Why Disclosure of Emissions from Deforestation Protects Investors

Deforestation Financial Risks

Emissions from deforestation are increasingly being seen as a material risk for investors. This isn’t only because the emissions released by deforestation drive climate change, threatening global economic stability and the lives of millions. It is because society’s reactions to climate change, known as climate transitions, will impact a wide range of holdings investors may hold. Each tree felled or burned contributing to climate change will potentially hold a bigger impact on an investor’s strategy as government policies, corporate climate goals, and consumer preferences react to a warming world.

Forest loss and degradation is a major driver of climate change responsible for 11% of emissions. As such, more governments and consumers are changing how they respond to companies that contribute to climate change. For corporations whose long-term business plans rely on further deforestation, they are accepting additional risks that investment in their business plans will fail to provide returns to investors.

Financial risk from deforestation is why Climate Advisers requested that the U.S. Securities and Exchange Commission (SEC) address deforestation emissions specifically in its final climate emissions disclosure transparency rule. The SEC’s climate emissions disclosure rule aims to provide reliable and useful data about corporations’ climate emissions so investors can navigate a world with growing climate-related financial risks.

In our comment to the SEC, we highlighted why deforestation-related climate emissions in particular need to be addressed in the final rule and disclosed to investors for their benefit. Our recent analysis found that 40% of US GDP is exposed to deforestation-specific risks.

Why should the SEC include special rules around deforestation in their final rule? We’ve laid out the basis for why deforestation is a material risk to investors.

  • Why deforestation matters in the context of climate change
  • How deforestation emissions pose financial risks for investors
  • Additional financial risks for investors from climate change
  • What the SEC can do about deforestation in its final rule on emission disclosures

The nature of climate-related financial risks

Climate-related financial disclosures would be ineffective in protecting investors without specific requirements directed to agriculture, forestry, and other land use (AFOLU). Globally, the forest, food, and land sector is responsible for almost a quarter (23%) of net anthropogenic global greenhouse gas (GHG) emissions, according to the Intergovernmental Panel on Climate Change (IPCC). Moreover, in the food sector alone, if activities in the pre- and post-production systems such as processing, distribution, consumption, and food waste are included, the contribution to net anthropogenic global GHG emissions from AFOLU emissions could be as high as 37%.

A major reason that the forest, food, and land sector contributes so substantially to anthropogenic GHG emissions is through deforestation, which alone is responsible for 11% of global emissions.  Maintaining healthy forests and reforesting degraded forest land are critical to achieving the goals of the Paris Agreement and the United Nations Sustainable Development Goals. Every IPCC pathway leading to average temperature increases of 1.5 degrees Celsius or less compared to pre-industrial temperatures is premised on no new deforestation after 2030. In fact, an estimated 16% to 30% of climate mitigation needed to limit global emissions to 1.5-2 degrees Celsius is based on halting deforestation by 2030 and a quarter of the 2030 climate mitigation promised in countries’ Nationally Determined Contributions comes from land-based mitigation options.

Combating deforestation is so important that the AFOLU sector is the only economic sector with its own chapter in the Paris Agreement. Political support for conserving and restoring forests globally was also on display in 2021 when President Biden joined more than one hundred and forty world leaders in endorsing the Glasgow Leaders Declaration on Forests and Land Use, which committed nations representing more than 90% of the world’s forests to ending natural forest loss this decade.

The impacts of deforestation are diverse and far reaching, and their emissions have a cascading effect on climate change resilience going forward for the following reasons:

  1. Indigenous People and Local Communities (IPLC): Displacement of Indigenous People risks the loss of traditional cultures and valuable expertise in maintaining healthy ecosystems that aid in mitigating climate change. Receding tropical forests have already led to frequent land disputes between commodity producers and IPLCs. Illegal encroachment onto Indigenous territories and land insecurity have also heightened violence against environmental defenders defending their homes. IPLCs are the most effective protectors of forest carbon and biodiversity, which is vital for investors given that intact ecosystems are worth $44 trillion to the global economic sector. The traditional knowledge of IPLCs continues to be the basis for medicines and foods of incalculable value. All climate mitigation measures should include these groups as important partners because at least 36% of the world’s large, unbroken swaths of natural forests, known as “intact forests,” are held by Indigenous People, along with about 80% of remaining biodiversity.
  1. Carbon storage: Terrestrial ecosystems release 10% to 20% of total global CO2 to the atmosphere and sequester 30% annually. Of this, gross emissions and sequestration in the tropics is about four times larger than temperate and boreal ecosystems combined. If deforestation emissions are conflated with those of other sectors in climate-related financial risk calculations, their role in sequestering carbon in future years is likely to be undervalued.
  1. Illegal activity: The lack of transparency into complex supply chains provides a cover for illegal activities, including deforestation, intentional fires, and human rights abuses. Most deforestation in the developing world linked to internationally traded commodities is illegal (violates local law) or is connected to organized crime. Corruption, bribery, money laundering, illegal logging, and other illegal acts referred to as “forest crimes” are common in the forest and land use sectors in many developing countries. The potential consequences, which can be widespread, include social conflict, injustice, poverty, economic stagnation, and carbon emissions.
  1. Biodiversity loss: Habitat loss is causing a biodiversity crisis and threatening valuable ecosystem services. Nowhere is this more apparent than in tropical forests, which are home to more than 80% of animal, plant, and fungi biodiversity. Wildlife populations, including mammals, birds, fish, amphibians, and reptiles, have been reduced by 68% since 1970 and about one million animal and plant species face the threat of extinction. The agriculture sector is responsible for about 80% of deforestation globally, but it is also among the sectors most reliant on ecosystem services, particularly pollination. Pollinator loss is currently placing USD 235 billion to USD 577 billion of annual agricultural production at risk. The economic cost of biodiversity loss is already estimated to be between USD 2.0 trillion and 4.5 trillion per year.
  1. Soil Degradation: Soil degradation costs an estimated USD 400 billion every year and has been linked to a potential 12% reduction in global food productivity and a 30% increase in food prices by 2030. Degradation is driven by the loss of organic matter and soil erosion, excessive use of fertilizers and pesticides, other types of contamination, salinization, acidification, and a loss of genetic diversity. Soil erosion, for example, is a major consequence of tropical deforestation because soil can no longer rely on intricate root structures to hold it in place or canopies to protect it from drying in the sun. Although recently deforested land may support productive agricultural activity, soil fertility decreases over time as topsoil is blown or washed away. For example, a study of deforested land in Iran measured a 70% to 82% drop in soil productivity of cultivated land and a 50% drop in organic matter overall.
  1. Global water cycles: As deforestation and land use change lead to the conversion of tropical forests to grasslands or savanna, less moisture is stored and released into the atmosphere. Thus, the hydrological cycle is disrupted with a major ripple effect on precipitation patterns around the world. Climate scientists have predicted a tipping point when 20% to 25% of the Amazon is cut down, warning that the rainforest’s hydrological cycle will be unable to support itself and the biome will convert to a savanna. Since the Amazon provides water to a region in South America responsible for 70% of the continent’s GDP, the risk to the continent’s financial sector is sizeable. This problem is not limited to South America. Deforestation in the Amazon could lead to a 25% reduction in rainfall in Texas, for example. Meanwhile, deforestation in Central Africa could reduce rainfall in the U.S. Midwest by 5% to 35%, and deforestation in Southeast Asia can influence rainfall in Europe.
  1. Clean Drinking Water and Flood Mitigation: Deforestation and land use change can have devastating implications to the availability and quality of clean drinking water to populations both locally and regionally. Forested land covers about 31% of watersheds worldwide and provides essential storage and filtration services. By absorbing nutrients and sediment, forests provide clean drinking water to large populations in urban centers downstream and can reduce infrastructure investments and water management costs. By storing water in roots, branches, and canopies, forests can also reduce the intensity of flooding and mitigate irregular rainfall patterns. Conversely, deforestation and land use change can lead to devastating flooding, increased need for costly infrastructure, and significant pollution because of the loss of ecosystem services and preventing the previously discussed runoff of agricultural fertilizers and pesticides.
  1. Infectious disease outbreak: Deforestation and land use change lead to habitat loss and increase the likelihood of zoonotic infectious diseases that result from proximity between humans and animals. Since infectious disease emergence is driven primarily by land use change (31%), followed by agriculture (15%), commodity-driven deforestation is a primary risk factor for future pandemics. Furthermore, according to some studies, 75% of emerging infectious diseases are zoonotic compared to 60% of all existing infectious diseases, which indicates that habitat loss resulting from land use change is playing an increasing role in infectious disease emergence over time. The Covid-19 pandemic has provided some insight into the potential costs of infectious diseases to both humans and the economy. In addition to the millions of lives lost, as early as October of 2020, the International Monetary Fund estimated that the pandemic would cost the global economy USD 28 trillion in lost output.  Without halting deforestation, the likelihood of society being exposed to more costly zoonotic diseases we are unprepared to manage will continue to increase.
  1. Pollution: In addition to absorbing CO2, trees absorb toxic chemicals and filter the air providing noteworthy benefits to human health. Despite only covering 6% of land, tropical forests produce 40% of the world’s oxygen alongside the absorption of harmful pollutants. Furthermore, particulate matter from fires linked to longer dry seasons and land clearing for agricultural use has been shown to increase pollution-related hospitalizations by 65% and to cost the Brazilian public healthcare system the equivalent of USD 660,000 during the 2019 fire season. With wildfire seasons increasing in severity and longevity, driven by climate change and the effects of global deforestation, a major step in mitigating the potential pollution impacts must include curbing global deforestation.
  1. Environmental refugees and local conflict: By depleting the ecosystem services that millions of people rely on for food, clean water, and energy, deforestation and land use change are likely to create climate change refugees and exacerbate geopolitical conflict. The inevitable floods, droughts, and repeated crop failures are likely to destabilize economies as they become unable to support their populations. Over 1.2 billion people could become climate change refugees by 2050. The world is already experiencing climate refugees and will continue to see an increase of this tragedy in the near-term. For example, the 90% reduction in the size of Lake Chad has provided some insight into the scale of potential migration patterns with 2.4 million displaced people and increased geopolitical conflict in the region.
  1. Medical Innovation: Future medical breakthroughs are dependent on the conservation of plant biodiversity today. The market for medicinal plant products is valued at over USD 100 billion and approximately 80% of the global population is reliant on botanical drugs. Moreover, a quarter of modern medicine originates in tropical forests. Yet, scientists have only scratched the surface of cataloging and understanding the vast biodiversity of the world’s forests. It is estimated that up to 100 species of animal and plant species disappear per day as tropical forest habitats are destroyed. A loss of plant biodiversity before medicinal impacts are understood is likely to lead to adverse impacts on human health and a slowdown in innovation in the pharmaceutical industry globally.


(JSL039T) 20" 36V 250/350W rear motor steel folding ebike electric bicycle with best affordable price

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Entertaining Indian Films You Need to See

21 July 2022

Entertaining Indian Films You Need to See

Bollywood dominates the global cinema market and is still expanding. Regarding productivity and audience, Bollywood surpasses Hollywood in the United States, and its films are popular with viewers worldwide.

But what precisely makes Bollywood movies so epic and unique? Some claim that all good Bollywood movies follow a tried-and-true formula: romance, music, and narrative twists you can guess. When you have access to the right DTH plans, you can have all the fun you want.

In any case, these movies are definitely worth the two or more hours you'll need to set aside in your schedule to watch them. Whether you enjoy romance, tragedy, or humour, this vibrant world has a movie for you. Here is a list of Bollywood films you must add to your watch list, ranging from sweeping historical dramas to action-packed blockbusters.

Some Awesome Bollywood Movies You Must Check Out

· Insaf Ka Tarazu: The title Insaf Ka Tarazu, which means "the balance of justice," refers to the film's in-depth exploration of what justice is and how to achieve that balance. In this gripping story, a wealthy man, Ramesh, meets Bharti, a beauty queen, and invites her onto his yacht. Bharti accuses him of sexually assaulting her. Two years later, when he is killed, Bharti is detained and placed on trial for the crime.

· Love Aaj Kal: It's fitting that this love story premiered on Valentine's Day. The drama centers on Zoe and Veer, a young couple who meet in a pub and click right away. However, their relationship struggles to advance since their work and life goals keep tearing them apart.

· Mother India: This sweeping drama centers on a lady who, after her husband abandons the family in disgrace, is left to care for her two sons alone. Mother India, regarded as one of the most prestigious films in Bollywood history, was one of the most expensive Hindi films ever produced at the time it was made.

· Dil Chahta Hai: This is a comedic romance, mixed with drama. It's a coming-of-age story. The National Film Award-winning Hindi feature film Dil Chahta Hai showcases the experiences of three college buddies as they struggle to find love and success after graduation.

· The Lunchbox: One of such moving films, The Lunchbox offers a tale you didn't know you indeed needed until you've seen it. In the movie, a character named Ila makes a special lunch for her spouse. The lunch is served to a widower instead. Ila adds a note to the next lunch she makes (which also goes to the widower) after her husband fails to mention his special meal, and an odd friendship develops.

So, if you want to spend some time relaxing while getting entertained after a long day of work or weekends, these are some movies you can check out. If you still haven’t checked out d2h, then get a connection and browse for the right DTH recharge plans to watch these movies.


Finance & economics | Roll up, roll up and write down

How can governments recover faster from insolvency?

Proposals from the G20 and the IMF may ease the pain for both borrowers and lenders

“THE PROCEDURES for resolving an international debt crisis”, wrote Alexis Rieffel, a former American Treasury official, in 1985, “resemble a three-ring circus”. In the first ring, the bankrupt country negotiates with the IMF, which must decide how much the country can repay and what belt-tightening it must endure. In the second ring, the country asks for leniency from other governments to whom it owes money. And in the third, it seeks a “comparable” deal from private lenders.

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The circus sometimes, however, struggles to hold it all together. After Argentina defaulted in May, for example, the IMF failed to play its customary role in the first ring. It could not provide new supervision and finance, because the country was still reeling from the failure of its previous IMF bail-out. The second ring has also suffered from some absent performers. In the past decade China has become a far bigger lender to poor countries than other governments combined (see chart 1). But it is not a member of the Paris Club, which has tended to oversee debt renegotiations between countries and their official creditors. As for the third ring, when the Latin American debt crisis struck in the early 1980s, it took commercial lenders (and their governments) almost seven years to find a lasting solution. The juggling went on and on.

Many fear another series of defaults is looming. Government revenues and export receipts have plunged in many poor countries (though efforts by America’s Federal Reserve to calm financial panic have lowered their cost of borrowing). On November 13th Zambia became the sixth country this year to default on its bonds. Eight spend over 30% of their fiscal revenues on interest payments, reckons Fitch, a rating agency, more than in the early 2000s when Bono and other debt-relief campaigners were at their clamorous best. Fitch gives 38 sovereigns a rating of B+ or worse, where B denotes a “material” risk of default (see chart 2). According to its projections, governments with a junk rating—BB+ or worse—may soon outnumber those classed as investment-grade.

Will the circus handle any new crisis better than it did in the 1980s? In some ways its task is even harder now. Poor countries owe a wider variety of liabilities to a broader range of creditors. For many emerging economies, bonds have eclipsed bank loans. And loans themselves are far from uniform. Some are secured against state assets, such as a stake in a public enterprise, or oil revenues; the creditor might prefer to seize the collateral rather than write off the debt. Others are syndicated, or parcelled out among many banks, which means that no single creditor can forgive the loan at its own discretion.

This gnarly mix of instruments is matched by an equally tangled bunch of creditors: public, private and everything in between. In April, for example, the G20 group of big economies called on member governments to provide a repayment holiday on loans to the world’s poorest countries. China was unhappy that private creditors did not share in the effort. Others complained that China Development Bank, which is owned and directed by the state but not synonymous with China’s government, did not take part.

There has, however, also been progress. On November 21st-22nd, G20 leaders will sign off on a “common framework” for renegotiating debts with the world’s poorest countries. The framework, in effect, extends the principles of the Paris Club to those G20 members who are not already in it, widening the second ring of the circus. It applies only to countries with unsustainable debts, and any borrower that receives relief from the G20 must seek a similar deal from other creditors. Because all lenders must do their bit, little hangs on whether they are classified as official or private. That is perhaps why the framework has met little opposition from China.

There has been progress in contracts as well as clubs. After Argentina defaulted in 2001, it offered to exchange its unpayable bonds for new securities with easier terms. Some bondholders rejected the deal, seeking full payment in New York’s courts instead. That made life harder for both Argentina and its other creditors. Since 2003, most bonds issued under New York law have contained “collective-action clauses”, which compel all bondholders to go along with any deal accepted by the majority. Such clauses helped Ecuador resolve its default this year with “hardly any real grumbling”, notes Clay Lowery of the Institute of International Finance, a bankers’ association. They also helped Argentina reach a deal with its main bondholders in August (albeit with “a fair amount of grumbling”).

A review of the “architecture” for resolving sovereign debt, published by the IMF in September, pondered other contractual innovations that might ease future restructurings. Lenders might insist on wider use of “negative-pledge clauses” that prevent a borrower pawning vital assets as collateral to other creditors. Syndicated loans might add “yank the bank” provisions that allow a lender to be kicked out of the syndicate if it blocks a deal. The fund is also paying renewed attention to “contingent” debt instruments that are more sensitive to the ups and downs that befall poor countries. Barbados, for example, has issued bonds that repay less in the event of an earthquake or tropical cyclone.

One idea, proposed by Ben Heller and Pijus Virketis of HBK Capital Management, an investment fund, is “bendy bonds”. In most cases, these would behave like ordinary bonds. But in a crisis the issuer could extend the maturity and defer interest for a couple of years in return for paying additional interest at the end of the bond’s life. The issuer could benefit from the kind of payment holiday envisaged in the G20’s April initiative without any help from the great powers. As the long history of debt restructurings attests, “fixed” income liabilities are often anything but. Solemn commitments to pay in full and on time cannot always be kept. Lenders and borrowers alike might therefore welcome instruments that specify up front when and how fixed income will become more flexible.

This article appeared in the Finance & economics section of the print edition under the headline "Roll up, roll up and write down"

The China strategy America needs

From the November 21st 2020 edition

Discover stories from this section and more in the list of contents

Explore the edition

More from Finance & economics

Why central banks are stockpiling gold

The metal offers a hedge against inflation—and a way to circumvent sanctions

Developing countries take tax talks to the UN

They want more of a say


Coelogyne Bird in Flight

coelogyne bird in flight orchid care key

Coelogyne Bird in Flight is a hybrid between Coelogyne Usitana and Coelogyne Lawrenceana. The flower opens with very dark lip colors and then it fades to lighter orange, rusty red/orange. The petals and sepals range from a very light green to almost white. The spike generally produces one flower at a time, but every three weeks or so it produces another flower on the same spike. The inflorescence can bear 20-30 or more flowers over a long period of time, with only one or two blooms open at a time.

CARE key

coelogyne bird in flight orchid care key

0 to 2300 M

water 4.png
somewhat bright sunlight.png
high humidity.png

A foot-candle, is a measurement of light intensity. One foot-candle is defined as enough light to saturate a one-foot square with one lumen of light. Rather than measuring the amount of light that leaves a light source (lumen output), foot-candle measurements focus on the amount of light that reaches a surface area. Well, lets not get too much technical.

Let's understand it quickly with some comparisons,

  1. Typical foot-candle reading for unobstructed sunlight - 10,000 fc ( imagine you are at the beach under clear sky )

  2. Sunshine on a cloudy day - 100 fc ( imagine a day when you feel its about to rain but there is diffused sunshine )

  3. Residential space (living) - 5-40 fc ( when your doors and windows are shut as you are using air conditioner )

That means, when we use a 50% shade net under the clear sky conditions, it is around 5000 fc that the surface receives. Under all weather conditions the 50% shade net on an average will allow 2500 - 3000 fc to fall on a square-foot of surface area.

And for those who have a lux meter (light meter) at home, remember that One foot-candle equals 10.76 lumens/lux.

Relative humidity is the ratio of how much water vapour is in the air and how much water vapour the air could potentially contain at a given temperature. It varies with the temperature of the air: colder air can hold less vapour. So changing the temperature of air can change the relative humidity.

Relative humidity is normally expressed as a percentage; a higher percentage means that the air–water mixture is more humid. At 100% relative humidity, the air is saturated.

Relative humidity only considers the invisible water vapour. Mists, clouds, fogs and aerosols of water do not count towards the measure of relative humidity of the air.

  • The factual information given on dynamic variable such as watering is irrespective of the media used for planting the orchid. That is, Imagine that the watering frequency given above is for a bare rooted specimen of this variety. Therefore, this watering frequency varies and has to be followed in accordance with the media used. More the quick drying tendency of the media more frequent the watering shall be. For example, charcoal/wood dries quicker than coconut husk/sphagnum Moss.

  • The elevation (above sea level) provided is to understand the level of Humidity available at the original natural habitat of the particular Species or Hybrid. Generally, higher the altitude, lesser will be the natural levels of relative humidity available for your plant to utilise. Thus, there might be a need to improve humidity artificially.

  • The best things about orchids are, these wonders are capable of adapting to any extreme changes in weather conditions provided their time, energy and initial support to assimilate to the new growing conditions. Thus there is no universal care guide for Orchid care. Their potency cannot be contained within these generalisations and the factual information. But we hope these details help you understand the basic requirements and natural demand of a particular Species or Hybrid.

Except as permitted by the copyright law applicable to you, you may not reproduce or communicate any of the content on this page, including files downloadable from this website, without the permission of the copyright owner.

© Guna Orchids [2019-2022]. All Rights Reserved.


The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC) and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS). Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

1. Introduction

Muscarinic acetylcholine receptors (mAchRs) are integral membrane proteins that belong to the rhodopsin family of G-protein-coupled receptors (GPCRs). These proteins play a pivotal role in the regulation of many physiological functions both in the central and in the peripheral nervous systems [1]. Like other GPCRs, these proteins exhibit a basal activity that is increased by the action of its natural agonist acetylcholine. In the central nervous system, mAChRs are known to regulate motor control, temperature homeostasis and are involved in the process of memory and learning whereas, in the peripheral system, they induce cardiovascular activity, smooth muscle contraction, gland secretion, regulation of cardiac activity, decrease blood pressure, and meiosis. The physiological actions the natural agonist acetylcholine are mediated by at least five subtypes of receptors known as M1-M5 that exhibit high sequence identity across mammalian species and exhibit different tissue distribution [2, 3].

Binding of agonists and competitive antagonists to GPCRs occurs at the orthosteric site, a hydrophobic pocket located at the extracellular side of the helix bundle that is highly conserved among the members of a subfamily [4]. However, the binding of diffusing ligands to the orthosteric site may be modulated by residues located at the extracellular loops that flank the entrance [5]. Interestingly, while residues defining the orthosteric binding site are well conserved within the different GPCR subfamilies, those of the extracellular loops are remarkably diverse within a subfamily, providing a good possibility to design selective allosteric antagonist [6]. Although a wealth of information about these receptors has been accumulated in the past, there are still many questions open. Issues like the effect of the same mutation on the function of different mAchRs subtypes or the actual mapping of the binding site, as well as the mechanism of activation, are poorly understood [7]. A deeper understanding of the structure-activity relationships of the different subtypes will help designing new selective ligands with various pharmacological profiles that may be useful for therapeutic intervention. In this sense, due to the scarcity of crystallographic structures available, molecular modeling methods can provide a deeper insight into the flexibility and the role that extracellular loops may play in ligand recognition [8].

In the present work, we focus on the human M3 muscarinic acetylcholine receptor (M3R), which is involved in modulation of neurotransmitter release, temperature homeostasis, and food intake in the central nervous system, as well as in the induction of smooth muscle contraction, gland secretion, indirect relaxation of vascular smooth muscle in the peripheral nervous system [1]. M3R is arranged in the distinctive seven-transmembrane (TM) helix bundle architecture like all GPCRs, with its N-terminus located on the extracellular side of the plasma membrane and the C-terminal tail in the cytoplasmic side. Helices are connected by intracellular and extracellular loops of different lengths exhibiting preserved features of the rhodopsin family, like the two highly conserved cysteine residues C221 and C141, responsible for a disulfide bond between the second extracellular loop (ECL2) and the TM3 helix [9].

We undertook a combined approach involving computational and site-directed mutagenesis studies to further dissect M3R-ligand interactions with the aim of getting a deeper insight into specific structural features that can modulate the binding of the competitive antagonist N-methylscopolamine (NMS) to the orthosteric site of the receptor. For this purpose, we constructed an atomistic model of the M3R that was further refined using extended molecular dynamics (MD) simulations of the protein embedded in a lipid bilayer. The analysis of the MD trajectories provides a better understanding of the structural features that characterize the ligand-receptor interaction at the orthosteric pocket, the dynamics of the extracellular loops, as well as its putative involvement in ligand binding [10].

Present modeling studies suggest that residues F222 located in the ECL2 and T235 located at the edge of TM5 participate in the recognition of NMS. The former had already been shown to be important for the binding of allosteric modulators [11] but has not yet been shown to modulate the binding of ligands to the orthosteric site. On the other hand, the latter had already shown to be important in conferring biding specificity to the physiological ligand acetylcholine [12, 13]. Accordingly, in order to demonstrate the prediction capabilities of the model, we designed and expressed mutants F222A and T235A. Moreover, in order to have a reference, other residues of the ECL2 not expected to modulate the binding of NMS to M3 according to the atomistic model but playing key roles in the binding of allosteric modulators such as gallamine and alcuronium in the M2 receptor [14] were also investigated. Thus, the following mutants Y209F, K213 V, and E228N were also constructed and expressed.

Saturation binding assays were performed to determine the affinity of [3H]-NMS for these mutants. Dissociation assays on the mutants were also performed in order to get insight about the kinetics of the binding process. These results suggest that residues F222 and T235 modulate the binding of NMS [15] that show a slight decrease of its binding affinity in contrast with the other mutants constructed and tested.

2. Materials and Methods

2.1. Theoretical Methods
2.1.1. Construction of a M3 Receptor Model

A model of the M3 receptor was constructed by homology modeling using the crystal structure of bovine rhodopsin as a template (entry 1GZM of the Protein Data Bank) [16] by means of the Modeller software [17]. The N-terminus as well as a long stretch of the C-terminus was not included in the model, due to the low sequence identity exhibited between the two sequences and that it does not presumably influence the model for the purpose of the present work. Residues 264–479, located in the third intracellular loop (ICL3), were also removed and substituted by stretch of alanine residues resulting into a shorter loop with the same length as that found in rhodopsin. The model includes the full remaining intradiscal and cytoplasmic loops, the conserved disulphide bridge between the cysteine residues located in TM3 and ECL2 (involving C141 and C221). All amino acids were modeled in the protonation state; they would have as free amino acids in water at pH 7 yielding a net charge of +17. This net charge is later compensated in the simulation box by adjusting the balance between sodium and chloride ions to give an electroneutral system, as described in detail later.

Fifty different models were generated following this procedure. These were subsequently ranked ordered using a scoring function based on structural compactness and embedded in Modeller [17]. Structures were processed by Procheck in order to assess the stereochemical quality of the models generated [18]. The structure finally selected exhibits the highest score with the minimum number of residues out of the preferred regions of the Ramachandran plot.

Although the constructed model provides a reasonable first approximation of the receptor, it still exhibits some limitations for its use in drug discovery [19]. First, consideration of the rearrangement due to the bound ligand and, second, the effect of the lipid bilayer on the protein structure are disregarded in the model constructed using a crystal structure. These two effects can be accounted for in the model by means of molecular dynamics simulations.

2.1.2. MD Simulations

In order to understand the structural features of M3R, four 50 ns molecular dynamics simulations with the receptor embedded in a lipid bilayer were carried out. Specifically, four trajectories were run: with and without the antagonist NMS bound into the orthosteric binding pocket and using two different bilayer compositions: a one-component palmitoyl-oleyl phosphatidylcholine (POPC) and a two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC/POPS), where POPS lipids were added at the cytoplasmic surface of the M3R at a POPC/POPS 10 : 1 ratio. The former bilayer was considered in the present paper because it has been widely used in MD simulations; its available force field parameters are known to be carefully calibrated and it is a good representative of the membrane composition together with the fact that its gel/liquid-crystalline phase transition is above the temperature of the simulation (300 K). Selection of the latter—a two-component bilayer—was done in order to investigate the role of the inclusion of anionic lipids to neutralize the net positive charge located in the cytoplasmic region of the M3 receptor.

The M3R was placed in the center of an equilibrated POPC box of dimensions 8.5 × 8.5 × 10 nm (XYZ) containing 256 molecules, a thick layer of water molecules on each side of the bilayer, and NaCl ions at physiological concentration of 0.2 M taken from a previous study [20]. Overlapping molecules were removed following the procedure described previously [20], in which all water molecules, with oxygen atoms closer than 0.40 nm to a nonhydrogen atom of the protein, as well as all lipid molecules with at least one atom closer than 0.25 nm to a nonhydrogen atom of the protein, were removed. This resulted in final system containing 189 lipids and ca. 14 600 water molecules. For convenience, the system is organized in such a way that the bilayer plane was oriented on the XY plane. For the construction of the POPC/POPS box, the 10% of the POPC molecules exhibiting the largest electrostatic potential were replaced by POPS molecules. For those simulations with N-methylscopolamine (NMS) bound into the orthosteric site, the ligand was manually docked, using information provided by site-directed mutagenesis on relevant residues involved in the binding [21]. Figure 1 shows the system showing a cartoon of the M3 receptor with NMS bound, surrounded by POPC molecules. Water molecules have been removed for clarity.

Calculations were carried out by means of the GROMACS 3.3.2 package [22] using periodic boundary conditions. The all-atom OPLS force field [23] was used for all the molecules of the system except for the lipids, for which a specific parameterization was used [24]. This combination has been previously employed [20]. Systems were subjected to periodic boundary conditions in the three-coordinate directions. Calculations were done at 300 K using separate thermostats for the protein, water, ions, and lipid molecules. The time constant for the thermostats was set to 0.1 ps, except for water, for which a value of 0.01 was used. Equations of motion were integrated using the leapfrog algorithm using a time step of 2 ps.

For equilibration, the system was subjected to 0.5 ns MD simulation to allow for the removal of voids present between the protein and the lipid or water molecules. The simulation was performed with the atomic coordinates of the protein restrained to their crystallographic positions and allowing the three periodic box dimensions to change size according to a pressure of 0.1 MPa in each coordinate direction [20]. Next, the restrains were released and the four MD trajectories containing rhodopsin were computed up to 20 ns each. As a reference, eight additional simulations were performed without protein and either with or without ions lasting 20 ns each. For all simulations, coordinates were collected every 10 ps and were stored for further analysis. In all cases, the first 10 ns were considered as equilibration period, and therefore they are not included in the analysis. All the bonds in the protein and lipid molecules were kept frozen using the LINCS algorithm. Electrostatic interactions were treated using the particle mesh Ewald summation procedure [25] and nonbonded interactions were computed using a cutoff of 1.0 nm.

3. Construction, Expression, and Binding Assays for Mutant M3R

3.1. Materials and Mutant Construction

1-[N-methyl-3H] scopolamine methyl chloride ([3H]-NMS; 84 Ci/mmol) was purchased from Perkin Elmer. Oligonucleotides were designed and obtained from Sigma Aldrich (for the sequence of the primers, see Figure 1(S) of Supplementary Materials available online at http://dx.doi.org/10.1155/2012/789741). The Quick Change kit was from Stratagene (La Jolla, CA, USA). Atropine was purchased from Sigma Aldrich.

Residues F222 and T235 of the human M3R were mutated to alanine using the Quick Change method. Mutant receptors, cloned into the pCD expression vector and confirmed by dideoxy sequencing, were transiently expressed in COS-7 cells by electroporation (Bio-Rad Gene Pulser, 15 μg of DNA, 260 V, 960 μF). In the case of the T235A mutant, its expression levels were improved by treatment of the cultured cells with atropine (10-6  M) for 48 h, before washing and harvesting for membrane preparation. The ability of atropine to act as a “pharmacological chaperone” has been described previously [26]. After transfection, media was removed and cells were washed twice with phosphate buffered saline (PBS) (in the case of mutant T235A, four additional washes were needed to remove atropine), then harvesting buffer (20 mM Hepes, 10 mM EDTA) was added to the flasks. The cells were incubated for about 10 min and then scraped from the flask. The cells were pelleted and homogenized in a glass pestle (×20 plunges on ice). This homogenate was transferred to JA-17 tubes and centrifuged at 17,000 rpm at 4°C for 30 minutes. The pellet was resuspended in storage buffer (20 mM Hepes, 0.1 mM EDTA). The cells were homogenized on ice using a Polytron homogenizer setting 12,2 × 15 s, the membranes resuspended, snap frozen, and stored at −80°C. Protein concentration was determined using the Bradford assay [27].

3.2. Radioactive Ligand Assays

[3H]-NMS saturation binding assays were performed by binding [3H] NMS (0.01–3 nM) to membrane preparations (10 μg/mL of membrane protein). Binding was measured at 30°C in a buffer containing 20 mM Hepes, 100 mM NaCl, and 1 mM MgCl2, pH 7.5, using an assay volume of 1 mL and an incubation time of 2.5 h. Nonspecific binding was determined with 1 μM atropine. Assays were performed in quadruplicate. The binding reaction was terminated by rapid filtration on a Brandel cell harvester (Brandel Model M-245, Serial 9219). Dissociation kinetic assays were carried out to investigate the time course of [3H]-NMS dissociation from the receptor. COS7 cell membranes expressing the M3R (10 μg/mL) were equilibrated with 0.2 nM [3H]-NMS in 1-mL total volume of buffer containing 20 mM Hepes, 100 mM NaCl, and 1 mM MgCl2, pH 7.5 for 60 min at 30°C. Atropine (10 μM) was added at various time points to prevent radioligand re-association to the mAchRs. Data analysis was carried out by means of Graph Pad Prism 3.0 (Graph Pad Software, San Diego, CA, USA). Saturation binding data were fit with hyperbolae (one-site binding); Bmax and Kd values were derived from these curves. Dissociation kinetic data were normalized and fit to for a monoexponential decay curves. Experiments were repeated 4 times. Log affinities were tabulated as mean ± SEM. Statistical comparison of affinity and dissociation rate constants for mutants and WT M3R controls were carried out by one-way analysis of variance followed by Dunnett’s post hoc test.

4. Results and Discussion

As described in the Materials and Methods section, we used rhodopsin as template [28] for the generation of a starting structure of the M3R by homology modeling. Comparison of the transmembrane helices yields a 21.7% sequence identity and a 42.9% sequence homology. Obviously, any of the available crystal structures could have also been used for this purpose, being the choice of a template to construct the initial atomistic model of a GPCR matter of debate in the scientific community [29, 30]. Since for several years rhodopsin was the only high-resolution GPCR structure available, most of the homology modeling made use of it as template [31]. The recently published squid rhodopsin structure [32] and a few ligand-activated structures the human β2 adrenergic receptor (β2AR) [33, 34], the avian β1 adrenergic receptor (β1AR) [35], the human A2A adenosine [36], and more recently the crystal structure of the CXCR4 chemokine [37] and the dopamine receptor [38] have brought the opportunity to increase the repertoire of templates that can be used for modeling purposes, opening the question of which is the most suitable structure to be used. Indeed, although the overall structure of the six proteins is similar, there are notable differences, mostly in the extracellular loops and in the ligand-binding region [39, 40]. For example, in contrast to the other structures, the human β2 adrenergic receptor as well as of the avian β1 adrenergic receptors exhibit an alpha helix in the second extracellular loop. Similarly, TM5 and TM6 in squid rhodopsin are more extended than in the other receptors, or the inverse agonist in rhodopsin is more deeply inserted inside the hydrophobic TM domain of the protein. One of the conserved structural features, the so-called ionic lock, is weaker in the case of the β1AR, β2AR and A2A adenosine receptors than in the case of rhodopsin and involves the ICL2. This latter difference may be responsible for the higher basal activity and structural instability of these receptors and may contribute to the challenges in obtaining diffraction crystals of nonrhodopsin GPCRs [34].

However, there are still inconclusive results about the choice of a template. Thus, at the community-wide assessment of GPCR structure modeling hold in 2008 [41] aimed at understanding of the performance of GPCR homology modeling, 206 models of the A2A adenosine receptor submitted and assessed for their accuracy prior the release of the solved crystal structure. These models were basically assessed by the accuracy of the ligand-binding mode. The results show that among the best models there are some that were constructed using the structures of the β-adrenergic receptors and some using rhodopsin. On the other hand, in a recent report an accurate model of the transmembrane region of the β2-adrenergic receptor was reported using rhodopsin as template [42].

Two recently studies address the problem of template selection for GPCR homology modeling [29, 30]. The two studies conclude that the selection of the template should not exclusively be based on sequence identity but on an a priori assessment of the expected structural features of the receptor wished to model and that more high-resolution structures are still required. Moreover, the latter study concludes that the best template needs to be considered case by case, recommending the use of chimeras constructed by selecting special features from the different receptors available. Unfortunately, the six crystallographic structures available nowadays do not cover all the structural diversity necessary to have adequate templates to model a specific GPCR of the rhodopsin family leaving the question still open.

5. MD Simulations

Deviations from the initial 7TM homology-based structure were monitored through the time evolution of the root-mean-square deviation (rmsd) of the α-carbon atoms as displayed in Figure 2(a). Smaller deviations are observed when the protein is embedded in the two-component lipid bilayer (POPS-POPC) than when the protein is embedded in the one-lipid environment (POPC) (Figure 2(a)). This behavior is probably due to the presence of the anionic lipid (POPS) in the cytoplasmic area of the receptor, neutralizing the cluster of positive charges located in that region, and it is consistent with the experimental finding that this part of the protein is difficult to crystallize due to its flexibility, as previously found in MD simulations of bovine rhodopsin [43]. The time evolution of the RMSD also permits to assess the influence of the ligand on the protein structure. The evolution of the α-carbon rmsd shows that the trajectories with the ligand bound display lower deviations in comparison to those simulations of the protein alone.

Deviations per residue were also computed for a better characterization of the contribution of each segment to the overall rmsd. Figure 2(b) shows the rmsd per residue, highlighting the lower values found in the TM regions, compared to the connecting loops. Specifically, the larger deviations correspond to residues on the ICL2 and ICL3 of the protein (deviations larger than 1 nm), whereas the smaller ones correspond to the residues located in the TM region of the protein (about 0.25 nm). This differential behavior can be correlated with the fact that receptor/G-protein coupling occurs through the intracellular loops in all mAChRs [44]. Furthermore, the structures embedded into the two-component lipid bilayer (POPS/POPC) exhibit, in general, smaller deviations as compared to those embedded into one-component lipid bilayer with exceptions such as residues in the intracellular loop ICL1 (135–140), in ICL2 (225–233), and residues in the ICL3 (520–535).

The root mean square fluctuations (RMSF) of the α-carbon atoms, taken as reference the average structure computed from the last 25 ns were also calculated for the four trajectories. The largest fluctuations are located on the second and third intracellular loops as anticipated in the rmsd calculations (for residue rmsf see Figure 2(S) of Supplementary Materials). Fluctuations were relatively small due to the restrictions imposed by the lipid bilayer, except on the non-TM regions. In the case of TM4, TM5 and TM6 show an overall larger fluctuation in comparison to the other TM helices. These results agree with the fact that some residues within TM3-TM7 participate in the binding of the antagonist ligand NMS [26, 45, 46].

Dihedral angles were also computed using four consecutive α-carbons for the four MD simulations and disregarding the first 12 ns (Table 1), in order to identify specific conformations adopted by the extracellular loops when the NMS antagonist is bound and in the ligand-free receptor. The analysis of the M3-NMS-POPC MD trajectory shows that ECL1 adopts three different conformations, whereas it adopts only one conformation in the other three simulations. In contrast, ECL3 adopts only one conformation in the four MD trajectories. On the other hand, the time evolution of the dihedral angles defined using four consecutive Cα of ECL2 shows that it attains two different conformations along the trajectories M3R-NMS-POPC and M3R-NMS-POPC/POPS, whereas in the M3-NMS-free POPC/POPS simulation exhibits three different conformations.

This observation agrees with the putative contribution of ligands to generate microdomains on the extracellular loops that are important for the conformational changes that accompany receptor activation [47, 48]. Interestingly, we can also see that most notable changes in the variation of the dihedral angle average occur in the preceding part of the residues 218–221; therefore it seems that residues near the C221, the one that forms a disulphide bond with C141, play an important role in the conformation of this second extracellular loop. Figure 3 shows the superposition of two snapshots at different times taken from the POPC simulation, where it can clearly be seen the distinctive features the conformation adopted by the ECL2 before the first 20 ns and after 20 ns of the simulation.

In order to study different specific structural features, we focused on residue-residue hydrogen-bonding interactions, and lipid-protein interactions. For the protein-protein intramolecular interactions, we focused on hydrogen bond formation and breaking between pairs of residues of the protein along the MD trajectory (Figure 4(a)). The analysis was carried out by counting all possible atoms involved in a hydrogen/charge interaction around one residue at a time using a cutoff of 0.4 nm. Figure 7 lists the interactions between residues and their persistence along the MD trajectories. They are classified in charged-charged, charged-noncharged and noncharged-noncharged. Among the residues listed in Figure 7, those forming the so-called ionic-lock between TM3 and TM6, in subscript the Ballesteros-Weinstein numbering is used [49]: D1653.49, R1663.50, Y1673.51 and E4866.30, R2535.60 and R1804.37 are indicated. Electrostatic interactions at these positions are known to be critical for restraining the receptor from adopting a constitutively active GPCR [50, 51]. Some of the interactions are only found in the two POPS/POPC simulations: D142-K213, D165-R166, E259-R480, E220-Y128, E220-Y530, and N548-T50. Other interactions can only be found in the POPC simulations: E257-R253, E486-R177, S121-Y534, T534-S121, T550-Q98, and T550-T554. Furthermore, some of the interactions are found in the simulations without ligand: D165-R184, E257-Y167, N104-Y544, Y149-T235. Finally, some residues are only found when the ligand is bound to the receptor: D165-R180, E220-K523, D148-S152, N104-T101, and S537-S152 (Figure 7).

On the other side, specific residues have been shown to be involved in protein-lipid interactions. These include charged like K and R, neutral like N, Q, T, and S, and aromatic like H, Y, and W [20]. Previous analysis of hydrogen bonding among protein donors of bovine rhodopsin and lipid oxygen atoms revealed the importance of these interactions for the anchoring of the protein to the membrane. In rhodopsin, residues R2526.35, Y962.63, Y2746.57, H1524.41 and T1083.23, and to a lower extent K661.61, R691.64, Y1353.50, and W351.30 are found to participate in hydrogen bond linkages with lipid molecules, and, therefore, they can be considered to be putative hooks of the protein to the bilayer [20]. In this work, specific hydrophilic lipid-protein interactions have been analyzed along the different trajectories using the criteria of any of the residue atoms of the receptor was within a cutoff of 0.4 nm of a lipid oxygen. Following this approach, we identified common interactions for the four trajectories studied (Figure 4(b)). Most of the lipid-protein interactions are detected at the cytoplasmic half of the receptor, similarly to what is found in the case of rhodopsin. Moreover, some of the residues in M3R, W661.30, K1001.64, T1272.63, Y1673.51, and R1844.41, correspond to homologous residues in the rhodopsin sequence that confers the visual photoreceptor the property of anchoring the protein onto the lipid bilayer [20].

Another point of interest arises from the comparison of the interactions using the two different bilayers. In this case, the location of POPS lipids in the cytoplasmic vicinity of the receptor would provide a larger number of these lipid-protein interactions due to the fact that carboxylate groups of POPS lipids can interact with positively charged residues such as K260, R261, K263, R480, K487, and K488 located at ICL3 of the receptor.

6. The Orthosteric Binding Pocket of M3R

NMS was docked onto the binding pocket of M3R taking into account the residues known to affect its binding from site-directed mutagenesis studies, specifically D3.32 known to interact with the ligand quaternary nitrogen [52], N6.52 putatively involved in a polar interaction with the ligand [5355], and the homologous hydrophobic residues implicated in the binding of NMS at the M1 muscarinic receptor [56]. Once docked, the complex was energy minimized and the MD simulation started. This process was done for the two different bilayer compositions studied in the present work, POPC and a 10 : 1 POPC/POPS mixture. In order to understand the stereochemical features of the binding pocket, we monitored all the residues within a cutoff of 0.35 nm of any of the atoms of the ligand along the MD trajectories. Residues involved in the binding of NMS are shown in Figure 5. Simultaneously, we monitored the different conformations the ligand attained during the simulations. The analysis reveals that the ligand actually adopts different conformations along the trajectories; however, all of them conserve the interaction between the quaternary nitrogen of NMS with the side chain of D1483.32 and the hydroxyl moiety with N5086.52 (for definition of NMS dihedral angles and their values along the MD trajectory see Figures 3(S) and 4(S) of the Supplementary Material). Two different representative conformations of the ligand along the MD trajectories are depicted in Figure 6.

In a detailed analysis of hydrogen bonding interactions between different residues of the protein (those within a cut-off of 0.35 nm) and atoms of NMS, we computed the average angle formation of each of the interactions along the dynamics, and we found that residues N5086.52, W5046.48, F222, and T235 of M3R participate in hydrogen bond formation (angle between 120°–180°) with specific atoms of the antagonist ligand NMS in the MD simulations. In agreement with previously published results literature [57], N6.52 is considered to be important for the binding of some muscarinic antagonists [53], and also W6.48 has also been shown to be important for the binding of NMS [45, 51].

We have constructed, expressed, and characterized the alanine mutants of F222 and T235 and studied them by means of saturation binding assays in order to determine the affinity of NMS for these mutants. Dissociation binding assays have also been performed to study the release of NMS as a function of time from the M3R, in 20 mM Hepes buffer. In fact, the corresponding homologous positions have shown to modulate binding of NMS to the M1 receptor [47, 58]. Thus, mutant Y177 (the corresponding position of F222) reduced the affinity of NMS about 2.2-fold in comparison to WT M1 [47]. Furthermore, the mutant T1925.42 (the corresponding position of T235) showed a reduction of the affinity of NMS about 1.69 compared to the WT M1 [58].

7. Expression and Experimental Characterization of WT M3R and Mutants

The effect of the mutations studied on the expression of mAChR was measured by saturation binding assays using [3H] NMS (for representative saturation binding curves see Figure 5(S) of the Supplementary Materials). Mutant F222A showed about 55% of expression level in comparison to the WT M3R. In the case of the T235A mutant, it showed lower expression levels that were improved after atropine treatment. Alanine substitution of residues F222 and T235 somehow resulted in reduced affinities for NMS when compared to WT M3R. Mutant F222A decreased the affinity for NMS by 1.7-fold and mutant T235A by 2.6 fold with regard to WT M3R (Table 2). However, Y209F, K213 V, and E228N mutants assayed in parallel did not show any significant difference with regard to WT M3R (data not shown) which is consistent with the proposed role for these position in allosteric—but not in orthosteric—ligand binding [14]. The measurement of dissociation time courses was carried out in order to provide information about the transition from the conformation of the receptor bound to the radioligand and the free state of the receptor. [3H] NMS dissociated from the WT M3 receptor with a monoexponential time course time course corresponding to a half-time of 10 min under this condition (20 mM Hepes buffer, 30°C) which is in good agreement with previously published values [59]. Dissociation rates for the mutants F222A and T235A were not significantly different from the WT M3 receptor (data not shown). The effect observed in the binding affinities, together with the lack of effect in the dissociation analysis, suggests different structural environment requirements for the binding and dissociation of the ligand to and from the receptor.

8. Conclusions

The present paper addresses the effect of lipid composition and the antagonist ligand on the structure of the human M3R. The atomic resolution model of the M3R constructed by homology modeling using the crystal structure of bovine rhodopsin is compatible with experimental data from site directed mutagenesis studies and represents the inactive form of the receptor. This model was refined by MD simulations and the effect of the ligand on the conformation of the protein analyzed. Therefore, MD trajectories of different component lipid bilayers (POPC and the two-component POPC/POPS lipid bilayer), and also with or without NMS within the orthosteric binding site of M3R receptor, were analyzed.

We could determine that POPS lipid molecules provide additional stabilization to the protein structure of human M3R, especially for ICL3 because charged residues localized in this area of the receptor interact with the lipid oxygen atoms of the POPS molecule. Furthermore, our lipid-protein analysis suggests that some specific residues in the GPCR family (positions 1.30, 1.64, 2.63, 3.51, and 4.41) are responsible for anchoring the membrane protein onto the lipid bilayer (e.g., rhodopsin and M3R), as previously hypothesized [20, 60].

Our modeling identified many polar interactions that could play a role as activation microswitches [61, 62]. This includes also interactions associated to the “ionic lock” located at the cytoplasmic ends of the third and sixth transmembrane helices, respectively, (positions: 3.49, 3.50, 3.51, 6.30, and 6.34 in the GPCR family) play an important role in the receptor activation mechanism [50, 63].

In agreement with previous molecular modeling and mutagenesis for other muscarinic receptors (Avlani et al., 2007) [5], we found that ECL2 has a requisite of flexibility promoting the binding of GPCR ligands despite of the presence of the disulphide bridge between this loop and TM3. As seen in our average dihedral angle calculations, this loop showed more flexibility in comparison to ECL1 and ECL3. This loop fluctuates in two different conformations (most notable changes occur in the residues preceding C221—involved in the disulfide bridge—in the two MD simulations in which the ligand NMS was inside the binding pocket.

On the other hand, our experimental ligand binding and dissociation results suggest that, although F222 and T235 are not critical in the binding of NMS, these residues may participate in the conformation of a optimal binding pocket of NMS, where every small contribution in the environment confers stability to the NMS-receptor complex. Further studies, using other experimental conditions, will be needed to determine the potential role of these residues in binding of other ligands, for example, agonists or allosteric modulators.

M. Martinez Archundia wishes to acknowledge CONACYT (Mexico) for a predoctoral fellowship. This work has been supported by the Spanish Ministry of Science and Innovation through the Grants SAF2008-04943-C02-01 (to J. J. Perez) and SAF2008-04943-C02-02 (to P. Garriga).

Supplementary Materials

Figure 1S. Forward and reverse primers for the mutants F222A and T235A.

Figure 2S. Root mean square fluctuations (rmsf) of Cα atoms, from an average structure computed from the last 25ns of the trajectories, were also calculated for the four trajectories.

Figure 3S. Definition of the dihedral angles of the antagonist NMS that were measured along the simulations.

Figure 4S. Values of the four dihedral angles of NMS measured in the different simulations: (a) POPC and (b) POPC/POPS.

Figure 5S. Representative Saturation Binding Curves of WT M3R and mutants F222A and T235A expressed in COS7 cells. The membranes were incubated with increasing concentrations of [3H] NMS in Hepes buffer at 30°C for about 2.5 hours. The protein concentration used was ca 10μg/mL. The specific and non-specific binding are expressed in fmol/mg protein.

  1. Supplementary Material


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This article is part of Redesigning EU Fiscal Rules After COVID-19

Designing an EU fiscal governance framework that ensures long-run fiscal sustainability, while pursuing appropriate counter-cyclical policies and contributing to sustainable growth, social and climate objectives, is a challenging task. Fiscal policy is multi-dimensional, complicated and uncertain. Political pressures often lead to short-termism and excessive risk-taking. In addition, the EU and euro area frameworks need to work for very different economies and manage the risk of spillovers.

The shortcomings of the existing framework in terms of weak compliance, procyclicality and excessive complexity are well known. Many proposals to reform EU fiscal governance have looked to strengthening the role of national independent fiscal institutions (IFIs).

This contribution draws on a recent analysis prepared through the EU Network of Independent Fiscal Institutions (EUIFI, 2021) to offer a perspective of national independent fiscal institutions. IFIs in the EU member states are independent official bodies with mandates to oversee different aspects of fiscal policy, including forecasting, compliance with fiscal rules and assessing various aspects of the fiscal position. While some EU IFIs are long-standing institutions, many were created in their existing roles by the EU governance reforms of the early 2010s.

National IFIs have a unique perspective and experience on EU fiscal governance, both in terms of the role that IFIs could play and on fiscal policy more widely. IFIs bring a practical perspective on the realities of national fiscal policies and how fiscal frameworks work in practice.

This paper sets out a three-pillar approach to strengthening the EU fiscal framework based on: improving the numerical fiscal rules, strengthening the role of national IFIs and ameliorating statistics and data on the public finances.

A three-pillar architecture

The fiscal framework needs in practice to achieve a balance between, on the one hand, effective budgetary rules that allow governments to commit to sound policies and, on the other, judgements and discretion in how these are applied so that the framework adequately reflects the complexity of the situations that countries may find themselves in. Alternative approaches to setting fiscal rules may require more or less judgement or discretion, but no simple set of rules alone is likely to be able to deliver the required outcomes across all states of the world, taking into account national specificities.

A three-pillar architecture would support effective EU fiscal governance by providing (Figure 1) continued reliance on simpler numerical fiscal rules, an enhanced role for IFI analyses and assessments in the context of the EU surveillance cycle, and improved information on budgetary-relevant metrics.

Figure 1
Three-pillar architecture to strengthen the EU fiscal framework

Three-pillar architecture to strengthen the EU fiscal framework

Note: IFIs stands for national independent fiscal institutions.

Source: Author’s own compilation.

The three pillars are mutually reinforcing. Numerical rules can provide a transparent benchmark and objective basis for setting and assessing policy. They allow politicians to commit to long-run budgetary goals. However, rules require monitoring and interpretation. The Commission and the Council currently play this role at the EU level. An enhanced role of the national IFIs as independent expert bodies could help on specific issues in the application of the rules to national circumstances, such as the assessment of discretionary revenue measures and improving budgetary forecasts, and provide assessments of fiscal sustainability. Adequate data and information is needed both to implement rules effectively and to support oversight of budgetary policy.

Relying on rules alone is likely to be unduly blunt. Simple rules would risk being either too strict to enforce or too lenient, while richer frameworks quickly become highly complex without necessarily resolving all issues that may arise. For example, structural balance-based approaches aim to get closer to a full economic assessment than using headline measures but open up a complicated set of problems around the measurement of the output gap. Conversely, relying entirely on policy discretion or on IFIs’ assessments, for example, without any numerical framework that sets high-level policy objectives or that provides benchmarks against which policy can be compared, is unlikely to result in a robust fiscal framework. While some have proposed moving away from rules altogether (Blanchard et al., 2021), such an approach is likely to face a number of practical changes, including the difficulty of making standards binding in many EU political systems.

Improving the numerical rules

While fiscal rules should remain at the heart of the EU framework, the numerical rules need to be better designed to improve compliance, reduce risks of procyclicality and be simpler. There are a number of ways this could be achieved and many different proposals have been made, each with different advantages and disadvantages.

However, what may matter more than high-level design choices is that any set of rules is well designed. The EUIFI (2021) paper sets out four main criteria for the new set of fiscal rules:

  • simplicity
  • transparency
  • reduced procylicality
  • a multi-annual focus.

In terms of how fiscal policy is managed, the most critical of these for achieving fiscal sustainabilitly is arguably moving towards a genuinely medium-term framework rather than one focussed on achieving fiscal goals one year at a time, which risks both policy drifting over time and being too inflexible in the near term.

Most reform proposals focus on two approaches to overhauling the rules. The first approach is the “debt anchor, expenditure ceiling” concept that aims to bring debt levels below a certain threshold deemed sustainable (debt anchor) by using a single operational rule (expenditure ceiling) as a lever. This approach has been advocated by the European Fiscal Board (EFB, 2020) and others (including Bénassy-Quéré et al., 2018; Constancio, 2020; Martin et al., 2021). Most proposals retain the current 60% public debt reference value over some horizon while others set the debt anchor through a formula or politically.

The second approach is to modify the existing EU Stability and Growth Pact framework with some simplification in the number of rules and their application (Feld et al., 2020). This could be achieved by focussing more on the existing expenditure benchmark, continuing the trend in recent years towards growing emphasis on this measure. The expenditure benchmark sets a ceiling for allowable expenditure growth (adjusted for discretionary revenue measures) based on the ten-year average growth of potential output and a convergence term intended to progress towards the medium-term objective for the structural balance, which itself partly depends on the deviation of debt from the 60% ceiling. While still reliant on measures of potential output, the expenditure path is less sensitive to measurement error along the path of potential output. Furthermore, the benchmark does not rely on estimates of the cyclical elasticity of tax revenues and so comes closer to tracking a bottom-up measure of the structural balance based on spending growth and tax policy changes than the standard approach of removing cyclical components from the headline balance.

In theory, the two proposed approaches can be viewed as achieving a similar outcome for the public finances. Both could be used to set a multiannual expenditure target, whether anchored directly in a debt objective or through the structural balance. In practice, however, there may be advantages and disadvantages to how each performs. This will depend critically on specific design features of their implementation.

The use of an expenditure ceiling as operational tool is attractive because government expenditures are mostly under the direct control of governments, largely independent of the business cycle, and have smaller forecast errors (Feld et al., 2018) than structural balances. A well-designed multi-year expenditure ceiling should avoid the risk of increasing spending in a procyclical way during good times. It would strongly support domestic medium-term budgetary frameworks.

A key design challenge is to ensure that the expenditure ceiling sets a stable path for the medium-term spending. The current EU expenditure benchmark does not do this: It is prone to revision, even with ten-year averaging, because of its reliance on a volatile measure of potential output (Barnes and Casey, 2019). Furthermore, the expenditure benchmark spending level depends on spending in the previous year, including locking in small overruns that raise the base or underspends that limit future spending. Setting the expenditure ceiling based on a level of spending of multiple years would be a significant step towards a more long-term and stability-oriented fiscal policy. The expenditure ceiling or benchmark needs to be predictable yet flexible enough to allow some adjustment if the economy follows a very different path from what was originally planned. This could imply fixing the nominal expenditure ceiling several years at a time, for example for at least three years. As discussed below, appropriate estimates for the medium-term growth path of the economy are a necessary underpinning for this approach.

Several other design issues around numerical EU fiscal rules also need to be addressed (EUIFI, 2021). First, the treatment of deviations from spending rules in specific circumstances could be addressed, including through an adjustment margin for overruns and a rainy-day fund for governments that want to build up more savings in good times to manage any future downturns. Second, growth-enhancing government investment may warrant special treatment and recognition in the design of the rules. Darvas and Wolff (2021) make a similar proposal with respect to “green” investment. Across all these areas, the advantages and drawbacks of these provisions need to be carefully weighed to ensure that they enhance the fiscal framework rather than undermine it.

Any proposal to reform the rules should be carefully tested and evaluated rather than just relying on a priori reasoning (Barnes and Oliinyk, 2021). This has been a weakness in the implementation of past reforms, as well as in some current proposals. Most strikingly, the one-twentieth debt reduction target has proven very difficult to apply at face value for high debt countries because it frontloads debt reduction and can imply an excessive fiscal tightening. This problematic issue would have been apparent had the rule been properly tested at the time. There are many other examples of such issues, such as the procyclicality of the structural balance requirements in real time for some countries (Barnes and Casey, 2019). Ideally, the rules should be tested against a wide range of possible outcomes, starting with medium-term forecasts for each country’s public finances. The testing should examine the pseudo-real time performance of the model, the risks and the opportunities for countries to game the rules.

Strengthening the role of IFIs

The capacity of national IFIs to make independent objective assessments of national fiscal dynamics and to increase transparency could be used more effectively in the EU fiscal framework. IFIs can help to produce more accurate and less biased fiscal forecasts (Beetsma et al., 2018). By improving monitoring and transparency, IFIs improve compliance with fiscal rules (Lledo, 2018). IFIs can help to make the case for sound fiscal policies (Beetsma et al., 2017) and to contribute to deficit reduction (Capraru et al., 2020). As increasingly noted, IFIs have a strong ability to engage with the public through national media (European Commission, 2021) and with domestic institutions such as national parliaments. This can help to anchor compliance with the fiscal framework in the national political landscape.

In terms of the EU fiscal governance, the capacity of IFIs is currently limited to a number of specific areas. First, IFIs monitor compliance with domestic fiscal rules that mirror EU requirements (EU Directive 2011/85), particularly for countries that adhere to the Fiscal Compact (the Treaty on Stability, Coordination and Governance) and with regard to the related national corrective mechanisms. Second, for euro area members, IFIs undertake or endorse macroeconomic forecasts used in national budgets and stability or convergence programme updates (“two-pack” Regulation 473/2013).

There should be a legal obligation at the EU level to take into account national IFI assessments across a range of areas (EUIFI, 2021). The “obligation to take into account” the IFIs’ work more widely would codify existing practice where Commission teams regularly draw on IFIs, taking it a step further by making it a legal requirement. The obligation would not require the Commission and Council to follow the same approach or conclusions as the IFIs, but rather to consider the assessments of the IFIs. This could strengthen the legitimacy of EU-level decisions, where these already align with the IFIs’ assessment, and provide some basis to take into account national specificities that are justified by the assessment of the national IFIs. This approach would reduce the risk that national IFIs are unduly undermined by contradictory assessments at the EU level.

There are a number of specific areas where IFIs are well placed to understand and assess specific national circumstances, as well as implications for the overall fiscal position (EUIFI, 2021):

  • Measurement issues. This could include the identification of one-offs, estimation of potential output and structural balances and the measurement of discretionary revenue measures.
  • Improving budgetary forecasting, where IFIs could have a greater role in the oversight of medium-term budgetary forecasts, extending their endorsement role from macroeconomic forecasts only.
  • Compliance with rules that mirror requirements at the EU level. The current scope of assessment of compliance with some rules could only be extended across all the EU rules.
  • Assessment at the national level, on a regular basis, of overall fiscal sustainability and the underlying fiscal position. This would help to ensure that formal compliance with the rules is supportive and sufficient for sound fiscal policy.

In all cases, assessment by the Commission and the Council would remain the formal legal benchmark. However, taking into account assessments by the IFIs could help to improve judgements at the EU level and to anchor them more firmly in the domestic institutional framework.

Situations may arise where Commission assessments differ from those of the national IFI, either on compliance with numerical benchmarks under the fiscal rules, the treatment of specific issues such as one-offs, or the overall fiscal position. The obligation for the EU institutions to take IFIs’ assessments into account could help to narrow undue differences in interpretation of these assessments by providing some leeway for the Commission to consider national specificities. Providing clearer and more comprehensive common principles and guidance could further help to avoid these differences.

At the same time, it is vital that national IFIs retain their independence, including from judgements made at the EU level, so they are able to provide objective assessments of national circumstances. Many IFIs also have separate mandates connected to national fiscal frameworks that should not be compromised. National mandates of IFIs are a key part of their legitimacy and ability to engage with national stakeholders. The obligation of EU institutions to take IFIs’ assessments into account would – by leaving the ultimate decisions in EU hands – enhance the role of national IFIs without compromising it.

A key condition for IFIs to play an enhanced role in the fiscal governance of the EU is to strengthen national independent fiscal institutions and their mandates. At present, there is a wide range of capacity across EU IFIs, reflecting differences in mandates and resources. The proposals in this paper would require IFI mandates to be extended in some cases, notably around monitoring compliance with the fiscal rules and in providing an overall assessment of the fiscal stance.

EUIFIs (2021) identifies a number of areas where minimum standards set at the EU level for national institutions would help to strengthen many IFIs in their role at the EU level and domestically:

  • A mandate to address government and parliament, and mandate to publicly disclose reports and recommendations. IFIs should be able to directly address national authorities and disclose reports and recommendations on their own initiative within their mandates.
  • Adequate level of resources and management flexibility. IFI budgets should be protected from political interference, potentially through a multiyear budgetary appropriation that stretches beyond the national electoral cycle. IFIs should also have adequate flexibility to manage their resources, to ensure their independence is preserved.
  • Good and timely access to information. IFIs should be able to obtain accurate information on demand from national statistical offices, national governments and the Commission without an undue delay and at no cost. Any restrictions on access to information should be clearly defined in law.
  • Effective implementation of the “comply or explain” principle. This is a key instrument in the current IFI toolbox. The details on implementation of this principle should be included and clearly defined in national regulations.
  • Sufficient safeguards against political pressures. It is essential that board members are selected under transparent procedures. The hiring process should be subject to strict rules on conflict of interest, and terms of board members should be independent of an electoral cycle.

Improved statistics and data on public finances

The setting and enforcement of fiscal rules and the assessment of fiscal sustainability should be supported by better statistics and information. Existing requirements to provide reliable statistical data on the general government fiscal position in a timely and comparable way and the independent oversight of macroeconomic forecasts are major achievements of the current EU fiscal governance.

The experience of national IFIs suggests a number of specific improvements to economic and budgetary information (EUIFIs, 2021):

  • The requirement for member states to produce credible medium-term fiscal projections in all budgetary documents/Stability Programme Updates based on clear and credible policy assumptions covering at least four years ahead. This could include extending the requirement for projections to be endorsed by an independent body from macroeconomic to budgetary forecasts.
  • Simplification, increased transparency and methodological improvements in the assessment of potential output. The existing potential output measures under the EU Commonly Agreed Methodology are procyclical (Darvas, 2019), especially at the end of the sample or of the forecast period. Consequently, output gaps are under-estimated (in absolute terms), particularly in some countries (Barnes and Casey, 2019). Any method to estimate the output gap or potential output is likely to face challenges, including the procyclical nature of many economic forecasts, and imply trade-offs. However, some methods perform better than others in terms of cyclicality and stability in real time.
  • Medium-term debt sustainability analyses at the national level, either by governments subject to national IFI oversight or by national IFIs.
  • Improved treatment of one-off measures and discretionary revenue measures in monitoring underlying fiscal positions. Net debt and net worth statistics should be published, as well as gross and net financing needs.

The future of the EU fiscal governance

This contribution, drawing on the experience of the national IFIs, lays out a three-pillar approach to strengthening the EU fiscal governance based on improving the numerical budget rules around a medium-term expenditure ceiling, strengthening the role and institutional framework of the IFIs, and improving statistics and data on fiscal policy.

Reform of the rules should be based on the experience both of what has worked and what has not worked. That is why this paper emphasises the criteria for effective rules, rather than any particular design choices. This calls for careful testing and evaluation of reforms before they are implemented. While there have been many challenges and weaknesses, the acceptance of a rules-based framework underpins most reform proposals. The creation of independent fiscal institutions in many EU countries has been a significant achievement and improved outcomes, although there is scope to use the IFIs more effectively in the EU context and to further strengthen IFIs in many countries. Improved statistics and data have also been a significant achievement of the EU governance framework.

Better fiscal policy in EU countries requires that the different elements support each other. Creating the conditions for national IFIs to develop by strengthening their mandates and institutional design, while assigning them a more explicit role at the EU level, could be a key part of this framework.

Barnes, S. and E. Casey (2019,17 June), Euro area budget rules on spending must avoid the pro-cyclicality trap, VoxEU.

Barnes, S. and I. Oliinyk (2021), The EU’s economic governance review: Road-testing alternative fiscal rules?, CEPS Policy Insights, 2021-19.

Beetsma, R. and M. Larch (2018, 10 May), Risk reduction and risk sharing in EU fiscal policymaking: The role of better fiscal rules, VoxEU.

Beetsma, R., X. Debrun and R. Sloof (2017), The Political Economy of Fiscal Transparency and Independent Fiscal Councils, IMF Working Paper, WP/17/195.

Bénassy-Quéré, A., M. Brunnermeier, H. Enderlein, E. Farhi, M. Fratzscher, C. Fuest, P. Gourinchas, P. Martin, J. Pisani-Ferry, H. Rey, I. Schnabel, N. Véron, B. Weder di Mauro and J. Zettelmeyer (2018, 17 January), How to reconcile risk sharing and market discipline in the euro area, VoxEU.

Blanchard, O., A. Leandro and J. Zettelmeyer (2020), Redesigning the EU Fiscal Rules: From Rules to Standards, 72nd Economic Policy Panel Meeting, Federal Ministry of Finance, Germany.

Capraru, S., G. Georgescu and N. Sprincean (2020), An Evaluation of IFIs Impact on EU Countries Budget Deficits, Romanian Fiscal Council Working Paper, 2.

Constancio, V. (2020), The Return of Fiscal Policy and the Euro Area Fiscal Rule, Comparative Economic Studies, 62(3), 358-372.

Darvas, Z., P. Martin and X. Ragot (2018), European fiscal rules require a major overhaul, Bruegel Policy Contribution, 18.

Darvas, Z. (2019, 1 October), Why structural balances should be scrapped from EU fiscal rules, Bruegel Blog.

Darvas, Z. and G. Wolff (2021, 9 September), A green fiscal pact: climate investment in times of budget consolidation, Bruegel Blog.

European Commission (2021), European Economic Forecast – Spring 2021, Institutional Paper, 149.

European Central Bank (2016), Public investment in Europe, ECB Economic Bulletin, 2.

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EU Network of Independent Fiscal Institutions (2019), Network Statement on the Need to Reinforce and Protect EU IFIs.

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Feld, L., C. Schmidt, I. Schnabel and V. Wieland (2018, 12 September), Refocusing the European fiscal framework, VoxEU.

Hagemann, R. (2011), How Can Fiscal Councils Strengthen Fiscal Performance, OECD Publishing, 2011(1), 1-24.

Martin, P., J. Pisany-Ferry and X. Ragot (2021, 26 May), A new template for the European fiscal framework, VoxEU.

© The Author(s) 2022

Open Access funding provided by ZBW – Leibniz Information Centre for Economics.

DOI: 10.1007/s10272-022-1023-z


National Celiac awarness day

 Every 13 September, a National Celiac Disease Awareness Day is held for those who have celiac disease. About 3 million people are affected by this disease.

Those who have celiac disease avoid a gluten diet and take a gluten-free diet because it will damage villi in the small intestine if they take a gluten diet. It also damages the immune system so that nutrients are not absorbed in the body. The gluten diet is found in wheat, barley, rye and some other products. It is a very harmful disease, and the solution to this problem is to avoid a gluten diet.

The national celiac awareness is held and helps to promote diagnosis and support those who have already been diagnosed. The Doctor also advised taking proper diet style to absorb in the body and live a happy and healthy life.

What is Celiac Disease?

It is an auto-immune condition in which the immune system is weak to absorb protein such as gluten. If you have celiac disease, then a gluten diet causes issues in your small intestine. Suppose the person suffering from Celiac and do not avoid from gluten diet so that there will be an issue to absorb nutrients and other vitamins in the body. The celiac issue can be diagnosed at any age, but avoiding a gluten diet is the best treatment.

How do celiac issues affect anyone?

Suppose you see the small intestine under the microscope so that you will see finger-like projections on the surface of the small intestine. These fingers like projections are called villi. The function of villi is to absorb nutrients in the body. The person who is suffered from Celiac has a problem in the villi and cannot absorb nutrients, and a minimal amount of nutrients is passed into the bloodstream.

What is the history of national celiac disease awareness day:

The United States passed a resolution in 2005 to support celiac disease on 13 September (National Celiac Disease Awareness Day).

The day is held to remember the birthday of Dr Samuel Gee, and he is a physician who first discovered in 1888 and wrote about celiac disease and published the treatment of this disease. Then Senate decided to hold this day as National Celiac Disease Awareness Day every year.

Causes of Celiac disease:

Many theories are given on the celiac issue, but primarily doctors believe that it is caused due to our genes. It is generated through genes from one another, but a specific reason is not discovered yet.

How is celiac disease diagnosed?

First of all, Doctors ask for the entire history and then advised about further issues, which are given below:

  • Blood tests: The blood test is taken if Doctor thinks that he is suffering from Celiac. It has also been taking when gluten diet is taking.

  • Endoscopy: if the blood test is positive, then the further procedure is involved, which endoscopy is, and if the blood test is negative but has the chances of Celiac, then endoscopy is taken. It is a thin tube passed through the mouth and then into the small intestine, and then biopsies took. This procedure is uncomfortable, but it is necessary to take. The biopsies confirm the diagnosis. Sometimes, the second endoscopy is also accepted if there is any uncertainty in the first endoscopy. Endoscopy is the only way to confirm the diagnosis.

Gynecologist, Gyn, Vagina, Uterus

Which treatment is available for celiac disease?

The only way to treat celiac disease is to avoid a diet that contains gluten. It's mean you have to eat a gluten-free diet, and you spend your life eating a gluten-free diet for the rest of your life. Suppose it confirms that you are suffering from Celiac; then, the Doctor advised you to consult a dietitian and get knowledge about a gluten-free diet and how you can eat a balanced diet. The dietitian is also given the best advice for a balanced diet.

What is a Gluten-free diet?

Gluten is found in all foods in which wheat, barley is present. Bread cakes contain a large amount of gluten because it made from flour. Most breakfast of cereals has a large amount of gluten because it is made from wheat and barley. For example, flour is used too thick; barley is used in many things, such as driving in all beers.

It is believed that oats are not harmful to celiac patients, but one thing remembers that some oats products contain gluten, and it can create an issue for celiac patients. All celiac patients must check the ingredients before eating any products.

Many products are present of a gluten-free diet for people who are suffered from celiac issues. According to Advisory Committee on Borderline Substances (ACBS), suggest to Department of Health to approve a list on a gluten-free balanced diet. These foods contain vitamins, proteins and other beneficial nutrients which are lack in a gluten-free diet.

gluten free breads on wood background a gluten free breads on wood background celiac stock pictures, royalty-free photos & images

Is it necessary monitoring in celiac disease?

 Suppose you want to stay healthy and happy life, so you have to keep your eye on your health. It also thinks to consult with your doctor once a year. It is good to take blood tests to understand that everything, such as proteins and vitamins, is in balance.

Your Doctor also scans your bones if your bones are thin so that you can work on them early. It is also estimated that people who suffer from Celiac have less capacity to produce antibodies against infections. So that vaccination against flu and for other issues is also recommended.

How is Celiac Disease celebrated?

The best way to celebrate this day is to support those suffering from this issue and help those who don't know how to prepare a gluten-free diet.

1% out of 100% is suffering from this issue on Earth, so it is possible your friend, family, and another person will suffer. The person eats exceptional food and food called a gluten-free diet.

National Celiac Awareness Day is held to see the pain of that person suffering from Celiac and a chance to learn from those who are facing this issue.

Writer: Sakeena Laghari

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