0 mm. After that, we moved the robot according to the average height of the welded bead in each layer. Therefore, the arc distance increases linearly.
# Porosity analysis
In the case of titanium alloy, there is always a certain proportion of porosity, and the content significantly depends on the use conditions [bib_ref] Microstructural evolution and mechanical property of Ti-6Al-4V wall deposited by continuous plasma..., Lin [/bib_ref] [bib_ref] XCT analysis of the influence of melt strategies on defect population in..., Tammas-Williams [/bib_ref]. [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] shows porosity in a sample specimen in the AM part: (a) front area; (b) back area. The porosity formed in the titanium alloy samples produced over a range of melt scan speeds, from 100 to 1000 mm/s, were investigated in [bib_ref] Influence of manufacturing parameters on microstructure and hydrogen sorption behavior of electron..., Pushilina [/bib_ref]. The pore sizes in [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] were measured using computerized tomography scans (CT scans) and a measuring gauge. A higher density of porosity was mainly distributed at the beginning of the welding process. The volume of pores measured in the part was 47 porous in the consolidated material, per area at a scale of 21:1 mm, but the number density of the pores was lower in the back region. The front area had a maximum porous size of 1.129 mm, and a minimum porous size of 0.45 mm. The back area had a maximum porous size of 0.44 mm, and a minimum porous size of 0.25 mm. In the front area, the porosity occurred the most because it is often opened by the shielding device as welding begins. After several trials building walls, the shielding gas cover was improved, the gas content was increased, and the porosity was reduced.
## Vickers microhardness
The tested Vickers microhardness coupons were incised from the formed thin wall, with a steady value of approximately 206. [bib_ref] Experimental and numerical investigation of laser hot wire welding, Liu [/bib_ref] [fig_ref] Table 5: Results of the Vickers microhardness test for hot-wire plasma welding [/fig_ref]. As reported for titanium alloy grade 2, US specs (AMS 4951), the average Vickers microhardness values of the deposited layers was around 160-200 HV. The average of all the results was 180.05-210.00 HV. When compared with all hardness values, we found that the area with the least hardness was at the top area. The table shows that the hardness values are similar and consistent with the standard values of titanium.
For testing the surface hardness, a microhardness indenter was used. The edge effect was also reported, which may result in lower hardness values. This work involved nanohardness indentations performed very close to the surface [bib_ref] The edge effect in nanoindentation, Jakes [/bib_ref]. A maximum microhardness value of micro plasma wire deposition material of 616 HV in the heat effect zone was The pore sizes in [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] were measured using computerized tomography scans (CT scans) and a measuring gauge. A higher density of porosity was mainly distributed at the beginning of the welding process. The volume of pores measured in the part was 47 porous in the consolidated material, per area at a scale of 21:1 mm, but the number density of the pores was lower in the back region. The front area had a maximum porous size of 1.129 mm, and a minimum porous size of 0.45 mm. The back area had a maximum porous size of 0.44 mm, and a minimum porous size of 0.25 mm. In the front area, the porosity occurred the most because it is often opened by the shielding device as welding begins. After several trials building walls, the shielding gas cover was improved, the gas content was increased, and the porosity was reduced.
## Vickers microhardness
The tested Vickers microhardness coupons were incised from the formed thin wall, with a steady value of approximately 206. [bib_ref] Experimental and numerical investigation of laser hot wire welding, Liu [/bib_ref] [fig_ref] Table 5: Results of the Vickers microhardness test for hot-wire plasma welding [/fig_ref]. As reported for titanium alloy grade 2, US specs (AMS 4951), the average Vickers microhardness values of the deposited layers was around 160-200 HV. The average of all the results was 180.05-210.00 HV. When compared with all hardness values, we found that the area with the least hardness was at the top area. The table shows that the hardness values are similar and consistent with the standard values of titanium.
For testing the surface hardness, a microhardness indenter was used. The edge effect was also reported, which may result in lower hardness values. This work involved nanohardness indentations performed very close to the surface [bib_ref] The edge effect in nanoindentation, Jakes [/bib_ref]. A maximum microhardness value of micro plasma wire deposition material of 616 HV in the heat effect zone was mentioned in a previous study [bib_ref] Development of micro-plasma wire deposition process for layered manufacturing, Jhavar [/bib_ref]. The hardness results of the three specimens are plotted in [fig_ref] Table 5: Results of the Vickers microhardness test for hot-wire plasma welding [/fig_ref]. From the table, for all specimens, hardness decreases with increasing deposit height. Specimens 1, 2, and 3 were completed with a waiting time of 10 s, and all specimens were completed with 200 g force. The average deposit hardness for Specimen 3 was greater than that of Specimen 2 and Specimen 1.
# Tensile strength
The tensile test is one of the most common tests used for material evaluation. In this paper, we used random sampling for the tensile test. The sampling positions of each wall were sectioned. Three tensile test samples along the vertical direction were equidistantly taken from the middle to the end of the wall. Another three tensile test samples in the horizontal direction were evenly taken from the top to the root of each wall [bib_ref] The strengthening effect of inter-layer cold working and post-deposition heat treatment on..., Gu [/bib_ref]. Tensile testing perpendicular to the build direction produces significantly reduced ductility in comparison to testing along the build direction [bib_ref] Mechanical properties of Ti-6Al-4V specimens produced by shaped metal deposition, Baufeld [/bib_ref] [bib_ref] Microstructure and Mechanical Properties of Wire and Arc Additive Manufactured Ti-6Al-4V, Wang [/bib_ref]. The tensile strength of the deposited titanium alloy in the vertical directions was less than it was in the other directions. Tensile tests for specimens were carried out on a Universal Testing Machine: Instron 8872 (Instron, Norwood, MA, USA). The results of the tensile strength tests for the as-deposited hot-wire plasma welding are shown in [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref]. mentioned in a previous study [bib_ref] Development of micro-plasma wire deposition process for layered manufacturing, Jhavar [/bib_ref]. The hardness results of the three specimens are plotted in [fig_ref] Table 5: Results of the Vickers microhardness test for hot-wire plasma welding [/fig_ref]. From the table, for all specimens, hardness decreases with increasing deposit height. Specimens 1, 2, and 3 were completed with a waiting time of 10 s, and all specimens were completed with 200 g force. The average deposit hardness for Specimen 3 was greater than that of Specimen 2 and Specimen 1.
# Tensile strength
The tensile test is one of the most common tests used for material evaluation. In this paper, we used random sampling for the tensile test. The sampling positions of each wall were sectioned. Three tensile test samples along the vertical direction were equidistantly taken from the middle to the end of the wall. Another three tensile test samples in the horizontal direction were evenly taken from the top to the root of each wall [bib_ref] The strengthening effect of inter-layer cold working and post-deposition heat treatment on..., Gu [/bib_ref]. Tensile testing perpendicular to the build direction produces significantly reduced ductility in comparison to testing along the build direction [bib_ref] Mechanical properties of Ti-6Al-4V specimens produced by shaped metal deposition, Baufeld [/bib_ref] [bib_ref] Microstructure and Mechanical Properties of Wire and Arc Additive Manufactured Ti-6Al-4V, Wang [/bib_ref]. The tensile strength of the deposited titanium alloy in the vertical directions was less than it was in the other directions. Tensile tests for specimens were carried out on a Universal Testing Machine: Instron 8872 (Instron, Norwood, MA, USA). The results of the tensile strength tests for the as-deposited hot-wire plasma welding are shown in [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref]. GPa. It can, however, be noted that, for these specimens, graph number 3 has the highest tensile strength value compared to other graphs, since the first layers inherit the original fine-grained structure of the substrate, so their strength is higher than it is in the rest of the sample.
The sample of the fractured specimen is shown in [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] , which indicates that the rupture occurred, and that the specimen suffered acute forming before fracturing. Ten pieces were examined, and they all behaved similarly. In addition, the average tensile It can, however, be noted that, for these specimens, graph number 3 has the highest tensile strength value compared to other graphs, since the first layers inherit the original fine-grained structure of the substrate, so their strength is higher than it is in the rest of the sample.
The sample of the fractured specimen is shown in [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] , which indicates that the rupture occurred, and that the specimen suffered acute forming before fracturing. Ten pieces were examined, and they all behaved similarly. In addition, the average tensile strength value of titanium alloy grade 2 is 485 MPa. When compared with the standard tensile strength, the tensile values were similar and consistent with the standard values of titanium alloy grade 2. strength value of titanium alloy grade 2 is 485 MPa. When compared with the standard tensile strength, the tensile values were similar and consistent with the standard values of titanium alloy grade 2.
## Microstructure
The analysis of the deposited microstructure showed a number of features. The staggered individual deposited tracks could be observed at low magnification. [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] present the as-deposited microstructure in a cross-section vertical to the plasma scanning direction. The weld metal deposit grains change from having a relatively fine microstructure and a small grain size near the substrate to a very coarse microstructure with a large grain size as the distance from the substrate increases.
## Microstructure
The analysis of the deposited microstructure showed a number of features. The staggered individual deposited tracks could be observed at low magnification. [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] present the as-deposited microstructure in a cross-section vertical to the plasma scanning direction. The weld metal deposit grains change from having a relatively fine microstructure and a small grain size near the substrate to a very coarse microstructure with a large grain size as the distance from the substrate increases. strength value of titanium alloy grade 2 is 485 MPa. When compared with the standard tensile strength, the tensile values were similar and consistent with the standard values of titanium alloy grade 2. [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref]. The tensile test specimens (Position 4).
## Microstructure
The analysis of the deposited microstructure showed a number of features. The staggered individual deposited tracks could be observed at low magnification. [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] present the as-deposited microstructure in a cross-section vertical to the plasma scanning direction. The weld metal deposit grains change from having a relatively fine microstructure and a small grain size near the substrate to a very coarse microstructure with a large grain size as the distance from the substrate increases. We used 3D measuring laser microscope OLS4100 (Olympus) analysis to evaluate the microstructure of hot-wire plasma deposited titanium alloy grade 2. To study the phase transformations of titanium alloy grade 2 during hot-wire plasma metal deposition, we transversely cut the sample, polished them with silica suspension, and examined them with an optical and 3D measuring laser microscope. We used 3D measuring laser microscope OLS4100 (Olympus) analysis to evaluate the microstructure of hot-wire plasma deposited titanium alloy grade 2. To study the phase transformations of titanium alloy grade 2 during hot-wire plasma metal deposition, we transversely cut the sample, polished them with silica suspension, and examined them with an optical and 3D measuring laser microscope.
Titanium alloy has a two-phase (α + β) microstructure. Titanium undergoes α to β phase transformation. In Position 1, the microstructure becomes relatively fine with a small grain size. In [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] , the darker regions are the β phase, which remains between the α plates that have developed. The microstructure consists of parallel plates of α characterized by the β phase between them. The microstructure of the substrate was characterized by alpha (lighter part) and beta grains (darker parts). [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] (Positions 4-6) also shows the microstructure in different areas of the top surface of the sample. As can be seen the microstructure varies in different areas. Some areas (Positions 4 and 5) contain mainly primary α grains surrounded by a coarse β phase. The primary α forms through nucleation and growth during the α + β working operation, and its morphology can vary from elongated plates in lightly worked material to equiaxed globular morphology in heavily worked material. The microstructure once again becomes very coarse with a relatively large grain size near the top region of the weld metal deposit and becomes relatively uneven, and expansion of the dendrite is found.
# Discussion
# Porosity analysis
The pore or cave pocket is a characteristic feature of welding and occurs frequently during the welding process. Gas comes out of solution in the form of bubbles since the solubility for gas decreases in a liquid metal upon cooling. Porosity will be increased because the dendritic solidification interface and some inclusions can be used as heterogeneous particles for pores. Pores serve as stress risers that are brittle fractures and that increase the susceptibility to failure [bib_ref] The strengthening effect of inter-layer cold working and post-deposition heat treatment on..., Gu [/bib_ref]. Several studies have shown that advanced tools can help detect faults or other defects that occur in the welding process such as ultrasonic non-destructive testing (NDT) techniques [bib_ref] Porosity measurements and analysis for metal additive manufacturing process control, Slotwinski [/bib_ref] , X-ray CT methods [bib_ref] XCT analysis of the influence of melt strategies on defect population in..., Tammas-Williams [/bib_ref] , and radiographic testing (RT) [bib_ref] Finite element analysis of the effect of porosity on residual stress in..., Poolperm [/bib_ref]. The slow welding speeds and the welding in the flat position, or uphill in the vertical position, encourage the escape of pores [bib_ref] Investigation of the overlapping parameters of MPAW-based rapid prototyping, Aiyiti [/bib_ref]. The variation in the arc length and the arc distance was large enough to affect the shielding gas (Argon), and porosity started to show in some layers.
## Vickers microhardness
The decrease in hardness with increasing deposit height is a direct result of the observed increase in dendritic structures with increasing deposit height. The mechanisms differ between the α-phase and β-phase. During the deposition, the melted area can be hardened by a solid solution (contamination from the atmosphere or base material), dislocation, or boundary hardening (a smaller grain or α-colony size, phase transformation, or a high dislocation density. A hardness gradient at deposits is generally expected, as each layer has a different thermal history. The hardness difference between the top region and bottom region is not equal in height. The hardness of the top layers (Position 1) is below more than other layers. In addition, the total heat induced is larger and homogeneously distributed through the specimen. This leads to a heterogeneous material and finally to a slight decrease in hardness from top to bottom. Therefore, the only three measurements (very few) are strongly dependent on the amount of αand β-phase at the particular location.
# Tensile strength
The location used to measure the longitudinal tensile strength, the transverse tensile strength, and the baseplate of hot-wire PAW for AM deposited titanium alloy grade 2 are presented in [fig_ref] Figure 6: Schematic illustration of microhardness measurement on the profile of the microhardness of... [/fig_ref]. The transverse direction refers to samples taken across the layers of the building, while the longitudinal direction refers to those taken along the layers [bib_ref] Wire+ arc additive manufacturing of aluminium, Gu [/bib_ref]. [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] shows the tensile properties of the specimens deposited by the hot-wire method, which combines plasma welding. Specimens for the tensile test were machined from the bottom to the top of the deposited sample. The test results showed a tensile strength of approximately 426. MPa. The tensile strength steadily declined from the bottom to the top part of the deposited sample. The results reveal that the percentage elongations of all specimens remained almost constant (14.46%). Compared with the longitudinal direction, the structural solidarity of parts in the transverse direction was worse due to the HAZ of the adjacent deposited tracks acting on each other. The voids (porosity or crack) were the main cause of the bad tensile strength in tensile specimens. Moreover, the nearby, inadequately fused areas were torn away, and the cracks then extended. At the same time, voids reduced the effective sectional areas of tensile specimens, and the tensile strength was worse [bib_ref] Investigation of the overlapping parameters of MPAW-based rapid prototyping, Aiyiti [/bib_ref]. The coating was able to increase the tensile strength of the test part surface, which was mentioned in [bib_ref] Material properties and fabrication parameters in selective laser sintering process, Gibson [/bib_ref]. Therefore, the tensile strength and elongation in the longitudinal direction were higher than those in the transverse direction.
## Microstructure
The analysis of the deposited microstructure showed a number of features. The staggered individual deposited tracks could be observed at low magnification. The formation of the microstructure and the phase of titanium alloy occur as a result of melting at 1900 - C and subsequent rapid cooling [bib_ref] Effect of build geometry on the β-grain structure and texture in additive..., Antonysamy [/bib_ref]. [fig_ref] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing [/fig_ref] presents the as-deposited microstructure in the transverse section of the baseline at deposit heights. Regarding the microstructure of the specimen near the surface, the light-colored regions of the microstructure are the alpha grain, and the dark regions are the beta grain. It was observed that the prior β-grains and α-grains have a globular shape at the bottom, i.e., near the base and the substrate. In addition, with increasing height, the microstructure of the specimen near the top region had many large dendritic structures, and the number of grains decreased. The heat input led to the growth of these grains in the build. The microstructure of the titanium alloy deposited alloy depends on the time at the temperature, the peak temperature, and the cooling rate of each layer of deposited material during the multiple thermal cycles. As regards the microstructure of titanium-based deposits, the needle-like dendritic microstructure of alpha-phase titanium was found to develop from bead formation, along with rapid cooling of the melt. According to, the main microstructural features of the welding process are, in addition to the β and α grain size, the conditions under which the high-temperature β and α phases transform during cooling. During the plasma welding, the heat flow direction of the solidification of the molten pool was about perpendicular to the surface of the substrate or the pre-deposited layers. The plasma transfer arc-assisted deposition technology can be also used to directly manufacture a real component [bib_ref] Fundamental study on plasma deposition manufacturing, Zhang [/bib_ref]. In [bib_ref] Microstructural Evolution and Mechanical Properties of Inconel 625 Alloy during Pulsed Plasma..., Xu [/bib_ref] , it was found that the microstructure of the sample was mostly a fine columnar dendritic structure and that grew epitaxially along the deposition direction. The growth rate, temperature gradient, melt pool shape, welding speed, and the alloy constitution will all control the final microstructure of a solidifying melt pool in AM [bib_ref] Effect of build geometry on the β-grain structure and texture in additive..., Antonysamy [/bib_ref].
# Conclusions
The following conclusions can be drawn based on the analytical and experimental investigations.
## 1.
The variations in welding speed and wire feeding speed that resulted from the in-consistent angular velocity led to inconsistent overall deposit geometry. To develop reliable hot-wire PAW process parameters, an accurate welding speed, and wire feeding speed must be determined for consistent weld metal deposit build-up. Nevertheless, a moving speed of the welding that is too great, could cause humping. The appropriate hot-wire plasma welding parameters are a welding speed of 1.83 mm/s and an arc current of 120 A. A hot-wire current (amperage) of 35 A, a wire feeding speed of 0.85 m/min, and a reverse feeding speed of 5 m/min (0.99 s) gave the appropriate hot-wire temperature in the weld pool range. In a single run, wall widths were approximately 7-13.25 mm after welding.
## 2.
The Vickers microhardness of as-deposited samples was in the range of 180.05-210.00 HV and the tensile strength was 426.42-578.86 MPa depending on the orientation and location of the specimens. The microstructure evolution, tensile strength, and yield strength decreased gradually from the bottom to the top part of the deposited sample because of the observed increase in grain size with increasing deposit height. The percentage elongations of all specimens were at an average of 14.67%. This indicates that the as-deposited sample exhibits improved mechanical properties for the hot-wire PAW welding process.
## 3.
The microstructure of the specimen near the bottom region (or substrate) consists of alpha (lighter part) and beta grains (darker parts). Cross-sections of the weld metal specimens showed an overall increase and a large size in the dendritic structure (top region of the part). The dendrite spread out until all solid metals and then stopped growing. This caused the alloy to enter into a non-equilibrium condition because the hot metal alloy cooled instantly or too quickly and suddenly solidified, whereas the inside remained hot and soft. Furthermore, the coarsening microstructure, as the height of the weld metal specimen increased, grew epitaxially along the deposition direction. However, there was not little effect found on the strength. Each crystal was often unequal because the growth of each dendrite was independent. 4.
The titanium alloy is highly susceptible to oxidation during welding. Oxidation and distortion can cause problems in particular when the deposition takes place outside of the chamber. During the manufacture of the first weld, a metal specimen found oxidation was observed.
## 5.
Further experiments are necessary to optimize technical parameters for a suitable deposition condition and improve the quality of the hot-wire PAW process. Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
# Data availability statement:
The data presented in this study are available on request from the corresponding author.
[fig] Figure 1: Setup of the hot-wire plasma welding for the additive manufacturing (AM) process system. [/fig]
[fig] Figure 2: Deposited single beads for a designed experiment for the hot-wire plasma welding process (Numbers 1-8). [/fig]
[fig] Figure 3: The flow-purge welding chamber. [/fig]
[fig] Figure 4: Positions of the weld bead height and bead width measurement for the hot-wire plasma welding process. [/fig]
[fig] Figure 5: Photographs of the deposited walls: 36 layers. [/fig]
[fig] Figure 6: Schematic illustration of microhardness measurement on the profile of the microhardness of layer bands. [/fig]
[fig] Figure 7: Dimensions of the tensile specimens (mm). [/fig]
[fig] Figure 8: Each part of the workpiece. [/fig]
[fig] Figure 9: Arc voltage (final) values of the deposition of the hot-wire PAW process per layer. [/fig]
[fig] Author: Contributions: Conceptualization, P.P., W.N., and N.N.; methodology and experimental, P.P., W.N., and N.N.; writing-original draft preparation, P.P., W.N., and N.N.; writing-review and editing, W.N., and N.N.; supervision, W.N. and N.N.; funding acquisition, P.P., W.N., and N.N. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Thailand Graduate Institute of Science and Technology (TGIST), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Thailand. [/fig]
[table] Table 1 Table 2: The chemical composition of the titanium alloy grade 2. The mechanical properties of the titanium alloy grade 2. [/table]
[table] Table 3: The welding experimental conditions of eight welding specimens for hot-wire plasma welding. [/table]
[table] Table 4: The parameters used for the hot-wire plasma welding process. [/table]
[table] Table 5: Results of the Vickers microhardness test for hot-wire plasma welding. [/table]
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10.3389/fpubh.2023.1152876
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CCBY
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10643213
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38026409
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s2orc_pubmed_articles
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The impact of the two-year COVID-19 pandemic on hospital admission and readmissions of children and adolescents because of mental health problems
Gatta M ( ) The impact of the two-year COVID-pandemic on hospital admission and readmissions of children and adolescents because of mental health problems.
# . introduction
At the beginning of 2020, the whole world was forced to deal with the COVID-19 pandemic and the subsequent security measures, which seemed to be the main source of psychological stress among children and adolescents [bib_ref] Risk and protective factors for prospective changes in adolescent mental health during..., Magson [/bib_ref] , who reported higher levels of isolation and loneliness during the government lockdowns [bib_ref] Loneliness and mental health in children and adolescents with pre-existing mental health..., Hards [/bib_ref].These factors probably led to an increase in research on ways to connect with peers, resulting in the higher usage of technological devices and social media and a subsequent decrease in sports activities [bib_ref] School closures during social lockdown and mental health, health behaviors, and well-being..., Viner [/bib_ref].The COVID-19 pandemic has strongly affected children's and adolescents' mental health, with increases in symptoms of anxiety and depression, internalizing and externalizing psychopathology, and stressful environmental situations [bib_ref] Loneliness and mental health in children and adolescents with pre-existing mental health..., Hards [/bib_ref] [bib_ref] School closures during social lockdown and mental health, health behaviors, and well-being..., Viner [/bib_ref] [bib_ref] Parental buffering of stress in the time of COVID-19: family-level factors may..., Cohodes [/bib_ref] [bib_ref] Predictors of mental health worsening among children and adolescents during the coronavirus..., Caffo [/bib_ref] [bib_ref] Effects of COVID-19 pandemic on mental health of children and adolescents: a..., Theberath [/bib_ref].Additionally, based on their gender and age, studies suggest that youths reacted differently to the COVID-19 pandemic [bib_ref] Predictors of mental health worsening among children and adolescents during the coronavirus..., Caffo [/bib_ref] [bib_ref] Global prevalence of depressive and anxiety symptoms in children and adolescents during..., Racine [/bib_ref] [bib_ref] Pediatric mental health presentations and boarding: first year of the COVID-19 pandemic, Ibeziako [/bib_ref].Regarding referrals to mental health services, national and international literature corroborates detecting different trends according to different phases of the pandemic: a "freezing" of emergency psychiatric visits during the first pandemic period, followed by increased use of emergency services and stabilization at higher levels in the following summer months .It was found that, despite a decline in emergency department presentations during the first lockdown, a greater proportion of assessed children and adolescents met the admission criteria compared to the previous year, with an increase in the acuity of mental health disorders [bib_ref] A 5-year retrospective study of demographic, anamnestic, and clinical factors related to..., Millner [/bib_ref].
Considering that the scientific literature documents a constant increase in psychiatric hospitalizations of youths over the last decade and that, even before the outbreak of the COVID-19 pandemic (13-16), there was already a significant shortage of beds in child neuropsychiatry units (compared to the estimated needs in the pre-pandemic period) in Italy, the COVID-19 pandemic caused a breakdown in mental health inpatient and outpatient services.However, quite a few studies have investigated the effect of the COVID-19 pandemic on mental health-related hospital admissions among youths in the 2 years since the pandemic first began.
In light of the daily clinical and nursing experience of the past 3 years, which has shown an increase in psychopathological complexity and some clinical pictures, such as self-harm [bib_ref] Review: Mental health impacts of the COVID-19 pandemic on children and youth..., Samji [/bib_ref] and eating disorders (18), we aimed to identify eventual specific risk factors and psycho-social and clinical features of young persons with psychiatric diseases to better target diagnostic and therapeutic interventions.To reach our goal (aim 1), we analyzed the psycho-social and clinical variables of hospitalized neuropsychiatric patients during the COVID-19 pandemic, particularly the phenomenon of hospital readmission (aim 2).The latter is considered an indicator of clinical severity and greater complexity in case management.This phenomenon appears to have increased in frequency during the early pandemic period, according to some local and international studies [bib_ref] Sociodemographic and clinical characteristics of paediatric patients admitted to a neuropsychiatric care..., Gatta [/bib_ref] [bib_ref] Higher admission and rapid readmission rates among medically hospitalized youth with anorexia..., Matthews [/bib_ref] an unprecedented total number of rehospitalizations/patients.We expected to find a greater impairment of the inpatient population during the COVID period in terms of personal and environmental risk factors, psychopathological severity, and management complexity during hospitalization.
. Material and methods
## . . procedures and participants
We conducted an observational retrospective cohort study by reviewing medical materials (clinical records, medical examination reports, neuropsychiatric interviews with patients and their parents, discharge reports, and clinical reports sent to territorial mental health and social services).The data were treated anonymously and in accordance with the guidelines of the Declaration of Helsinki 2013.This study was approved by the Institutional Ethics Committee of University Hospital of Padua (CESC Prot.N. 0044914, 13.07.2021).
With regard to the first study aim, two groups of neuropsychiatric inpatients were enrolled.The inclusion criteria for both groups were children and young adults who were aged between 0 and 17 years and had been hospitalized for more than 24 h in the Child and Adolescent Neuropsychiatric Unit, University Hospital of Padua between 1 February 2018 and 31 March 2022.Outpatients admitted to neuropsychiatric visits, inpatients with 1 day hospital stay, or those who prematurely self-discharged before 24 h were excluded from the study.In total, the number of patients enrolled was 375.The majority of patients were girls: 265 girls vs. 110 boys.The mean age was 13.9 years (SD 2.30 years).For study purposes, the total sample was divided into two groups: the pre-COVID-19 group, which included 160 inpatients hospitalized between February 2018 and February 2020, and the COVID-19 group, which enrolled 215 inpatients hospitalized between March 2020 and March 2022.To explore the phenomenon of readmission (second aim), we selected participants from the two groups of patients with at least one hospital readmission within 365 days after the first discharge.
In total, we included 38 patients, nine of whom belonged to the "pre-COVID-19 Hospital Readmission (HR) group, " with an average age of 14.3 years (SD 1.66 years), and 29 of whom belonged to the "COVID-19 HR group, " with an average age of 14.4 years (SD 1.76 years).Considering gender, we enrolled 30 female and eight male patients.The two samples of relapsing patients were compared based on risk factors (well-known in the literature) linked to hospital readmission in a psychiatric ward to investigate possible sociodemographic and clinical differences.Figure shows the flowchart of the two study aims.
## . . data collection
A Microsoft Excel database was created and scrupulously compiled to collect data.Multiple variables, which are described below, were collected for each patient: sociodemographic variables-gender (girls vs. boys), age at the time of hospitalization (quantitative data collected), ethnicity (Caucasian, Latin, African, Asian, or "other" when parents belong to different ethnicities), history of immigration to Italy (yes or no), educational level (primary, middle, or high school), academic or school behavioral problems (yes or no), peer socialization (good, difficult, or social withdrawal), and bullying/cyberbullying (yes or no).From the collection of family anamnesis, information was obtained regarding the number of siblings (single child, one sibling, ≥two siblings), the marital status of parents (married/separated/widowed/other), familiarity with psychiatric disorders (yes or no), other health problems in family members (yes or no), and intra-family conflict (yes or no).The collected clinical anamnesis variables were the presence of chronic diseases (yes or no), traumatic life events (yes or no), previous requests for psychological assistance or admittance to neuropsychiatric services (yes or no), high-risk behavior such as alcohol use (yes or no) and substance use (yes or no), and the usage of technological devices (more or <4 h per day); suicidal self-injury: suicidal ideation (SI; yes or no), suicide attempt (SA; yes or no), and method of suicide attempt, such as drug or substance poisoning/self-cutting/other; nonsuicidal self-injury (NSSI; yes or no), frequency of self-harming acts (more or less than five times per year in accordance with DSM-5 criteria), self-injured body parts (one or more than one), and age of the first self-injurious act (quantitative data); and eating problems: focus on food and body image (yes or no), diagnoses of eating disorders (anorexia nervosa, bulimia nervosa, or binge-eating disorder), and age of onset.Variables related to hospitalization were the reason for hospitalization (suicidal and non-suicidal self-injury, anxiety symptoms and/or functional symptoms, eating disorders, psychomotor agitation and/or aggression, psychotic symptoms, and other); hospitalization modality (urgent or scheduled), method of hospitalization access [Emergency Department (ED); neuropsychiatric consulting, outpatient service, scheduled hospitalization, and transfer from another medical unit or from another hospital]; length in days of the hospitalization; post-discharge relapse (yes/no and number of readmission to the hospital); diagnosis according to ICD-10 criteria (psychotic disorders F20-29, affective syndromes F30-39, neurotic stress-related and somatoform disorders F40-48, syndromes and disorders associated with abnormal physiological functions F50-59, behavioral and emotional syndromes and disorders with onset in childhood and adolescence, and personality disorders F90-98/F60 or other F70/F80/Z); psychiatric comorbidity (yes or no); pharmacological therapy (monotherapy or polytherapy and pharmacological combination: neuroleptics, antidepressants, benzodiazepines, and mood stabilizers); and post-discharge services (public and private territorial outpatient care, residential or semi-residential care, intensive hospital monitoring, social services, or family counseling).Additional variables were collected only for patients with at least one hospital readmission: readmission time (in days), defined as the difference between the date of index discharge and date of second rehospitalization; the presence of relapse within 1 year after discharge and the first 3 months after discharge (revolving door); and the length in days of the second hospitalization.
## . . statistical analysis
Data were analyzed using Jamovi Statistic Software (the Jamovi project, version 1.6.23.0).The statistical significance level was set at a p-value of ≤0.05.Mean, standard deviation (SD), and frequency were used for conducting a descriptive analysis.Contingency tables and the chi-squared test were used for the categorical variables to test for the distribution in terms of gender, psychiatric familiarity, school problems, psychiatric comorbidity, SI, SA, NSSI, and relapse within 1 year and within 3 months from discharge to highlight the risk factors and psycho-social and clinical features of hospitalized young patients in the two groups.After verifying the validity of the assumptions, we performed a t-test for independent samples.The test aimed to identify differences between the two groups in several continuous variables.These variables included the age at which the first self-injurious act occurred, the age of onset for eating disorders, the duration of the second hospitalization in days, and the number of readmission times.
. Results
## . . demographic, socio-familial, and academic variables
The results of demographic, socio-familiar, and educational variables are shown in Table .
In the two periods, female patients used neuropsychiatric services more than male patients.
## . . anamnesis-clinical variables and hospitalization-related variables
Regarding the anamnesis data, there were no significant changes when comparing the two periods.A total of 48.4% of patients in the pre-COVID-19 group and 41.1% of those included in the COVID-19 group suffered from one or more chronic diseases (asthma, allergies, diabetes mellitus, IBD, etc.).Over 80% of inpatients in both groups had previously accessed neuropsychiatric (NPI) services or received other types of support (psychological, psychotherapeutic, or psychiatric).Almost half of the patients (44%) of both groups experienced at least one traumatic event, such as parental divorce, family mourning, or a scholarly failure.As shown in Table , a statistically significant increase [χ 2 (1, N = 368) = 23.1, p < 0.001, an increase of 10%, 95% CI (13%, 28%)] was found by comparing the use of electronic devices (more than 4 h per day: from 8.8% in the pre-COVID-19 group to 29.2% in the COVID-19 group).The percentages more than tripled in the pandemic period, showing how the pandemic significantly affected young people's lifestyles from this point of view.
## . . modality, reason for hospitalization, and diagnosis
Regarding the modalities of hospitalization, whether planned or compulsory, the results were uniform in the two periods (planned hospitalizations reached 14.5% in the pre-COVID-19 period vs. 14.1% in the COVID-19 period, while urgent hospitalizations reached 85.5% in the pre-COVID-19 period vs. 85.9% in the COVID-19 period).There was an increase in admissions from the Emergency Department following a visit to the hospital's outpatient service.
The reasons for hospitalizations are reported in Table , showing increased numbers of patients with eating disorders, suicidal and non-suicidal self-injury, and psychomotor agitation/aggression.
Data on the first and second ICD-10 (International Classification of Disease System) diagnoses at discharge are shown in Table .A total of 17 inpatients did not receive a complete diagnosis (early discharge due to the parents' choice or transfer to another medical unit).
Between the two periods, there was a decline in all diagnoses, except for ICD-10 F50-59 codes, which include eating disorders.Nearly 75% of patients with these diagnoses were hospitalized during the 2-year pandemic period, compared with only 25% of patients during the 2-year pre-pandemic period.With regard to comorbidity (the presence of two or more psychiatric diagnoses in the same patient), we observed a statistically significant increase from 66.4% of inpatients in the pre-pandemic period to 77.0% in the pandemic period [χ 2 (1, N = 358) = 4.91, p = 0.027, increase of 11%, 95% CI (1%, 20%)].
Hospitalization lasted an average of 20.8 days (SD 19.0) in the pre-COVID-19 period and 19.9 days (SD 16.3) in the COVID-19 period.
## . . suicidal and non-suicidal self-injury
With regard to suicidality, no significant results were found; from a descriptive point of view, small increases in suicidal ideation (from 44.0 to 53.1% between the two periods) and in suicide attempts (from 22.0 to 27.1%) were detected.Suicidal methods changed: in the COVID-19 period, we registered increasing suicidal attempts through drugs or substance poisoning (from 44.7 to 56.7%) but not through wrist cutting, whose percentages were comparable (7.9% pre-COVID-19 and 7.3% COVID-19).Suicidal methods classified as "other" (defenestration, falling from heights, choking, and being hit by fast vehicles) dropped from 47.4 to 36.4%.With regard to NSSI, the percentage of NSSI inpatients was 40.6% in the pre-COVID-19 period and 44.2% in the COVID-19 period.Meanwhile, for NSSI frequency acts, 52.3% of inpatients had an occasional history of NSSI in the pre-COVID-19 period (less than five acts per year), compared to 62.2% of inpatients in the COVID-19 period.In comparison, 37.8 vs. 47.7% of inpatients had a history of repetitive NSSI (more than five times a year), respectively, in Statistically significant changes [χ 2 (4, N = 148) = 11.7,p = 0.019] in the reasons for self-injuring acts (self-punishment, relief from negative emotions, both known and unknown) have been observed.Specifically, the reason "relief from suffering" both alone and grouped with "self-punishment" increased (from 40 to 53.8% and from 1.8 to 10.8%, respectively).Moreover, we found a significant increase [t (127) = -3.90;p < 0.001] in the age of the first self-injurious act from an average age of 12.7 years (SD 1, 41 years) in the pre-pandemic period to an average age of 13.8 years (SD 1.43 years) in the pandemic period.
## . . eating disorders
Regarding patients with eating disorders, particularly anorexia nervosa, we registered three times more patients with anorexia nervosa in the COVID-19 period than in the pre-COVID-19 period; respectively, the percentages were 73.9 vs. 23.8%.The increase related to eating problems had statistical significance [χ 2 (1, N = 374) = 9.63, p = 0.002, increase of 13%, 95% CI (5%, 20%)], with an increase from 11.3% in the pre-COVID-19 group to 23.8% in the COVID-19 group.We observed a significant increase [t (67) = 3.59; p < 0.001] in the age of the onset of eating disorders from the pre-pandemic period (a mean age of 12.6 years) to the pandemic period (a mean age of 14.1 years).
## . . post-relapse discharge
Data regarding post-discharge hospital readmission showed that, in the pre-pandemic period, relapses reached 16.7%, while in the pandemic period, this percentage increased to 26.2, meaning that, in the pandemic period, compared to the pre-pandemic period, more inpatients had already been hospitalized at least once (Figure .This increase had statistical significance [χ 2 (1, N = 304) = 3.92, p = 0.048; an increase of 10%, CI (0%, 19%)], from 16.7% in the 2-year pre-COVID-19 period to 26.2% in the COVID-19 period.
Considering the number of total hospitalizations per patient, 75% of patients hospitalized three or more times were part of the COVID-19 group.This percentage in the COVID-19 period also included patients hospitalized up to five, six, or seven times; these numbers were never reached by the pre-COVID-19 group.Furthermore, patients with at least one readmission within 1 year after discharge reached 76.3% in the COVID-19 group and only 23.7% in the pre-COVID-19 group [χ 2 = 6.73, df = 1, p = 0.009, an increase of 11%, 95% CI (3%, 18%)].More than half (55.3%) of these patients were "revolving doors, " as hospital readmission occurred within 3 months after discharge.Since these data showed that the phenomenon of hospital readmissions (HR) was much more frequent in the pandemic period than in the prepandemic one, it was considered appropriate to carry out further analyses only on patients affected by this phenomenon.It was found that the "pre-COVID-19 HR group" and the "COVID-19 HR group" did not differ significantly with regard to the following: gender (∼78% girls and 22% boys in both groups), the presence of psychiatric familiarity (66.7 vs. 58.6%),school problems (88.9 vs. 62.1%), psychiatric comorbidity (77.8 vs. 72.4%),SI (88.9 vs. 72.4%),SA (33.3 vs. 31.0%),NSSI (66.7 vs. 62.1%), and revolving door nature, i.e., readmission within 3 months after discharge (33.3 vs. 62.1%).Interestingly, 85.7% of "revolving door" patients were part of the COVID-19 HR group.Regarding the readmission time, the average number of days before the second hospitalization was considerably lower for the COVID-19 HR group, 97 days (SD 105.0), compared to the pre-COVID-19 HR group, 157 days (SD 93.1).
The average length of the second hospitalization of relapsing patients was 25.3 days (SD 23.5) in the pre-COVID-19 HR group and 19.2 days (SD 15.5) in the COVID-19 HR group.
# . discussion
This study investigated the phenomenon of NPI admissions during the COVID-19 pandemic, comparing them with admissions in the immediately preceding period, focusing on the psycho-social risk factors connected with hospitalization and on readmissions during the first year after discharge, in regard to which there is little data in the literature, to improve the diagnostic-therapeutic care of adolescents with psychiatric problems.
## . . educational level
Compared to the pre-COVID-19 group, it is possible that the excess share of patients during the COVID-19 pandemic were those who were exclusively attending high school.Consistent with the literature on the subject [bib_ref] Age-related differences in psychological distress during the COVID-19 pandemic, Rega [/bib_ref] , during the pandemic period, the severity of psychopathology appeared to be higher in middle school students than in primary school students, and it was the highest in high school students.Indeed, considering the average age of inpatients in the pandemic 2-year period, i.e., 14.3 years, it is coherently rather plausible that a good percentage of them were experiencing a school transition period (secondary school), which implies changes in habits and the relational environment.These changes can be added up and contextualized into those of adolescence, including physical and hormonal ones, as well as those related to the processes of one's own identity and self-efficacy.Additionally, the long or short suspension of face-to-face teaching and the quarantine may have hindered teenagers' achievement of a new balance or even deprived them of stable peer relationships in the school environment, which are important protective factors from the onset of mental health disorders [bib_ref] Social isolation, psychological health, and protective factors in adolescence, Hall-Lande [/bib_ref].
## . . substance abuse
With regard to lifestyles, the increasing trends in the consumption of alcohol, psychotropic substances, and tobacco in the COVID group already observed in Gatta et al.'s study [bib_ref] Sociodemographic and clinical characteristics of paediatric patients admitted to a neuropsychiatric care..., Gatta [/bib_ref] 12 months after the beginning of the pandemic were also confirmed 24 months after its start.In line with this, the longitudinal study by tracked alcohol and cannabis consumption during alternating periods of confinement and re-release for a total of 14 months, showing a gradually increasing use of these substances.
## . . technologic devices
Our study investigated the role of devices and, thus, social networks in the context of psychopathological disorders in hospitalized patients.Electronic device abuse is undoubtedly one ./fpubh. .
of the main consequences of the deleterious effects of COVID-19 pandemic measures on children's mental health.Children and adolescents confined at home have attempted to alleviate negative feelings and experiences related to the pandemic period by spending more time online.However, if media use can be identified as a coping strategy and a protective factor for children's mental health during the pandemic, then it is also an important risk factor.The association between excessive Internet use (more than 3 h per day) and internalizing mental problems, particularly anxiety and depression, is well known (24).We would especially like to highlight the risk of developing body image, diet, and exercise concerns associated with increased social media use (25).This may have contributed to the increase in the number of eating disorders recorded during the pandemic.A previous study [bib_ref] Sociodemographic and clinical characteristics of paediatric patients admitted to a neuropsychiatric care..., Gatta [/bib_ref] found no significant differences in the increased use of electronic devices during the first year of the pandemic compared to the previous one.Conversely, 2 years after the pandemic outbreak, the percentage of children using these devices for more than 4 h/day quadrupled.This suggests that, despite the weakening of isolation measures as the pandemic progressed, electronic devices remained a habit and the primary means of social connection and spending time.
A study conducted in Ferrara on a sample of Italian children and adolescents showed that, after the second pandemic wave, not only did the number of children with smartphone addiction increase but so did the number of children at greater risk of addiction compared to before the pandemic [bib_ref] Smartphone use and addiction during the coronavirus disease 2019 (COVID-19) pandemic: cohort..., Serra [/bib_ref].When studying why our patients come to the hospital, we found that, compared to the 2 years before COVID-19, a higher number of patients experienced suicidal ideation and attempted suicide.
## . . self-injury
Despite what was expected and the data in the literature, our study did not reveal an increase in suicidal or non-suicidal selfharm as an incidence of the phenomenon, but rather, differences were found in relation to features of the NSSI in terms of the number of self-injured body parts, which decreased during the pandemic period, and in the reasons for self-injuring acts, with predominantly relief from negative and blaming emotions.Moreover, we found a significant increase in the age of the first selfinjury act during the pandemic period, which might suggest that the onset of the disorder was recent in young self-injurers of the pandemic period compared with pre-pandemic young self-injurers.With regard to the above results, one of the possible explanations is that young people were more often under adult control due to periods of confinement.
## . . eating disorders
Finally, the statistically significant increase in patients with eating disorders during the pandemic is consistent with the international and national literature.A Canadian study reported that ED visits and hospitalizations of pediatric patients suffering from these disorders increased significantly (by 66 and 37%, respectively) as soon as COVID-19 started, compared to the expected rates before the pandemic, and remained well above the expected levels during the first 10 months of the pandemic.Specifically, hospitalizations of adolescents aged 14-17 years were significantly higher than expected, in line with our results [bib_ref] Acute care visits for eating disorders among children and adolescents after the..., Toulany [/bib_ref].Particularly, with regard to anorexia nervosa, the COVID-19 pandemic seems to have had a deep impact in terms of symptom severity, which is mirrored by a large increase in admission rates across Europe [bib_ref] Higher admission and rapid readmission rates among medically hospitalized youth with anorexia..., Matthews [/bib_ref] [bib_ref] Increase in admission rates and symptom severity of childhood and adolescent anorexia..., Gilsbach [/bib_ref].
## . . multiple diagnoses
It is also important to underline that, in agreement with the increase in severity and clinical complexity, independent of the psychiatric diagnosis, the number of patients with at least one psychiatric comorbidity increased over the 2 pandemic years (from 66.7 to 77%).
## . . post-discharge relapses
It is striking how, within this phenomenon, cases with three or more close hospitalizations after the first admission increased.However, the comparison between the two groups of patients rehospitalized before and during the COVID-19 pandemic did not reveal any differences in sociodemographic (gender, age, the presence of psychiatric familiarity, and school problems) and clinical (the presence of SI, NSSI, previous SA, and psychiatric comorbidity) characteristics.This suggests that this phenomenon mainly depended on the inability of territorial services to ensure adequate patient care after hospital discharge, together with increased severity and clinical complexity [bib_ref] A 5-year retrospective study of demographic, anamnestic, and clinical factors related to..., Millner [/bib_ref] [bib_ref] Higher admission and rapid readmission rates among medically hospitalized youth with anorexia..., Matthews [/bib_ref].With regard to the findings, we observed an increase in comorbidities related to eating disorders during the COVID-19 period.There are probably no specific services equipped to handle such a large influx of patients.Additionally, there was a rise in admissions among adolescents (high school students), particularly high school students.This could align with the notion that a delicate transition period is needed involving both child neuropsychiatric and adult psychiatric services.Furthermore, the reorganization of services due to the COVID-19 pandemic meant reduced access to in-person treatment, restricted entry into intensive psychiatric treatment programs, the stopping of services such as group therapies and daily centers, and prolonged wait times for those seeking to establish care.Further confirmation of the above considerations would lie in the significant increase in the use of pharmacotherapy during the 2-year pandemic period, specifically neuroleptic associations with other pharmaceuticals.
This study has some limitations, such as patients' heterogeneity in age and other correlated variables.Moreover, the SARS-CoV-2 sanitary infection is still ongoing, although not in a state of emergency, and it is not yet possible to have a complete overview of its effects.
. /fpubh. .
# . conclusion
Given the results of this study, we can conclude that the 2 years after the pandemic outbreak, adolescents, especially high school students, have been the most affected.The increased use of electronic devices, a known risk factor for mental health, stands out as one of the main consequences of the pandemic period on the lifestyles of adolescents [bib_ref] Pediatric preventive care in middle-high resource countries-the padova chart for health in..., Galderisi [/bib_ref].The increase in hospital readmissions, without any significant pre/post-pandemic differences in terms of sociodemographic and clinical characteristics of relapsed patients, prompts consideration of better coordination between hospital and territorial services to prevent the need for hospitalization and contain relapses requiring rehospitalization.Certainly, further research, hopefully polycentric, is needed to better delineate the roots of this phenomenon.The consequences arising from the SARS-CoV-2 health emergency undoubtedly highlight the weakness of the resources needed to provide primary and secondary prevention measures to protect the mental health of tomorrow's adults.
[fig] FIGURE: Percentages of patients with post-discharge relapse between the two periods. [/fig]
[table] TABLE Socio -: demographic, socio-family and academic variables. [/table]
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The Impact of COVID-19 on the Neck of Femur Fracture Service in a Tertiary Care Hospital in the United Kingdom
IntroductionThe emergence of the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in late 2019 ushered in a global crisis that profoundly impacted healthcare systems worldwide.In the United Kingdom, COVID-19 resulted in a significant toll on public health and the National Health Service (NHS).As the virus surged, the NHS faced unprecedented challenges, including surges in COVID-19 cases, a dire need for medical equipment, and a strain on intensive care units.Simultaneously, stringent nationwide lockdowns were imposed to curb the virus's spread, disrupting daily life and healthcare access.Amid this crisis, the interactions between COVID-19 and other prevalent health conditions came to the forefront of medical research, sparking interest in understanding their connections.This study delves into the intriguing interplay between COVID-19 and neck of femur (NoF) fractures, exploring shared risk factors, resource implications, and potential alterations in patient pathways.Given the severity of both conditions and their impact on the vulnerable elderly population, elucidating these connections is crucial for comprehensive patient care and resource allocation within the healthcare system.MethodsThis study used data from the National Hip Fracture Audit (NHFA) database, focusing on NoF fracture patients at Wythenshawe Hospital.We examined two cohorts: pre-pandemic (from March 2019 to March 2020) and pandemic (from March 2020 to March 2021).We compared key parameters and incorporated COVID-19 data.Graphs showed trends and cohort similarities.We also analyzed demographic data (age, gender, fracture type, times, COVID-19 status, and mortality), removing outliers for accuracy.ResultsThe data revealed that while certain factors such as patient age and mobilization remained largely unaffected, there was a modest association between COVID-19 incidence and NoF fracture patients.Notably, regional lockdown measures had a substantial impact on patient care.The initial lockdown effectively reduced COVID-19-positive cases upon admission but led to prolonged intervals and surgical delays.However, the second lockdown showed improvements, attributed to lessons learned, increased resource allocation, and better familiarity with hospital-specific lockdown measures.This research sheds light on the intricate relationship between a global pandemic and orthopedic patient care, highlighting the importance of adapting healthcare systems to evolving challenges.ConclusionThis study explores the impact of COVID-19 on neck of femur (NoF) fracture patients, highlighting key findings from Wythenshawe Hospital.It uncovers a dynamic relationship between the pandemic and patient care, with increased COVID-19 cases coinciding with reduced NoF fracture rates.Lockdowns influenced outcomes, with the first causing delays and higher post-discharge mortality, while the second improved efficiency and safety.These insights extend beyond Wythenshawe Hospital, offering implications for healthcare practices in the United Kingdom and beyond, especially in countries with limited vaccination resources.This research underscores the need for tailored strategies to optimize NoF fracture patient outcomes during pandemics and lockdowns.
# Introduction
In December 2019, the novel coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or COVID-19, was first identified in the United Kingdom [bib_ref] Understanding of COVID-19 based on current evidence, Sun [/bib_ref].This highly transmissible virus spread rapidly throughout the country, resulting in over four million positive tests and 124,000 deaths within 15 months.While COVID-19 cases were observed across all demographics, individuals who were elderly, frail, immunocompromised, or had multiple comorbidities faced an increased risk of contracting COVID-19 and experiencing severe outcomes [bib_ref] Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection:..., Collaborative [/bib_ref].This had profound repercussions on both the UK population and the National Health Service (NHS).
The NHS faced unprecedented demands for medical equipment, including ventilators, oxygen therapy, and personal protective gear for both patients and healthcare workers.The surge in COVID-19 cases also led to a substantial need for intensive care unit space [bib_ref] COVID-19 pandemic in the United Kingdom, Flynn [/bib_ref].Consequently, the United Kingdom implemented nationwide lockdowns, restricting non-essential activities and work.These restrictions, while essential for controlling the virus's spread, had significant effects on public physical and mental health due to reduced physical activity and drastic lifestyle changes [bib_ref] Indirect acute effects of the COVID-19 pandemic on physical and mental health..., Mansfield [/bib_ref].Furthermore, general practices (GPs) became less accessible than previously, with the NHS.uk website stating, "You'll only be asked to visit the surgery if absolutely necessary".Therefore, the COVID-19 pandemic had far-reaching implications for various health conditions, affecting incidence rates, mortality, and the quality of hospital care.
Neck of femur (NoF) fractures are a significant medical issue throughout the United Kingdom, predominantly affecting older individuals.NoF fractures are the most common serious injuries among older adults, often requiring emergency surgery and anesthesia.Patients with these fractures typically have longer in-patient stays, with approximately one in 45 hospital beds occupied by hip fracture patients in 2019.These fractures are more prevalent in the elderly and are associated with various risk factors, such as low mobility, female sex, vitamin D deficiency, falls, comorbidities, corticosteroid use, and trauma.Given the systemic nature of these risk factors and the comprehensive impact on patients' quality of life, a multidisciplinary approach involving various healthcare professionals is crucial for holistic patient care.
Notably, there is considerable overlap between COVID-19 and NoF fractures in terms of shared risk factors.The demographic most susceptible to NoF fractures is also at a higher risk of contracting COVID-19 [bib_ref] Why does COVID-19 disproportionately affect older people?, Mueller [/bib_ref].Moreover, both conditions impose substantial demands on NHS resources, potentially affecting the quality of care for NoF fracture patients during their hospitalization.Lockdown measures may have influenced the incidence of NoF fractures by promoting a sedentary lifestyle, which could lower mobility and lead to vitamin D deficiency, both risk factors for these fractures.However, lockdowns may have also reduced the probability of falls by limiting non-essential activities [bib_ref] Decrease in incidence of proximal femur fractures in the elderly population during..., Oulianski [/bib_ref].Consequently, a theoretical correlation exists between COVID-19 and NoF fractures, as the virus may have altered the patient pathway for these fractures.
# Materials and methods
## Aims
The aim of this study is to assess the extent of the correlation between COVID-19 and NoF and identify whether COVID-19 has had any impact on NoF fractures in clinical practice within Wythenshawe Hospital.Elucidating the association between the two can enhance the understanding of the virus and its impact on the United Kingdom.
## Literature review
The limited literature on the impact of COVID-19 on patients with NoF fractures presents conflicting evidence, emphasizing the need for more comprehensive research.Wang et al.'s systematic review investigated early mortality risk in COVID-19-positive NoF fracture patients undergoing surgery, revealing a fivefold increase in mortality risk, especially among those with comorbidities such as obesity [bib_ref] Early mortality after hip fracture surgery in COVID-19 patients: a systematic review..., Wang [/bib_ref].However, this study solely focused on direct mortality without considering broader effects such as incidence, demographic changes, or quality of care.To gain a more holistic understanding, future research should adopt a broader perspective.
Rasidovic et al.'s multicenter cohort study in the United Kingdom during the pandemic's early phases showed a marked increase in mortality among COVID-19-positive NoF fracture patients (32.5% versus 7.2% in COVID-19-negative patients) [bib_ref] Impact of COVID-19 on clinical outcomes for patients with fractured hip: a..., Rasidovic [/bib_ref].Nevertheless, this study also concentrated solely on mortality and did not explore other variables or the evolving pandemic context.The introduction of personal protective equipment (PPE), increased testing, and vaccination distribution could potentially alter these findings.
Conversely, Craig et al.'s audit of hip fracture management and mortality during the pandemic in a regional trauma center found the maintenance of service efficacy and similar outcomes compared to pre-pandemic data [bib_ref] The effects of COVID-19 on hip fracture management and mortality in a..., Craig [/bib_ref].The study indicated improvements in surgical timing despite reduced theatre capacity.However, this audit spanned only a two-month period during the lockdown, and outcomes may differ over a longer period and with changing restrictions and public attitudes.Additionally, being conducted in an Irish regional facility, differences from the United Kingdom's experience are likely.
Williams and Kumar's cohort study showed a 64% increase in admission-to-surgery time during and after the introduction of COVID-19 contingency plans.Like Craig et al.'s [bib_ref] The effects of COVID-19 on hip fracture management and mortality in a..., Craig [/bib_ref] study, it has limitations due to its small sample size and a short observation period within the evolving pandemic context.
In summary, existing research on the impact of COVID-19 on NoF fracture patients is scarce, specific, and often contradictory.Clear conclusions are challenging to draw.To better understand the consequences of COVID-19 on these patients' health, further research is imperative for future public health considerations.
# Methodology
Data was collected from the National Hip Fracture Audit (NHFA) database for NoF fracture patients from two separate cohorts within Wythenshawe Hospital.One dataset contained all patients who presented with a neck of femur fracture in the year prior to the pandemic between 18 March 2019 and 17 March 2020.The other dataset comprised all patients who presented with neck of femur fractures during the COVID-19 pandemic, from 18 March 2020 to 17 March 2021.Certain parameters were collected from each dataset for comparison.The final dataset consisted of the government website for the incidence of COVID-19 between 18 March 2020 and 17 March 2021; this acted as a marker of the impact of the virus on population health, burden on healthcare services, and public opinion toward COVID-19 during the year.For each parameter, a graph was created, within which both trends from the NHFA cohorts were plotted alongside the incidence of COVID-19.This allowed for insight into whether the March 2020/2021 NoF cohort mirrored the COVID-19 pattern or the previous NoF more.
The following parameters were collected from both NHFA cohorts: incidence, delay in theatre, delay in mobilization, and mortality.The March 2020/2021 cohort was compared against both the March 2019/2020 cohort and the national incidence of COVID-19 to demonstrate any trends and, if present, which dataset they followed more closely.Further analysis of changes during periods of lockdown was conducted to identify any impact this might have had.
Additional demographic data was collected from the March 2020/2021 cohort including patient age at presentation, patient sex, type of fracture, time from presentation to admission, time from presentation to theatre, time from presentation to discharge, patients testing positive for COVID-19, and positive patient mortality.These were plotted and compared against COVID-19 incidence to investigate any variations throughout the year and during lockdown, with outliers removed to prevent skew.
# Results
## Comparing 2020/2021 data to see a correlation with 2019/2020 data and covid-19 data
## Incidence of nof fractures
The analysis of graph data from Figure [fig_ref] FIGURE 1: FIGURE 1 [/fig_ref] shows that the March 2020/2021 cohort and the March 2019/2020 cohort at Wythenshawe Hospital were observed to have a similar pattern, deviating from the COVID-19 incidence trend.Throughout the year, there were no significant differences in patient presentations to the emergency department.However, a minor 4.23% decrease in presentations was noted.Although the statistical significance of this slight variation may be debatable, it could be linked to the regional lockdown measures imposed in the area.These restrictions may have led to individuals spending more time at home, potentially reducing their risk of falls, a common factor contributing to neck of femur fractures.
## Timing of theatres
During the pre-COVID-19 cohort (2019/2020), a total of 397 surgeries were undertaken, with 61.7% of them adhering to the stipulated 36-hour punctuality threshold.In contrast, the COVID-19 cohort (2020/2021) witnessed 366 surgeries, of which 70.8% met the same punctuality criteria.This marked a notable 9.1% increase in punctuality during the COVID-19-affected year.
Remarkably, COVID-19 had a limited direct impact on surgery delays, as only 2.8% of cases were attributed to COVID-19-related delays.The primary cause of delay across both cohorts was identified as "administrative/logistic, cancelled due to list over-run," accounting for 46.7% of delays in 2019/2020 and increasing to 55.1% in 2020/2021.This heightened occurrence of delays due to list over-runs, despite a reduced surgical caseload, suggests that a decrease in the number of theatres did not affect improved punctuality.Instead, this phenomenon can be attributed to a broader reduction in other causes of delay, for example, "awaiting orthopedic diagnosis/investigation."
As seen in Figure , the improvement in punctuality following the initial lockdown, when COVID-19 incidence was low, may be attributed to regional public sentiment regarding the virus.Lower incidence rates resulted in reduced admissions of COVID-19-positive patients, but paradoxically, fewer individuals sought health services during the pandemic due to COVID-19 concerns.This created an environment in which health services across all specialties in the hospital experienced reduced strain, providing more time, resources, and attention to urgent procedures, such as surgeries for hip fractures (NoF).A further examination of Figure reveals distinct spikes during the initial lockdown period, surpassing anticipated levels.These spikes, potentially linked to increased demands for hospital resources, shed light on the pandemic's influence on surgical punctuality.After the initial lockdown, a modest correlation emerged between delayed theatre frequency and COVID-19 incidence, particularly leading up to the peak in COVID-19 cases on January 4. As seen in both Figure [fig_ref] FIGURE 4: FIGURE 4 [/fig_ref] and Figure [fig_ref] FIGURE 5: FIGURE 5 [/fig_ref] about the mobilization of patients following their surgery, the 2020/2021 cohort followed the 2019/2020 trajectory far more closely than the COVID-19 incidence.Additionally, the lockdowns had no apparent effect.There was an overall decrease in delayed mobilization of 2% between the years, but such a small figure is unlikely to hold significance.These graphs exhibit no clear relation between COVID-19 and the mobilization of patients in the northwest center.The 2020/2021 cohort exhibited a noteworthy 50% increase in hospital mortality when compared to the 2019/2020 cohort.As seen in Figure [fig_ref] FIGURE 6: FIGURE 6 [/fig_ref] , these fatalities were uniformly distributed across the year and displayed no apparent correlation with lockdown measures or COVID-19 incidence rates.In contrast to expectations stemming from a reduced incidence of hip fractures (NoF fractures), a decline in mortality rates was not observed.This anomaly suggests that the cause of these deaths is more likely associated with deficiencies in healthcare staff rather than patient-related activities.The pandemic imposed considerable strain on healthcare workers due to increased workloads, evolving protocols, and constrained resources, potentially resulting in a compromised quality of care and subsequently elevating hospital mortality rates.This shift in mortality rates may be attributed to the resumption of risk-taking behaviors and activities detrimental to health following the easing of lockdown restrictions.With the reinstatement of non-essential activities, it is conceivable that some patients engaged in behaviors such as excessive alcohol consumption or strenuous physical activity, potentially leading to further medical complications, especially in individuals with a history of joint fractures.Subsequent to this peak, few residential mortalities were noted, indicating that measures implemented by care homes, including the use of personal protective equipment, social distancing, symptom monitoring, and the early vaccination of residents, effectively mitigated mortality following discharge.This is corroborated by the decline in the mean age of mortality from 87 in 2019/2020 to 85 in 2020/2021, signifying improved care for the very elderly, a population predominantly residing in care homes.
## Evaluating the effect of covid-19 incidence and lockdowns on 2020/2021 nof fracture demographics
Age Patient age varies from 61 years to 103 throughout the year.By plotting a trend line as in Figure , it is evident that the mean age of presentation did not vary with time; however, the age of presentation plot became more erratic following the increase in COVID-19 incidence during October.
## Figure 8: age of the presentation of neck of femur fracture cases in 2020/2021 compared to the incidence of covid-19 cases and lockdowns
This phenomenon could be due to COVID-19 contributing to frailty.NoF fractures are often preceded by falls, a significant risk factor for frailty.Frailty can be caused by existing comorbidities or illness.Therefore, with more of the population contracting COVID-19, more of the population is at risk of developing NoF fractures.This would impact both older and younger patients in the local region alike, causing no variation in the mean age of presentation.
## Patient sex
As seen in Figure and Figure [fig_ref] FIGURE 1: FIGURE 1 [/fig_ref] , both female and male patients presented reasonably consistently throughout the year, with no noticeable temporal variations.Therefore, neither COVID-19 nor lockdowns had any impact on either gender differently.
## Time from presentation to admission
Initially, as seen in Figure [fig_ref] FIGURE 11: FIGURE 11 [/fig_ref] , the plot fluctuates, peaking several times.However, after lifting lockdown restrictions, the time taken significantly reduces and stabilizes.The time taken remains fairly consistent until late December when it begins to peak again, coinciding with COVID-19 incidence.Vital data in earlymid January was not recorded; however, when the plot resumes, the time has once again reduced and stabilized.
## Cases and lockdowns
This graph depicts that the first lockdown was ineffective at reducing the time taken in Wythenshawe Hospital from presentation to admission; however, the second lockdown may have improved this.This could be caused by physician unfamiliarity with the altered patient pathways introduced during lockdown.Many local guidelines were changed within the hospital regarding patient isolation, the use of aerosol-generating procedures, and other aspects; initially, healthcare workers would not be accustomed to these, resulting in a delay in admitting patients.However, by the time the second lockdown was introduced, as these measures had previously been introduced, the healthcare workers had a far better understanding of these changes, resulting in faster admission.
## Time from presentation to surgery
Figure [fig_ref] FIGURE 12: FIGURE 12 [/fig_ref] shows that immediately prior to the introduction of the second lockdown, the COVID-19 incidence rate was at its highest; at the same point in time, the time taken from the presentation of a NoF fracture to theatre had been comparatively quick.
## Time from presentation to discharge
Much like the time from presentation to admission graph (Figure [fig_ref] FIGURE 11: FIGURE 11 [/fig_ref] , Figure [fig_ref] FIGURE 13: FIGURE 13 [/fig_ref] shows an initial fluctuating plot until the termination of the first lockdown.However, unlike the other graphs, there is no clear correlation with COVID-19 incidence as the time taken remains consistently lower.The second lockdown succeeded in reducing the time taken and preventing peaks from occurring.These findings are likely also to be due to healthcare worker familiarity with altered patient pathway during periods of lockdown.These findings may be explained by public engagement with lockdowns.During the first lockdown, the public engaged with measures strictly.A slight latency could explain the initial cases, as COVID-19 can be contracted and remain asymptomatic for a period before presentation; halfway through this lockdown, the positive results stopped for a period.However, the same was not seen for the second lockdown.This could be due to public disillusion and the loss of enthusiasm; people followed the preventative measures far more loosely when compared to the first lockdown, causing increased contraction in patients who then went on to present.
## Covid-19-positive results after theatre
Much like the previous graph (Figure [fig_ref] FIGURE 14: FIGURE 14 [/fig_ref] , in Figure [fig_ref] FIGURE 15: FIGURE 15 [/fig_ref] , many cases were seen during the first lockdown.However, upon lifting these measures, positive results appeared to cease until 9 September 2020.No further cases were seen during the November spike, but more positive cases were seen in the lead-up to the January spike.Despite this, during the second lockdown, no positive cases after theatre were recorded.This could be explained again by healthcare workers' familiarity with altered patient pathways, as seen in time from presentation to admission and discharge.During the first lockdown, healthcare workers were not accustomed to new changes that had been implemented for COVID-19-positive patients, allowing for transmission during admission to the hospital, resulting in nosocomial infection.However, by the second lockdown, they will be less likely to make the same mistakes, resulting in minimal transmission within the center.
## Death of covid-19-positive patients in hospital
In Figure [fig_ref] FIGURE 16: FIGURE 16 [/fig_ref] , cases are seen in the early months, with one further case seen in December.While not obviously coherent with the COVID-19 incidence plot, it follows the COVID-19-positive result graphs.
# Discussion
On exploring the intricate relationship between COVID-19 and the care of patients with NoF fractures within the Wythenshawe Hospital orthopedic department, most parameters remained largely unaffected by the pandemic itself.The data generated from our study suggests that COVID-19 incidence has only a modest association with NoF fracture patients, with correlations primarily observed in patient age and the time elapsed from presentation to admission.
The graphical representations in our study underscore the substantial impact of regional lockdown measures on patient care.The initial lockdown appeared effective in reducing the number of COVID-19-positive cases upon admission, possibly due to the rigorous adherence of the public to restrictions in the northwest region.However, adverse consequences became evident during these lockdown periods, including prolonged intervals from presentation to admission and discharge, along with surgical delays.These challenges could be attributed to heightened demands on healthcare resources, as well as the adjustment period required for hospital staff to become familiar with new COVID-19-related protocols.
Conversely, the second lockdown witnessed a reversal of these trends, as the time from presentation to admission and discharge improved, and no COVID-19-positive cases were recorded post-surgery, with no fatalities following discharge.This improvement may be attributed to lessons learned from the initial lockdown, resulting in increased local resource allocation and greater familiarity with hospital-specific lockdown measures.
While our study is limited to patients at Wythenshawe Hospital, its implications extend beyond this single institution.The insights gleaned from our data could be extrapolated to inform healthcare practices across the entire United Kingdom, especially in anticipation of future waves of the pandemic.Our findings suggest that most parameters are likely to remain stable, but a potential decrease in NoF fracture incidence, greater variability in patient age, and extended timeframes from presentation to surgery may be expected.Furthermore, we anticipate that additional lockdowns may yield diminishing returns as public compliance wanes, potentially exacerbating the negative consequences observed, such as increased COVID-19-positive admissions, greater age variation, and elevated mortality rates post-discharge.By anticipating these trends, preemptive measures can be implemented to mitigate their impact.
While the United Kingdom is progressing toward the end of the pandemic with widespread vaccine distribution, it is imperative to recognize that many other countries may not have the same resources to provide vaccination to their populations promptly.Consequently, they may grapple with the virus for an extended duration.The findings from this study, conducted in a single center, can be extrapolated and applied in these nations to anticipate how NoF fracture patient care may be affected by the pandemic and associated preventive measures.This study has several limitations that need to be addressed.Primarily, we only used data from one specific medical center, so the trends we found may not apply to other regions in the United Kingdom or other countries.It would be valuable to expand the study to include data from different places to make our findings more widely applicable.
Certain gaps in data remained since data was collected retrospectively from an audit database.Having more complete data would have made more continuous graphs pointing toward any missed trends.
Moreover, we could have gained a better understanding of how COVID-19 affected these patients by considering additional factors.For example, we could have included a measure of their quality of life, which would have given us a more complete picture of their well-being during and after their experience with the virus.
Lastly, to make our results more reliable, we could have used more rigorous statistical tests.These tests would have helped us determine whether the patterns we observed were statistically significant.However, we have used graphical patterns to determine trends and are not drawing causality in this study.It would be something for future studies with larger datasets to explore and draw stronger correlations from.
# Conclusions
In conclusion, the COVID-19 pandemic has exerted a profound and multifaceted influence on various facets of our lives.This study has explored its potential ramifications on neck of femur (NoF) fracture patients, acknowledging the scarcity of comprehensive research on this subject and the incongruities present in the existing literature.Leveraging data from the National Hip Fracture Audit and focusing on Wythenshawe Hospital as a microcosm, this investigation sheds light on the intricate relationship between the pandemic and the care of NoF fracture patients.The findings reveal a dynamic interplay between COVID-19 incidence and NoF fracture rates, with increasing national COVID-19 cases coinciding with decreased NoF fracture incidence.Notably, this period witnessed enhancements in theatre punctuality and reduced hospital mortality, greater age diversity among patients, prolonged intervals between presentation and admission and discharge, and an elevated prevalence of COVID-19-positive test results at admission and post-surgery within the hospital.
Additionally, the study underscores the significant influence of lockdown measures on patient care.The initial lockdown correlated with a rise in COVID-19-positive test results at admission but posed challenges in terms of delayed surgeries and extended timeframes for admission and discharge, leading to a spike in post-discharge mortality.Conversely, the second lockdown demonstrated improved efficiency in terms of timing and safety, as evidenced by expedited processes, the absence of COVID-19-positive results following surgery, and zero post-discharge fatalities.These findings collectively highlight the intricate relationship between the COVID-19 pandemic and the care of NoF fracture patients, emphasizing the need for further research and tailored strategies to optimize patient outcomes during pandemics and associated lockdowns.
[fig] FIGURE 1: FIGURE 1: Cases of neck of femur fractures presenting in 2020/2021 compared to that in 2019/2020 and to the incidence of COVID-19 cases and lockdowns [/fig]
[fig] FIGURE 2 FIGURE 3: FIGURE 2: Cases of neck of femur fractures operated on within 36 hours in 2020/2021 compared to that in 2019/2020 and to the incidence of COVID-19 cases and lockdowns [/fig]
[fig] FIGURE 4: FIGURE 4: Cases of neck of femur fractures where postoperative mobilization was done within the same day in 2020/2021 compared to that in 2019/2020 and to the incidence of COVID-19 cases and lockdowns [/fig]
[fig] FIGURE 5: FIGURE 5: Cases of neck of femur fractures where postoperative mobilization was delayed in 2020/2021 compared to that in 2019/2020 and to the incidence of COVID-19 cases and lockdowns [/fig]
[fig] FIGURE 6: FIGURE 6: Mortality rate of neck of femur fractures while in-patient in 2020/2021 compared to that in 2019/2020 and to the incidence of COVID-19 cases and lockdowns [/fig]
[fig] FIGURE 7: FIGURE 7: Mortality rate of neck of femur fractures within 120 days after discharge in 2020/2021 compared to that in 2019/2020 and to the incidence of COVID-19 cases and lockdowns [/fig]
[fig] Limitations2023: Nath et al.Cureus 15(10): e47298.DOI 10.7759/cureus.47298 [/fig]
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Increase in Th17 and T-reg Lymphocytes and Decrease of IL22 Correlate with the Recovery Phase of Acute EAE IN Rat
Experimental autoimmune encephalomyelitis (EAE), a well-established model of multiple sclerosis, is characterised by microglial activation and lymphocyte infiltration. Induction of EAE in Lewis rats produces an acute monophasic disease characterised by a single peak of disability followed by a spontaneous and complete recovery and a subsequent tolerance to further immunizations. In the current study we have performed a detailed analysis of the dynamics of different lymphocyte populations and cytokine profile along the induction, peak, recovery and post-recovery phases in this paradigm. MBP-injected rats were sacrificed attending exclusively to their clinical score, and the different populations of Tlymphocytes as well as the dynamics of different pro-and anti-inflammatory cytokines were analysed in the spinal cord by flow cytometry, immunohistochemistry and ELISA. Our results revealed that, during the induction and peak phases, in parallel to an increase in symptomatology, the number of CD3+ and CD4+ cells increased progressively, showing a Th1 phenotype, but unexpectedly during recovery, although clinical signs progressively decreased, the number and proportion of CD3+ and CD4+ populations remained unaltered. Interestingly, during this recovery phase, we observed a marked decrease of Th1 and an important increase in Th17 and T-reg cells. Moreover, our results indicate a specific cytokine expression profile along the EAE course characterized by no changes of IL10 and IL17 levels, decrease of IL21 on the peak, and high IL22 levels during the induction and peak phases that markedly decrease during recovery. In summary, these results revealed the existence of a specific pattern of lymphocyte infiltration and cytokine secretion along the different phases of the acute EAE model in Lewis rat that differs from those already described in chronic or relapsing-remitting mouse models, where Th17-cells were found mostly during the peak, suggesting a specific role of these lymphocytes and cytokines in the evolution of this acute EAE model. Citation: Almolda B, Costa M, Montoya M, González B, Castellano B (2011) Increase in Th17 and T-reg Lymphocytes and Decrease of IL22 Correlate with the Recovery Phase of Acute EAE IN Rat. PLoS ONE 6(11): e27473.
# Introduction
Experimental autoimmune encephalomyelitis (EAE) is a useful animal model for the study of multiple sclerosis (MS) that is produced by the immunisation of susceptible animals with myelin proteins. Among the different EAE models, immunisation of susceptible Lewis rats with Myelin Basic Protein (MBP) induced an acute monophasic disease characterised by weight loss and hindlimb paralysis, followed by a spontaneous recovery, after which animals became resistant to further immunisations with MBP [bib_ref] Studies on the refractoriness to reinduction of experimental allergic encephalomyelitis in Lewis..., Macphee [/bib_ref] [bib_ref] Modulation of experimental allergic encephalomyelitis (EAE): suppression of active reinduction of EAE..., Namikawa [/bib_ref]. This EAE model is of special interest because provides a unique tool to study the cellular and molecular mechanisms involved not only in the induction of the disease, but more interestingly, those participating in the recovery process and the establishment of immunological tolerance.
Although there are a wide number of studies published about different aspects of EAE in Lewis rats, the major part of these studies are focused on specific time-points, mainly at the peak of the disease, whereas molecular and cellular changes taking place during the induction and recovery phases remain poorly defined.
To address this lack of information and gain insights into the mechanisms involved in the initiation and resolution of the pathological process, in the last years we have performed a series of detailed studies in which animals were sacrificed attending exclusively to their clinical score along the different phases characteristics of this model. In these studies we have reported changes in the morphology and phenotype of microglial cells, in terms of antigen presenting capacity, along the different phases of EAE showing that during the induction and peak phases, microglia exhibited a phenotype of immature dendritic cells, characterised by MHC-class I and class-II expression, no costimulatory molecules and CD1 expression, whereas during the recovery and even post-recovery phases, activated microglia maintained MHC expression and a subpopulation of cells expressed B7.2 [bib_ref] Activated microglial cells acquire an immature dendritic cell phenotype and may terminate..., Almolda [/bib_ref] and/or CD4 [bib_ref] CD4 microglial expression correlates with spontaneous clinical improvement in the acute Lewis..., Almolda [/bib_ref].
In addition to microglial reactivity it is well established that Tcell infiltration is a crucial feature of EAE [bib_ref] T cell response in experimental autoimmune encephalomyelitis (EAE): role of self and..., Kuchroo [/bib_ref]. Specifically, CD4+ Th1 cells are widely accepted as the key players in leading to the immune response associated with EAE [bib_ref] Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years..., Gold [/bib_ref]. However, in the last years, upon the emergence of new subsets of CD4+ T-cells with different cytokine profiles and functions [bib_ref] T helper cell effector fates-who, how and where?, Reinhardt [/bib_ref] [bib_ref] CD4 T cells: Balancing the coming and going of autoimmunemediated inflammation in..., Dittel [/bib_ref] [bib_ref] New complexities in helper T cell fate determination and the implications for..., Takatori [/bib_ref] , this classical assumption has been reconsidered and nowadays it is suggested that, in addition to Th1 cells, other subtypes of CD4+ T-cells may also be involved in EAE pathogenesis. Indeed, it has recently been demonstrated that injection of Th17 lymphocytes, a new characterised subset of CD4+ T-cells [bib_ref] Expanding the effector CD4 Tcell repertoire: the Th17 lineage, Harrington [/bib_ref] [bib_ref] Th17 Cells and autoimmune encephalomyelitis (EAE/MS), Aranami [/bib_ref] , are able to induce EAE in mice [bib_ref] Th1, Th17, and Th9 effector cells induce experimental autoimmune encephalomyelitis with different..., Jager [/bib_ref]. Moreover, accumulation of T-regulatory Foxp3+ cells (T-regs) was reported within the mouse CNS concurrent with EAE recovery [bib_ref] The dynamics of effector T cells and Foxp3+ regulatory T cells in..., Korn [/bib_ref] [bib_ref] Cutting Edge: Anti-CD25 monoclonal antibody injection results in the functional inactivation, not..., Kohm [/bib_ref] [bib_ref] Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells..., Mcgeachy [/bib_ref]. Although an important number of reports can be found in the literature about infiltration of T-cells in different EAE models [bib_ref] Presence of T cells with activated and memory phenotypes in inflammatory spinal..., Barten [/bib_ref] [bib_ref] The role of regulatory T cells in Lewis rats resistant to EAE, Sun [/bib_ref] [bib_ref] Migratory activity and functional changes of green fluorescent effector cells before and..., Flugel [/bib_ref] [bib_ref] Autoreactive T cells persist in rats protected against experimental autoimmune encephalomyelitis and..., Conant [/bib_ref] [bib_ref] Live imaging of effector cell trafficking and autoantigen recognition within the unfolding..., Kawakami [/bib_ref] [bib_ref] Chronological changes of CD4(+) and CD8(+) T cell subsets in the experimental..., Sonobe [/bib_ref] [bib_ref] Actively induced EAE in Lewis rats: characterization of spleen and spinal cord..., Rigolio [/bib_ref] [bib_ref] Differential immune cell dynamics in the CNS cause CD4+ T cell compartmentalization, Siffrin [/bib_ref] , it is important to take into account that major part of these studies are focused specially in the peak of the disease, and do not characterized the different subtypes of T-cells in the induction and recovery phases. Moreover, some of these studies analysed only changes occurring in lymphatic organs, especially spleen and lymph nodes [bib_ref] The role of regulatory T cells in Lewis rats resistant to EAE, Sun [/bib_ref] [bib_ref] Autoreactive T cells persist in rats protected against experimental autoimmune encephalomyelitis and..., Conant [/bib_ref] , or described changes in the CD4+T-cell population but not in the subtypes of these cells [bib_ref] Presence of T cells with activated and memory phenotypes in inflammatory spinal..., Barten [/bib_ref] [bib_ref] Chronological changes of CD4(+) and CD8(+) T cell subsets in the experimental..., Sonobe [/bib_ref] [bib_ref] Actively induced EAE in Lewis rats: characterization of spleen and spinal cord..., Rigolio [/bib_ref] [bib_ref] Differential immune cell dynamics in the CNS cause CD4+ T cell compartmentalization, Siffrin [/bib_ref]. Nevertheless, little is known about the dynamics of the specific lymphocyte subtypes inside the spinal cord along the course of the disease.
In this way, the aim of the present study was to perform a careful analysis of CD4+ T-cell subsets dynamics and their cytokine profile along the induction, peak, recovery and postrecovery phases in the acute EAE model induced in Lewis rats.
Our results indicate that the population of T-cells remained unaltered during all the phases of the acute EAE but showing specific phenotypes during the induction and recovery phases. Interestingly, and in contrast to studies in EAE mice models, in this acute model the population of Th17 cells was higher during the recovery phase.
# Materials and methods
# Ethics statement
All experimental animal work was conducted according to Spanish regulations in agreement with European Union directives (86/609/CEE, 91/628/CEE i 92/65/CEE) and was approved by the Ethical Committee of the Autonomous University of Barcelona (CEEA-UAB 569). The presence of clinical signs was evaluated daily in all animals, using the following clinical score test: 0, no clinical signs; 0.5, partial loss of tail tonus; 1, tail paralysis; 2, paraparesis of hindlimb; 3, paraplegia; 4, tetraparesis; 5, tetraplegia and 6, death. Paralysed animals were afforded easier access to food and water.
## Animals and eae induction
## Experimental groups
As in previous studies [bib_ref] Activated microglial cells acquire an immature dendritic cell phenotype and may terminate..., Almolda [/bib_ref] [bib_ref] CD4 microglial expression correlates with spontaneous clinical improvement in the acute Lewis..., Almolda [/bib_ref] , EAE-induced animals were sacrificed according to their clinical score, at different phases along the EAE course, as detailed: 1) before the appearance of symptomatology (2, 4, 6 and 8 days post-immunisation); 2) during the induction phase: at score 0.5, score 1 and score 2; 3) at the peak of the disease (score 3); 4) during the recovery phase: at score 2 of recovery (score 2R), score 1 of recovery (score 1R) and 0 of recovery (score 0R) and 5) during the post-recovery phase, at 28, 32, 40 and 90 days post-immunisation (referred to as score 0R-28dpi, score 0R-32 dpi, score 0R-40 dpi and score 0R-90 dpi). A total of 56 EAE-induced rats and 7 shams injected with vehicle were used for flow cytometry and ELISA studies. For immunohistochemistry, 68 EAE-induced rats and 9 sham animals were processed.
# Flow cytometry analysis
As already described in previous studies [bib_ref] CD4 microglial expression correlates with spontaneous clinical improvement in the acute Lewis..., Almolda [/bib_ref] , for flow cytometry analysis, animals were anesthetised with 0.015 ml/g ketamine (80 mg/kg)/xylazyne (10 mg/kg) and intracardially perfused with phosphate buffer solution (PBS). Quickly, the entire spinal cord was dissected out and the meninges were carefully removed. In order to obtain cell suspensions, tissue was dissociated through meshes of 140 mm and 70 mm and digested with a mixture of DNase I (28 U/ml; 10 104 159 001; Roche) and collagenase (0.2 mg/ml; LS004194, Worthington). Subsequently, each cellular suspension was centrifuged for 20 min at 600 g at room temperature in a discontinuous-density Percoll gradient (17-0891-02; Amersham-Pharmacia) between 1.08 g/ml and 1.03 g/ ml. Myelin in the upper layer was removed. Cells in the interphase and in the upper-phase were collected, washed in PBS +2% serum and labelled during 30 min at 4uC with different combinations of the following surface markers: anti-CD4-PECy5 (1:400; 554839; BD Pharmingen; San Diego, CA), anti-CD4-APC.Cy7 (1:400; 201518; Biolegend), anti-CD3-FITC (1:400; 557354; BD Pharmingen, San Diego, CA), and anti-CD45RC-PE (1:400; 554888; BD Pharmingen, San Diego, CA). Subsequently, for the detection of intracellular markers, samples were permeabilised for 40 min using the Foxp3 staining buffer set (00-5523-00; eBiosciences; San Diego, CA) and labelled for 30 min at 4uC with anti-Tbet-PerCP.Cy5.5 (1:400; 45-5825; eBiosciences; San Diego, CA), anti-GATA3-PE (1:400; 560074; BD Pharmingen; San Diego, CA), anti-RORc-APC (1:400; IMG-6275G; IMGENEX; San Diego, CA) and anti-Foxp3-PE.Cy7 (1:400; 25-5773; eBiosciences; San Diego, CA) following the instructions specified in the manufacturer's protocol. In parallel, isotype-matched control antibodies for the different fluorochromes were used as negative controls and spleen samples as positive control. Spleen samples were also used to determine the gates for CD3+ and CD3+CD4+cells. In order to perform the quantification of the total number of cells, a known volume of fluorescence beads (CytoCountTM, S2366, DakoCytomation) was added and mixed with each sample. Finally, cells were acquired using a FACScalibur or FACsCanto flow cytometer (Becton Dickinson; San Jose, CA), and the results were analysed using FlowJoH software. Quantification of total number of cells was performed following the methodology specified in the manufacturer's data-sheet (CytoCountTM, S2366, DakoCytomation). Spinal cords from each animal in both sham and EAE groups were analyzed separately. Only in the case of the transcription factors study spinal cords were pooled (a minimum of three animals per clinical score).
## Tissue processing for histological analysis
Animals processed for immunohistochemistry were sacrificed under deep anaesthesia (ketamine/xylazyne) as previously described and perfused intracardially with 4% paraformaldehyde in 0.1 M PBS (pH 7.4)+5% sucrose. Spinal cords (cervical and thoracic part) were dissected out immediately, postfixed for 4 h at 4uC in the same fixative, and series of parallel longitudinal sections (40 mm thick) were obtained using a Leica VT 1000 S vibratome. Series were stored at 220uC in the Olmos antifreeze solution until their later use.
## Single immunohistochemistry
Some parallel free-floating vibratome sections were processed for the visualisation of different subtypes of lymphocytes: CD3 for all T-cell populations and CD4 for T-helper cells. After endogenous peroxidase blocking with 2% H 2 O 2 in 70% methanol for 10 min, sections were blocked in 0.05 M Tris-buffered saline (TBS), pH 7.4, containing 10% foetal calf serum, 3% bovine serum albumine (BSA) and 1% Triton X-100 for 1 h. Afterwards, sections were incubated overnight at 4uC with either anti-CD3 (1:500; A0452; Dakopatts, Denmark) or anti-CD4 (1:1000; MCA55G; AbD Serotec) antibodies diluted in the same blocking solution. Sections incubated in media lacking the primary antibody were used as negative controls, and spleen sections as positive control. After washes with TBS +1% Triton, sections were incubated at room temperature for 1 h with either biotinylated anti-mouse secondary antibody (1:500; BA-2001; Vector Laboratories, Inc; Burlingame, CA) or biotinylated anti-rabbit secondary antibody (1:500; BA-1000; Vector Laboratories, Inc; Burlingame, CA). After 1 h in streptavidin-peroxidase (1:500; SA-5004; Vector Laboratories, Inc; Burlingame, CA), the reaction was visualised by incubating the sections in a DAB kit (SK-4100; Vector Laboratories, Inc; Burlingame, CA) following the manufacturer's instructions. Finally, sections were mounted on slides, some of them counterstained with toluidine blue, dehydrated in alchohol and after xylene treatment, coverslipped in DPX. Sections were analysed and photographed with DXM 1200F Nikon digital camera joined to a Nikon Eclipse 80i microscope.
## Double immunohistochemistry
Double-immunolabelling was carried out by firstly processing the sections with either CD3 or CD4 immunolabelling as described above, but using, as secondary antibodies, AlexaFluorH 488-conjugated anti-rabbit (1:1000, A-21206; Molecular Probes) in the case of CD3, or AlexaFluorH 555-conjugated anti-mouse (1:1000, A31570; Molecular Probes) in the case of CD4. After several washes, these sections were incubated overnight at 4uC with either rabbit anti-Iba1 (1:3000; 019-19741; Wako), mouse anti-CD4 (1:1000; MCA55G; AbD Serotec), rabbit anti-Tbet for demonstration of Th1 cells (1:1000; sc-21003; Santa Cruz Biotechnology), rabbit-anti-Foxp3 for demonstration of T-reg cells (1:2000; sc-28705; Santa Cruz Biotechnology) or rabbit anti-RORc for demonstration of Th17 cells (1:2000; ab78007; AbCam) followed, by AlexaFluorH 555-conjugated anti-mouse (1:1000; A31570; Molecular Probes) in the case of CD4, and by AlexaFluorH 488-conjugated anti-rabbit (1:1000, A-21206; Molecular Probes) for Iba1. In the cases of Tbet, Foxp3 and ROR-c after the primary antibody, sections were incubated with biotinylated anti-rabbit secondary antibody (1:500; BA-1000; Vector Laboratories, Inc; Burlingame, CA) followed by Streptavidin-Cy3 (1:1000; PA-43001; Amersham). Finally, sections were mounted on slides, dehydrated in graded alchohol and coverslipped in DPX. Sections were analysed using a fluorescence Nikon Eclipse E600 confocal microscope and photographed with a Leica DMIRE 2 confocal camera.
## Tissue processing for elisa analysis
For ELISA studies, animals at different clinical scores (3 animals per score) were anesthetised using ketamine/xylazyne solution, as previously described, intracardially perfused with phosphate buffer solution (PBS) and samples corresponding to the part between the cervical and the thoracic spinal cord (C7-T2 levels) snap frozen in liquid nitrogen and stored at 280uC. Total protein were extracted by solubilization of spinal cords in Lysis buffer containing 250 mM HEPES (pH7.4), 1% Igepal, 5 mM MgCl 2 , 1.3 mM EDTA, 1 mM EGTA, 1 mM PMSF and protease and phosphatase inhibitor cocktails (1:100, Sigma Aldrich). Following solubilization, samples were clarified by centrifugation at 13000 rpm for 5 min and the supernatant retained. Total protein concentration was determined with a commercial Pierce BCA Protein Assay kit (23225, Thermo Scientific) using manufacturer's protocol. Protein lysates were stored aliquoted at 280uC until their posterior used for ELISA.
## Cytokines elisa
Cytokine ELISA kits for IFN-c (KRC4021, Invitrogen), IL10 (KRC0101, Invitrogen), IL17 (E90063Ra, USNC Life Science Inc), IL21 (E91688Ra, USNC Life Science Inc) and Quantikine ELISA kit for IL22 (M2200, R&D Systems) were used according to the manufacturer's instruction. A standard curve was generated with each assay with the limit of detection for IFN-c = 13 pg/ml, IL10 = 5 pg/ml, IL17 = 6.1 pg/ml, IL21 = 5.8 pg/ml and IL22 = 3.2 pg/ml.
## Cd3+ cell quantification
For CD3+ cell quantification, a total of three EAE-induced animals per clinical score and three sham were used. Analysis was performed using one entire longitudinal section of the cervical spinal cord, per animal. This section corresponds to a medial section containing both the grey and the white matter areas. Each section was photographed at 4x using a DXM 1200F Nikon digital camera joined to a Nikon Eclipse 80i microscope, and pictures taken were merged using Adobe Photoshop software. The total number of CD3+ cells per section was counted by the use of analySISH software.
# Statistical analysis
All statistics along the study were performed using the Graph Pad PrismH software. Either standard two-tailed, unpaired Student's-T test, to compare two specific groups of animals, or one-way ANOVA with Tukey's post-hoc test, to compare between the different scores, were used to determine statistically significant differences.
# Results
MBP-induced Lewis rat developed the first signs of EAE around 10 days post-immunisation (dpi), displaying loss of tail tonus (score 0.5). Afterwards, during the induction phase, animals progressively presented tail paralysis (score 1) followed by hindlimb paraparesis (score 2) until reaching the maximum clinical symptomatology at score 3 (approximately around 12-14 dpi) when animals showed complete hindlimb paralysis. Animals remained at this maximum score for 1-2 days and afterwards spontaneously started to recover. During the recovery phase, animals progressively improved their mobility achieving scores 2R and 1R, and around 21 dpi, were completely recovered (score 0R) and no clinical signs were observed. During the post-recovery phase (28 dpi, 32 dpi, 40 dpi and 90 dpi), the animals did not show any visible sign of disease.
It is important to highlight here that, as we have done in previous works [bib_ref] Activated microglial cells acquire an immature dendritic cell phenotype and may terminate..., Almolda [/bib_ref] [bib_ref] CD4 microglial expression correlates with spontaneous clinical improvement in the acute Lewis..., Almolda [/bib_ref] , animals to be analysed for flow cytometry, ELISA and immunohistochemistry were selected not based on the days post-immunisation, but rather exclusively according to their clinical score, during the induction, peak and recovery phases. This criterion has demonstrated to be more useful because, as already stated in our previous studies, the variability among animals in the same score, if any, is very low.
## Cd3+ cells
The study of sections immunolabelled for CD3 revealed that there was no presence of T-lymphocytes in the spinal cord of sham animals [fig_ref] Figure 1: Dynamics of CD3+ cells [/fig_ref]. In contrast, in EAE animals, from score 0.5, few little round CD3+ cells were observed in the parenchyma [fig_ref] Figure 1: Dynamics of CD3+ cells [/fig_ref].
The number of CD3+ cells increased progressively during the induction phase, mainly accumulating around blood vessels [fig_ref] Figure 1: Dynamics of CD3+ cells [/fig_ref] , D and J), reaching their maximal at scores 2 and 3. At score 3, CD3+ cells in addition to remain in close proximity to blood vessels, were also found extensively distributed throughout the parenchyma, in both the grey and the white matters [fig_ref] Figure 1: Dynamics of CD3+ cells [/fig_ref].
During the recovery phase, from score 2R to 1R, although the number of CD3+ cells remained without significant changes [fig_ref] Figure 1: Dynamics of CD3+ cells [/fig_ref] , the density of cells around blood vessels increased, whereas a progressive decrease in the number of CD3+ cells within the parenchyma of the cervical spinal cord was observed [fig_ref] Figure 1: Dynamics of CD3+ cells [/fig_ref]. The major part of positive cells at score 0R was accumulated in the surroundings of blood vessels of both grey and white matters [fig_ref] Figure 1: Dynamics of CD3+ cells [/fig_ref]. Quantitative analysis showed that at score 0R, the number of CD3+ cells apparently decreased, although the value did not reach statistical significance.
During the post-recovery phase, at score 0R-40 dpi, a high reduction of CD3+ cells was observed [fig_ref] Figure 1: Dynamics of CD3+ cells [/fig_ref] although few perivascular CD3+ cells were still detected in the cervical spinal cord [fig_ref] Figure 1: Dynamics of CD3+ cells [/fig_ref]. At score 0R-90 dpi, CD3+ cells completely disappeared [fig_ref] Figure 1: Dynamics of CD3+ cells [/fig_ref].
## Cd3+cd4+cells
The number and proportion of CD4+ cells within the gated CD3+ cell population was analysed by flow cytometry along the different phases of EAE evolution [fig_ref] Figure 2: Figure 2 [/fig_ref]. In contrast to sham animals, where no presence of CD3+CD4+ cells was found, in EAE animals, from score 1 in the induction phase, a high number of CD4+T-helper lymphocytes was observed [fig_ref] Figure 2: Figure 2 [/fig_ref]. The number of CD3+CD4+ cells remained without significant changes along the induction and peak phases and also at score 2R, and only significantly decreased from score 1R. During the postrecovery phase, at score 0R-32dpi, an important and marked decrease in the number of these cells was found [fig_ref] Figure 2: Figure 2 [/fig_ref]. In addition to the number of cells, we also analysed the proportion of CD3+CD4+ cells. It should be noted that CD4+ T-cells represented around 70% of the total CD3+ lymphocytes [fig_ref] Figure 2: Figure 2 [/fig_ref]. This high percentage of CD4+ T-cells was maintained without significant changes at the different scores analysed during the induction, peak and recovery phases [fig_ref] Figure 1: Dynamics of CD3+ cells [/fig_ref]. Only during the post-recovery phase, at score 0R-32dpi, a slight decrease in the proportion of CD4+ cells was observed [fig_ref] Figure 2: Figure 2 [/fig_ref].
In all phases analysed, around 90% of CD3+CD4+ cells showed a CD45RC-phenotype, characteristic of effector/memory lymphocytes, and only few CD45RC+ naïve lymphocytes were detected [fig_ref] Figure 2: Figure 2 [/fig_ref].
## Subtypes of cd4+ t-lymphocytes
The different subpopulations of CD4+ T-helper lymphocytes were determined by flow cytometry using specific antibodies against lineage-specific transcription factors. After gating in the CD3+ cell population, combinations of CD4 with Tbet (for Th1 cells), RORc (for Th17 cells) and Foxp3 (for T-regulatory cells) were analysed.
Th1 cells. As shown in [fig_ref] Figure 3: Dynamics of Th1 cell population [/fig_ref] , in EAE animals, a progressive increase in the number of CD4+Tbet+ cells in the gated CD3+ cell population was detected from score 1, reaching the maximum value at scores 2 and 3 [fig_ref] Figure 3: Dynamics of Th1 cell population [/fig_ref]. From score 2R, a marked decrease in the number of CD4+Tbet+ cells was observed [fig_ref] Figure 3: Dynamics of Th1 cell population [/fig_ref]. This population of CD4+Tbet+ cells represented, at scores 2 and 3, around 7% of CD3+ lymphocytes [fig_ref] Figure 3: Dynamics of Th1 cell population [/fig_ref]. During the post-recovery phase (score 0R-28dpi and score 0R-40dpi) Th1 cells were absent [fig_ref] Figure 3: Dynamics of Th1 cell population [/fig_ref]. As expected, levels of IFNc were high during the inductive phase and peak and decreased during the recovery phase [fig_ref] Figure 3: Dynamics of Th1 cell population [/fig_ref].
Double immunohistochemistry demonstrated the presence of Th1 cells in the spinal cord mainly accumulated around blood vessels [fig_ref] Figure 2: Figure 2 [/fig_ref].
Th17 cells. In EAE animals, some CD4+RORc+ cells were observed during the induction phase, the peak and at score 2R in the recovery phase [fig_ref] Figure 4: Dynamics of Th17 cells [/fig_ref]. The number of CD4+RORc+ cells highly increased from score 1R in the recovery phase, reaching the maximum value at score 0R [fig_ref] Figure 4: Dynamics of Th17 cells [/fig_ref]. During the post-recovery phase, an important decrease in the number of CD4+RORc+ cells was detected [fig_ref] Figure 4: Dynamics of Th17 cells [/fig_ref]. In terms of percentage of this population, it should be noted that, whereas during the induction and peak phases the subpopulation of CD4+RORc+ lymphocytes represented around 7% of CD3+ T-cells, from score 1R in the recovery phase the percentage of these lymphocytes increased until a value of around 50% at score 0R [fig_ref] Figure 4: Dynamics of Th17 cells [/fig_ref]. During the post-recovery phase, a decrease in the percentage of these cells was found although levels remained higher than those observed during the induction and peak phases [fig_ref] Figure 4: Dynamics of Th17 cells [/fig_ref]. In addition to CD4+RORc+ cells, our analysis also demonstrated the presence of a population of CD4-RORc+ cells (less than 10%) [fig_ref] Figure 4: Dynamics of Th17 cells [/fig_ref].
The study of double immunolabelled sections indicated the presence of CD4+RORc+ cells mainly accumulated around blood vessels in the spinal cord of EAE animals [fig_ref] Figure 2: Figure 2 [/fig_ref].
T-regulatory cells. As shown in [fig_ref] Figure 5: -F [/fig_ref] , during the induction phase, the peak, and at score 2R in the recovery phase, only few CD4+Foxp3+ cells within the gated CD3+ population were observed [fig_ref] Figure 5: -F [/fig_ref]. From score 1R during the recovery phase, a substantial increase in the number of CD4+Foxp3+ cells was observed until score 0R, when the maximum quantity of these cells was found [fig_ref] Figure 5: -F [/fig_ref]. The number of these CD4+Foxp3+ cells significantly decreased during the postrecovery phase from score 0R-28dpi to score 0R-40dpi [fig_ref] Figure 5: -F [/fig_ref]. This population of CD4+Foxp3+ cells represented less than 10% of CD3+ T-cells at score 1R, and approximately 15% at score 0R [fig_ref] Figure 5: -F [/fig_ref]. Remarkably, at score 0R-28dpi, the proportion of these cells remained unchanged, though the total number of CD4+Foxp3+ cells decreased considerably [fig_ref] Figure 5: -F [/fig_ref]. It was not until score 0R-40dpi when the proportion of this population underwent a substantial decline [fig_ref] Figure 5: -F [/fig_ref].
Double immunofluorescence analysis showed the accumulation of T-reg cells around blood vessels [fig_ref] Figure 5: -F [/fig_ref].
## Expression of th17 and t-reg related cytokines
The analysis of different cytokines such as IL17, IL21 and IL22 commonly related with the subpopulation of Th17 lymphocytes, as well as IL10, related with both Th17 and T-reg populations were studies using ELISA assays. Our results indicated that interestingly, there are no changes in IL10 and IL17 protein levels along CD4 T-helper cells. A) Dot-plots exemplifying how the analysis of CD3+CD4+ cells was performed. CD3+ T-cells were gated (circle in left dot-plot) and the percentage and number of CD3+CD4+ cells were analysed in this gated population (square in right dot-plot). Spleen samples were used to determine CD3+ and CD3+CD4+ gates. B) Representative dot-plots showing the dynamics of the CD3+CD4+ cell population (square) in both sham and EAE animals during the induction phase (score 1 and 2), peak (score 3), recovery phase (score 2R, 1R and 0R) and post-recovery phase (score 0R-32dpi). Note that in relation to shams, there is an important and persistent population of CD3+CD4+ cells in EAE animals. C and D) Histogrammes showing the values corresponding to CD3+CD4+ cell number (C) and relative percentage (D) along EAE evolution (ANOVA and Tukey's post-hoc test, *p#0.01 and # p#0.001 with respect to the previous score, $ p#0.05 with respect to score 1 and score 3). E) On the left side, representative dot-plot of CD3+CD4+ cells of EAE animals. In the middle, representative histogramme where populations of CD45RC-cells (activated/ effector lymphocytes) and CD45RC+ cells (naïve lymphocytes) were defined. Isotype control is represented in grey. On the right, histogramme showing the values of the percentages of CD45RC-(white columns) and CD45RC+ cells (black columns) in the different phases along EAE evolution (Student's-T test, * p#0.0001 when compare CD45RC+ vs CD45RC-in each score). Spinal cords of each animal were analysed separately (from three to seven animals per group were used). doi:10.1371/journal.pone.0027473.g002 the different phases of the disease [fig_ref] Figure 6: Cytokine profile along EAE [/fig_ref]. In contrast, a decrease in IL21 was found from score 2 of the inductive phase of EAE showing the lowest levels of expression of this phase at score 3 [fig_ref] Figure 6: Cytokine profile along EAE [/fig_ref]. A slight increase of this cytokine was found during the recovery phase although it was only at score 1R when the level of expression was similar to the observed in sham animals.
The analysis of IL22 indicated that levels of this cytokine increased in the induction phase showing a high level of expression at the peak of disability (score 3). Subsequently, a marked decrease of IL22 was found since score 2R in the recovery phase. Like in sham animals, levels of this protein were undetectable at score 0R [fig_ref] Figure 6: Cytokine profile along EAE [/fig_ref].
# Discussion
In this study a detailed analysis of the different subtypes of Thelper lymphocytes that infiltrated the CNS along the diverse phases of the acute EAE model in Lewis rat is performed. Our results show that during the induction and peak phases, the number of CD3+ cells increased in close relationship to disease severity. Nevertheless, during the recovery phase, although clinical signs decreased, the number of CD3+ lymphocytes remained very high without significant variations. Even during the post-recovery phase, some CD3+ cells were still found within the parenchyma of the spinal cord. Similarly we found that the proportions of CD4+ T-helper cells remained unchanged along the evolution of EAE, displaying a phenotype of activated/effector cells (CD45RC-cells) throughout all phases analysed. These findings are striking as traditionally T-lymphocytes, especially those with a CD4+ Thelper phenotype, are considered pathogenic cells in EAE. This assumption is partly due to the capacity of CD4+ lymphocytes to induce the disease when injected into susceptible animals [bib_ref] The rapid isolation of clonable antigen-specific T lymphocyte lines capable of mediating..., Ben-Nun [/bib_ref] and their presence at the peak of the disease in different models of EAE [bib_ref] Migratory activity and functional changes of green fluorescent effector cells before and..., Flugel [/bib_ref] [bib_ref] Chronological changes of CD4(+) and CD8(+) T cell subsets in the experimental..., Sonobe [/bib_ref] [bib_ref] Actively induced EAE in Lewis rats: characterization of spleen and spinal cord..., Rigolio [/bib_ref] , and partly due to studies reporting an improvement of clinical symptomatology of EAE after treatments that produced a decrease in lymphocyte infiltration [bib_ref] Beneficial effect of modified peptide inhibitor of alpha4 integrins on experimental allergic..., Van Der Laan [/bib_ref]. Although in this line, a decrease in the number of these cells could be also expected during the spontaneous recovery phase of this acute EAE model, noticeably, the findings obtained in this study show that the number of CD4+ T-lymphocytes remained elevated during this phase. These observations correlate with our previous findings showing that microglia/macrophages in this EAE model remained activated also during the recovery and post-recovery phases of this acute EAE model [bib_ref] Activated microglial cells acquire an immature dendritic cell phenotype and may terminate..., Almolda [/bib_ref] [bib_ref] CD4 microglial expression correlates with spontaneous clinical improvement in the acute Lewis..., Almolda [/bib_ref] , and point towards a cross-talk between microglia and lymphocyte populations along the evolution of the disease, not only during the induction and peak, but also during the recovery phase [bib_ref] Antigen presentation in EAE: role of microglia, macrophages and dendritic cells, Almolda [/bib_ref].
It is important to take into account that, CD4+ T-cells comprise a wide range of subpopulations which not only play pathogenic functions but also may play regulatory/suppressive roles [bib_ref] T helper cell effector fates-who, how and where?, Reinhardt [/bib_ref] [bib_ref] CD4 T cells: Balancing the coming and going of autoimmunemediated inflammation in..., Dittel [/bib_ref] [bib_ref] New complexities in helper T cell fate determination and the implications for..., Takatori [/bib_ref] [bib_ref] T cells in multiple sclerosis and experimental autoimmune encephalomyelitis, Fletcher [/bib_ref] that may explain why in our study, these CD4+T cells remained high also during all the recovery phase. In this sense, by using lineage-specific transcription factors, we carried out an accurate study of different subsets of CD4+ T-helper lymphocytes along EAE evolution, determining the specific temporal pattern of infiltration of Th1 (Tbet+), Th17 (RORc+) and T-reg (Foxp3+) cells. These specific transcription factors were shown to regulate genes encoding the signature cytokines of these different subpopulations of T-cells. Thus, Th1 cytokines such as IFN-c and TNF-a are regulated by Tbet [bib_ref] A novel transcription factor, T-bet, directs Th1 lineage commitment, Szabo [/bib_ref] , Th17 cytokines IL17, IL22 or IL21 by RORc and cytokines associated with T-reg cells such as IL10 and TGF-b were regulated by Foxp3 [bib_ref] Common themes emerge in the transcriptional control of T helper and developmental..., Miller [/bib_ref]. Our findings revealed that these different subsets of T-helper lymphocytes are present in the spinal cord in specific phases along the course of the disease. In our study it was clearly demonstrated that the number of Th1 lymphocytes (CD3+CD4+Tbet+ cells) and the expression of the pro-inflammatory cytokine IFNc, parallels the disease evolution, increasing progressively during the induction phase, reaching the maximum at the peak of the disease and decreasing thereafter during the recovery phase.
In parallel to a decrease in Th1 cells, during the recovery phase, a high increase in Th17 and T-reg cell populations was found. Th17 cells are commonly considered as a pathogenic population of lymphocytes, as they have been detected at the onset of EAE in mice [bib_ref] Infiltration of Th1 and Th17 cells and activation of microglia in the..., Murphy [/bib_ref] , infiltrated the CNS parenchyma after the initial influx of Th1 lymphocytes [bib_ref] T cells in multiple sclerosis and experimental autoimmune encephalomyelitis, Fletcher [/bib_ref] [bib_ref] Cutting edge: Th1 cells facilitate the entry of Th17 cells to the..., O'connor [/bib_ref] and, when injected into susceptible animals, they are able to induce EAE [bib_ref] Th1, Th17, and Th9 effector cells induce experimental autoimmune encephalomyelitis with different..., Jager [/bib_ref] [bib_ref] Cutting edge: Th1 cells facilitate the entry of Th17 cells to the..., O'connor [/bib_ref]. However in our study, the number of these cells greatly increased at the end of the recovery phase (from score 1R), peaking at score 0R. One plausible explanation for these distinct results could be the differences in the EAE model used. In contrast to the aforementioned studies describing Th17 cells as pathogenic lymphocytes, which used relapsing-remitting or chronic models induced in mice, in this study we analysed the acute EAE model induced in Lewis rats. This model has a unique feature: the complete and spontaneous recovery, that was absent in both the relapsing-remitting or chronic models in mice, and whose nature has not yet been established. Thus, it is reasonable to think that cells involved in the evolution of this acute model, including Th17 cells, can play different roles in the progressive or chronic models of EAE. Indeed, this is the first study describing the specific dynamics of different subsets of T cells in correlation to clinical symptomatology in an acute EAE model with spontaneous recovery. Therefore, the pathogenic role attributed to Th17 cells in mice, could be not applicable to this acute rat model. In fact, the exact role played by Th17 cells in autoimmunity has been under active discussion [bib_ref] Translational mini-review series on Th17 cells: are T helper 17 cells really..., Koenders [/bib_ref] , specially after the publication of some interesting studies providing data that bring in question the pathogenic potential of IL17, the signature cytokine of Th17 cells. In one hand IL17 treatment has been shown to induce an amelioration of experimental autoimmune uveitis in Lewis rats [bib_ref] Anti-inflammatory role of IL-17 in experimental autoimmune uveitis, Ke [/bib_ref] and, in the other hand, mice with a conditional deletion of IL17 develop EAE normally [bib_ref] IL-17A and IL-17F do not contribute vitally to autoimmune neuro-inflammation in mice, Haak [/bib_ref]. In agreement with this last statement, the analysis of IL17 protein levels in our study demonstrated that despite the high increase in Th17 lymphocytes during the recovery phase, protein levels of this cytokine remained unaltered along the different phases of EAE, suggesting that may be IL17 is not the key cytokine secreted by these lymphocytes in this acute model or that their function is not as relevant as though. In this way, it has been demonstrated that beyond to produce proinflammatory cytokines such as IL17, Th17 lymphocytes are also able to secrete anti-inflammatory cytokines such as IL10 [bib_ref] TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T..., Mcgeachy [/bib_ref] with a suggested beneficial effect specifically in acute EAE in Lewis rat [bib_ref] Suppression of acute and protracted-relapsing experimental allergic encephalomyelitis by nasal administration of..., Xiao [/bib_ref] , and others such as IL22 [bib_ref] New complexities in helper T cell fate determination and the implications for..., Takatori [/bib_ref] and IL21 whose role in EAE is still not well established. Addition of IL21 before the onset of EAE symptoms aggravates the disease [bib_ref] Differential effects of IL-21 during initiation and progression of autoimmunity against neuroantigen, Vollmer [/bib_ref] , but the blockade of IL21/ IL21R pathway induced an enhancement of EAE severity [bib_ref] IL-21 modulates CD4+ CD25+ regulatory T-cell homeostasis in experimental autoimmune encephalomyelitis, Piao [/bib_ref] [bib_ref] IL-21 receptor expression determines the temporal phases of experimental autoimmune encephalomyelitis, Liu [/bib_ref]. Furthermore, exposure of dendritic cells to IL21 induced an from score 1R, a great increase in the subpopulation of CD4+RORc+ cells was observed (ANOVA and Tukey's post-hoc test, *p#0.001 with respect to the previous score). immature phenotype of these cells [bib_ref] Interleukin 21: a cytokine/cytokine receptor system that has come of age, Leonard [/bib_ref] that cannot induce T-cell responses [bib_ref] Interleukin-21 inhibits dendritic cell activation and maturation, Brandt [/bib_ref] [bib_ref] Interleukin-21 inhibits dendritic cell-mediated T cell activation and induction of contact hypersensitivity..., Brandt [/bib_ref]. In this regard, it is interesting to highlight that we have previously reported that parenchymal microglial cells acquire an immature DC phenotype during the recovery phase, characterised by the expression of CD1 (an immature marker of dendritic cells) and MHCs but not co-stimulatory molecules [bib_ref] Activated microglial cells acquire an immature dendritic cell phenotype and may terminate..., Almolda [/bib_ref]. Thus, we can speculate that cytokines secreted by Th17 lymphocytes during the recovery phase can be involved in the induction of changes in microglial phenotype during this phase. Following this hypothesis, we analysed the pattern of expression of IL10, IL21 and IL22 along the different phases of acute EAE. Interestingly, in contrast to our initial hypothesis and as already mentioned for IL17, the pattern of expression of these three cytokines did not correlate with the presence of Th17, indicating together that, at list in this acute EAE model, Th17 cells are not producing IL17, IL10, IL21 or IL22. It is interesting to remark the marked decrease observed in the levels of IL22 in the initiation of the recovery phase. This striking result open a new and interesting way of study pointing to this cytokine as a putative key factor involved in the evolution of EAE. One possibility is that this cytokine may drive the inflammatory events occurring during the inductive and peak phases, and therefore a decrease in IL22 production may lead to stop inflammation and initiate the recovery. In another hand, also we can argue that certain levels of this cytokine are required to initiate the recovery phase, therefore when reach the correct threshold triggers the recovery mechanisms. Further studies in this sense are however necessary to completely understand the role played by this cytokine in the acute EAE model in Lewis rat. At this time, it is also interesting to point out that in general, there is a lack of information in the literature regarding the specific pattern of cytokine expression along the different phases of the different EAE models. Moreover, most studies linking lymphocytes with the secretion of various cytokines are based on the isolation of these cells and their subsequent activation in vitro, results that indicate the ability of these lymphocytes to produce these cytokines but, as already demonstrated in this study, does not necessarily implicate that they are doing the same function in vivo in the CNS. A better understanding of the real scenario occurring within the CNS parenchyma in terms of cytokine profile may be very helpful to understand the processes leading to the resolution and/or chronicity of this disease in the different animal models.
In addition to the Th17 lymphocyte population, during the recovery phase we also found a significant increase in the number of Foxp3+ T-reg cells. Accumulation of these T-regs in the CNS has already been reported during recovery in mice EAE models [bib_ref] Cutting Edge: Anti-CD25 monoclonal antibody injection results in the functional inactivation, not..., Kohm [/bib_ref] [bib_ref] Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells..., Mcgeachy [/bib_ref] [bib_ref] Myelin-specific regulatory T cells accumulate in the CNS but fail to control..., Korn [/bib_ref] , albeit to our knowledge this is the first study demonstrating accumulation of Foxp3+ cells within the spinal cord of acute EAE-induced rats. Some studies have demonstrated the beneficial role played by these cells in EAE pathogenesis in mice. As such, injection of Foxp3+ T-reg cells, derived from EAErecovered mice or in vitro-expanded, ameliorates EAE symptomatology when injected into MOG-induced mice [bib_ref] Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells..., Mcgeachy [/bib_ref] [bib_ref] Cutting edge: CD4+CD25+ regulatory T cells suppress antigen-specific autoreactive immune responses and..., Kohm [/bib_ref]. In the same way, a decrease or inactivation of Foxp3+ cell numbers in vivo by the use of anti-CD25 antibody treatment, makes these treated animals more vulnerable to EAE induction [bib_ref] IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+..., Zhang [/bib_ref]. Since we found the major proportion of T-reg cells during the recovery phase of EAE, we can speculate that Foxp3+ cells in this model may play also a role in the resolution of the immune response and can be one of the critical factors involved in the spontaneous recovery characteristic of the model.
Noticeably, during the post-recovery phase, although the number of both Th17 and T-reg cells declined, the proportion of these cell populations remained high, mostly at score 0R-28dpi. This long-time permanence suggests that these cells can still play an active role even after the animals have fully recovered and do not show any clinical symptom, may be being involved in the tolerance mechanism that, after EAE induction, renders these Lewis rats resistant to further immunization with the same antigen [bib_ref] Studies on the refractoriness to reinduction of experimental allergic encephalomyelitis in Lewis..., Macphee [/bib_ref].
Several studies have demonstrated that recovery from acute EAE is commonly associated with apoptotic elimination of pathogenic lymphocytes [bib_ref] Apoptosis in the nervous system in experimental allergic encephalomyelitis, Pender [/bib_ref] [bib_ref] Apoptosis of alpha beta T lymphocytes in the nervous system in experimental..., Pender [/bib_ref] [bib_ref] Apoptosis of T lymphocytes in experimental autoimmune encephalomyelitis. Evidence for programmed cell..., Schmied [/bib_ref] [bib_ref] Apoptosis of V beta 8.2+ T lymphocytes in the spinal cord during..., Mccombe [/bib_ref]. As our findings showed that when the recovery phase started the number of Th1 cells abruptly decrease, we hypothesise that this decrease may be due to an induced apoptotic elimination of these lymphocytes. In fact, we detected a high amount of apoptotic lymphocytes, especially during the peak of the disease, in near proximity to microglial cells [fig_ref] Figure 3: Dynamics of Th1 cell population [/fig_ref]. This hypothesis fits well with our previous findings [bib_ref] Activated microglial cells acquire an immature dendritic cell phenotype and may terminate..., Almolda [/bib_ref] showing that during the induction and peak phases, microglial cells displayed an immature dendritic-cell phenotype (MHC-class I and II+/CD1+/B7.1-/B7.2-) which may provide an anergic or apoptotic signal to the Th1-infiltrated lymphocytes [bib_ref] Antigen presentation in EAE: role of microglia, macrophages and dendritic cells, Almolda [/bib_ref] and is in agreement with a recently published study [bib_ref] Myeloid-Derived Suppressor Cells Limit the Inflammation by Promoting T Lymphocyte Apoptosis in..., Moline-Velazquez [/bib_ref] showing that myeloid-derived suppressor cells, an heterogeneous population of immature myeloid cells involved in the regulation of immune responses in tumour microenvironments [bib_ref] Myeloid-derived suppressor cells as regulators of the immune system, Gabrilovich [/bib_ref] , can induce the apoptosis of infiltrated T-cells also in a chronic mouse model of EAE. Nevertheless, we cannot exclude the possibility that the different T-cell populations observed and their dynamics are the result of a phenomenon of lymphocytic plasticity, bearing the interconversion between T-cell subtypes, as has recently been postulated by some authors [bib_ref] Plasticity of CD4+ T cell lineage differentiation, Zhou [/bib_ref] [bib_ref] Mechanisms underlying lineage commitment and plasticity of helper CD4+ T cells, O'shea [/bib_ref].
# Conclusion
In conclusion, we clearly demonstrate in this study that, although the number of T-lymphocytes inside the spinal cord parenchyma remains constant along the three main phases of EAE (induction, peak and recovery) the lymphocytic phenotype along the different phases undergo major changes. During the induction and peak phases major part of lymphocytes exhibited a phenotype of Th1 cells, whereas during the recovery and post-recovery phases Tlymphocytes mainly displayed a phenotype of Th17 or T-reg cells. Moreover, our results demonstrated a specific cytokine profile along the different phases of this acute EAE model, which differs from the described in chronic and relapsing-remitting models, characterized by no changes of IL10 and IL17 levels, decrease of IL21 on the peak phase, and high levels of IL22 during the induction and peak phases that markedly decrease during recovery.
These results suggest that the general view of lymphocyte infiltration in the CNS as a detrimental process should be reviewed. Our results demonstrated that it is not a question of presence or absence of T-cells, but the scenario is more complicated, being necessary to consider the specific subtype of infiltrated lymphocytes, their function and the specific interactions that these lymphocytes established with resident cells within the CNS. Further studies to analyse these interactions are necessary to understand the specific role played by these lymphocytes along EAE evolution. In this context, actions played by secreted cytokines in particular situations should also be more carefully reviewed. [fig_ref] Figure 1: Dynamics of CD3+ cells [/fig_ref] A-D) Cells immunolabelled with CD3 and CD4 were observed at the different scores of EAE evolution (arrows). Note that, in addition to these double positive cells, also few CD3+CD4-cells (arrowheads in B and D) and CD3-CD4+ cells (asteriscs in A, B and C) were also observed. Bar scale = 30 mm (TIF) [fig_ref] Figure 2: Figure 2 [/fig_ref] A-C) Photographs showing the CD4 (A) and Tbet (B) immunostatining at score 0R. Note that the few Tbet+ cells did not colocalize with CD4 (arrows in A-C). D-F) Colocalization between CD4 and ROR-c was found at score 0R in some cells located near blood vessels (arrows). Bar scale = 30 mm (TIF) [fig_ref] Figure 3: Dynamics of Th1 cell population [/fig_ref] Apoptotic lymphocytes. A-C) Double immunolabelling combining the microglial marker Iba1 and CD4, and counterstained with DAPI. Iba1+ microglial cells (green) were observed closely related to apoptotic cells that were identified as CD4+ lymphocytes (arrows in A, B and C). Bar scale = 20 mm (TIF)
## Supporting information
[fig] Figure 1: Dynamics of CD3+ cells. Immunohistochemistry for CD3 in the cervical spinal cord of sham animals (A) and in EAE animals during the induction (B-D), peak (E), recovery (F-H) and post-recovery phases (I). Arrows in B and I point to the few CD3+ cells observed in these scores. Note the progressive increase in the number of CD3+ cells in both the grey (GM) and white matter (WM) during the induction and peak phases and the maintenance of these cells during the recovery phase. Bar scale = 30 mm. Inserts in C-I represents panoramic views showing the localization of CD3+ cells in the spinal cord. Bar scale = 100 mm. J) Histogramme showing analysis of CD3+ cell density observed in the different clinical scores (ANOVA and Tukey's post-hoc test *p#0.05 with respect to sham and score 0.5; $ p#0.05 with respect to score 2, 3, 2R and 1R). doi:10.1371/journal.pone.0027473.g001 [/fig]
[fig] Figure 2: Figure 2. CD4 T-helper cells. A) Dot-plots exemplifying how the analysis of CD3+CD4+ cells was performed. CD3+ T-cells were gated (circle in left dot-plot) and the percentage and number of CD3+CD4+ cells were analysed in this gated population (square in right dot-plot). Spleen samples were used to determine CD3+ and CD3+CD4+ gates. B) Representative dot-plots showing the dynamics of the CD3+CD4+ cell population (square) in both sham and EAE animals during the induction phase (score 1 and 2), peak (score 3), recovery phase (score 2R, 1R and 0R) and post-recovery phase (score 0R-32dpi). Note that in relation to shams, there is an important and persistent population of CD3+CD4+ cells in EAE animals. C and D) Histogrammes showing the values corresponding to CD3+CD4+ cell number (C) and relative percentage (D) along EAE evolution (ANOVA and Tukey's post-hoc test, *p#0.01 and # p#0.001 with respect to the previous score, $ p#0.05 with respect to score 1 and score 3). E) On the left side, representative dot-plot of CD3+CD4+ cells of EAE animals. In the middle, representative histogramme where populations of CD45RC-cells (activated/ effector lymphocytes) and CD45RC+ cells (naïve lymphocytes) were defined. Isotype control is represented in grey. On the right, histogramme showing the values of the percentages of CD45RC-(white columns) and CD45RC+ cells (black columns) in the different phases along EAE evolution (Student's-T test, * p#0.0001 when compare CD45RC+ vs CD45RC-in each score). Spinal cords of each animal were analysed separately (from three to seven animals per group were used). doi:10.1371/journal.pone.0027473.g002 [/fig]
[fig] Figure 3: Dynamics of Th1 cell population. A) Representative dot-plots of the population of CD4+Tbet+ cells of EAE animals. Dot-plots were obtained by previously gating in the CD3+ T cell population. Different quadrants were defined by application of the appropriate isotype control. A minimum of three animals per group was pooled and three replicates per score were analyzed. B and C) Histogrammes showing, respectively, the values corresponding to the total number and the percentage of CD4+Tbet+ cell population along EAE. Note that CD4+Tbet+ lymphocytes are found during the induction and peak phases and markedly decreased at score 2R of the recovery phase (ANOVA and Tukey's post-hoc test, *p#0.05 with respect to the previous score). D) Histogramme showing the IFNc protein levels detected by ELISA assay at the representative scores during the induction, peak and recovery phases of EAE (ANOVA and Tukey's post-hoc test). E-G) Photographs of double immunostained sections showing a representative CD4+Tbet+ cell found around blood vessels (BV) (arrows point to double-immunolabelled cell). Bar scale = 20 mm. doi:10.1371/journal.pone.0027473.g003 [/fig]
[fig] Figure 4: Dynamics of Th17 cells. A) Representative dot-plots of CD4+RORc+ cells in the different phases along EAE evolution. Dot-plots were obtained after gating in the population of CD3+ T-cells. Different quadrants were defined by application of the appropriate isotype control. A minimum of three animals per group was pooled and three replicates per score were analyzed. The total number and percentage of CD4+RORc+ cells along the different phases of EAE are represented in histogrammes B and C, respectively. Note that during the recovery and the post-recovery phases, Th17 Lymphocytes in Acute EAE PLoS ONE | www.plosone.org [/fig]
[fig] Figure 5: -F) Photographs of double immunolabelled sections showing a representative CD4+RORc+ cell (arrow) observed around blood vessels (BV). Bar scale = 30 mm. doi:10.1371/journal.pone.0027473.g004 Dynamics of T-regulatory cells. A) Representative dot-plots of the CD4+Foxp3+ cell population along the different phases of EAE evolution. Dot-plots were obtained after gating in the population of CD3+ T-cells. Quadrants were defined by application of the appropriate isotype control. A minimum of three animals per group was pooled and three replicates per score were analyzed. B and C) Histogrammes showing, respectively, the total number and percentage value of CD4+Foxp3+ cells along the different phases of EAE. Note that although the number of CD4+Foxp3+ cells decreased at 0R-28dpi, their percentage remained high until score 0R-40dpi (ANOVA and Tukey's post-hoc test, *p#0.001 with respect to the previous score). D-F) Photographs of double immunolabelled sections showing a representative CD4+Foxp3+ cell (arrows) found around blood vessels (BV). Bar scale = 30 mm. doi:10.1371/journal.pone.0027473.g005 [/fig]
[fig] Figure 6: Cytokine profile along EAE. Histogrammes showing the IL10 (A), IL17 (B), IL21 (C) and IL22 (D) protein levels along the different phases of EAE. Note that while IL10 and IL17 cytokines remained unaltered along EAE evolution (A and B), IL21 levels decreased in score 2, 3 and 2R (C) (Student's-T test, **p#0.01 and *p#0.05 with respect to sham; $ p#0.05 with respect to score 1 and 2) and IL22 levels were higher during the inductive phase and markedly decreased during the recovery phase (D) (ANOVA and Tukey's post-hoc test, $ p#0.05 with respect to sham; *p#0.05 with respect to score 3). doi:10.1371/journal.pone.0027473.g006 [/fig]
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s2orc_pubmed_articles
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Vascular CT and MRI: a practical guide to imaging protocols
Non-invasive cross-sectional imaging techniques play a crucial role in the assessment of the varied manifestations of vascular disease. Vascular imaging encompasses a wide variety of pathology. Designing vascular imaging protocols can be challenging owing to the non-uniform velocity of blood in the aorta, differences in cardiac output between patients, and the effect of different disease states on blood flow. In this review, we provide the rationale behind-and a practical guide to-designing and implementing straightforward vascular computed tomography (CT) and magnetic resonance imaging (MRI) protocols. Teaching Points - There is a wide range of vascular pathologies requiring bespoke imaging protocols.- Variations in cardiac output and non-uniform blood velocity complicate vascular imaging.- Contrast media dose, injection rate and duration affect arterial enhancement in CTA.- Iterative CT reconstruction can improve image quality and reduce radiation dose.- MRA is of particular value when imaging small arteries and venous studies.
# Introduction
Non-invasive cross-sectional imaging plays a crucial role in the assessment of the varied manifestations of vascular disease, and in both the planning and follow-up of minimally invasive interventional techniques. Designing vascular imaging protocols can be challenging owing to the non-uniform velocity of blood in the aorta, differences in cardiac output between patients and the effect of different disease states on blood flow that cannot be predicted pre-scan. The complexity of the disease under investigation also influences vascular imaging protocols; for example, the need for a delayed phase to detect endoleaks in patients post endovascular aortic aneurysm repair (EVAR). In this review, we endeavour to provide the rationale behind and a practical guide to designing and implementing straightforward vascular computed tomography (CT) and magnetic resonance imaging (MRI) protocols [fig_ref] Table 1: Sample CT vascular protocols Peak kilovoltage [/fig_ref].
## Technical
## Computed tomography angiography (cta)
CT is a quick, non-invasive imaging modality with excellent spatial and temporal resolution. Modern CT scanners can provide sub-millimetre isotropic threedimensional (3D) datasets within a single breath-hold during the first past of intravenous (IV) iodinated contrast medium (CM). One of the minimum requirements for more advanced CTA applications, such as coronary CTA, is a 64-channel CT; for many of the other less challenging vascular CT protocols, such as abdominal aorta or visceral aneurysm assessment, a 16-channel CT is adequate. The continued evolution of CT technology is based in no small part on the demands that cardiovascular imaging places in terms of speed, temporal resolution and scan volume. To help cope with the demands of cardiovascular imaging, manufacturers have made significant improvements in z-axis volume coverage, detector and tube technology, with different emphasis depending on the vendor [bib_ref] Recent developments in the use of computed tomography scanners in coronary artery..., Aghayev [/bib_ref]. State of the art widearea detector CT scanners, with up to 320-detector rows, can provide up to 16-cm z-axis detector coverage in a single gantry rotation; this allows for large volume coverage in both helical and axial (step and shoot) acquisition modes [bib_ref] The Bpost-64^era of coronary CT angiography: understanding new technology from physical principles, Otero [/bib_ref]. Dual-source CT scanners provide the maximum temporal resolution available, as the temporal resolution is equal to a quarter of the gantry rotation time; this is as low as 66 milliseconds (ms) in the third-generation scanners. Maximising temporal resolution is advantageous when imaging structures prone to cardiac motion artefact, such as the aortic root [bib_ref] Computed tomographic assessment of coronary artery disease: state-of-the-art imaging techniques, Flohr [/bib_ref] , or when imaging patients prone to motion, such as trauma patients or poor breath-holders.
Obtaining satisfactory arterial enhancement is crucial in the assessment of intravascular pathology. One of the important scan parameters that can influence arterial enhancement is scan acquisition time. A short acquisition time is preferable once the scan begins (in most situations) to ensure uniform arterial opacification on the acquired images. For helical scans, the acquisition time is equal to the gantry rotation time multiplied by the number of gantry rotations required to cover the anatomical area. The number of gantry rotations is determined by the scan range divided by the product of the detector bank width and the pitch. Axial scanning acquires multiple volumes, and is used in particular when performing ECG-gated studies such as coronary and aortic CTA, to help reduce radiation dose [bib_ref] Artifacts at cardiac CT: physics and solutions, Kalisz [/bib_ref]. For axial acquisitions of volumes smaller than the width of detectors, the scan time is equal to the gantry rotation time. When axial scanning is used All phases are reconstructed with a slice thickness of 1 mm at an interval of 0.8 mm and sent to a 3D post-processing workstation AA abdominal aorta, TA thoracic aorta, C chest, A abdomen, P pelvis, I iodinated contrast, Arch aortic arch, MPA main pulmonary artery, CTA CT angiogram, CTV CT venogram, DIEP deep inferior epigastic perforators, SGAP superior gluteal artery perforators, ROI region of interest All protocols receive 100 ml of iodinated contrast, except for venogram protocols a , which receive 125 ml b Optional for volumes larger than the detector width, the total scan time is equal to the total number of volumes required to cover the desired anatomical area multiplied by the gantry rotation time, added to the sum of the interscan time intervals required for table repositioning.
Optimising IV contrast medium (CM) administration is important in obtaining strong arterial enhancement during CTA. The degree of enhancement of a system is proportionally related to the concentration of iodine within it. There is variation in the relationship between enhancement and iodine concentration in different CT scanners, but it is the range of approximately 25-30 Hounsfield units (HU) per milligram (mg) of iodine per millilitre (ml) at 120 peak kilovoltage (kV) [bib_ref] Intravenous contrast medium administration and scan timing at CT: considerations and approaches, Bae [/bib_ref]. The easily adjustable factors that determine arterial enhancement in CTA are the concentration of iodine in the CM used, the injection rate and the injection duration.
There is an almost linear relationship between enhancement and iodine concentration, which makes CM preparations with a high iodine concentration ideal (preferably 350-400 mg/ml) for CTA when the injection rate is fixed, resulting in a higher iodine delivery rate. The concept of iodine delivery rate (IDR, mg/s) is a method of standardising the rate of iodine delivery across CM with different iodine concentrations, and is calculated from the following formula: IDR = [CM
[formula] - - Axe + Cor - - - - - Cor Cor Cor - - - Cor - - - Axe Axe Axe + Cor - - - - - - Lower extremity MRA 5 ml 5 ml 0.1 Foot Calf Abdo/pelvis/ thigh - - - - - - - - Cor (3 stn) Sag Cor - - - Cor (3 Stn) - - - - - Axe + Cor - - - - - - Poplitea entrapment ** 0.1 Knee Axe + Cor - Cor Cor Cor - Axe + Cor - - [/formula]
T2 SSFSE T2-weighted single shot fast spin echo, GRE gradient echo, DIR double inversion recovery, TR-MRA time-resolved MRA, CE-MRA T1 spoiled gradient echo contrast-enhanced MRA, T1FS GRE T1-weighted 3D spoiled gradient echo sequence with a fat selective prepulse, HRT1FS GRE ± C high resolution T1-weighted 3D spoiled gradient echo sequence with a fat selective prepulse pre-and post-contrast, PC phase contrast C Gadolinium-based contrast agent, Abdo abdomen, Axe axial, Cor coronal, Sag sagittal, Sag Obl sagittal oblique, AV aortic valve, Stn station a Contrast dose is expressed in mmol/kg unless otherwise specified and is injected at a rate of 1.5 ml/s followed by a saline chaser b Protocol is performed with the feet in the neutral, dorsiflexed and plantarflexed positions iodine concentration (mg/ml)/1,000] × flow rate (ml/s) [bib_ref] CT of the abdomen: degree and quality of enhancement obtained with two..., Paparo [/bib_ref]. High iodine delivery rates are important in providing diagnostic image quality in CTA [bib_ref] Impact of iodine delivery rate with varying flow rates on image quality..., Hansmann [/bib_ref]. If CM preparations with lower concentrations of iodine are used, for example 300 mg/ml, the injection parameters can be adjusted to ensure a similar iodine delivery rate to that of a higher iodine concentration CM preparation [bib_ref] Intravascular enhancement with identical iodine delivery rate using different iodine contrast media..., Mihl [/bib_ref] ; for example, in a porcine model undergoing CT pulmonary angiography, CM with an iodine concentration of 300 mg/ml injected at a rate of 5 ml/s provided an identical IDR of 1.5 g/s to 370 mg/ml CM injected at a rate of 4.1 ml/s [bib_ref] Identification of the iodine concentration that yields the highest intravascular enhancement in..., Behrendt [/bib_ref]. CM with lower iodine concentrations have lower viscosity, with reduced injection pressures, which may potentially reduce extravasation risk [bib_ref] CT of the abdomen: degree and quality of enhancement obtained with two..., Paparo [/bib_ref] [bib_ref] Intravascular enhancement with identical iodine delivery rate using different iodine contrast media..., Mihl [/bib_ref] ; however, for a fixed scan duration, the higher injection rate required to keep the same iodine delivery rate would result in an increase in total CM volume required [bib_ref] CT of the abdomen: degree and quality of enhancement obtained with two..., Paparo [/bib_ref]. Experimental models suggest that an IDR of 1.5-2 g/s provides adequate arterial opacification (>200 HU) in CTA protocols, regardless of the concentration of CM used [bib_ref] Relationship between low tube voltage (70 kV) and the iodine delivery rate..., Lell [/bib_ref] [bib_ref] Coronary CT angiography using low concentrated contrast media injected with high flow..., Mihl [/bib_ref]. The strength of arterial enhancement (peak HU) is proportional to the injection rate, and the duration of enhancement to the injection length [bib_ref] Improved uniformity of aortic enhancement with customized contrast medium injection protocols at..., Fleischmann [/bib_ref]. Increasing the injection rate leads to a faster accumulation of contrast in the aorta, increasing peak aortic enhancement. For a fixed contrast volume, however, this reduces injection duration, and in turn reduces the available time window to acquire the CT within. With modern scanners, an injection rate of 4-5 ml/s is usually sufficient in providing excellent arterial opacification for most vascular studies; venous imaging does not require as high injection rates. The traditional approach to determine injection duration was to match it to scan duration time; however, with modern multi-detector, fast CT scanners, this may result in inadequate opacification due to a lower volume of CM being delivered. One approach for estimating injection duration is to set a minimum duration of 10 s, and to add on the estimated scan duration time, which is available from all vendors after the scan range has been chosen. CTA requires use of a power-injector to allow uniform high injection rate CM bolus delivery. Use of a saline flush should be routine to help push the tail of the CM bolus into the central blood volume, as without it, the bolus tail would remain unused in the peripheral veins [bib_ref] Improvement of parenchymal and vascular enhancement using saline flush and power injection..., Schoellnast [/bib_ref]. The saline chaser also helps reduce intravascular CM dispersion and reduces streak artefact from dense contrast in the brachiocephalic veins and superior vena cava, which is especially important in thoracic CTAs [bib_ref] Reduction of contrast material dose and artifacts by a saline flush using..., Haage [/bib_ref].
Obtaining high-quality arterial enhancement depends on various CT scanner, CM and patient-related factors. Even when CM and scanner use remain constant, patient factors such as body size, cardiac output and disease state can influence inter-individual variation in arterial enhancement. For example, large calibre and diseased vessels may take longer to opacify than normal. Reduction in cardiac output means that the CM bolus is slower to arrive and clear, resulting in delayed, but stronger peak arterial enhancement. These differences mean that the same scan timing delay cannot be used for everyone, and it needs to be tailored to the individual. Two methods commonly used to provide accurate CTA scan timing are the test bolus and bolus tracking methods. The test bolus method is based on injecting a small quantity (10-20 ml) of CM, then obtaining multiple low radiation dose images at a fixed time interval. By placing a region of interest (ROI) over the target vessel, a time-enhancement curve can be plotted to determine the time to peak enhancement. This can then be used to estimate the scan delay for the CTA. The bolus tracking technique involves acquiring a precontrast image at a reference level with placement of an ROI over a target vessel. After the CM injection is started, a l ow-dose m onitoring scan is performed at a predetermined level after a fixed time delay, usually 5 s, and thereafter every 1-3 s until the enhancement in the ROI reaches a specified level (typically 150 HU). The CTA then begins after a pre-specified adjustable delay to allow peak arterial enhancement (approximately 8 s); this delay must also take into account time for table repositioning. The two methods are comparable in terms of satisfactory CTA timing, with bolus-tracking frequently used due to its reduced examination time and ease of use [bib_ref] Intravenous contrast material administration at 16-detector row helical CT coronary angiography: test..., Cademartiri [/bib_ref]. The test bolus method is useful in patients with challenging anatomy, such as congenital heart disease patients post complex surgical repair [bib_ref] Computed tomography in congenital heart disease: how generic principles can be applied..., Loughborough [/bib_ref] [bib_ref] State-of-the-art CT imaging techniques for congenital heart disease, Goo [/bib_ref].
The role of CM as a causative agent in acute kidney injury (AKI) is currently a topic of debate, with recent studies suggesting the risk of contrast-induced nephropathy (CIN) may not be as high as previously thought [bib_ref] Risk of intravenous contrast material-mediated acute kidney injury: a propensity score-matched study..., Mcdonald [/bib_ref] [bib_ref] Time to revisit the problem of CIN? The low incidence of acute..., Hemmett [/bib_ref] [bib_ref] Intravenous contrast material-induced nephropathy: causal or coincident phenomenon?, Mcdonald [/bib_ref]. The use of CM in patients with normal renal function is safe, with no evidence of a significant drop in glomerular filtration rate (GFR) post CM administration [bib_ref] Glomerular filtration rate in evaluation of the effect of iodinated contrast media..., Becker [/bib_ref]. To mitigate against the possible risk of CIN, CM should only be given to patients with severe renal dysfunction (GFR <30 ml/min) or AKI on a case-by-case basis after a risk-benefit analysis [bib_ref] Canadian Association of Radiologists consensus guidelines for the prevention of contrast-induced nephropathy:..., Owen [/bib_ref]. There is no evidence available that reducing CM volume in patients with mild-to-moderate renal impairment (GFR 60-30 ml/ min) has an effect on development of CIN.
Many modern scanners automate peak kilovoltage (kV) selection based on the topogram. Reducing the kV, for example from 120 to 100 in patients with a body mass index (BMI) of <25, can help improve image quality, and may potentially reduce radiation dose [bib_ref] Effect of tube voltage (100 vs. 120 kVp) on radiation dose and..., Khan [/bib_ref]. Much of the radiation dose reduction achieved by reducing kV is offset in the presence of automatic exposure control (AEC), which increases the tube current to maintain a user-specified noise level [bib_ref] 320-row coronary computed tomography angiography (CCTA) with automatic exposure control (AEC): effect..., Di Cesare [/bib_ref] ; for example, to maintain diagnostic image quality, the tube current approximately doubles for a reduction from 120 to 100 kV [bib_ref] Computed tomography angiography: a review and technical update, Fleischmann [/bib_ref]. Small radiation dose reductions are still achievable with the use of model-based iterative reconstruction techniques [bib_ref] Low-contrast and low-radiation dose protocol in cardiac computed tomography: usefulness of low..., Iyama [/bib_ref] , but most of the benefit from reduced kV scanning in the presence of AEC in CTA comes from improved vessel contrast. Lower kV CTA has higher vessel HU values due to relatively increased attenuation of iodine as the kV nears its kedge of 33 kV, improving image signal to noise and contrast to noise ratios [bib_ref] 320-row coronary computed tomography angiography (CCTA) with automatic exposure control (AEC): effect..., Di Cesare [/bib_ref]. Lower injection rates should be used in reduced kV CTA (for example, 4 ml/s at 100 kV, 3 ml/s at 80 kV) to prevent the vessels appearing too high density, like bone; this has the added benefit of reducing the overall volume of CM required [bib_ref] Relationship between low tube voltage (70 kV) and the iodine delivery rate..., Lell [/bib_ref] [bib_ref] Low kV settings CT angiography (CTA) with low dose contrast medium volume..., Ippolito [/bib_ref] [bib_ref] Low-dose CT angiography of the abdominal aorta and reduced contrast medium volume:..., Nijhof [/bib_ref]. There is, however, a cost to low kV CTA: the higher tube current required to reduce noise requires a larger focal spot, reducing spatial resolution [bib_ref] Efficacy of Bfine^focal spot imaging in CT abdominal angiography, Oh [/bib_ref]. Blooming artefact from calcified atherosclerotic plaque or metal stents is also exaggerated at lower kV, which can be problematic in CTA interpretation [bib_ref] Artifacts at cardiac CT: physics and solutions, Kalisz [/bib_ref].
Dual-energy CT (DECT) is a state-of-the art technology that can improve image contrast in CTA by providing monoenergetic lower-energy reconstructions closer to the k-edge of iodine, with improved image contrast by a relatively increased contribution of the photoelectric effect [bib_ref] Value of monoenergetic low-kV dual energy CT datasets for improved image quality..., Apfaltrer [/bib_ref] [bib_ref] Evaluation of different keV-settings in dual-energy CT angiography of the aorta using..., Beeres [/bib_ref] [bib_ref] Advanced image-based virtual monoenergetic dual-energy CT angiography of the abdomen: optimization of..., Albrecht [/bib_ref] [bib_ref] Dual-energy computed tomography angiography of the lower extremity runoff: impact of noise-optimized..., Wichmann [/bib_ref]. This can be accomplished using dual-source dual-energy (DSDE) CT systems that employ two separate X-ray tubes situated 90°apart that can operate at two different voltages, single-source dual-energy (SSDE) CT systems with fast kV switching or with single-source CT systems with a dual-layer of detectors [bib_ref] Dual-energy spectral CT: various clinical vascular applications, Machida [/bib_ref]. Low-energy monoenergetic reconstructions in CTAs with suboptimal vessel opacification can improve iodine attenuation to levels similar to conventional polyenergetic images obtained with higher volumes of contrast, allowing 'rescuing' of a suboptimal CTA [bib_ref] Spectral detector CT for cardiovascular applications, Rajiah [/bib_ref] ; this also has the potential to reduce the iodine load required to obtain a diagnostic CTA, allowing the use of reduced concentration CM preparations and/or a lower volume [bib_ref] Iodine concentration and optimization in computed tomography angiography: current issues, Faggioni [/bib_ref]. Virtual monoenergetic datasets reconstructed at a high kV can help reduce blooming artefact, allowing improved assessment of vascular stent patency [bib_ref] Metal artifact reduction by dual energy computed tomography using monoenergetic extrapolation, Bamberg [/bib_ref] , and of heavily calcified vessels [bib_ref] Evaluation of image quality of coronary artery plaque with rapid kVp-switching dualenergy..., Ohta [/bib_ref]. DECT allows reconstruction of virtual non-contrast images from post-contrast CT acquisitions by excluding iodine-containing pixels, thus enhancing water attenuation [bib_ref] Dual-energy spectral CT: various clinical vascular applications, Machida [/bib_ref] ; this has the potential to reduce radiation dose in multiphase vascular CT protocols, by obviating the need to acquire a separate non-contrast CT [bib_ref] Dual-source dual-energy CT angiography with virtual non-enhanced images and iodine map for..., Sun [/bib_ref] [bib_ref] Virtual Monoenergetic imaging and iodine perfusion maps improve diagnostic accuracy of dual-energy..., Sommer [/bib_ref]. DECT can provide an assessment of organ perfusion using iodine map imaging. This is often presented using a colour look-up table, and can improve the detection of embolic disease by detecting areas of parenchymal hypoperfusion; this technique has been shown to improve the diagnostic accuracy of CTA in the detection of pulmonary emboli [bib_ref] CT pulmonary angiography of adult pulmonary vascular diseases: technical considerations and interpretive..., Taslakian [/bib_ref].
Cardiac motion artefact can be problematic when assessing the aortic root, and the use of ECG-gating in thoracic aorta CTA can help to address this. Motion artefact at the aortic root is dependent on several factors; chief among them, the gantry rotation time of the CT scanner and the patient's heart rate [bib_ref] Artifacts at cardiac CT: physics and solutions, Kalisz [/bib_ref]. ECG-gating can help to reduce the ill-effects of cardiac motion on the aortic root, but it does not eliminate it. Pre-scan beta-blockade is another step that can help to reduce motion artefact, and is commonly used in coronary CTA; in practice, the administration of beta-blockers to patients undergoing routine thoracic aorta CTA is not often necessary to obtain satisfactory image quality, particularly with the improved temporal resolution of modern CTs [bib_ref] Impact of betablockade premedication on image quality of ECG-gated thoracic aorta CT..., Entezari [/bib_ref]. The available ECG-gating techniques include prospective, retrospective or high pitch gating, with the optimum choice largely scanner dependent [bib_ref] Recent developments in the use of computed tomography scanners in coronary artery..., Aghayev [/bib_ref]. Scanners with large banks of detectors can cover the thorax quickly, making prospective gating ideal. Smaller detector-width scanners are more suited to retrospective gating with tube current modulation. High pitch gated acquisitions are suited to dual-source scanners. The use of ECGgating does increase radiation dose, primarily determined by the number of cardiac phases, rather than the type of ECGgating used. The optimum phase (percent of the R-R interval) for image acquisition is heart-rate dependent [bib_ref] Narrowing the phase window width in prospectively ECG-gated single heart beat 320-detector..., Steigner [/bib_ref]. When the heart rate is less than 75 beats per minute (bpm), a diastolic acquisition window of approximately 70-80% is preferred, with a systolic phase acquisition (30-40%) used in patients with higher heart rates.
There is no single best 'one size fits all' CTA protocol. Depending on the specific indication, it may be useful to obtain a non-contrast phase first; this can be of particular use when assessing calcified plaque, in postoperative patients, and in cases of suspected active haemorrhage. One approach described for a 64-channel CTA is to: (1) fix the scan duration to 10s for all CTAs; (2) adjust the pitch depending on the volume of coverage required; (3) fix the injection duration to 18 s; (4) operate a constant scan delay time of 8 s after CM arrival; (5) adjust the injection rate according to patient weight (5.0 ml/s for a 75-kg patient, ±0.5 ml/s for every 10 kg of body weight) [bib_ref] Computed tomography angiography: a review and technical update, Fleischmann [/bib_ref]. With this protocol, the long injection duration, combined with the extra 8 s delay post CM arrival, allows adequate time for arterial filling in nearly every patient. A delayed phase may also be helpful depending on the indication, with the timing measured from the end of the CM injection.
## Magnetic resonance angiography (mra)
MRA is a multiparametric imaging modality, with excellent contrast resolution. Contrast-enhanced MRA (CE-MRA) involves the administration of a gadolinium based contrast agent (GBCA), which shortens blood longitudinal relaxation (T1). A rapid 3D T1-weighted spoiled gradient echo (GRE) pulse sequence with a short repetition time (TR) and echo time (TE) is ideal for CE-MRA. This provides images with high signal-to-noise ratio (SNR), good spatial resolution and is free from flow-related artefacts [bib_ref] Contrast-enhanced peripheral MRA: technique and contrast agents, Nielsen [/bib_ref]. Subtraction techniques improve contrast resolution in CE-MRA. This reduces signal from background tissues by acquiring a mask image prior to GBCA injection, and subtracting it from the post-contrast imaging.
In general, an injection rate of 1.5 ml/s provides arterial imaging with high vessel to background contrast; this can be improved by increasing the injection rate, but similar to CTA, this reduces the available time window to acquire the scan for a fixed volume of contrast. Two methods are commonly used to appropriately time CE-MRA imaging. Similar to the method described for CTA, a test bolus method can be performed, administering 1-2 ml of GBCA and acquiring a series of rapid two-dimensional (2D) images of the vessel in question to determine the optimum time to start imaging post injection. In fluoroscopic triggering, the full bolus of contrast is administered and fluoroscopic-like images of the area of interest are obtained, and when the bolus is detected within the vessel, the technologist can trigger scan acquisition.
Two different acquisition modes are common in CE-MRA, single phase and time-resolved MRA. Single phase MRA captures vascular images at a single point in time. Timeresolved MRA consists of multiple acquisitions of an imaged volume over successive time points post GBCA administration. It is often known under vendor-specific acronyms such as TWIST (Siemens, Erlangen, Germany), TRICKS (General Electric, Chicago, IL, USA), 4D-TRAK (Philips, Best, Netherlands), TRAQ (Hitachi, Tokyo, Japan) and Freeze Frame (Toshiba, Otawara, Japan). This technique is particularly useful in displaying the passage of the contrast bolus through smaller vessels, such as the hands and feet. The core of time-resolved MRA is a 3D-spoiled GRE sequence employing k-space filling tricks to quicken image acquisition, such as non-Cartesian k-space filling, oversampling the centre of k-space (responsible for image contrast) and under sampling of the periphery (responsible for spatial resolution) [bib_ref] Recent advances in 3D time-resolved contrast-enhanced MR angiography, Riederer [/bib_ref]. These techniques, aligned with the use of parallel imaging, delivers ultra-fast imaging [bib_ref] 4D time-resolved MR angiography with keyhole (4D-TRAK): more than 60 times accelerated..., Willinek [/bib_ref].
Paramagnetic contrast agents shorten the T1 and T2 relaxation times of water protons in their immediate surroundings, creating a locally increased magnetic field strength. This change in the local magnetic field strength results in increased local field inhomogeneity, driving the shortening of T1 and T2 relaxation. The resultant increased signal intensity (SI) on T1weighted images provides the basis behind the use of contrast agents in MR. GBCAs are the most commonly used in MRA, and there are currently nine available GBCAs licensed by the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) in the USA for clinical use. GBCAs can be divided into two different groups, linear and macrocyclic, based on how the ligand chelates the gadolinium ion [bib_ref] Extracellular gadoliniumbased contrast media: an overview, Bellin [/bib_ref]. In the linear agents, the ligand wraps around the gadolinium (Gd 3+ ) ion, but does not completely enclose it. The macrocyclic agents consist of a chelator, which completely surrounds the Gd 3+ ion in a cage-like structure. The latter agents demonstrate greater stability in vivo than the linear agents, with little (if any) free Gd 3+ ion dissociation, reducing the risk of nephrogenic systemic fibrosis (NSF) [bib_ref] Safety of gadoterate meglumine (Gd-DOTA) as a contrast agent for magnetic resonance..., Ishiguchi [/bib_ref] [bib_ref] Safety of meglumine gadoterate (Gd-DOTA)-enhanced MRI compared to unenhanced MRI in patients..., Deray [/bib_ref]. The recent discovery of cerebral gadolinium deposition in patients with normal renal function is also of concern, although the clinical significance of this phenomenon is yet to be determined [bib_ref] Increasing signal intensity within the dentate nucleus and globus pallidus on unenhanced..., Stojanov [/bib_ref]. Linear GBCAs are thought to confer a higher risk of cerebral deposition, and their use is now discouraged by the EMA, although this guidance has not been reciprocated by the FDA. Cerebral gadolinium deposition is not only associated with linear GBCA use, however, and has been demonstrated in macrocyclic GBCAs in both animals [bib_ref] T1-weighted Hypersignal in the deep Cerebellar nuclei after repeated administrations of gadolinium-based..., Robert [/bib_ref] and humans, in particular the macrocyclic agent gadobutrol [bib_ref] Signal enhancement of the dentate nucleus at unenhanced MR imaging after very..., Bjørnerud [/bib_ref].
The majority of GBCAs in routine clinical use are extracellular fluid (ECF) agents. After injection, they initially distribute in the intravascular space, before rapidly diffusing across the vascular membranes into the interstitial space, eventually establishing an equilibrium between the intravascular and interstitial compartments after approximately 10 min. ECF GBCA agents that demonstrate weak plasma protein binding, such as gadobenate dimeglumine (Gd-BOPTA), help increase relaxivity compared to the other ECF GBCAs [bib_ref] 25 years of contrastenhanced MRI: developments, current challenges and future perspectives, Lohrke [/bib_ref]. Gadofosveset trisodium (Gd-DTPA-DO3A/MS-325) is currently the only intravascular GBCA licensed by the FDA. It was licensed by the European Medicines Agency for distribution in the European Union (EU) in 2005, but was voluntarily withdrawn from commercial use in the EU by the manufacturer in 2011. It is a linear ionic agent, which binds strongly to albumin, limiting its diffusion into the extravascular space [bib_ref] MS-325: albumin-targeted contrast agent for MR angiography, Lauffer [/bib_ref].
GBCAs with high relaxivity that remain within the bloodpool are the most attractive from an image quality point of view, however, safety considerations should be taken into account when choosing an agent. Gadobenate dimeglumine has the highest relaxivity of the ECF GBCAs, and gadofosveset trisodium is the only intravascular GBCA. These are both linear ionic agents, placing them in the intermediate risk category for NSF in susceptible patients, and at potentially higher risk for cerebral deposition. The current commercially available GBCA with the most favourable risk profile in terms of NSF and cerebral deposition is the macrocyclic ionic agent gadoterate meglumine. This agent has inferior protein binding and relaxivity compared with some of the other GBCAs, but this is offset by its safety profile. There have to-date been no reported cases of NSF with this agent, even in patients with severe renal dysfunction [bib_ref] Safety of gadoterate meglumine (Gd-DOTA) as a contrast agent for magnetic resonance..., Ishiguchi [/bib_ref] [bib_ref] Safety of meglumine gadoterate (Gd-DOTA)-enhanced MRI compared to unenhanced MRI in patients..., Deray [/bib_ref] , and it is also the only GBCA in which cerebral deposition has not been demonstrated [bib_ref] T1-weighted Hypersignal in the deep Cerebellar nuclei after repeated administrations of gadolinium-based..., Robert [/bib_ref].
For routine MRAs, a straightforward protocol is to begin with localisers of the anatomical area in question, followed by coronal and axial T2-weighted single-shot fast spin echo sequences, which allow a global anatomic assessment. This is then followed with 3D CE-MRA with two successive arterial phase acquisitions, providing anisotropic images, which allows reconstruction of the dataset on a 3D workstation. Finally, an axial T1-weighted 3D spoiled GRE sequence with a fat selective prepulse of the anatomical area can be acquired, allowing for an assessment for significant incidental findings. This basic MRA protocol can then be modified/added to according to the clinical question, as outlined in the anatomical site-specific sections below. It is our practice to administer weight-based GBCA dosing to all patients with GFR >30 ml/ min, followed by a saline chaser [fig_ref] Table 2: Sample MRI vascular protocols [/fig_ref]. There is no evidence available to support GBCA dose reduction in patients with mild to moderate renal impairment (GFR 60-30 ml/min). The risk of NSF remains in patients with severe renal dysfunction (GFR <30 ml/min); in these patients the decision to administer GBCA should be made on a case-by-case basis [bib_ref] Current status of nephrogenic systemic fibrosis, Besheli [/bib_ref].
For patients who cannot have gadolinium, such as those with severe renal impairment (GFR <30 ml/min), noncontrast imaging of the aorta and larger vessels can be performed. In these circumstances, a bright blood imaging, such as a balanced steady state free precession (SSFP) sequence can be used; this is a coherent gradient echo sequence that provides bright blood imaging without gadolinium. In the setting of acute aortic syndromes, this can detect the presence of an aortic dissection with high accuracy when compared with CE-MRA [bib_ref] Image quality and diagnostic accuracy of unenhanced SSFP MR angiography compared with..., Krishnam [/bib_ref] [bib_ref] Non-contrast-enhanced MR angiography of the thoracic aorta using cardiac and navigatorgated magnetization-prepared..., Amano [/bib_ref]. The major disadvantage of this sequence, however, is the presence of off-resonance artefact; this artefact is more pronounced at higher magnetic field strengths. To overcome this, where possible we perform non-contrast MRAs on 1.5 T rather than at 3.0 T.
## Cta and mra post-processing
In order to obtain the spatial resolution required, vascular imaging techniques tend to produce large datasets, which can be intimidating and difficult to negotiate. A minimum requirement of any post-processing software package is the ability to perform multiplanar reformats (MPR) of 3D CT or MRI datasets to create 2D images in coronal, sagittal, oblique or curved planes [bib_ref] Optimizing analysis, visualization, and navigation of large image data sets: one 5000-section..., Andriole [/bib_ref]. As the course of vessels tends to not follow along anatomical axial, coronal or sagittal planes; this hinders accurate measurement. Routine use of MPRs to perform measurements in a plane short axis to the centerline of a vessel is the most reliable and reproducible method of performing measurements [fig_ref] Figure 2: A 45-year-old man with Marfan's syndrome post aortic valve replacement with a... [/fig_ref] , ESM 1) [bib_ref] Proximal thoracic aortic diameter measurements at CT: repeatability and reproducibility according to..., Quint [/bib_ref]. The use of semi-automated tools to determine the vessel centerline improves measurement time [bib_ref] Reliability of semiautomatic centerline analysis versus manual aortic measurement techniques for TEVAR..., Rengier [/bib_ref].
Maximum intensity projection (MIP) reconstruction is an algorithm that selects and displays only the voxels with the highest HU (CT) or SI (MRI) of a selected slab in the imaged plane [bib_ref] Informatics in radiology (infoRAD): introduction to the language of three-dimensional imaging with..., Dalrymple [/bib_ref]. MIPs allow a global assessment of the imaged vasculature, and are useful in the rapid detection of vascular stenosis and occlusion. Readers must, however, be aware of limitations with this technique such as the overestimation of stenosis due to calcified plaque on CT, and findings should be confirmed with the thin-slice raw data [bib_ref] Volume rendering versus maximum intensity projection in CT angiography: what works best,..., Fishman [/bib_ref].
Segmented volume-rendered (VR) images can be created using most modern post-processing software packages. Volume rendering operates by assigning opacity values to image data on a scale from 0 to 100% along an artificial line of sight projection [bib_ref] Informatics in radiology (infoRAD): introduction to the language of three-dimensional imaging with..., Dalrymple [/bib_ref] [bib_ref] Volume rendering versus maximum intensity projection in CT angiography: what works best,..., Fishman [/bib_ref] [bib_ref] Three-dimensional volume-rendered CT angiography of the renal arteries and veins: normal anatomy,..., Urban [/bib_ref]. VR images are visually attractive, can be useful in pre-procedure planning and are an excellent means of displaying complex anatomy, especially to clinicians with varying knowledge of cross-sectional anatomy (Figs. 3, ESM 2 and 4, ESM 3) [bib_ref] Threedimensional aortic aneurysm model and endovascular repair: an educational tool for surgical..., Wilasrusmee [/bib_ref].
## Notes on specific protocols
## Aorta
In the emergency setting, imaging of the aorta is primarily focused on the assessment of the acute aortic syndrome (dissection, intramural haematoma or penetrating atherosclerotic ulcer) or for active haemorrhage, and CT is generally the preferred modality [fig_ref] Figure 3 a: Three-dimensional segmented volume rendered [/fig_ref]. Common elective indications for thoracic or abdominal aorta imaging includes the assessment and follow-up of aortic aneurysms, pre-and post-procedure assessment of endovascular aneurysm repair (EVAR) and in the setting of suspected aortitis. Aorta MRA can be used to assess thoracic aortic aneurysms, is the preferred modality in cases of suspected aortitis and aortic coarctation, and can be used to image acute aortic syndromes in selected cases, for example those with a severe iodinated contrast allergy [fig_ref] Figure 6 A: MR angiogram in a 45year-old woman who presented with acute onset of... [/fig_ref].
Acquiring a non-contrast CT prior to aorta CTA is useful to look for high-density intramural haematoma, which can be difficult to visualise after contrast administration. In the post-operative setting, it helps distinguish high-density surgical material such as felt pledgets routinely from a pseudoaneurysm [bib_ref] Ascending thoracic aorta: postoperative imaging evaluation, Prescott-Focht [/bib_ref] [bib_ref] MDCT angiography after open thoracic aortic surgery: pearls and pitfalls, Hoang [/bib_ref] [bib_ref] MDCT evaluation of postoperative aortic root pseudoaneurysms: imaging pearls and pitfalls, Chu [/bib_ref]. Using ECG-gating in thoracic aorta CTA helps to overcome the effect of cardiac motion on the aortic root, which can hide or mimic significant pathology, such as aortic dissection.
For patients undergoing consideration for EVAR, an additional non-contrast CT of the abdomen and pelvis can help delineate calcified plaque (CT AA pre-EVAR, [fig_ref] Table 1: Sample CT vascular protocols Peak kilovoltage [/fig_ref]. Following aneurysm endovascular stent graft repair (EVAR), [fig_ref] Figure 5: Axial [/fig_ref] -year-old man with Marfan's syndrome post aortic valve replacement with a fusiform ascending aortic aneurysm. Measurement of ascending thoracic aorta dimension (double arrows) in an axial plane (a) will yield erroneous values due to oblique orientation relative to the centerline of the aorta, as demonstrated on sagittal (red line, b) and coronal (red line, c) multiplanar reformats (MPRs). Threedimensional segmented volume rendered (VR) image of the thoracic aorta (d) demonstrates the site of measurement (line) [fig_ref] Table 1: Sample CT vascular protocols Peak kilovoltage [/fig_ref] can assess both the size of the excluded aneurysm and for the presence of an endoleak [fig_ref] Figure 8: Selected images from a CT angiogram in a 55-year-old man undergoing imaging... [/fig_ref] [bib_ref] Leakages after endovascular repair of aortic aneurysms: classification based on findings at..., Görich [/bib_ref] [bib_ref] Helical CT of aorta after endoluminal stent-graft therapy: value of biphasic acquisition, Golzarian [/bib_ref]. This identical protocol can also be used to assess for the presence of active bleeding, with the arterial and delayed phases demonstrating active extravasation, and can help triage patients for IR embolisation [bib_ref] Common and uncommon CT findings in rupture and impending rupture of abdominal..., Ahmed [/bib_ref] [bib_ref] CT findings of rupture, impending rupture, and contained rupture of abdominal aortic..., Schwartz [/bib_ref]. When performing multiphasic aortic CTA using a DECT system, reconstruction of a virtual non-contrast image may obviate the need to acquire a separate non-contrast CT [bib_ref] Dual-source dual-energy CT angiography with virtual non-enhanced images and iodine map for..., Sun [/bib_ref] [bib_ref] Virtual Monoenergetic imaging and iodine perfusion maps improve diagnostic accuracy of dual-energy..., Sommer [/bib_ref]. For patients undergoing post-EVAR CTA follow-up to detect endoleak, use of a split-bolus injection technique has the potential to reduce radiation dose, by and sagittal (c) arterial phase images from an ECG-gated CT thoracic aorta angiogram in a 62-year-old woman with chest pain demonstrates a type A dissection in the ascending thoracic aorta (a-c, arrow) with evidence of prior stent graft repair of the descending thoracic aorta (a-c, curved arrow). Threedimensional segmented VR image of the thoracic aorta (d) delineates the ascending aorta dissection flap (arrow) and descending thoracic aorta stent graft (curved arrow) allowing acquisition of a simultaneous arterial and delayed phase. The split-bolus technique involves injecting two sequential CM boluses separated by a time delay (approximately 35 s), and acquiring a simultaneous arterial and venous phase CTA after the second CM bolus. This can be further combined with DECT's capability to reconstruct virtual non-contrast images, reducing the protocol down to a single CT acquisition, with significant radiation dose saving [bib_ref] Endoleak detection using single-acquisition split-bolus dual-energy computer tomography (DECT), Javor [/bib_ref].
Performing a CTA prior to re-do cardiac surgery, with extra-cranial coverage to include the origin of the internal mammary arteries, allows identification of the location and can help surgeons alter surgical strategy, reducing the risk of intra-operative injury and improve outcomes [bib_ref] Does preoperative computed tomography reduce the risks associated with re-do cardiac surgery?, Khan [/bib_ref] [bib_ref] Preoperative computed tomography is associated with lower risk of perioperative stroke in..., Lapar [/bib_ref].
In thoracic aorta MRA (MR TA, [fig_ref] Table 2: Sample MRI vascular protocols [/fig_ref] , in addition to the routine MRA protocol, six to eight cine GRE short-axis slices can be acquired through the aortic valve, allowing characterisation of the aortic valve morphology (tricuspid/bicuspid/ unicuspid), valve leaflet opening and coaptation. Using ECG-gating for the sagittal oblique 'candy-cane' CE-MRA helps combat aortic root motion [fig_ref] Figure 6 A: MR angiogram in a 45year-old woman who presented with acute onset of... [/fig_ref] [bib_ref] Quantitative analysis of ECG-gated high-resolution contrast-enhanced MR angiography of the thoracic aorta, Groves [/bib_ref]. ECG gating is not required for imaging the abdominal aorta. For combined MRA of the thoracic and abdominal aorta, for example in suspected aortitis, we perform separate gadolinium injections for each. In patients in whom there is a clinical suspicion of large vessel vasculitis, or in cases of suspected aortic infection, acquisition of additional pre-and postcontrast high-resolution T1-weighted 3D spoiled GRE sequences with a fat selective prepulse allows an assessment for arterial mural enhancement (Aortitis, [fig_ref] Table 2: Sample MRI vascular protocols [/fig_ref] [bib_ref] Multimodality imaging of aortitis, Hartlage [/bib_ref] [bib_ref] Crosssectional imaging of aortic infections, Murphy [/bib_ref]. T1 double inversion recovery (DIR) ECG-gated, breath-held images provide excellent images of the aortic wall. DIR involves two successive 180°r adiofrequency (RF) inversion pulses to null signal from moving blood in the aortic lumen, preserving the magnetisation of stationary tissues, and is excellent in delineating the structural anatomy of the aorta, particularly the aortic wall. Turbulence or slow flow within the lumen will result in incomplete nulling of the lumen, which can be difficult to distinguish from thrombus. Each slice requires a single breath-hold, which adds a considerable time penalty to the study when imaging of the entire aorta is required; therefore we recommend reserving DIRs for equivocal cases.
In cases of suspected aortic coarctation, additional phase contrast (PC) imaging is helpful (Coarctation, [fig_ref] Table 2: Sample MRI vascular protocols [/fig_ref]. This technique allows for a functional assessment of coarctation including quantification of peak gradient and collateral flow. PC is based on the principle that the spin phase of moving protons will change in proportion to their velocity. A bipolar magnetic gradient is applied to a volume of tissue; stationary spins will experience no net phase shift, but moving spins will experience a phase shift proportional to their velocity [bib_ref] Cardiovascular flow measurement with phase-contrast MR imaging: basic facts and implementation, Lotz [/bib_ref]. By applying a flow-sensitive PC sequence orthogonal to the direction of blood flow, flow can be quantified as either velocity or volume per unit time. PC imaging is performed at the site of the coarctation, immediately distal to the coarctation, and in the distal descending thoracic aorta immediately above the diaphragm. The severity of coarctation can be assessed by measuring the volume of collateral flow present, or by estimating the pressure gradient across the stenosis. The flow volume of the collateral circulation is calculated by subtracting the total flow volume in the proximal descending thoracic aorta immediately distal to the site of the coarctation from the volume in the distal descending thoracic aorta [bib_ref] Velocity-encoded cine MR imaging in aortic coarctation: functional assessment of hemodynamic events, Hom [/bib_ref]. The percentage increase in flow volume from the collateral circulation increases linearly with the severity of stenosis at the coarctation site, and is the most useful measurement in the assessment of coarctation severity [bib_ref] Collateral flow in coarctation of the aorta with magnetic resonance velocity mapping:..., Holmqvist [/bib_ref]. The PC acquisition through the site of maximal stenosis can be used to measure the peak velocity (v) across the site of coarctation and the maximal pressure gradient across the coarctation (ΔP) can then be measured by use of the modified Bernoulli equation (ΔP = 4v 2 ) [bib_ref] Velocity-encoded cine MR imaging in aortic coarctation: functional assessment of hemodynamic events, Hom [/bib_ref]. In patients with previous coarctation repair, the percentage increase in flow from the proximal to distal descending thoracic aorta is the most reliable indicator of haemodynamically significant restenosis [bib_ref] MR findings of collateral circulation are more accurate measures of hemodynamic significance..., Araoz [/bib_ref]. Selected axial images (ad) from an arterial phase CT angiogram of the abdominal aorta in a 55-year-old man with hypotension and abdominal pain demonstrates a large retroperitoneal haematoma (curved arrow) and active extravasation from the infrarenal abdominal aorta (arrow) consistent with aortic rupture. A ruptured mycotic aneurysm was found in the operating room
## Mesenteric vessels
In the setting of suspected gastro-intestinal (GI) haemorrhage, endoscopy remains the initial test of choice, with CTA the imaging test of choice, reserved for those in whom endoscopy fails, or in the unstable patient with lower GI bleeding [bib_ref] The value of multidetector-row computed tomography for localization of obscure acute gastrointestinal..., Chang [/bib_ref]. The same protocol used in post-EVAR assessment is suitable (CT post-EVAR, [fig_ref] Table 1: Sample CT vascular protocols Peak kilovoltage [/fig_ref] , and oral contrast should not be administered, as this reduces the ability to detect intraluminal haemorrhage [bib_ref] Preexisting oral contrast from lanthanum carbonate: a confounding factor in CT mesenteric..., Bull [/bib_ref]. CTA has a high diagnostic accuracy in detecting and localising the source of acute GI bleeding, both upper and lower, with a sensitivity of approximately 85% [bib_ref] Accuracy of CT angiography in the diagnosis of acute gastrointestinal bleeding: systematic..., García-Blázquez [/bib_ref] , and it can detect bleeding rates of as little as 0.3 ml/ min [bib_ref] Detection of active colonic hemorrhage with use of helical CT: findings in..., Kuhle [/bib_ref]. Performing CTA prior to catheter angiography can increase the ability to successfully localise the bleeding source at catheter angiography [bib_ref] Arteriography for lower gastrointestinal hemorrhage: role of preceding abdominal computed Tomographic angiogram..., Jacovides [/bib_ref]. DECT has the potential to improve the detection of active GI haemorrhage, with iodine map reconstructions providing additional diagnostic information regarding the presence and source of active bleeding [bib_ref] Dual-source dual-energy CT angiography with virtual non-enhanced images and iodine map for..., Sun [/bib_ref].
In patients with suspected acute mesenteric ischaemia, CTA is the first-line imaging test (CT mesenteric angiogram, [fig_ref] Table 1: Sample CT vascular protocols Peak kilovoltage [/fig_ref] [bib_ref] ACR Appropriateness Criteria ® imaging of mesenteric ischemia, Oliva [/bib_ref]. Whilst occlusive arterial disease is causative in 85% of cases, 15% are secondary to mesenteric venous thrombosis, which makes the acquisition of delayed venous phases helpful [bib_ref] Radiology and mesenteric ischaemia, Mccarthy [/bib_ref]. CT can also detect the ancillary findings of mesenteric ischaemia, such as bowel wall hypoenhancement, bowel wall thickening, fat stranding, pneumatosis intestinalis, portal venous gas, intra-peritoneal free gas and ascites [bib_ref] Radiology and mesenteric ischaemia, Mccarthy [/bib_ref] [bib_ref] Radiological evaluation of bowel ischemia, Dhatt [/bib_ref].
MRI is useful in the evaluation of chronic mesenteric ischaemia, and when CT is contraindicated (MRA mesenteric, [fig_ref] Table 2: Sample MRI vascular protocols [/fig_ref]. Mesenteric MRA has a high sensitivity and specificity in evaluating the proximal coeliac and superior mesenteric arteries [bib_ref] Gadolinium-enhanced MR angiography of visceral arteries in patients with suspected chronic mesenteric..., Meaney [/bib_ref] , but is limited in its ability to detect distal mesenteric stenosis and occlusions compared to CTA [bib_ref] Comparison of noninvasive imaging modalities for stenosis grading in mesenteric arteries, Schaefer [/bib_ref].
## Renal vasculature
Dedicated renal vessel imaging is primarily performed in patients with suspected renovascular hypertension, and in the workup of potential donors and recipients in the live renal transplant program.
A standard renal CTA protocol [fig_ref] Table 1: Sample CT vascular protocols Peak kilovoltage [/fig_ref] consists of a single post-contrast bolus tracked arterial phase acquisition, with CTA performing well in the assessment of haemodynamically significant renal artery stenosis and fibromuscular dysplasia with high sensitivity and specificity [bib_ref] Imaging modalities for renal artery stenosis in suspected renovascular hypertension: prospective intraindividual..., Rountas [/bib_ref] [bib_ref] How to measure renal artery stenosis-a retrospective comparison of morphological measurement approaches..., Andersson [/bib_ref] [bib_ref] Fibromuscular dysplasia: what the radiologist should know: a pictorial review, Varennes [/bib_ref]. Anatomic coverage should include the adrenals superiorly and extend inferiorly to the aortic bifurcation to exclude the presence of a pheochromocytoma arising from the adrenal medulla or an extra-adrenal paraganglioma, the most common site for which [fig_ref] Figure 5: Axial [/fig_ref] -year-old man with oesophageal cancer was referred to CT following discovery of a mesenteric haematoma during exploratory laparoscopy. Axial non-contrast (a), arterial phase (b) and 70-s post contrast (c) images demonstrate an ill-defined mesenteric haematoma (arrow), a pancreaticoduodenal arcade pseudoaneurysm (curved arrow), with no active extravasation. The patient was taken to the interventional radiology suite and the pseudoaneurysm was occluded with coils; selected spot fluoroscopic image (d) demonstrates the occluded pancreaticoduodenal arcade filled with coils (curved arrow)
is the organ of Zuckerkandl, extra-adrenal chromaffin tissue near the origin of the inferior mesenteric artery [bib_ref] Extraadrenal pheochromocytoma in the organ of Zuckerkandl: diagnosis and treatment strategies, Kahraman [/bib_ref].
Laparoscopic living donor nephrectomy requires accurate pre-procedural vascular mapping (CT renal donor, [fig_ref] Table 1: Sample CT vascular protocols Peak kilovoltage [/fig_ref]. There is a large number of potential normal variants in renal arterial vascular anatomy. CT provides a better depiction of small renal arteries than MRI, and is the generally preferred modality [bib_ref] Pre-operative assessment of living renal transplant donors with state-of-the-art imaging modalities: computed..., Engelken [/bib_ref]. CTA has a very high accuracy in identifying accessory renal arteries and pre-hilar arterial branching, which are important variants that the surgeon needs to be aware of [fig_ref] Figure 2: A 45-year-old man with Marfan's syndrome post aortic valve replacement with a... [/fig_ref] [bib_ref] Three-dimensional volume-rendered CT angiography of the renal arteries and veins: normal anatomy,..., Urban [/bib_ref] [bib_ref] CT angiography of potential renal transplant donors, Pozniak [/bib_ref]. The non-contrast CT phase identifies renal calculi, with the arterial and nephrographic phases providing an accurate assessment of renal size, vascular anatomy and for any renal parenchymal lesions such as incidental renal tumours, an exclusion criterion for donors [bib_ref] Multidetector row CT evaluation of living renal donors prior to laparoscopic nephrectomy, Kawamoto [/bib_ref]. The urographic phase is used to evaluate for any anomalies of the renal collecting systems or ureters.
Although it does not offer the spatial resolution of CT, the lack of radiation makes renal artery CE-MRA [fig_ref] Table 2: Sample MRI vascular protocols [/fig_ref] an attractive modality, especially in younger patients. In a prospective comparison of 58 patients with suspected renovascular hypertension, Rountas et al. [bib_ref] Imaging modalities for renal artery stenosis in suspected renovascular hypertension: prospective intraindividual..., Rountas [/bib_ref] found that CE-MRA has a slightly lower sensitivity than CTA for the detection of renal artery stenosis or fibromuscular dysplasia, 90% versus 94% respectively; this is likely due to the lower spatial resolution of MRI compared with CT.
For patients who cannot have gadolinium, a non-contrast renal MRA be performed with a respiratory-gated inflow An 85-year-old woman developed severe abdominal pain two days post percutaneous aortic valve replacement and was referred for a CT mesenteric angiogram. Axial (a) and coronal (b) images from an arterial phase CT mesenteric angiogram demonstrate hypoenhancement of distal ileal loops (straight arrow) compared with adjacent proximal ileal and jejunal loops with normal mural enhancement (curved arrow). Axial (c) and coronal oblique images from the same study demonstrate focal occlusion of the mid-superior mesenteric artery with calcified plaque (arrow). Coronal oblique maximum intensity projection (MIP) reformat (d) is useful in demonstrating the point of SMA obstruction (arrow). Exploratory laparoscopy revealed extensive small bowel ischaemia, and the patient unfortunately expired [fig_ref] Figure 2: A 45-year-old man with Marfan's syndrome post aortic valve replacement with a... [/fig_ref] Three dimensional segmented volume rendered image of the kidneys and their arterial supply in a renal donor volunteer, segmented from an arterial phase renal artery angiogram CT, demonstrates bilateral accessory renal arteries supplying the lower renal poles [fig_ref] Figure 3 a: Three-dimensional segmented volume rendered [/fig_ref] -year-old man with bilateral leg swelling underwent an MR venogram of the abdomen and pelvis. Coronal image from a T2-weighted sequence (a) demonstrated an expanded suprarenal IVC with heterogeneous T2 high signal material, likely thrombus (arrow). Coronal (b) and axial (c) images from a venous phase T1weighted fat-saturated postgadolinium sequence demonstrates heterogeneous enhancement of the intraluminal IVC material, concerning for tumour thrombus. A coronal image from a T1-weighted fat-saturated postgadolinium sequence of the abdomen (d) demonstrates a horseshoe kidney with an exophytic mass arising from the right lower moiety (curved arrow), which was subsequently confirmed as a papillary renal cell carcinoma on biopsy [bib_ref] Free-breathing renal MR angiography with steady-state free-precession (SSFP) and slabselective spin inversion:..., Katoh [/bib_ref]. Firstly, the volume of interest is saturated with a 180°radiofrequency pulse, inverting signal from background tissue and venous blood. Fresh, unsaturated arterial blood then flows into the slab, and a rapid 3D SSFP sequence is acquired after an appropriate inversion time to null signal from background tissue [bib_ref] Non-contrast-enhanced MR imaging of renal artery stenosis at 1.5 tesla, Wilson [/bib_ref] [bib_ref] Non-contrast enhanced MR angiography: established techniques, Miyazaki [/bib_ref]. This is a useful technique in patients suspected of having renovascular hypertension, with good agreement with CE-MRA and CTA for the presence of renal artery stenosis or fibromuscular dysplasia [bib_ref] Non-contrast renal artery MRA using an inflow inversion recovery steady state free..., Glockner [/bib_ref] [bib_ref] Non-enhanced MR angiography of renal arteries: comparison with contrast-enhanced MR angiography, Angeretti [/bib_ref] [bib_ref] High-resolution 3D unenhanced ECG-gated respiratory-navigated MR angiography of the renal arteries: comparison..., Mohrs [/bib_ref] [bib_ref] An international multicenter comparison of time-SLIP unenhanced MR angiography and contrast-enhanced CT..., Albert [/bib_ref].
## Thoracic venous imaging
The most common reason for dedicated imaging of the thoracic venous circulation is in the evaluation of suspected superior vena cava (SVC) obstruction or thrombosis. Contrastenhanced MR venography (CE-MRV) allows better differentiation of intraluminal thrombus from contrast mixing than CT venography (CTV), is equivalent to conventional venography in the assessment of central venous obstruction and is the noninvasive imaging modality of choice (CT SVC, [fig_ref] Table 1: Sample CT vascular protocols Peak kilovoltage [/fig_ref] [bib_ref] Three-dimensional gadolinium-enhanced MR venographic evaluation of patency of central veins in the..., Shinde [/bib_ref] [bib_ref] 3D gadolinium-enhanced MRI venography: evaluation of central chest veins and impact on..., Oxtoby [/bib_ref]. In patients who have difficulty with breath-holding, the post-contrast imaging can be performed free-breathing with respiratory gated navigator MRA [bib_ref] Comprehensive imaging review of the superior vena cava, Sonavane [/bib_ref].
## Abdominal and pelvic venous imaging
CT/MR venography of the abdomen and pelvis is commonly performed to assess for extension of lower limb deep vein thromboses (DVTs) or for a compressive venous syndrome, such as May-Thurner syndrome (obstruction of the left common iliac vein by the crossing right common iliac artery) [bib_ref] Multidetector CT of vascular compression syndromes in the abdomen and pelvis, Lamba [/bib_ref]. MRV is preferable to CTV due to the presence of contrast mixing artefact with the latter, and imaging of the thighs can be included [fig_ref] Figure 3 a: Three-dimensional segmented volume rendered [/fig_ref]. CTV is preferred when assessing potential inferior vena cava (IVC) filter complications, such as malposition, migration, tilting, caval thrombosis or perforation [bib_ref] Inferior vena cava filter-associated abnormalities: MDCT findings, Rao [/bib_ref].
## Lower-limb angiography
CTA and MRA both provide a highly accurate vascular map, and have largely replaced catheter angiography in the diagnosis of lower limb ischaemia, acute and chronic [bib_ref] Computed tomography angiography and magnetic resonance angiography imaging in critical limb ischemia:..., Iglesias [/bib_ref]. A meta-analysis of the diagnostic performance of CTA and CE-MRA to detect haemodynamically significant arterial stenosis or occlusion demonstrated equal diagnostic accuracy of both techniques [bib_ref] Diagnostic performance of computed tomography angiography and contrastenhanced magnetic resonance angiography in..., Jens [/bib_ref].
Acquiring a non-contrast CT phase allows for the assessment of calcified plaque, whilst the bolus-tracked CTA is This study demonstrates occlusion of the left dorsalis pedis in the left mid-foot (c, arrow), the likely cause of the patient symptoms highly accurate in depicting arterial occlusions and haemodynamically significant stenosis (CTA lower limbs, [fig_ref] Table 1: Sample CT vascular protocols Peak kilovoltage [/fig_ref] [bib_ref] Diagnostic performance of computed tomography angiography and contrastenhanced magnetic resonance angiography in..., Jens [/bib_ref] [bib_ref] Diagnostic performance of computed tomography angiography in peripheral arterial disease: a systematic..., Met [/bib_ref] [bib_ref] Peripheral arterial occlusive disease: diagnostic performance and effect on therapeutic management of..., Napoli [/bib_ref]. This may be omitted in DECT systems with the capability to perform virtual non-contrast reconstructions [bib_ref] Dual-energy computed tomography angiography of the lower extremity runoff: impact of noise-optimized..., Wichmann [/bib_ref]. The smaller below-the-knee runoff vessels are more challenging to interrogate due to their small size and difficulty opacifying adequately. Due to the speed of modern CT scanners, the CT often outruns the contrast bolus, affecting opacification of the below-the-knee arteries; acquiring an additional CTA phase with coverage from the knees through to the toes immediately after the bolus-tracked CTA can help assess these hard to image vessels. Providing separate reconstructions of each leg with a small field of view is also useful in improve spatial resolution.
CE-MRA provides an accurate luminal map of the arterial tree, with equal performance to CTA in the detection of arterial stenosis and occlusion (MRA lower limbs, [fig_ref] Table 2: Sample MRI vascular protocols [/fig_ref] [bib_ref] Diagnostic performance of computed tomography angiography and contrastenhanced magnetic resonance angiography in..., Jens [/bib_ref]. CE-MRA is often preferable to CTA, especially in patients with distal disease, and in those with heavily calcified vessels, which can hinder stenosis assessment on CTA [bib_ref] Computed tomography angiography and magnetic resonance angiography imaging in critical limb ischemia:..., Iglesias [/bib_ref]. MRA is limited in the assessment of stent patency due to local susceptibility artefact, where CTA is the preferred technique.
Time-resolved CE-MRA is an excellent method of assessing small vessel patency, and is particularly useful when imaging the below-the-knee vessels, where it is superior to standard CE-MRA [bib_ref] Peripheral arteries in diabetic patients: standard bolus-chase and timeresolved MR angiography, Andreisek [/bib_ref] [bib_ref] Time-resolved MR angiography of the calf arteries using a phased array cardiac..., Eshed [/bib_ref]. Both calves can be imaged simultaneously in the coronal plane following gadolinium injection, and when used, should be performed first in a lower limb CE-MRA. After this, a standard three-station (abdomen and pelvis, thighs, calves) bolus CE-MRA can be performed with a separate gadolinium injection. If foot arterial imaging is required, a separate time-resolved foot MRA is performed at the start of the examination before the calf MRA, using a boot coil with a sagittal acquisition separate gadolinium injection for the calves and for each foot [fig_ref] Figure 5: Axial [/fig_ref].
For patients with suspected popliteal artery entrapment syndrome, MRA is the preferred technique (MRA popliteal entrapment, [fig_ref] Table 2: Sample MRI vascular protocols [/fig_ref]. Typically, these are young patients, and definitive diagnosis requires imaging in multiple degrees of plantar and dorsiflexion. MRA allows precise analysis of local muscle anatomy, making it an ideal modality for diagnosis [bib_ref] Popliteal artery entrapment syndrome, Tercan [/bib_ref] [bib_ref] Clinical evaluation and MR imaging features of popliteal artery entrapment and cystic..., Elias [/bib_ref] [bib_ref] CT angiography and MRI in patients with popliteal artery entrapment syndrome, Hai [/bib_ref]. Time-resolved CE-MRA with the toes in the neutral position, in plantar flexion and then in dorsiflexion, with separate gadolinium injections for each.
## Upper limb angiography
Upper limb CTA and MRA is usually limited to one upper limb, and IV cannulation should be performed in the contralateral arm to avoid injection-related artefact. CTA is preferred in cases of suspected acute upper limb ischaemia due to its relatively quick time of acquisition, with MRA preferred in the chronic setting [bib_ref] Upper limb ischemia: clinical experiences of acute and chronic upper limb ischemia..., Bae [/bib_ref]. Where possible, upper limb CTAs should be acquired with the arm of interest raised above the head, with imaging extending from the aortic arch through the fingers (Upper extremity CTA, [fig_ref] Table 1: Sample CT vascular protocols Peak kilovoltage [/fig_ref]. A delayed CTA [fig_ref] Figure 5: Axial [/fig_ref] -year-old man with a previous history of left radial artery thrombosis post coeliac artery stenting with persistent arm claudication. Time resolved MRA (a-d) of the left forearm demonstrates opacification of the left brachial artery, and left ulnar artery (arrows) with occlusion of the proximal left radial artery (curved arrows) (b, c), followed by venous filling (d) phase can help in assessment of the small arteries of the forearm, which may not be adequately opacified on the bolustracked CTA due to the scanner out-running the CM bolus. In upper extremity MRA [fig_ref] Table 2: Sample MRI vascular protocols [/fig_ref] , time-resolved MRA is preferred for the forearm and hand vessels, with a standard CE-MRA for the proximal vessels [fig_ref] Figure 6 A: MR angiogram in a 45year-old woman who presented with acute onset of... [/fig_ref].
In cases of suspected thoracic outlet syndrome (TOS), MRA [fig_ref] Table 2: Sample MRI vascular protocols [/fig_ref] with positional manoeuvres is the preferred imaging modality for diagnosis, pre-operative planning and post-surgical follow-up [bib_ref] Vascular thoracic outlet syndrome: protocol design and diagnostic value of contrastenhanced 3D..., Ersoy [/bib_ref] [bib_ref] State-of-the-art magnetic resonance imaging in vascular thoracic outlet syndrome, Aghayev [/bib_ref]. CE-MRA with coverage including the bilateral subclavian and axillary vessels is performed with the arms abducted approximately 150-160°, and repeated with the arms adducted, with separate contrast injections at each position.
## Pulmonary arteries
Acute pulmonary embolism (PE) is the third most common acute cardiovascular disorder after myocardial infarction and stroke [bib_ref] Pulmonary embolism and deep vein thrombosis, Goldhaber [/bib_ref]. CT pulmonary angiography (CTPA) is the noninvasive reference standard for PE diagnosis and risk stratification, identifying adverse prognostic indicators such as right ventricular dilatation, interventricular septal bowing and a high embolus burden [bib_ref] Computed tomography of acute pulmonary embolism: state-of-the-art, Zhang [/bib_ref]. DECT, in particular iodine map reconstructions, have been shown to improve the accuracy of pulmonary emboli detection on CT, by demonstrating areas hypoperfused lung [bib_ref] CT pulmonary angiography of adult pulmonary vascular diseases: technical considerations and interpretive..., Taslakian [/bib_ref]. MR pulmonary angiography (MRPA, [fig_ref] Table 2: Sample MRI vascular protocols [/fig_ref] is a suitable alternative modality to diagnose PE in patients who cannot undergo CT, and is useful in the follow-up of pulmonary artery aneurysms [fig_ref] Figure 17 A: pulmonary artery MRA in a 55-year-old woman with pulmonary hypertension [/fig_ref]. In our experience, a timeresolved CE-MRA of the chest performed in the coronal plane with approximately nine phases yields diagnostic pulmonary arterial imaging in the majority of cases.
## Breast reconstruction flap planning
The deep inferior epigastric perforator (DIEP) flap is the most common flap used in breast reconstruction. Raising a DIEP [fig_ref] Figure 5: Axial [/fig_ref] -year-old woman with breast cancer undergoes a CT deep inferior epigastric artery perforator (DIEP) protocol prior to breast reconstruction. Coronal oblique 3D segmented VR image from the CT displays the abdominal wall musculature and superficial arterial supply. The location of the largest DIEP relative to the umbilicus is annotated with craniocaudal and mediolateral distance measurements to help the surgeon locate the vessel during surgery flap requires meticulous dissection of the DIEP vessels; however, there is significant heterogeneity in their branching pattern and location [bib_ref] Optimising the preoperative planning of deep inferior epigastric perforator flaps for breast..., Santiago [/bib_ref]. CTA is the current 'gold standard' for pre-operative vascular mapping, reducing operative time and postoperative complications [bib_ref] Does the preoperative imaging of perforators with CT angiography improve operative outcomes..., Rozen [/bib_ref] [bib_ref] Abdominal wall CT angiography: a detailed account of a newly established preoperative..., Phillips [/bib_ref] [bib_ref] CTguided deep inferior epigastric perforator (DIEP) flap localization-better for the patient, the..., Malhotra [/bib_ref]. A standard CT DIEP protocol is a single bolus-tracked CTA of the abdomen and pelvis, acquired in the caudo-cranial direction to mirror the direction of blood flow in the DIEP arteries (CT DIEP, [fig_ref] Table 1: Sample CT vascular protocols Peak kilovoltage [/fig_ref]. A segmented batch of 3D VR images displaying the number and site of DIEPs relative to the rectus sheath and overlying skin, providing the distance of the largest perforator from the umbilicus, is of particular use in surgical planning [fig_ref] Figure 8: Selected images from a CT angiogram in a 55-year-old man undergoing imaging... [/fig_ref]. CTA is also used in patients undergoing pre-operative assessment for a superior gluteal artery perforator (SGAP) flap [fig_ref] Table 1: Sample CT vascular protocols Peak kilovoltage [/fig_ref] ; in this protocol, the patient is scanned prone, with anatomical coverage from the umbilicus to the mid-thighs.
# Conclusions
A basic understanding of the technical, physiological and pathological challenges posed by vascular imaging allows the creation of bespoke, safe imaging protocols that can help improve diagnosis and impact outcomes. The challenges posed by cardiovascular imaging will continue to drive technological improvements in scanner technology, and it is important that radiologists continue to improve and streamline vascular imaging practices.
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[fig] Figure 2: A 45-year-old man with Marfan's syndrome post aortic valve replacement with a fusiform ascending aortic aneurysm. Measurement of the ascending thoracic aorta dimension (double arrows) in a plane double oblique to the vessel centerline (a) with sagittal (b) and coronal (c) MPRs demonstrating the plane of measurement (red lines). Threedimensional segmented VR image of the thoracic aorta (d) demonstrates the site of measurement (line) a CTA with additional 70 s delayed phase imaging [/fig]
[fig] Figure 3 a: Three-dimensional segmented volume rendered (VR) image of the thoracic aorta with b standard sites of measurement. a Sinuses of Valsalva, b sinotubular junction, c ascending thoracic aorta, d aortic arch, between origin of left common carotid and left subclavian arteries, e descending thoracic aorta, f aortic hiatusFig. 4 a Three-dimensional segmented VR image of the abdominal aorta with b standard sites of measurement. a Proximal abdominal aorta, b juxtarenal abdominal aorta, c infrarenal abdominal aorta, d right common iliac artery, e left common iliac artery [/fig]
[fig] Figure 5: Axial [/fig]
[fig] Figure 6 A: MR angiogram in a 45year-old woman who presented with acute onset of tearing chest pain. Sagittal oblique image from a ECG gated T1-weighted postgadolinium 3D acquisition (a) demonstrates a dissection flap (arrow) arising in the aortic arch distal to the origin of left subclavian artery consistent with a type B aortic dissection, with the dissection flap extending into the abdominal aorta. Corresponding 3D segmented volume rendered image of the thoracic aorta (b) from the thoracic MRA demonstrates the dissection flap (arrow). Coronal abdominal T1-weighted post gadolinium MRA image (c) demonstrates the distal extent of the dissection flap (arrow) into the common iliac arteries bilaterally, with a 3D segmented volume rendered image of the abdominal aorta (d) demonstrating the dissection flap in the abdominal aorta (arrow) [/fig]
[fig] Figure 8: Selected images from a CT angiogram in a 55-year-old man undergoing imaging surveillance post abdominal aortic aneurysm endovascular stent graft repair (EVAR). Axial non-contrast (a), arterial phase (b) and 70 s delayed phase (c) images demonstrate iodinated contrast material within the excluded aneurysm sac (arrow) consistent with an endoleak. Sagittal oblique MPR of the right common iliac artery (d) demonstrates the endoleak arising from the distal insertion point of the right iliac limb of the EVAR consistent with a type 1b endoleak [/fig]
[fig] Figure 14 A: 35-year-old woman underwent an abdominal MR venogram 5 days post dilatation and curettage for an intrauterine fetal death at 25 weeks. Coronal images from a post-contrast venous phase fat saturated T1 sequence of the abdomen demonstrates the post-partum uterus (a, curved arrow), with marked dilatation of the right (b, arrow) and left (c, arrow) gonadal veins with central filling defects consistent with bilateral gonadal vein thrombosis. The thrombus extends up to the juxtarenal IVC (c, curved arrow). Axial image from a T1 post-contrast venous phase fat saturated T1 sequence of the abdomen (d) demonstrates the expanded gonadal veins bilaterally with central filling defects balanced-SSFP sequence with inversion recovery saturation (examples: Inhance Inflow IR, General Electric; NATIVE TrueFISP, Siemens; B-TRANCE, Philips). These sequences accentuate the high signal of arterial blood by exploiting inflow-enhancement, similar to that used in time-of-flight (TOF) MRI [/fig]
[fig] Figure 15: Selected sagittal images from a time-resolved MRA of the left foot in a patient with diabetes and foot pain. The initial mask image (a) demonstrates only a vague outline of the foot, with subsequent enhancement of the arteries (b, c), followed by venous filling at a later time point (d). [/fig]
[fig] Figure 17 A: pulmonary artery MRA in a 55-year-old woman with pulmonary hypertension. Single coronal image from time-resolved pulmonary artery MRA (a) and axial T1-weighted fat-saturated post-gadolinium angiogram image (b) demonstrate fusiform aneurysmal dilatation of the main pulmonary artery (PA) [/fig]
[table] Table 1: Sample CT vascular protocols Peak kilovoltage (kVp) is chosen at 140/120/100/80 kVp dependent on BMI Tube current (mA) is determined by automatic exposure control Tube rotation time is maximum [/table]
[table] Table 2: Sample MRI vascular protocols [/table]
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