In order to examine the relationship between accumulation of residual DNA double-strand breaks (DSBs) and cell death, we have used a control and an ATM (Ataxia-Telangiectasia Mutated) defective cell line, as Ataxia-Telangiectasia (AT) cells tend to accumulate residual DSBs at long times after damage infliction. After irradiation, AT cells showed checkpoint impairment and a fraction of cells displayed an abnormal centrosome number and tetraploid DNA content, and this fraction increased along with apoptosis rates. At all times analyzed, AT cells displayed a significantly higher rate of radiation-induced apoptosis than normal cells. Besides apoptosis, 70–85% of the AT viable cells (TUNEL-negative) carried ≥10 γH2AX foci/cell, while only 12–27% of normal cells did. The fraction of AT and normal cells undergoing early and late apoptosis were isolated by flow cytometry and residual DSBs were concretely scored in these populations. Half of the γH2AX-positive AT cells undergoing early apoptosis carried ≥10 γH2AX foci/cell and this fraction increased to 75% in late apoptosis. The results suggest that retention of DNA damage-induced γH2AX foci is an indicative of lethal DNA damage, as cells undergoing apoptosis are those accumulating more DSBs. Scoring of residual γH2AX foci might function as a predictive tool to assess radiation-induced apoptosis.

1. Introduction

Following DNA double-strand breaks (DSBs) generation by ionizing radiation (IR), the cell undergoes an Ataxia-Telangiectasia Mutated (ATM) dependent p53 activation of the DNA damage response (DDR) cascade to activate the cell checkpoints in order to gain time for DNA repair [13]. If the DNA damage cannot be repaired during checkpoint arrest, cells are driven to undergo an irreversible fate by apoptosis or senescence [4, 5]. Sensing of the DNA damage involves the extensive phosphorylation of histone H2AX molecules at both sides of the DNA break [6]. Phosphorylated H2AX forms foci immediately after DNA damage induction by IR. These γH2AX IRIF (Ionizing Radiation-Induced Foci) are detectable with immunostaining or cytometry techniques as soon as 3 minutes afterwards, and the maximum number of foci is detected 30–60 minutes after irradiation [7]. The number of γH2AX foci has been found to closely correlate with the number of radiation-induced DSBs [8, 9]. Very soon after irradiation, γH2AX foci are numerous and small and they disappear along with resolution of DNA damage [8]. Nonetheless, several results have shown that radiosensitive cell lines retain γH2AX foci longer than radioresistant cell lines after exposure to radiation [1012]. The fraction of tumor cells that retain γH2AX foci 24 hours after irradiation has been correlated with the fraction of cells that fail to divide and form colonies [13, 14], suggesting that the H2AX assay can be used as an indicator of cell death. However, there are also negative studies that found no correlation between γH2AX and clonogenic cell survival [15, 16], demonstrating that it is yet unclear whether residual DSBs are ultimately related with apoptosis triggering.

Recently, apoptosis and mitotic catastrophe (MC) have been functionally linked [17, 18]. MC has been defined as an oncosuppressive mechanism resulting from a combination of deficient cell cycle checkpoints, persistence of DNA damage and mitotic failure, which can ultimately lead to cell death during mitosis or alternatively by apoptosis or senescence. This mechanism mainly operates in a cell-cycle-specific kinases-dependent and p53-dependent way to avoid accumulation of genomic instability and is prevalent in cancer cells that exhibit genomic instability and are devoid of proper checkpoint control [17, 19, 20].

The goal of this study was to examine the relationship between DNA damage accumulation and apoptosis. In radiosensitive ATM deficient cells, the defects in triggering the whole DDR following IR lead to checkpoint failure and to the accumulation of unresolved DSBs [1, 3], thus being an ideal model to study this relationship. Our results show that AT and normal lymphoblastoid cell lines undergoing apoptosis accumulate a higher number of γH2AX foci than those belonging to the viable fraction. Specifically, AT lymphoblasts accumulate a higher fraction of residual DSBs and undergo significantly higher levels of IR-induced apoptosis at all postirradiation (pIR) times analyzed. Also AT lymphoblasts display a strong G2/M arrest and tetraploidization, suggesting that MC contributes to apoptosis in ATM deficient cells.

2. Results and Discussion

2.1. AT Lymphoblasts Efficiently Trigger a p53-Dependent Apoptotic Response and Undergo High Levels of Radiation-Induced Apoptosis

In order to determine a relationship between persistent radiation-induced DSBs and apoptosis, we had to first determine the ability to undergo apoptosis in AT and normal lymphoblastoid cell lines (LCLs). To this end, Annexin-V (An) and propidium iodide (PI) incorporation in cells was measured by flow cytometry, and cells were analyzed at 0, 24, 48, and 72 hours after 5 Gy irradiation. Loss of plasma membrane asymmetry by exposing phosphatidylserine in the outer leaflet is an early event in the apoptotic process, previous to loss of membrane integrity and to DNA fragmentation. Thereby, cells positive for Annexin-V and negative for PI staining (An+/PI−) are considered to be undergoing early stages of apoptosis (EA) [2123]. Later in this process, cells lose membrane integrity, allowing PI staining. Therefore, cells that are An+/IP+ are considered to be in late apoptosis (LA), although necrotic cells can also be found in this fraction [21, 24].

As shown in Figure 1(a), the fraction of An+/PI− cells is higher in AT LCL at all times analyzed, even before irradiation. AT cells undergoing early apoptosis reach 8.0% and 12.5% at 24 hours and 48 hours pIR, respectively, while the fraction of normal cells An+/PI− during these time points is always lower than 5%. The overall fraction of Annexin-positive cells (sum of An+/PI− and An+/PI+ cells) reaches its maximum at 48 hours after irradiation, being 12.3% in normal LCL and 31.8% in AT LCL. At later time points, An+/PI− and An+/PI+ cells start to slowly decline, although they are still significantly higher in AT cells and far from the basal levels in both cell lines. These results demonstrate that this AT LCL not only efficiently triggers apoptosis, but also undergoes higher rates of radiation-induced apoptosis than its normal counterpart.

Because several works have reported contradictory results regarding apoptosis induction in AT cells [2531], we aimed to confirm the previous results obtained with Annexin-V/PI by analyzing radiation-induced apoptosis using TUNEL methodology. One characteristic feature of the later stages of apoptosis is the internucleosomal fragmentation of DNA into ~180 bp repeats often referred to as DNA laddering [32]. TUNEL allows the detection of these DNA fragments by labeling their 3′-OH end with a fluorescent molecule. AT and normal lymphoblasts were irradiated at the same dose of γ-rays (5 Gy), and apoptosis levels were analyzed at the same time points previously described. TUNEL was performed on slides and quantitation of TUNEL-positive cells was performed with an epifluorescence microscope. The majority of TUNEL-positive cells also displayed characteristic morphological features of apoptosis, such as nuclei shrinkage, DNA compaction, and nuclear fragmentation. All these features combined with TUNEL staining allowed certain detection of apoptotic cells (Figure 1(b)).

As shown in Figure 1(a), the levels of spontaneous apoptosis measured with TUNEL were higher in AT than in normal cells. Higher rates of spontaneous apoptosis in AT lymphoblasts have been described before [33] and are confirmed by the results obtained in the present study with both apoptotic cell detection assays (total Annexin-V-positive cells before irradiation: 5.2% in normal cells versus 10.9% in AT cells; test, ; TUNEL: 3.7% in AT cells versus 1.7% in normal cells; test, ). At early postirradiation times, the fraction of TUNEL-positive cells remains low in both cell lines, but they increase at 48 hours pIR and reach maximum levels at 72 hours pIR, being of 17.2% in normal and 32.4% in AT cells ( test, ). Although both Annexin-V/PI and TUNEL methodologies measure apoptosis, they seem to detect correlative stages of this process. At twenty-four hours after irradiation, there has been an increase of cells undergoing EA and evolving to a LA stage compared to unirradiated cells, while yet very few cells are positive for TUNEL staining. EA and LA fractions reach a plateau level at 48 hours pIR, while at this time there is an increasing frequency of TUNEL-positive events. Because TUNEL methodology detects extensive DNA fragmentation, TUNEL-positive cells might undergo a later apoptotic stage than those signaled with Annexin. In this way, the combination of the results obtained with the Annexin-V/PI and the TUNEL procedures renders a dynamic picture of the apoptotic process in the lymphoblast cells analyzed.

Lymphocytes are removed, both physiologically and after irradiation, by a p53- and caspase-dependent apoptotic pathway that leads to DNA cleavage [19, 34, 35]. The role of the ATM protein in triggering this IR-induced apoptotic response has been examined using different experimental systems in AT lymphoblasts, AT lymphoblastoid cell lines (LCLs), and Atm−/− mouse thymocytes with conflicting results. Lymphocytes from AT patients were found to have an increased spontaneous apoptotic level [33]. Also, a normal apoptotic response after IR was demonstrated in Atm−/− mouse cells [26] and in lymphocytes from AT patients [27]. Variable results have been described in AT LCLs, although most of them displayed a normal apoptotic response to IR [28, 36]. To ultimately determine p53 status, we analyzed p53 presence and its activation after IR. Levels of p21, a p53 effector involved in cell cycle arrest at G1 and S phases after DNA damage induction [37], have also been analyzed. As shown in Figure 1(c), despite ATM absence, p53 was effectively induced in normal and AT cells at 24 hours pIR, when the fraction of apoptotic cells starts to increase. Consistent with greater apoptotic induction, levels of activated p53 are still high in AT cells at 48 hours pIR. Induction of p21 is observed in both cell lines although higher expression is observed in normal than in AT cells. In this regard, it has been suggested that ATM regulates distinct p53-dependent pathways that selectively trigger checkpoint arrest or apoptosis. For example, effective p53 induction coupled with checkpoint failure and a normal apoptotic response after IR has been described in ATM deficient cells [26, 28, 38, 39]. In agreement with these works, normal cells efficiently arrest at G1 after irradiation while the AT lymphoblastoid cell line tested in this study undergoes high apoptosis rates along with G1 checkpoint failure (see Section 2). Bax, another p53 target involved in activation of caspases, shows a similar expression in both LCLs. The cleaved fragment of caspase 3 is detected only after irradiation in both cell lines but in AT cells its expression is still visible at 72 hours, consistent with higher frequency of apoptotic AT cells at this time point. Altogether, our results are in agreement with a role for ATM selectively activating p53 to regulate cell-cycle checkpoint but not apoptosis. In this regard, ATM- and Rad3-related (ATR), Chk2 and DNA-PKcs have been proposed as candidates to regulate IR-induced apoptosis in AT cells [3840].

2.2. Radiation-Induced Mitotic Catastrophe Is a More Relevant Cell Death Process in AT Lymphoblasts Than in Its Normal Counterparts

We proceeded by analyzing cell cycle progression after irradiation. As shown in Figure 2(a), normal lymphoblasts are efficiently arrested at G1, as demonstrated by a diminution of the S fraction at 24 h pIR that prevails up to 72 h. As expected, impairment of proper G1 arrest in the AT LCL was demonstrated by no noticeable decrease in the S fraction at 24 hours pIR, and later decreases were low when compared to normal cells. These results are consistent with the Western blot results showing a weak induction of p21 in AT cells after irradiation (Figure 1(c)) and are in agreement with the ATM deficiency cell phenotype, which is characterized by impairment of G1 and intra-S checkpoint activation upon DNA damage infliction. This leads to cell cycle progression of cells bearing unresolved DNA damage [3, 41]. These cells are efficiently arrested in G2 [42] unless the DNA damage has been inflicted during G2 phase, in which case ATM deficient cells proceed into mitosis [43]. In any case, damaged cells that surpass G1 and/or G2 checkpoints become later arrested in mitosis (M) because of spindle anaphase checkpoint (SAC). In this work, cells were irradiated during their exponential growth, implying that many AT cells will surpass G1 and intra-S checkpoints and will be arrested at G2, while those AT cells irradiated during G2 phase will surpass the G2 checkpoint and arrest in M by SAC. Remarkably, our results show that AT lymphoblasts significantly accumulate at G2/M after irradiation at all times analyzed (Figure 2(a)), suggesting that, besides cells arrested at G2 checkpoint, some of them might remain in M phase.

Persistent arrest at G2/M boundaries after DNA damage infliction is a first indicator of mitotic catastrophe (MC), so we aimed to analyze the possibility that MC contributes to cell death in AT cells. MC has been described as an oncosuppressive mechanism that, in order to avoid accumulation of genomic instability, senses this mitotic failure and responds to it by driving the cell to death during mitosis [20]. Sometimes, some of these cells do not die during mitosis and eventually overcome SAC signaling and “slip” into the next interphase without dividing. These cells may reenter the cell cycle and reduplicate its DNA content, turning into the accumulation of tetraploid cells within the population. Indeed, AT lymphoblasts showed an increasing frequency of cells with a 4N DNA content that reached 5.6% at 72 hours after irradiation (Figures 2(a) and 2(b)). Although small, this fraction was higher than in normal lymphoblasts, whose tetraploid population was lower than 0.7% at all times analyzed. To further confirm these results, we quantified the centrosome number, as cells that skip mitosis and reenter the cell cycle will reduplicate their centrosomes along with DNA. Centrosomes were scored by means of immunofluorescent pericentrin detection and cells were classified into those having a normal number of centrosomes (1 centrosome in interphase and 2 centrosomes in S, G2, and M phases) or an aberrant number of centrosomes (more than 2). As shown in Figure 2(b), the basal frequency of AT cells with >2 centrosomes was very low, but it strikingly increased at 48 hours after irradiation, when it reached almost 3% of the cell population and correlated with the appearance of a 4N cell population (Figure 2(b)). In contrast, the fraction of normal lymphoblasts with an abnormal number of centrosomes did not reach 1% of the population during the 48 h analyzed ( test, ). Thus, some AT cells are able to reach mitosis despite defective repair. Eventually, some of them skip M phase, giving raise to the appearance of a tetraploid population together with an increasing population of cells with an abnormal centrosome number. Polyploid cells with extra centrosomes are prone to form transient multipolar mitotic spindles, which can either directly trigger mitotic death or result in the generation of aneuploid daughter cells. A small fraction of these cells might survive and enter a mitotic round that is also likely to be catastrophic [20]. Because the MC mechanism can ultimately culminate in apoptotic cell death [20], we propose that this mechanism contributes to the radiation-induced apoptotic levels detected in AT lymphoblasts.

2.3. Viable AT Lymphoblasts Display Higher Levels of Radiation-Induced DNA Damage and Delayed DSB Repair at Long Times after Irradiation

We next aimed to examine the levels of radiation-induced DNA damage in both lymphoblast cell lines. To this end, we analyzed γH2AX foci corresponding to radiation-induced DSBs in viable cells—those negative for TUNEL (Figure 3(a)). Figure 3(b) shows that, 24 h after irradiation, 64.1% of the normal lymphoblasts have γH2AX foci, a 3.2-fold increase compared to unirradiated cells. Within the same time interval, viable AT lymphoblasts displayed a 7-fold increase, as 87% of them showed γH2AX foci ( test, ). At 72 h pIR normal cells have repaired most of their DSBs and only 22.2% of them have γH2AX foci, while this fraction is still around 50% in AT lymphoblasts ( test, ). All together, these results reflect the DNA repair impairment of AT cells, which repair most of the DSBs in a fast and efficient way, while a subset of breaks remains unrepaired for long times, even days, after DNA damage infliction [3, 1012, 44]. In agreement with that, our results show that AT cells, despite showing an initial decline in the fraction of cells displaying γH2AX foci, are unable to efficiently proceed to further diminish this population and accumulate high numbers of residual DSBs, even at very long times after DNA damage has been induced.

After that, we scored the number of γH2AX foci in the TUNEL-negative cells and classify them into two groups: cells with less than 10 γH2AX foci and cells with 10 or more γH2AX foci (Figure 3(b)). Only a small fraction of the normal cells accumulated 10 or more γH2AX foci, reaching a peak of ~18% at 24 hours pIR and declining thereafter. On the contrary, most of the irradiated AT cells accumulated 10 or more γH2AX foci, reaching a maximum of around 75% at 24 hours pIR ( test, ). From 24 to 72 hours after irradiation, 85 to 70% of the γH2AX-positive AT cells carry 10 or more DSBs while this frequency is much lower in normal cells (27 to 12%). Thus, after irradiation, AT cells accumulate more cells with DSBs and more DSBs/cell than their normal counterparts.

The results presented here suggest that DSB repair might be inversely correlated with apoptosis induction. Indeed, at 48 hours pIR, the percentage of normal cells with ≥10 γH2AX foci is low (6.3%) and it coincides with the stabilization of the TUNEL-positive rate (around 17%). On the other hand, at 48 hours pIR, still most of AT cells have ≥10 γH2AX foci (43.3%) and TUNEL rates continue to increase at 72 hours pIR (from 26 to 32%; Figure 3(b)). In agreement with that, recent studies have revealed that some residual 53BP1, Rad51, and γH2AX foci remain in cells for a relatively long time after irradiation and have indicated an inverse correlation between the number of residual foci and the surviving fraction of cells [4549]. Similarly, a correlation between a higher rate of foci loss and a higher clonogenic surviving fraction in ten different cancer cell lines has been described [50]. Finally, it is worth noting that the fraction of AT cells with less than 10 γH2AX foci remains stable before and after irradiation and is hardly changed along with the apoptotic rate (Figure 3(b)), thus discarding this subpopulation of cells as that with more probabilities of undergoing IR-induced apoptosis. This result is in agreement with other studies describing that low background levels of foci (<3 foci per cell) scored at 24 hours after irradiation were correlated with cell survival [14, 51].

2.4. Apoptotic AT Cells Accumulate More Residual DSBs Than Normal Lymphoblasts

To further analyze this possibility, we proceeded to analyze radiation-induced DSBs in normal and AT lymphoblasts undergoing apoptosis. The characteristic DSB-signaling processes of the DDR, such as phosphorylation of histone H2AX forming visible foci, are eventually abolished in cells undergoing last stages of apoptosis, probably due to DNA condensation [52]. Consequently, no γH2AX foci were scored in TUNEL-positive cells (Figure 3(a)). We reasoned that earlier apoptotic stages, such as those detected with Annexin-V/PI methodology, would better allow the detection of radiation-induced DSBs. To this end, AT and normal lymphoblasts were irradiated and enriched populations of early apoptotic (An+/PI−) and late apoptotic/necrotic (An+/PI+) cells were obtained by flow sorting at 48 hours after irradiation. Immediately after sorting, cells were fixed on slides and γH2AX immunofluorescence was performed. This procedure resulted in the loss of Annexin-V and PI staining, allowing for reliable identification of γH2AX signaling. In all the populations analyzed we found a fraction of cells displaying a pan-nuclear γH2AX staining (Figure 3(a)). This kind of staining has been related to apoptosis induced by several DNA damaging agents and is concurrent with the initiation of DNA fragmentation resulting from the apoptotic process [53, 54]. This fraction of cells was taken into account when calculating the percentages shown in the figures. At the time point selected after irradiation (48 hours), the fraction of cells undergoing early and late apoptosis was at its maximum in both cell lines. Cells undergoing early apoptosis (An+/PI−) were 4.8% in normal and 12.5% in AT cells, while those undergoing late apoptosis (An+/PI+) were 7.5% and 19.3% in normal and AT LCL, respectively (Figure 1(a)).

Within the above mentioned fraction of cells undergoing early apoptosis (An+/PI−), most of them had γH2AX foci—64.8% of the normal lymphoblasts and 51.2% of the AT cells (Figure 3(c)). Nonetheless, most of the cells undergoing early apoptosis had less than 10 γH2AX foci/cell in normal cells (47.6%) but ≥10 γH2AX foci/cell in AT cells (28.4%), demonstrating that also the AT cells that initiate apoptosis (An+/PI−) accumulate a significantly higher number of DSBs than normal cells ( test; ). As normal lymphoblasts enter in later apoptotic/necrotic stages (An+/PI+), the frequency of cells with less than 10 γH2AX foci is sharply reduced (from 47.6% to 19.9%; 2.4-fold reduction), while the fraction of cells with ≥10 γH2AX foci is more or less maintained (14%). Similarly, the frequency of AT lymphoblasts undergoing later apoptotic stages (An+/PI+) that present less than 10 γH2AX foci is reduced, while the population of cells with ≥10 γH2AX foci is increased with respect to early apoptosis and to normal cells (75% of the γH2AX-positive AT lymphoblasts). Thus, AT lymphoblasts accumulate more DSBs/cell than their normal counterparts, also while undergoing apoptosis ( test; ).

It is important to note that An+/PI− cells might be considered viable, as early apoptosis is believed to be reversible if the conditions inducing apoptosis are removed [5557]. Moreover, it has been suggested that DNA repair is involved in this reversibility [58]. Thus, while undergoing early apoptosis, cells might be able to perform some degree of DSB repair that leads to reduction of γH2AX foci-positive cells in later apoptotic stages. Most probably, cells that carry a larger amount of DSBs have a lower probability of eventually performing successful repair; thus cells with ≥10 γH2AX foci accumulate at later stages of apoptosis. In this work, cells undergoing late apoptosis are those accumulating more DSBs, and the cell line with impaired repair activity is the one carrying more residual DSBs and displaying higher rates of cell death. Similar results have been very recently described in which cells carrying Rad51 foci at 24 hours pIR are the ones more likely to die [44]. It cannot be concluded that accumulation of ≥10 γH2AX foci signals cells to undergo apoptosis, as γH2AX foci dose-response may depend on cell type, time allowed after irradiation, and the cell cycle phase in the moment of irradiation among other factors. Nonetheless, these results support the notion that persistence of residual DSBs signals those cells that are more likely to undergo cell death.

3. Conclusions

Annexin-V/PI and TUNEL methodologies have been used to analyze IR-induced apoptosis. These methodologies seem to detect subtle differences in apoptotic frequencies that might correspond to progressive apoptotic stages, as maximum levels of Annexin-V-positive cells are reached earlier than maximum levels of TUNEL-positive cells. Annexin-V/PI allows discrimination between cells undergoing early and late apoptosis as well as fast scoring of more cells. TUNEL detection on slides and subsequent microscope analysis allows the combination of TUNEL and protein immunodetection—like γH2AX—and the analysis of these events in the apoptotic and the healthy fraction at the same time.

The results in this work show that AT LCL efficiently undergoes IR-induced apoptosis to a higher level than its normal counterpart at all times analyzed. Along with accumulation of residual DSBs, indicators of mitotic catastrophe such as prolonged G2/M arrest and DNA and centrosomes reduplication are found in irradiated AT cells, which also contribute to the apoptotic levels scored. In these cells, apoptosis is accompanied with p53 induction and cleavage of caspase 3, while they show low levels of p21 induction that correlate with impairment of G1 and intra-S checkpoint activation after irradiation. These results agree with a role for ATM selectively activating p53 to regulate apoptosis and not cell cycle checkpoint. Accumulation of radiation-induced unrepaired DSBs contributes to cell death. For this reason, viable cells that are undergoing apoptosis (Annexin-positive) have been isolated in both cell lines and γH2AX foci have been scored. The results show that these are the cells accumulating more DSBs per cell. As cells progress from EA to LA, the fraction of cells with fewer γH2AX foci decreases in both AT and normal cells, while cells with more than 10 γH2AX foci accumulate in LA, especially in AT cells—consistent with their DNA repair defect. These results support the notion that persistence of residual DSBs signals those cells that are more likely to undergo cell death and that scoring of γH2AX foci might function as a predictive tool to assess radiation-induced apoptosis.

4. Material and Methods

4.1. Cell Culture and γ-Irradiation

EBV-transformed human lymphoblastoid cell lines (LCLs) GM08436A (derived from a child suffering from Ataxia-Telangiectasia) and GM09622 (derived from a sex and age matched control) were obtained from the Coriell Cell Culture Repositories. Cells were grown in suspension in RPMI 1640 medium with GlutaMAX-I (Life Technologies, CA, USA) supplemented with 15% fetal bovine serum and kept in the incubator at 37°C and 5% CO2 atmosphere. When indicated, cells were irradiated with 5 Gy γ-rays using an IBL-437C R-137 Cs irradiator, with a dose rate of 5.10 Gy/min.

4.2. Apoptosis Detection
4.2.1. Annexin-V-Fluorescein Assay

The Annexin-V/propidium iodide (PI) assay was performed following the manufacturer’s instructions (Annexin-V-FLUOS Staining Kit, Roche, Switzerland). Briefly, lymphoblast cells were collected, centrifuged, and washed in 1xPBS. The cell pellet was resuspended in freshly prepared Annexin-V-FLUOS labeling solution with PI and incubated for 15 minutes (min) at room temperature, in the dark. Cells were analyzed on a FACSCalibur flow cytometer (Becton-Dickinson, CA, USA) using 488 nm excitation and 530/30 nm band pass filter for fluorescein detection and a FL2 photomultiplier and band pass filter 585/42 nm for PI detection after electronic compensation. Flow cytometry analysis was done with the CellQuest software (Becton-Dickinson, CA, USA). Cells were classified into the following fractions: (a) viable cells (An−/PI−) were impermeable for PI and also did not bind Annexin-V (An); (b) early apoptotic cells (An+/PI−) bound An and were PI impermeable; (c) late apoptotic or, also called, secondary necrotic cells (An+/PI+) bound An and were PI permeable; (d) primary necrotic cells (An−/PI+) only displayed PI staining. When indicated, cell sorting of enriched populations of An+/PI− and An+/PI+ cells was performed with a FACS Aria SORP sorting cytometer (Becton-Dickinson Biosciences, CA, USA) using a 488 nm excitation laser and 525 nm band pass filter for fluorescein detection and a 350 nm excitation UV laser and 660/400 nm band pass filter for PI detection. Cells obtained after sorting were dropped on poly-L-lysine coated slides and allowed to attach for 3 min before proceeding with immunofluorescence.

4.2.2. TUNEL Assay

The TUNEL assay was performed following the manufacturer’s instructions (In Situ Cell Death Detection Kit, Fluorescein, Roche, Switzerland). Briefly, lymphoblasts were centrifuged, washed with 1xPBS, and dropped on poly-L-lysine coated slides. Cells were then fixed with 2% paraformaldehyde for 20 min at room temperature and permeabilized with 0.1% Triton-X100 and 0.1% sodium citrate in 1xPBS for 5 min in ice. The TUNEL mix was applied to the cells following the manufacturer’s instructions and allowed to be incubated at 37°C for 40 minutes. Before analysis, Vectashield Mounting Medium for fluorescence (Vector Laboratories Inc., CA, USA) supplemented with 4′,6-diamino-2-phenylindole (DAPI) was applied. TUNEL analysis was performed with an Olympus BX41TF epifluorescence microscope equipped with an Olympus U-TVIX digital camera using the Isis v5.4.9 software (MetaSystems, Germany).

4.3. Cell Cycle Analysis

Lymphoblasts were washed in 1xPBS, centrifuged, and kept in 70% ethanol at −20°C until analysis. Cells were then centrifuged, washed with 1xPBS, and resuspended in 1 mL of freshly prepared PI/Triton/RNAsa solution: 0.1%Triton-X100, 2 mg RNAsa A (DNAsa free), and 200 μL of 1 mg/mL PI. After 15 min incubation at 37°C, DNA content was measured on a FACSCalibur flow cytometer (Becton-Dickinson, CA, USA). Red fluorescence of PI-stained nuclei was excited at 488 nm with an argon laser and was collected through a 670 nm long pass filter detection into the FL3 photomultiplier tube on a linear scale, at a flow rate of 12 mL/min (low). Cell cycle analysis of the DNA histograms of integrated red fluorescence was performed with CellQuest software (Becton-Dickinson, CA, USA).

4.4. Immunofluorescence

Immunodetection of γH2AX was performed on the same samples previously analyzed for TUNEL or sorted after Annexin-V/PI staining. Pericentrin detection was performed on newly obtained samples of irradiated lymphoblasts allowed to attach onto poly-L-lysine slides. Cells were fixed for 15 min in 4% paraformaldehyde and permeabilized in 1xPBS-0.5% Triton-X100 solution for 15 min. After 30 minutes of blocking with 0.1% Tween20 and 5% FBS, mouse anti-γH2AX (Ser139) (Upstate/Millipore, MA, USA) or rabbit anti-pericentrin (Abcam, UK) was applied at a 1 : 1000 concentration and allowed to incubate for 1 hour at room temperature. Anti-mouse Cy3 (Amersham Biosciences/GE Healthcare, NJ, USA) and anti-rabbit A488 (Invitrogen/Molecular Probes, OR, USA) secondary antibodies were applied at 1 : 1000 final concentration for 45 minutes at room temperature, followed by extensive washing. Before analysis, Vectashield Mounting Medium for fluorescence (Vector Laboratories Inc., CA, USA) supplemented with DAPI was applied. Slides were analyzed using an Olympus BX41TF epifluorescence microscope equipped with an Olympus U-TVIX digital camera using the Isis v5.4.9 software (MetaSystems, Germany).

4.5. Western Blotting

Cells were collected by centrifugation, washed with PBS, treated with RIPA lysis buffer, and sonicated. Whole-cell extracts were loaded onto a 10% SDS-polyacrylamide gel that was run at 150 V for 50 minutes in a Bio-Rad mini-gel system. Proteins were transferred to a nitrocellulose membrane (30 V, 90 minutes) and blocked for 1 h at room temperature in 5% BSA brought to 37°C or with nonfat milk at 4°C. Primary antibodies used were mouse monoclonal anti-p53 (Santa Cruz Biotechnology, Inc., TX, USA), rabbit monoclonal anti-phospho-p53 (Ser15) (Thermo Fisher Scientific, MA, USA), rabbit monoclonal anti-p21 (Abcam, UK), rabbit monoclonal anti-Bax (Abcam, UK), rabbit polyclonal anti-active caspase 3 (Abcam, UK), and mouse anti-GAPDH (Abcam, UK). Membranes were washed with TTBS (Tris 10 mM, NaCl 150 mM, and 0.005% Tween20) and incubated for 1 hour at room temperature with secondary antibody, goat anti-rabbit or goat anti-mouse, conjugated to horseradish peroxidase (Upstate/Millipore, MA, USA). Proteins were visualized using Immobilon Western kit (Upstate/Millipore, MA, USA) and the signal was captured with ChemiDoc XRS (Bio-Rad, CA, USA).

4.6. Statistical Analysis

The statistical analysis was performed using GraphPad InStat version 3.05 (GraphPad Software Inc., CA, USA).

Conflict of Interests

The authors declare that they have no competing financial interests.

Authors’ Contribution

Marta Martín and Anna Genescà developed the study concept and design. Testing and data collection and analysis were performed by Teresa Anglada, Marta Martín, Mariona Terradas, and Laia Hernández. Marta Martín and Anna Genescà wrote the paper and approved its final version for submission.

This work was funded by grants from Consejo de Seguridad Nuclear (CSN 2012-0001) and EURATOM (Dark.Risk GA 323216). Anna Genescà Laboratory is supported by Generalitat de Catalunya (2009SGR-282). Laia Hernández is supported by the Universitat Autònoma de Barcelona Ph.D. programme fellowship. The authors would like to thank Dr. Dik van Gent and Dr. Humberto Sánchez for their valuable comments. They also thank the Radiological Protection Unit at Universitat Autònoma de Barcelona for sample irradiation and the Cytometry Service from the Centres Científics i Teconològics (CCiT), Universitat de Barcelona (UB), for cell sorting.


Cardi B Responds To Criticism That She’s Trying To Boost Album Sales With Old Songs

Cardi B is responding to criticism that she’s putting older songs on her sophomore album just to boost sales.

The rapper confirmed on the Playboy-housed Centerfold platform that both “WAP” and “UP” will be featured on her forthcoming album despite the songs being released in 2020 and 2021. Fans took to social media accusing her of adding the hits in order to get certifications for her album.

Cardi wrote on Twitter, “Lmaaaoooooo imagine me not putting my OWN records on my album.”

According to Complex, she also responded to a now-deleted tweet with, “Why haven’t you said nothing about other artists who recently put songs b4 the pandemic on their album or people that have to put other people songs on their sh*t ..Imagine my biggest song not being on a album.”


Britcher Leone Blog

What should I know about prenatal testing for Down syndrome?

On Behalf of | Dec 14, 2021 | Birth Defects |

Families who are preparing to welcome a baby should be aware of medical advances that help provide information on the health of both the mother and developing fetus. There are tests available to provide information on various medical issues that can help parents to determine the best course of medical intervention for their situation.

One example of prenatal testing that can provide important medical information involves tests used to determine the presence of Down syndrome. Down syndrome is a genetic condition caused by extra genes of the 21st chromosome. It presents with some degree of cognitive disability and other developmental delays. Some common physical traits include an upward slant of the eyes; flattened bridge of the nose; a single, deep crease on the palm of the hand; and decreased muscle tone. However, not every affected child will have these traits.

The chance of having a child with Down syndrome increases with the mother’s age. At age 35, the odds are about 1 in 350. At age 40, the odds are about 1 in 100. Unlike other genetic syndromes, there is no association between Down syndrome and culture, ethnic group, socioeconomic status or geographic region.

There are three known causes of Down syndrome:

  • Trisomy 21, a doubling of one of the genes of the 21st pair of chromosomes, accounts for an estimated 95 percent of children born with Down syndrome.
  • Translocation, where an extra part of the 21st chromosome gets stuck onto another chromosome, occurs in 3 to 4 percent of the cases.
  • Mosaicism, where the person with Down syndrome has an extra 21st chromosome in some of the cells but not all of them, is responsible for 1 to 2 percent of people with Down syndrome.

Prenatal Testing

Non-invasive testing can screen for markers that are known to show an increased risk for Down syndrome. These can be conducted at two times in the pregnancy. One test can be performed between 10 and 14 weeks of pregnancy by taking a blood sample and performing an ultrasound to measure a nuchal translucency (the amount of fluid at the back of the baby’s neck). The results of the blood test, the nuchal translucency measurement and the mother’s age are then used to estimate the risk for Down syndrome and trisomy 18.

The other test involves a maternal blood test between 15 to 20 weeks of pregnancy. When the results of this blood test are combined with the results from the first trimester blood test and nuchal translucency ultrasound, the detection rate for Down syndrome increases. This test, often called a quin-screen, also provides a personal risk assessment for other possible fetal issues including neural tube defects or an abdominal wall defect.

Based on risk assessment a mother can decide to undergo more definitive diagnostic testing.

Chorionic Villus Sampling (CVS) can be used to detect other genetic diseases like cystic fibrosis, Tay-Sachs disease and sickle cell disease in at-risk fetuses. However, CVS does not detect spinal cord defects. CVS is done at 10 to 12 weeks. CVS involves removing a tiny piece of tissue from the placenta, under ultrasound guidance, either with a needle inserted through the abdomen or a catheter inserted through the cervix. Results are usually received in about two weeks. There is a risk of miscarriage as a result of CVS — 1 in 100 or less, although some centers claim an even lower risk.

Amniocentesis can be used to identify chromosomal problems such as Down syndrome, cystic fibrosis, Tay-Sachs disease and sickle cell disease. An amniocentesis procedure for genetic testing is typically performed between 15 and 20 weeks of pregnancy. Under ultrasound guidance, a needle is inserted through the abdomen to remove a small amount of amniotic fluid.

Amniocentesis detects most chromosomal disorders, such as Down syndrome, with a high degree of accuracy. Testing for other genetic diseases and for neural tube defects, like spina bifida can also be performed at the same time. Again, there is risk of miscarriage as a result of amniocentesis. This risk is very dependent on the facility and doctor performing the procedure and the risk is quoted as high as 1 in 100 to as low as 1 in 800, depending on the facility and operator.

Ultrasound also has a role in both providing screening information for Down Syndrome, but also for other aspects of the pregnancy and non-chromosomal birth defects that can be determined at various points in the pregnancy. Modern 4D ultrasound and a survey of fetal anatomy can provide much more information than just the sex of the fetus.

If you receive positive results on a screening test, start by discussing the results with your doctor and a genetic counselor. Depending on the findings, a medical geneticist may also be helpful. Remember, the purpose of the screening tests and follow up discussions with medical professionals is to give you the greatest amount of information to enable you to make an educated decision for yourself.


• Andy Davis

Monarch caterpillars become frightened when touched by humans

UPDATE Feb 2020 - This blog entry was written ~2 years ago, using a small collection of monarch caterpillars, which were not directly handled. This is NOT a description of the latest research project from the Davis lab examining effects of handling on monarchs.

Greetings blog readers,

Are you read to have your mind blown? Get ready...

Today's post is about a topic that is of immense importance to the issue of captive-rearing, which has been hotly discussed as of late. Folks have been debating the pros and cons of this practice (hint - there are more cons than pros), with most of the talk being about the rates of tag recoveries, and migration success. I noticed that a lot of the discussions have revolved around whether or not captive rearing harms monarchs, and on this, no one seems to have an answer. Today's post may just change that. I'm going to tell you about some data I collected in the last year that were published in the recent study I spearheaded on road noise (Davis et al 2018, Biology Letters). The data I'm going to highlight were largely overlooked in all of the media frenzy around that study, probably because they were only presented in the supplemental file that accompanied the publication. Even so, keep in mind that these results are peer-reviewed, published science.

If you recall from a few months ago when this paper came out, my colleagues and I had set out to measure the effect of highway noise on monarch caterpillars - specifically on their heart rate! Elevations in heart rate are a universal signal of stress in the animal kingdom, even in insects! So my colleagues and I wanted to see if road noise causes stress in monarch larvae, by examining their heart rates. I had written a summary of that paper in a previous blog. Basically, I have developed a technique in my lab to monitor, in real-time, the beating hearts of caterpillars, using a very cool electronic device attached to a computer. It doesn't hurt them, because it senses the heart movement from about a half-inch away from the larva. I'll show a picture of this gizmo below, in case you didn't see the previous blog.

The device reads the beatings of the caterpillar's heart, and displays them in real-time on a computer screen, so it's a lot like those screens you see next to a hospital bed. In essence, I use it to take the caterpillar's pulse!

Recall that in that study, we determined that larvae exposed to road noise at realistic loudness causes their hearts to race (there was a 16% elevation in heart rate), which is a sign of a physiological stress reaction. In other words, the larvae were frightened by the noise.

Now, in the course of doing that project, we also conducted a side-experiment, to determine if monarch larvae become stressed simply by picking them up and handling them, and more importantly, how long does it take for their heart rates to increase. This was an important question, because we needed to know if the act of handling them compromised the data from the noise experiment. This is the part that was published in the supplemental file.

To answer this question we conducted a very simple experiment - at least, it was simple to do after we had all of the gear set up and running. We examined twelve 5th instar larvae for this experiment. They had all been reared in the lab (by us). They were reared from eggs in plastic containers, fed greenhouse-grown swamp milkweed, and most importantly, their handling was minimized. That means we did not physically handle the larvae whenever their food needed to be replenished in their containers. An in fact, we only changed out their food every other day so as to minimize disturbance.

The procedure we used to assess their stress reaction was pretty simple - we had the larvae containers in front of us to start - then we picked up a larva by pulling out the leaf it was sitting on, held it in front of us for 5 seconds, and then placed it (the leaf plus larva) on the heart monitor to get readings. As an aside, you will be surprised to know that the larvae usually hold still under the device if we allow them to hold onto their leaf. Every now and then one decides to wander off during the procedure, so then we just use another one. Anyway, this "procedure" was nothing more than us picking up the caterpillar.

Once the heart readings began, we allowed the device to continue monitoring for at least 10 minutes. We let it run for so long because we wanted to know how long it took for the heartbeats to speed up, or in other words, how long it took for the larva to be stressed from being picked up. We actually suspected that it might take a while, because I had been doing some similar research on a beetle species that showed it can take up to 8 minutes for their heart rates to max out after a stressor (link here to see that paper).

OK, now that I've told you about the project, and the procedures, now let's get to the results. Below is the primary graph from this experiment, showing the heart rates of the caterpillars over time. Each point here is the average of the 12 larvae.

There are a couple of things to point out here. First, there was indeed an elevation of heart rate - the initial rate of 46 beats per minute was typical for a "resting" heart rate of monarch caterpillars (I know this from experience in the lab). And recall that this initial rate was obtained pretty quickly after picking up the larva, so we can be confident that it was not itself elevated. Next, see that the heart rate maxed out at 5 or 6 minutes, at around 53 beats per minute. That time frame is pretty much what I expected, based on the work I do with other species. Importantly, the maximum elevation is an increase of about 15% over the baseline heart rate. I can tell you (also from experience) that a 15% elevation is consistent with a stress reaction in an insect. In fact, recall that when larvae are exposed to road noise, their heart rate goes up by 16%. Actually, if you're wondering, when humans become stressed, our heart rate increases between 15-20%.

Establishing this timecourse of the monarch caterpillar stress response was very important to our road noise study. This graph told us that the cardiac stress reaction in monarch larvae takes a few minutes to kick in after you pick up a given larva. So we figured that if we could work quickly enough during testing, then we should be able to obtain the heart rates of our test larva before they became stressed from our handling. But this timecourse is a little tangential to the fact that there WAS an increase, which is of importance here.

For the current discussion about captive rearing, these results are extremely relevant. In fact, these may change the way you think about how you interact with monarch larvae (if at all). What these data are telling us is that monarch caterpillars become frightened when someone picks them up. Let me repeat this, and more clearly - monarch larvae experience a stress reaction if a human picks them up. This result is pretty undeniable - we saw this pattern in all twelve of the larvae we tested. There was some variation in the magnitude of the elevation, but the point is they all became stressed.

Now, before you ask, we didn't continue monitoring the caterpillars after 10 minutes, so we don't really know how long the stress reaction lasts (that wasn't our intent). But I can tell you that in other insects, acute stress usually lasts for about a half hour. So that means that stressed monarch larvae probably return to normal after a stress event like this, provided they are returned to their milkweed where they feel safe. BUT (and this is a big but), in most captive rearing scenarios, larvae need to be repeatedly disturbed, moved, and/or picked up, typically on a daily basis for about a week. What happens over time to these larvae? Do they eventually acclimate to this repeated stress? Don't know. And if they do, does this mean they become acclimated to humans? Don't know. And is this daily handling stress similar to what they would face in the wild? Don't know.

There is one other thing that is important to discuss here. Recall that when we "picked up" the larvae to start the testing, we actually didn't even touch each one, but rather we pulled off the leaf it was sitting on, and carried the larvae and leaf to the device. And the heartrate device also didn't physically touch them. At no time was the larva ever directly touched. And yet it still became stressed. That means that the mere act of moving the larva away from its habitat, holding it in front of you, and then exposing it to an unfamiliar environment was enough to frighten it. Given this, one can imagine that the stress reaction would be even more pronounced if a larvae were actually handled.

So let's sum this up here. I've just presented results from a project that is up-to-date, peer-reviewed science, which shows that monarch larvae become stressed when picked up by a human. That's another way to say that they are frightened, or scared, when they are handled. This stress is probably transient, and likely goes away after 30-40 minutes (we don't really know), but it also probably happens each time larvae are handled. And we also don't know what the effect of repeated stressing is to monarchs in the long run. So this research really opens up a lot of questions - questions I hope to tackle in the coming years.

Now, back to the original question of this post - does captive-rearing harm monarchs? I guess that depends on what your definition of harm is. If by harm, you mean, do they die, then that's one thing. But if you consider scaring them (on a daily basis) to be harmful, then the answer is yes.

Before I sign off, please don't email me telling me that YOUR monarchs are not stressed. You can't tell just by looking at them.

Direct link to this blog entry:

The science of monarch butterflies


Most popular

Is it legal for a landlord to disconnect electricity?

Is it legal for a landlord to disconnect electricity?

It is illegal for the owners, landlords or real state firms to disconnect the power to the tenants even if they fail to pay the rent. There are many other legal ways to be followed to get their payment made by the tenants.

Can landlord disconnect electricity in UAE?

Landlords have no right to cut off water or electricity supply of tenants in UAE.

Can landlord disconnect electricity in India?

Under the law and also decided in various judgments, it is unlawful for a landlord to disconnect essential services such as water and electricity or to restrict a tenant from using common amenities for the recovery of rental dues or for any other reasons.

Can a landlord cut off electricity South Africa during lockdown?

Your electricity cannot be cut off, UNLESS: There is an emergency. The landlord needs to do maintenance. You are in arrears and fail to pay your arrears within 7 days of the landlord letting you know that you should.

Can a landlord disconnect electricity in Kenya?

Unlike in other parts of the country, where landlords are a bit lenient, in Nairobi, the deadline for most landlords is the fifth day of the month. If you are lucky not to have your house locked, some landlords disconnect electricity or water supply.

How can I evict a tenant fast in India?

Stage I – Send a Notice to the Tenant to Vacate: An eviction notice needs to be filed in a court under the appropriate jurisdiction mentioning the reason for eviction and the time and date by which the tenant has to vacate the property and is then sent to the tenant to vacate the rental property.

Is there a way to break a lease without penalty?

When does a tenant have the right to break the lease?

The Tenant May Have the Right to Break the Lease If: The landlord tries to enter the tenant’s rental for reasons that are not legally allowed. Harasses the tenant. The tenant must usually obtain a court order to get the landlord to stop the behavior.

Can a tenant break a lease due to domestic violence?

Provide the landlord with written notice of their desire to break the lease due to domestic violence. Notice must be at least 30 fays prior to desired date of termination. Some states require more than 30 days’ notice. The tenant is only responsible for paying rent up until the date of lease termination.

Can a landlord be constructively evicted for breaking a lease?

Depending on the state, if the landlord is given notice of violations & doesn’t remedy them in time, the tenant is considered “constructively evicted”. Depending on the state, if the action is serious enough, harassment by a landlord or their violation of a tenant’s privacy may be enough justification for breaking a lease.


I’m creating telepathy technology to get brains talking

Brain-to-brain communication is becoming a reality, says Andrea Stocco, who sees a future where minds meet to share ideas, or even to aid recovery from a stroke

Humans 2 March 2016

“A healthy person’s brain could be used in the treatment of depression or schizophrenia”

Brian Smale

You are working on brain-to-brain communication. Can one person’s thoughts ever truly be experienced by another person?

Each brain is different. And while differences in anatomy are relatively easy to account for, differences in function are difficult to characterise. And then we have differences in experience – my idea of flying could be completely unlike your idea of flying, for example. When you think about flying, a bunch of associated experiences come into your mind, competing for …

Register for FREE to read this article in full

Subscribe today

with a 12-week introductory offer

No commitment, cancel anytime*

Inclusive of applicable taxes (VAT)

2JRB8F9 Synthetic turf football field fifty, 50, yard line in white.

Athletes may have higher risk of concussion on artificial grass

Abstract visualisation of sound waves

Meta’s text-to-audio AI can create common sounds and generate music

Smoking and our body mass index may cause more than 200 medical conditions

Smoking and body mass are together linked to 226 medical conditions

London heatwave

Sign up to our newsletters


One Piece Ending – Analysis

First, it’s important to point out that most of the following analysis has been made based on the fact that arcs have an official beginning and an official ending (more or less), while Saga’s grouping is always on the readers so it’s far from being something that come directly from Oda.

For all different arcs I put down their duration (in chapters and in days) and the beginning time, that is the elapsed time (measured in days) since the beginning of that specific arc since the beginning of the series.

Arcs have been further divided into their length percentiles to make more meaningful description about their likely length.

PercentileOrdinal RankOrdinal Ranked ArcAverage LengthStandard DeviationVariation
25th8Amazon Lily7.372.610.35
75th24Fish-Man Island3811.280.30
  • Minimum Arcs: Logue Town, Reverse Mountain, Return to Sabaody, Reverie, Romance Dawn, Whiskey Peak, Post-Enies Lobby, Amazon Lily;
    • Minimum arcs are the arcs below the 25th percentile (or the 25% shorter arcs), all with 11 chapters or less. They are often transitional arcs and their length has been kept constant during the series. The sample is polydisperse for Minimum arcs.
  • Short Arcs: Orange Town, Little Garden, Post-War, Jaya, Long Ring Long Land, Syrup Village, Zou, Sabaody;
    • Short arcs are the arcs in between the 25th percentile and the 50th percentile (or the 50% shorter arcs), all within an interval given by 11 to 25 chapters. Their content is usually self-conclusive and act rarely as very small transitional arcs, but they are never the payoff arc within a saga.
  • Medium Arcs: Drum Island, Impel Down, Baratie, Arlong Parc, Marineford, Punk Hazard, Thriller Bark, Fish-Man Island;
    • Medium arcs are the arcs in between the 50th percentile and the 75th percentile (or the 75% shorter arcs), all within an interval given by 25 to 50 chapters. Their content is always self-conclusive, with Big Boss Battle for Luffy and, sometimes, for some of the other crew members. Arguably, as seen in the statistical analysis, this could have been split up in between short arcs and long arcs (putting 30-ish chapters as an arbitrary separation), but I prefer to kept the slip to the a priori chosen percentiles.
  • Long Arcs: Water 7, Enies Lobby, Alabasta, Skypiea, Wano*Whole Cake, Dressrosa;
    • Long arcs are the arcs above the 75th percentile (or the 25% longer arcs), all with more than 50 chapters. They are long arcs (except Water 7) that have a long conflict between the Strawhats and an enemy crew, with a main fight with the Big Boss and Luffy and most of the other Strawhats with a main enemy. Long arcs are payoff to long sagas.

This sub-classification is useful to look at different arcs from a descriptive point of view.

Since we have 165-ish chapters left, shouldn’t we just take the averages length and determine how many of those arcs we have left? Sadly, it’s not that easy. The main problem to determine how many arcs we have left is that, as the series went on, arc’s length has kept growing longer and longer.

Oda explained why Robin’s and Nami’s busts in Wano get hidden away

10 Characters That Could Die in Wano!


Genshin Impact is a popular game in recent years. It is available on many platforms. Unfortunately, some users get stuck on the loading screen while running this game. To fix the Genshin Impact loading screen stuck issue, MiniTool Partition Wizard puts together some solutions in this post.

Genshin Impact is an action role-playing game developed by miHoYo and released in 2020. This game is available on various platforms including Android, iOS, PlayStation 4, PlayStation 5, and Microsoft Windows. And it will be able to play on Nintendo Switch soon.

It is a quite popular game among game lovers. Unfortunately, some users encountered issues while playing the game. Genshin Impact stuck on the loading screen is one of the most common ones. This problem can be caused by many factors. If you are bothered by the same issue, you can try the following solutions.

Tip: Before you try the following solutions, you should make sure your Genshin Impact and the operating system have installed the latest updates.

Solution 1: Check Genshin Impact PC System Requirements

To run Genshin Impact properly, you should make sure your PC meets the minimum Genshin Impact system requirements. If you expect high performance, your PC needs to meet the recommended system requirements.

You can visit the news from miHoYo to get the detailed requirements. If your PC doesn’t meet the requirements, you might need to upgrade some hardware of your PC.

Solution 2: End Other Background Programs

If you are running too many programs at the same time, your programs might freeze or even get crashing. Therefore, you’d better close unnecessary programs running in the background while playing Genshin Impact.

Step 2: Under the Processes tab, right-click the program you want to end and select the End task option.

end task

Repeat the operation to end all the unnecessary programs. Then you can close Task Manager and check if the program is finished.

Solution 3: Update Your Graphics Card Driver

Outdated or missing device drivers could also lead to the Genshin Impact loading screen stuck issue. In this case, you need to update your device drivers regularly, especially the graphics card driver. And here are the detailed steps.

Step 2: Expand a category, right-click your device, and select Update driver.

Update driver

Step 3: In the pop-up window, select Search automatically for updated driver software. Then follow the on-screen instructions to finish the operation.

After that, restart your computer and run Genshin Impact again. If the loading screen stuck issue still exists, please move on to the next solution.

How to Get Genshin Impact Update? Version 2.8 Will Come Soon!

When is the next Genshin Impact update? How to get Genshin Impact update on PC, PS4, PS5, or mobile devices? This post introduces a detailed guide for you.

Read More

Solution 4: Disable Your Overlay Programs

Some overlay programs could cause certain programs with games, too. To solve the Genshin Impact loading screen stuck issue, you might also need to disable your overlay program, such as Discord, Xbox Game Bar, etc.

Solution 5: Check and Repair Game Files

To fix Genshin Impact loading screen stuck issue, you should also check for corrupted or missing game files.

Step 1: Run Epic Launcher and go to Library.

Step 2: Click the three-dot icon on Genshin Impact.

click three-dot icon

Step 3: Select the Verify option in the pop-up menu. Then wait until the process is finished.

verify game files

Where to Find the Genshin Impact Screenshot Folder? [Full Guide]

Do you take screenshots while playing Genshin Impact? Where is the Genshin Impact screenshot folder location? How to find screenshots? Here is a tutorial.

Read More

  • Linkedin
  • Reddit


Master Lumbar Puncture with Picmonic for Nursing RN

Lumbar Puncture

Recommended Picmonics

picmonic thumbnail
Tuberculosis Skin Mantoux Test (PPD)
picmonic thumbnail
picmonic thumbnail
Magnetic Resonance Imaging (MRI)
picmonic thumbnail
Oral Glucose Tolerance Test (OGTT)
picmonic thumbnail
Total Knee Replacement (TKR)

Lumbar Puncture

Lumber Punctured
A lumbar puncture is a commonly used method for obtaining cerebrospinal fluid (CSF) samples and measuring CSF pressure by inserting a needle between the 3rd and 4th lumbar vertebrae. The sample is collected in a series of tubes and must be numbered in the order of collection. Prior to the procedure, ensure that the patient empties their bladder. Although the patient is usually in a side-lying fetal position, a prone position is the alternative used if the lateral decubitus position cannot be used or is unsuccessful. Contraindications to lumbar puncture include increased intracerebral pressure (ICP) and infection at the site of the puncture.
CSF Sample
Brain-Spine-Fluid Sampler

A sterile needle is inserted between two lumbar vertebrae to withdraw CSF samples for diagnostic testing. The fluid is collected in a series of tubes and sent for analysis.

CSF Pressure
Brain-Spine-Fluid with Pressure Gauge

A manometer is attached to the hub of the sterile needle used to withdraw CSF samples. The manometer is used to measure CSF pressure. Normal CSF pressure is below 20 mm H2O (refer to the Picmonic on "CSF Lab Values: Normal").

Empty Bladder
Empty-gauge Bladder

Prior to beginning the procedure, have the patient empty their bladder. Voiding will help minimize patient discomfort and decrease the risk of accidentally perforating the bladder during the procedure.

Side-lying Position
Side-lying Position

Usually, the patient undergoing a lumbar puncture is placed in a side-lying (lateral decubitus), fetal position with the knees flexed and the back rounded for maximal exposure of the site. To prevent accidentally puncturing the wrong site, ensure the patient remains still during the procedure. If the patient cannot tolerate the side-lying position or if the initial lumbar puncture is unsuccessful, a prone position may be used. A sitting position may also be used if the lumbar puncture is unsuccessful in the side-lying position. The patient should sit on the side of the bed and lean forward with arms on a table in front.

Between 3rd and 4th Lumbar Vertebrae
Punctured Between (3) Tree and (4) Fork Vertebrae

During a lumbar puncture, a sterile needle is inserted in between the 3rd and 4th lumbar vertebrae. Inform the patient that they may feel a temporary pain radiating down the leg.

Number Tubes
Numbering Tubes

The CSF samples are withdrawn in a series of four tubes and must be numbered in the order of collection. Each tube is analyzed separately for components, such as glucose, protein, and bacteria.

Supine Position After Procedure
Supine-spine After Puncture

After a lumbar puncture, the patient should return to a supine position for at least two hours to minimize the risk of developing a spinal headache. Encourage the patient to drink plenty of fluids and monitor for headache and signs of possible infection.

Increased ICP (Intracranial Pressure)
Up-arrow Pressure-cooker Cranium

Lumbar puncture is contraindicated in patients with increased ICP. If the patient has increased CSF, puncturing the lumbar area will cause the fluid to leak and decrease CSF pressure in the spinal column. This will cause the CSF pressure in the brain to shift towards low CSF pressure in the spinal column and cause acute neurological damage.

Infection at Puncture Site
Infectious-bacteria at Puncture Site

Lumbar puncture is contraindicated in patients with an infection at the puncture site. The presence of skin infection may be transferred into the CSF as the needle is inserted into the lumbar area.

Take the Lumbar Puncture Quiz

It's worth every penny

Our Story Mnemonics Increase Mastery and Retention

Memorize facts with phonetic mnemonics

Memorize facts with phonetic mnemonics

Works better than traditional Registered Nurse (RN) flashcards.


In 2020, seeking to engage and contribute as a new member of the ASQ Board of Directors, I volunteered to participate in a diversity, equity, and inclusion task force launched by then-Chair Austin Lin. Working with the ASQ DEI task force members has proven to be one of the most fulfilling learning experiences of my career to date.

At the start of this journey, the task force set out to define what we mean when we talk about DEI:

Diversity is the presence and engagement of people with a wide range of perspectives and characteristics, such as ethnicity, abilities, education, age, gender identity, religion, and others (this is not an exhaustive list). We defined diversity broadly to include both demographic and cognitive diversity.

Equity is ensuring fair treatment and equal opportunity. Equity adds a flavor of justice to the concept of equality. Each person may arrive from different circumstances that may necessitate different actions and resources to achieve proportionate fairness, or equity, in the result.

Inclusion speaks to the organizational practices and governance that ensure equitable access and ensure that people feel they belong and can participate and engage freely within the organization.

“Let’s all challenge ourselves...”

One of my “ah ha” moments came from listening to the stories shared within our task force discussions as well as hearing personal stories from across ASQ. The more I engaged in hearing personal stories, the more fully I could understand that something we all share is our own individual “otherness”. Every person has their unique experiences, perspectives, and biases.

My own biases and perspectives have been largely shaped by the way I grew up. My parents were highly educated, intelligent, and deeply religious. We were a large loud and competitive family. Being a family of 15 on one income, we lived below the poverty line, which was just a matter of fact not something we thought of negatively or even thought much about at all.

My parents encouraged us in intellectual debate, balanced with respecting authority. While education was valued, it was not seen as completely necessary – those of us who wanted to go to college established our own paths and found scholarships to get there.

We had dinner as a family most nights. My father is an innovator and often shared what we all thought of as his crazy ideas at the dinner table. One memorable dinner was when he told us his idea to engineer basically a pulley system to transport materials between the earth and the moon. We thought it was hilarious. Now I can read some of the patents he worked on and realize some of his ideas might not have been as crazy as we thought. This makes me more open to the seemingly “crazy ideas” of others, I hope.

Our neighborhood and school system were predominantly people of Italian, Irish, German, and Polish ancestry. There was not a lot of racial diversity and that appears to be still largely true of my old hometown when I go looking for recent demographics. This is a gap in my early experiences that I became much more aware of in my professional life.

Being aware of my unconscious biases, formed by my life experiences, is critical in not letting those biases negatively affect my decisions and behaviors. The more I can make data-driven decisions and standardize processes, the less likely I am to let my biases skew my decision-making in unintended ways.

Applying this thinking into the world of Quality with a capital “Q”, I think of several of Deming’s 14 points, including driving out fear and breaking down barriers between staff areas, as well as self-improvement for everyone. Having psychological safety, where everyone can freely share their ideas, increases performance and innovation in a group.

Our task force considered inputs from a variety of sources regarding the value of diversity. For example, Harvard Business Review found that companies across industries whose leaders exhibited at least three “identity diversity” traits and three “experiential diversity” traits out-innovated and out-performed others. The National Council of Nonprofits describes why DEI matters as well stating, “…embracing diversity, equity, and inclusion as organizational values is a way to intentionally make space for positive outcomes to flourish, whether in direct services or in the nonprofit capacity building or public policy spheres…” Why Diversity, Equity, and Inclusion Matter for Nonprofits | National Council of Nonprofits.

ASQ is committed to providing an inclusive and safe environment. Fulfilling the promise of that commitment takes all of us across the ASQ Society. Let’s all challenge ourselves to be open to the stories and perspectives of others and to make sure everyone feels confident in saying, “ASQ is where I belong.”


On Monday, the Food and Drug Administration authorized baricitinib as the first oral tablet for treating severe alopecia areata, an autoimmune condition that affects over 300,000 people in the United States each year.

Alopecia areata is a condition that causes temporary or permanent patchy hair loss in any hair-bearing part of the body, causing emotional discomfort. High-profile examples including Hollywood actress Jada Pinkett Smith and congressman Ayanna Pressley have brought the issue to light recently.

"Access to safe and effective treatment options is crucial for the significant number of Americans affected by severe alopecia," FDA official Kendall Marcus said in a statement.

Baricitinib, marketed as Olumiant by Eli Lilly in the United States, belongs to a family of medications known as Janus kinase inhibitors. It operates by interfering with the inflammation-causing biological mechanism.

The findings of two randomized, controlled clinical studies involving a total of 1,200 people with severe alopecia led to its clearance for treatment against alopecia areata.

A placebo group, a group that got a 2-milligram dosage every day, and a group that received a 4-milligram dose every day were all assigned to each experiment.

After 36 weeks, over 40% of individuals on the higher dose had regrown 80% of their scalp hair, compared to roughly 23% of those on the lower dose and 5% of those on the placebo.

Around 45 percent of participants in the higher dosage group had substantial regeneration of their eyebrows and eyelashes.

Upper respiratory tract infections, headaches, acne, elevated cholesterol, and a rise in an enzyme called creatine phosphokinase were the most prevalent adverse effects.

Topical and oral medicines have been used in the past to treat alopecia areata, but they have all been considered experimental and none have been approved.

Baricitinib was originally licensed for the treatment of rheumatoid arthritis, and its license was extended during the COVID-19 pandemic to include the treatment of COVID-19 patients who were hospitalized.


The Sustainability Agenda

Hosted ByFergal Byrne

Exploring today's sustainability questions with leading experts.

Episode 40: Interview with Jay Coen Gilbert on the growth and success of B Corps and how the movement aims to re-define success in business

Jay Coen Gilbert is co-founder of B Lab, the non-profit organisation that serves a global movement of people using business as a force for good. B Lab certifies B Corporations, companies that meet the highest standards of verified, overall social and environmental performance, public transparency, and legal accountability.Since its inception in 2006, the B Corp movement has grown so that there are currently over 2,300 certified corporations operating within 150 industries in 50 countries. Jay also serves on the board of Investors’ Circle, an organisation dedicated to the acceleration of patient capital markets towards a sustainable future. Prior to founding B Lab, Jay enjoyed a successful career in the private sector, notably through AND 1, a 250 million dollar basketball footwear and apparel company which he co-founded and later sold.

In this encouraging interview, Jay provides some fascinating insights into an exciting global movement that aims to re-define success in business. He describes how institutions are typically slow to react to social change and how there is ample opportunity to cater to millennials’ greater concern for social and environmental issues, both as consumers and as a workforce. He outlines how B Corps unlock the full potential of business by harnessing the power of competition so that businesses not only compete to be the best in the world but “the best for the world.” He also describes the “B Impact Assessment,” the comprehensive accreditation system which accounts for supply chains, workers’ rights, the environment, community engagement and governance, thereby providing a holistic and genuine picture of a company’s impact. This assessment is fully transparent and is regularly updated through solicited public engagement. Joel also touches on B Corps’ relationship with finance by describing how larges investors can use B Corps as a blueprint for managing systemic risk. Joel is optimistic about the prospects for expansion of the movement, pointing to Danone as the first Fortune 500 Company with aspirations to join, but stresses how the ultimate goal is to change attitudes in the business community. Finally, he outlines the need for a more inclusive and distributive form of capitalism, stressing how the rise of populism is symptomatic of a growing disenchantment with an economic system that rewards an ever shrinking portion of the population.

The post Episode 40: Interview with Jay Coen Gilbert on the growth and success of B Corps and how the movement aims to re-define success in business appeared first on The Sustainability Agenda.


Planning a business trip to London is always stressful, not to mention it can always be costly as well. In London, your options for accommodation and transport are endless, so it can be difficult to know where to start. If you want to plan a business trip to London, you’re in the right place!

If an employee or yourself will be in London for a long day, you should probably consider accommodation. Maybe you have multiple meetings in one day, you could book a hotel so you can spread them across two days to make it less stressful. The last thing you want is to be rushing around the city centre to find the location of the next meeting.

Luckily in London, there are many places to stay. The power of AirBnB and are great for finding accommodation on the day. However, you should consider booking the accommodation a few weeks beforehand, because it will save you money, a lot more money than you may think!

Find the hotels around the area that you are visiting and then order them by price, or by recommendation if budget isn’t a concern. Make sure to double check the hotel has working wifi in case you need to follow up on anything after the meeting.

Check Your Transport

Transport to the city and around the city is really efficient, but it can be a bit complex if you haven’t used it before. London is a massive city, so the best way to get around is definitely on the London underground, both in terms of efficiency and affordability, so when you arrive, familiarise yourself with the Tube

Before arriving in London, you look at the local train station to your main destination(s). This includes the train/tube station from your accommodation or where you arrive in London. That also includes the stations you may be using for your meetings.

If you are driving to London, you want to ensure you can park either near your meetings or accommodation if you are stopping over. There are many places, such as Care Park, that will offer safe and secure 24-hour parking, so you can park for the day and not have to worry about it again! Parking on the day is expensive in city centres, especially London, so ensure you pre-book your slot to make it more affordable.

Time For Relaxing

Once you have completed your day’s work and are getting ready to go back to the hotel, find a place to unwind. This is a great opportunity to see some of the city and bond with a client or socialise with the work colleagues you have gone with. Maybe go for a nice dinner before you head back or have a couple of quiet drinks to unwind.

London is a massive city and there are many places which you can visit. If your train is not until later on in the day to go back home, be a tourist for the morning. Find a great brunch spot and then head to one of the landmarks in the city.

There are many things you need to consider when going on a business trip to London. If you leave everything to the last minute, it is going to be costly, so you need to pre-book any accommodation or parking spaces.

Business trips are great. Not only do they get the employee out of the office but it also helps build a better relationship with a client or get the deal over the line. So, invest in these face-to-face meetings and it will be a lot more beneficial than you might think.


3 Types Of Sand Used In Prototype Casting

Jan. 18, 2022

Discharge Head

Achieving consistently good results by sand casting is a complex process. Did you know, for instance, that three different kinds of sand are used in sand casting? Why is a particular type of sand used at what time? When should each type of sand be used?

We will discuss the three types of sand that are commonly used in sand casting, their usage, as well as the why's and when's of the different types.

What Is Sand Casting?

Non-ferrous alloy casts are commonly produced through sand casting, also known as sand mold casting. The origins of this process date back more than 6,000 years, and it has undergone a number of changes and modernizations over the centuries. Nonetheless, the basic principles have not changed. There have been numerous replications of this technique in everything from small DIY shops to large commercial foundries. There are many variations of this process, but one of the main differences is the type of sand used. Here, you’ll discover the types of sand used in sand casting.

What Type Of Sand Is Used For Sand Casting?

  • Green Sand

Green sand is made by molding wet sand which can also be termed clay. When the metal is being poured, the sand mold is uncured. Using green sand for casting is quick and economical, as the sand can be reused. However, the sand is soft, meaning that if that mold collapses or shifts during casting, the cast will be useless. Even so, the process has proven reliable enough to last for centuries and it remains in wide use today.

Advantages of Green Sand in Casting

Green sand is one of the most cost-effective materials to mold.

Mold is less likely to grow from heat-torn material.

Due to the fact that green sand has not been baked, it will deform less in the course of molding.

Disadvantages of Green Sand in Casting

Green sand is usually not smooth, in other words, it is rough and crude.

With an increase in weight, the surface gloss of products made from green sand will be damaged.

Having a soft texture leads to casting defects like sand leftovers and sand holes.

  • Resin Sand

Mixing resin sand and heating the surface produces a solid mold with a smooth surface. Castings from a solid mold are less likely to produce defects, but they also cost more and take longer to produce. Unlike green sand molds, resin sand molds require more time, since each of them must be mixed and burned to produce the perfect mold. In addition to its high cost, resin sand must constantly be replaced, despite its ability for re-use. Therefore, the process tends to be more expensive.

Advantages of Resin Sand

Sand made from resin has excellent fluidity and compaction properties. It is possible to adjust the time of ejection using the above feature.

Resin sand becomes extremely strong after hardening. In this way, the resin sand will be less likely to be deformed in the casting process.

Disadvantages of Resin Sand

When resin sand is molded and cast, it releases an irritating odor.

Compared to green sand, resin sand is significantly more expensive.

  • Sodium Silicate Or Water Glass

Mold castings can be created using sodium silicate as well. By passing carbon dioxide through Sodium Silicate, it quickly turns from a liquid into a solid. This leads to sodium silicate dehydrating. It is particularly useful when castings need a cavity within them. In general, it can be mixed with other materials to create a solid core but must be mixed with other materials, so it can break down upon casting so the core can be removed. An incorrect mixture will result in the core becoming lodged inside the casting, making the casting defective.

Advantages of Sodium Silicate in Casting

It is non-toxic, colorless, and odorless.

During casting, it will not emit gases that are irritating or harmful.

No black or acid pollution is produced by sodium silicate.

Disadvantages of Sodium Silicate in Casting

It is difficult to collapse or separate.

Reclaiming the material from the waste is difficult. In addition, the water glass will cause pollution if it is not handled correctly.

Zen Young's foundries implement investment casting,sand casting,and lost foam casting processing. Specialized in foundry technologies and solutions for worldwide manufacturers of pump, valve, electric motor, compressors, etc. We supply various castings and mechanical parts for various industries. Contact us now!


Catering With Cultural Flavors

Florida Atlantic University Dining Services served up diverse and hearty cuisine for ICASA, a business services conference for Florida public university associates. Hosted on the FAU campus, Executive Chef Shane Lynch and the catering team spiced up the first evening’s dinner at FAU Stadium with a smorgasbord highlighting “Flavors of Diversity.”

Guests tasted a variety of traditional foods with a modern twist from Morocco, Peru, Spanish Basque and the United States. For dessert they were treated to FAU Dining’s “Brain Freeze” ice-cream bicycle, combining liquid nitrogen ice cream with Belgium waffles. Pops of color in the food and décor represented Chartwells’ five pillars, thematically tying everything together.

As the icing on the cake, FAU Dining Services’ catering and culinary teams closed this event with a scrumptious dinner at The FAU Club. Guests tried snapper bao buns and tuna tacos during social hour and treated themselves to oysters and handcrafted sashimi at the cold bar.

When it was time for entrees, Chartwells’ chefs delivered a lively interactive presentation at the carving and pasta stations. The evening ended on a sweet note with a delectable array of cakes, pastries, and artisan desserts and drinks.


Allocate Synonyms

assign antonym

He was the boy who had lost his penis to a botched circumcision at 8 months in 1966, was reassigned from boy to girl at 18 months with further surgery to remove testes and estrogen treatment beginning at age 12. Although John Money had described the reassignment as a success in the 1970s, Reimer went public with his account of continuing to have a male gender identity despite the reassignment to a female sex of rearing. The view of gender as a social construct, and gender identity as a result of nurture rather than nature reached widespread acceptance, at least in liberal, progressive, and academic portions of Western society. The primary goal of assignment was to choose the sex that would lead to the least inconsistency between external anatomy and assigned psyche . Observation or recognition of an infant’s sex may be complicated in the case of intersex infants and children, and in cases of early trauma.

This article needs more medical references for verification or relies too heavily on primary sources. Charge is what a cavalry does when it rushes towards enemy lines. When you are “in charge,” it means that you are in command of everything. And when you are “in charge of,” it means that you have a specific responsibility. QuickBooks And when you are excited about something, you “get a charge out of it.” If you like power, you might get a charge out of being in charge. Assign all of the pre-built exercises in this program component. Create an interactive version of this PDF students can complete on any device.Easel is free to use!

  • In the majority of births, a relative, midwife, nurse or physician inspects the genitalia when the baby is delivered and sex is assigned without ambiguity.
  • You may want to encourage students to take advantage of these components to improve their vocabulary acquisition.
  • For example, “he was unregistered” is ambiguous because it can mean either “he was not registered” or “his registration was removed”, while “he was deregistered” is not.
  • Most of the time the prefix de- is used for verbs while un- is used for adjectives.

Changes to clinical recommendations in the current millennium do not yet address human rights concerns about consent, and the child’s right to identity, privacy, freedom from torture and inhuman treatment, and physical integrity. Elementary Secondary Independent Learning Teacher Guided Unit Passage Unit Passage Through teacher-led instruction, guide students in using context clues to the meaning of the vocabulary words introduced in the passage. Through teacher-led instruction, guide students in using context clues Certified Public Accountant to the meaning of the vocabulary words introduced in the passage. Definitions Definitions In teacher-facilitated discussions, students will determine and clarify the meaning of unknown words introduced in the unit. In teacher-facilitated discussions, students will determine and clarify the meaning of unknown words introduced in the unit. Use these 32 multiple choice task cards to reinforce antonyms. A student answer sheet for students to record their answers is included along with an answer key for self-checking.

Antonym List

After two/three units of study, have students complete the Review. At the end of the year of study, you may choose to administer the Final Mastery assessment as a summative assessment to determine students’ level of vocabulary acquisition. Vocabulary instruction should provide students with opportunities to encounter words repeatedly and in a variety of contexts.

assign antonym

In some of these cases, it is clear that the child will face physical difficulties or social stigma as they grow up, and deciding upon the sex of assignment involves weighing the advantages and disadvantages of either assignment. Intersex activists have criticised “normalising” procedures performed on infants and children, who are unable to provide informed consent. These may complicate the sex assignment of a newborn and can result a phenotypical sex assignment that is inconsistent with normal genotype. Research demonstrates the importance of periodically involving students in games that allow them to play with new vocabulary. These games are always accessible to students and don’t need to be assigned. However, it is best practice to provide time for students to engage with these games in a teacher-guided approach.

The only rare exception would be completely and unambiguously virilized XX infants with CAH, who might be raised as male with genital surgery deferred. In the 1950s, endocrinologists developed a basic understanding of the major intersex conditions such as congenital adrenal hyperplasia , androgen insensitivity syndrome, and mixed gonadal dysgenesis. The discovery of cortisone allowed survival of infants with severe CAH for the first time. New hormone tests and karyotypes allowed more confident diagnosis in infancy and prediction of future development. Cases of trauma include the famous John/Joan case, where sexologist John Money claimed successful reassignment from male to female at age 17 months of a boy whose penis was destroyed during circumcision. After every three units of study, students complete the Review and Word Study.

Use our search box to check present tense, present participle tense, past tense and past participle tense of desired verb. In some cases, a company may be able to assign the rights of these instruments to another party. If you “assign” users to an operator, you may “remove” users from the operator. Connect and share knowledge within a single location that is structured and easy to search. Stack Exchange network consists of 178 Q&A communities including Stack Overflow, the largest, most trusted online community for developers to learn, share their knowledge, and build their careers. Here’s a list of opposite words from our thesaurus that you can use instead.

assign antonym

A list of common terms used in programming and their respective antonyms. The definition of a grant is something given for a particular purpose. A characterization of the occurrence of the actual unique values in a set of data or of the theoretical unique values of a random variable .

Words That Rhyme With Assign

The synonyms ascribe and assign are sometimes interchangeable, but ascribe suggests an inferring or conjecturing of cause, quality, authorship.

Effective implementation and achievement of your instructional goals for this program require planning. There are a variety of considerations regarding purpose, time, resources, and instructional models before implementing VWIE in your classroom. Designated is defined as appointed someone to a particular position or gave something a particular purpose.

Assign the pre-built and secured Unit Test assessment to determine students’ level of vocabulary acquisition. A preposition is a word or set of words that indicates location or some other relationship between a noun or pronoun and other parts of the sentence . A verb is a word that expresses an action or a state of being. Because action verbs and linking verbs are strong enough to be used in sentences all by themselves, they are called main verbs. Synonyms are words that are similar, or have a related meaning, to another word. They can be lifesavers when you want to avoid repeating the same word over and over. Also, sometimes the word you have in mind might not be the most appropriate word, which is why finding the right synonym can come in handy.

The Cumulative Reviews are designed to ensure students know a word’s definition, its relationship with other words, and its function in different contexts. Additional resources, such as Flash Cards and iWords, are provided as Study Aids for students. You may want to encourage students to take advantage of these components to improve their vocabulary acquisition. If the program includes a focus on ACT or SAT preparation, teachers will assign the Additional Practice exercises appropriate for those standardized assessments. The answers to these questions will guide your planning and help you customize vocabulary instruction in your classroom to maximize student outcomes. Whether you are a one-to-one school, only have access to a computer connected to a projection device, or can only access a computer lab every once in a while, you can implement VWIE in your classroom.

assign antonym

In my case I had to find a well-known word since it was intended for use in software code. It won’t be if a person reading will have to look refer to a dictionary to understand function’s name. Raise nearly all XX infants with functional ovaries as female.

To Set Aside Or Distribute As A Share

Raise most ambiguous XY infants with testes as male unless the external genitalia are more female than male, marked androgen insensitivity is present, and testes are absent or non-functional. Raise as male any XY infant with functional testes and normal androgen sensitivity but atypically formed or absent penis. Gender identity is set by 2 years of age and reassignment after that age should not be attempted unless requested by the patient. A Dutch report on gender registration states that sex registration was introduced in 1811 as an intrinsic component in population registration, due to gender-specific rights and responsibilities, such as military conscription. Many discriminatory provisions in legislation no longer exist, but the provisions remain for rationales that include “speed of identification procedures”.

This resource includes a self-grading quiz students can complete on any device.Easel by TpT is free to use! Interactive resources you can assign in your digital classroom from TpT.

The remaining additional practice does not generate a grade and is designed to be accessible for students to use at their own discretion. You may recommend that students who need additional support take advantage of the Practice Quiz and Practice Worksheet to improve their vocabulary acquisition. Vocabulary Workshop Interactive Edition is easy to use and will save you time. The program introduces students to word lists drawn from the vocabulary that they will encounter in their reading. Exercises assign antonym provide the right number of exposures to the new vocabulary, enhancing students’ vocabulary acquisition and improving their reading comprehension. Transgender is an umbrella term for people whose gender identities, gender expressions, and/or behaviors are different from those culturally associated with the sex to which they were assigned at birth. Controversies over surgical aspects of intersex management, have often focused on controversies regarding indications for surgery and optimal timing.

Additional Synonyms

However, intersex and human rights organizations have criticized medical models as they are not based on the consent of the individuals on whom such irreversible medical treatments are conducted, and outcomes may be inappropriate or poor. Anne Tamar-Mattis, for example, states that, “The true choice is not between early and late , but early surgery versus patient autonomy.” Human rights institutions now refer to such practices as “harmful practices”. A large proportion of XY infants born with cloacal exstrophy and raised as female from early infancy have requested reassignment to male gender in late childhood and adolescence. Public disclosure by intersex individuals and organizations about unsatisfactory sexual function or incongruent gender identity, and criticism of infant surgery for “psychosocial rationales”.

Synonym Of Antonyms For Assign

The Beginning-of-Year Pre-Test and the End-of-Year Post-Test are provided as a tool for schools purchasing this program with federal or other funds requiring evidence of student outcomes based upon the use of VWIE. Administer the Pre-Test before beginning VWIE instruction if data is needed to meet funding requirements. Administer the Post-Test at the end of the year’s VWIE instruction. These tools are beneficial in all classrooms and will help you determine the effectiveness of your implementation of the program. It is important to provide students with opportunities for ongoing review of new words to store the meanings of these words in their long-term memory and can use them to comprehend the text they are reading and studying. You may also use Custom Assessments to build assessments or exercises tailored to the instructional needs of your students. Accountability is important to students’ acquisition of new vocabulary.

A person’s credit rating or creditworthiness, as represented by their history of borrowing and repayment . The assignment of a person by an official to perform a duty, such as a presidential appointment of a judge to a court. The definition of appoint is to choose an individual for a position or office. Our site contains antonyms of assign in 10 different contexts. We have listed all the opposite words for assign alphabetically.

The definition of an authorization is something that allows an action or event or an act of allowing some action or event to happen. To ascertain the exact rate of the gain or loss of as compared with true time. To name someone as a candidate for a particular role or position, including that of an office.

If sex assignment and gender identity do not align, the person may be transgender or gender non-conforming . The sex assignment of an intersex individual may also contradict their future gender identity. Transgender is an umbrella term for people whose gender identity and/or gender expression differs from the sex they were assigned at birth (Gay and Lesbian Alliance Against Defamation , 2007). There have been cases where a child with an intersex condition recording transactions has made known that they are of a gender identity that does not align with the sex they were assigned at birth, and has requested reassignment. Examples of this have occurred in adolescents with several forms of CAH and 5-alpha-reductase deficiency. Approximately 1 in 20,000 infants is born with enough ambiguity that assignment becomes a more drawn-out process of multiple tests and intensive education of the parents about sexual differentiation.

In such cases, sex assignment is generally taken to require medical treatment to confirm that assignment, but this is disputed in part due to the human rights implications of such treatment. It is best practice to discuss words and their definitions in class.

Any reassignment after the first month or two is no longer considered an “early reassignment”. Non-disclosure of the intersex condition to the individual on whom sex assignment treatment was conducted. Before the 1950s, assignment was based almost entirely on the appearance of the external genitalia. Vocabulary in Context is assignable additional practice and may be used if time or instructional focus permits.