BioQuakes

AP Biology class blog for discussing current research in Biology

Tag: antibodies (Page 2 of 2)

ARE WE DOOMED? Maybe not

     Well, this year has been a ride. Starting off with a potential WWIII, continuing with the tragic loss of hall of fame athlete Kobe Bryant, 2020 has been one roller coaster of a year. But the most bizarre of it all was the COVID-19 pandemic. The pandemic swept the nation way back in March and it still has its grasp on us today. At the time it started, there was very little information on this virus. But now, due to our vast intricate technologies, we were able to find out lots of information on this virus. But, specifically, I want to talk about life after contracting the virus. See, normally when you have a virus and successfully heal from it, you develop antibodies so you will not get this type of virus again. The case is a bit different for COVID-19, or it might be the same. Read to find out!

     This topic is very interesting because there have been more than 10 million people who have acquired the virus. The people that have successfully recovered from the virus want to know the main question: Will I be able to get this virus again? The answer isn’t so simple. Early on the data provided to us gave us hope that the immunity to this virus was possible, but numerous cases also suggest that this immunity to the virus is brief (on a larger scale). Nothing is definite as of now, there is more research to be done, but for now we remain hopeful. 

 

So why do we say the immunity to the virus is brief?

     We know there is hope because there is proof that people who have contracted COVID-19 produce antibodies that protect our immune system, but this production of antibodies lasts maybe 3 to 4 months based on the data provided. The length of time still remains unclear. 

 

So how does this actually work?

     Researchers from Massachusetts General Hospital tested three types of antibodies in blood samples: immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin B (IgB). Immunoglobulin is a large Y-shaped protein used in the immune system to detect foreign invaders in the blood such as viruses. These proteins bind to these foreign invaders in order to fight them off. We learned from our unit with proteins that antibodies are a form of proteins that can influence the life of a molecule/virus. The most important of all the immunoglobulins stated above is IgG. The reason is because IgG has the potential to sustain immunity in the body. This is because when all three of these antibodies were found in the blood after being infected by COVID-19, IgA and IgB were obliterated by the spike protein found in COVID-19. But, IgG lasted in the stream for up to four months! Now, the researchers could not test IgG for that long, but the four months that they could observe showed that these IgG antibodies do persist to beat the virus! A more long term study is definitely needed. This study is also confirmed by another research group from the University of Toronto. This group also showed how IgA and IgB levels dropped rapidly about 12 days after infection while IgG levels remained steady. 

 

So can you get COVID more than once?

     Although it is very rare, there have been some cases where people contracted the virus more than once. But, there is no evidence that suggests that immunity is or is not possible. All in all, evidence shows that immunity after acquiring the virus is generally protective and the persistence of the IgG antibody provides hope for immunity to the virus. – Ghohesion

How Reliable Are Covid-19 Tests and What Are The Different Types of Tests?

For my study of research, I’ve decided to learn more about Covid-19 testing and its effectiveness. In this article, How Accurate Are COVID-19 Tests? Many Factors Can Affect Sensitivity, Specificity of Test Results, it discusses several methods of testing, along with how accurate the results are. The article also goes into detail about what factors can affect the tests accuracy. 

Sensitive tests, which are positive results, are less likely to produce a false-positive outcome, and a specific test, negative results, are less likely to produce a false-negative outcome. Labs can provide the analytics of sensitivity and specificity for a test, which is concluded from confirmed specimens of positive and negative results. These results, however, come from when someone either had a great exposure, or none, so they are true under ideal conditions. Since there is so much variability between patients, the numbers are often lower when they are under real life conditions. 

There are two main types of testing for the novel Coronavirus. The first type of test detects RNA from the virus by using methods such as, polymerase chain reaction (PCR). I have never heard of this process before, so I decided to find a source explaining what it is. PCR is used to amplify, which is making many copies of a gene or DNA. Using this process, many copies can be created, just from a small part of the DNA taken for the sample. This process can help to identify a pathogen when trying to detect a virus, such as the novel Coronavirus. This past week in class, we learned about the immune system and about the characteristics of viruses. We learned that a virus has spike proteins on the outside, and it has RNA strands in the inside of the cell. This connects to what we learned about RNA and viruses, because this test actually tests for RNA to see if a patient has the virus. they are more accurate because they are from the genetic sequence from the virus itself, which is unique to it. If a test comes back positive, it is most likely accurate. The second type of test is molecular testing. The nasopharynx is said to have the largest concentration of a virus. Since using NP swab samples, nasopharyngeal swabs, are hard to get, the sensitivity of a test can be altered or tampered. This can create a false-negative result in a patient, who really could have it. Testing with saliva and blood has more of a likeliness to reduce the sensitivity. The article also mentions that swabbing the patient in the oropharynx or nose can also have a lower sensitivity. 

Antibody testing is through drawing blood from a vein, and it can detect whether or not someone was infected by Covid-19. The test uses enzyme immunoassays and rapid lateral flow immunoassays. By day 14 following symptoms appearing, most patients did have the IgG antibodies. I wasn’t exactly sure what an IgG antibody was, so I found a source to explain that in some more detail. It is an immunoglobulin and is found in all fluids within the body. They are the most common and small antibodies that are in the body. These antibodies help to fight bacterial infections and viruses. These antibodies are actually the only ones that can help protect a woman’s fetus, which is very interesting. As time goes on, it is less likely that the antibodies will be detected. There is some evidence, not confirmed yet, that suggests that children and asymptomatic or mild-symptom patients could be less likely to have detected antibodies. 

I found this article to be very fascinating because it went into detail about each test and its effectiveness. I didn’t know that children and asymptomatic or mild-symptom patients were less likely to have detectable antibodies. I am excited to research more as I continue to further my studies in Covid-19 testing. 

 

Can your common cold help you beat vicious COVID-19?

Season colds are quite common, and while they are inconvenient and make us feel icky, they may be our advantage for our battle with COVID-19. 

To start off, when reading this article, I noticed that the author used the term “coronavirus” more casually. He referred to a “coronavirus” as a common cold, which of course left me confused. So I dug a little deeper…

Here’s a fun fact that I learned from this:

Many of us having been thinking that COVID-19 is the same as what we call the “coronavirus.” After reading an article differentiating the difference between the terms, I found that the term coronavirus is actually the broad term to describe a whole range of viruses. SARS-CoV-2 is the specific virus that causes only COVID-19 and is causes what doctors call a respiratory tract infection.

Basic biology tells us that while there are many cells that make up our body, they are all interconnected. A pathogen, like the SARS-CoV-2 virus, is an enemy to the cell. We learned about how things enter the cell in biology: the pathogen enters the cell, travels through the cytoplasm, and enters the nucleus. Because the virus has genes, it is able to rapidly produce copies of itself to infect the other cells. And of course, we know how scary these infected cells are when they start spreading to the lives around us given our situation with a global pandemic.

What we now know is that the SARS-CoV-2 virus, our “bad guy,” can actually induce memory B cells. These memory B cells survive for quite a long time; they are important in identifying pathogens, and creating antibodies to destroy such pathogens. So when we got sick during the winter last year, chances are these memory B cells fought them off. The key part of the memory B cell in our fight against COVID-19 is the cell’s ability to remember the antibodies it created from past illness for the future.

What does this mean?

The belief is that anyone infected by COVID-19 already has the memory B cells from past common colds to fight the virus off.  Taking a further step, it is believed that since everyone already has the memory B cells, anyone who has had COVID-19 in the past is unlikely to get it a second time. If the SARS-CoV-2 virus were to enter your body a second time (which is likely considering the virus has not gone away and is literally all around us), our bodies would be prepared with former knowledge of the antibodies used to fight and win this time.

A study performed at the University of Rochester Medical Center is the first to demonstrate how this may be so.

Mark Sangters, Ph.D., is a research professor of Microbiology and Immunology at URMC; he has backed up his findings by comparing different blood samples. When looking at 26 blood samples of recovering moderate COVID- 19 patients (people who have had it for their first time now), it seems that many of them had a pre-existing pool of memory B cells that could recognize the SARS-CoV-2 virus and rapidly produce antibodies to destroy it. He also studied 21 blood samples of healthy donors, collected years before COVID-10 existed. What he found was that these B cells and antibodies were also already present.

When we are sick with a common cold, our antibodies are created by memory B cells to attack the Spike protein. This protein is what helps viruses infect our cells. What Sangters noticed, is that although each Spike protein is different for each illness, the S2 portion of the Spike protein is the same throughout all sickness. Our antigens can not differentiate the parts of the S2 subunit, so they attack the Spike protein regardless. This was his final piece in his conclusion that our common colds that caused our memory B cells to make antibodies, could be used to fight against COVID-19.

The Long Road Ahead:

My concern with this article is that this is the biggest issue we face with COVID-19 is patient outcome. As of right now, there is no way to fully prevent everyone from COVID-19 because it is still all around us. The issue the world is facing, is how to treat those who have already contracted the virus. This information just simply is not enough to help. How will these memory B cells help those who are currently sick? The answer: Scientists are unsure. There is still the uncertainty of the future vaccine and study of these memory B cells for a possibility of milder symptoms or shorter length of illness from COVID-19.

 

Despite all of this concern, this is still a step in the right direction. Any information about this terrorizing virus is still helpful given how little we know about COVID-19. If we were to expand more on this information, we could save the lives of those around the world!

 

 

Milking Scorpions yields $10,300/ml Antibiotic Venom

Why is it important?

Who would have thought that Scorpions could be providing useful antibiotics through their venom, something most would think of as a harmful substance. It was found that while testing the venom of a Mexican Diplocentrus melici scorpion, a previously studied venom, the venom could be split into two parts. The two types are called Escherichia coli and Mycobacterium tuberculosis bacteria” and when separated they both have specific uses. One of the parts was red and the other blue, it was found that one part was good at killing Staphylococcus, and the other was good at combatting Tuberculosis. Due to these findings, the scientists saw parts of the venom as potential antibodies.

(Scorpion Diagram) 

The Testing:

The two parts of the venom where both tested on rats. It was found that both parts respectively combatted the staph bacteria and the TB. The researchers, however, are skeptical as to whether the antibodies would work as well in humans, and how they would measure the correct amount to be administered to a human given that the substance is so difficult and/or expensive to obtain.

The High Pricetag:

“When electrical stimulation is used to “milk” the venom glands of scorpions, an average yield of anywhere from 0.006 mg to about 2.0 mg of venom can be obtained from a single scorpion.” Not a lot, right? But according to Scientific American, the “milk” from a single scorpion sells for about $10,300 per Milliliter, thats nearly $39 Million per gallon. The reason for its high price tag is its low quantity per scorpion and how inconvenient it is to extract the venom. Each scorpion also takes about two weeks to replenish its venom.

Conclusion:

While the thought of administering scorpion venom as a medical treatment, in my opinion, sounds awesome, it may not be the most realistic method. The testing proves that it does work for combating TB and Staph microorganisms, however, the tests were done on rats. Until the venom can be said for certain to be effective for humans, and there is a MUCH cheaper cost, I’m not sure how feasible it is to treat people with scorpion venom.

 

Strong Genes Equal Strong Immune System

Although scientists have long agreed that antibodies are in integral part of building up the body’s immune system, there is new evidence that strongly suggests genetic factors play a large role in determining how well the immune system builds and uses these antibodies when fighting disease.

https://commons.wikimedia.org/wiki/File:Redhead_twins.jpg

In a recent study, “researchers from James Cook University’s Australian Institute of Tropical Health and Medicine (AITHM) and the University of Queensland’s (UQ) Diamantina Institute have analyzed blood samples from 1835 twins and thousands of their siblings.” The team looked at the body’s immune response to “six common human viruses, including the Human Herpes virus, Parvovirus, Epstein Barr virus and the Coxsackie virus.” The team determined that genes passed down by parents are the major factor in how powerfully an immune system responds to diseases. “These genes determine whether you mount an intense or weak immune response when confronted with a viral infection,” says Associate Professor Miles.

“Demonstrating that antibody response is heritable is the first step in the eventual identification of individual genes that affect antibody response.” The researchers’ next goal is to identify the superior genes in order to, “imitate ‘super defenders’,” and “design next generation vaccines.”

 

IS THAT POISON IVY???

Scientists have discovered a possible new treatment to prevent itching from poison ivy, a painful nuisance that affects up to 50 million Americans annually. Using an antibody for the protein interleukin-33, scientists were able to drastically reduce itching in test mice. As treatment now generally doesn’t do much to combat the discomfort of itching, this is a major breakthrough in poison ivy treatment.

What is Poison Ivy?

 Poison Ivy is a plant that produces resinous oil called urushiol that covers the entire plant: stems, leaves, and roots. When this oil comes into contact with human skin, either directly or through a secondary source like an animal or tool, it triggers an allergic reaction in 85% of people, causing your immune system to attack and create an itchy rash. This rash can escalate into hives and blisters.

What is this discovery?

So far there is treatment such as antihistamines and corticosteroids that help swelling, pain and accelerate recovery, but there is no real cure for the itchiness and discomfort this rash can cause. However, by blocking a certain protein in the immune system with an antibody, one can block the signals to the brain communicating an itch on the skin. This protein is interleukin 33 (IL-33), a common protein that acts directly on the nerve fibers in the skin, exciting them and telling the brain that the skin is severely itchy. When blocking this protein, scientists conducting an experiment on lab mice were able to reduce not only inflammation, but the amount of scratching.

What does this mean?

If this treatment could be used on humans, it would provide a far more affordable and effective treatment for victims of this pesky condition, and eliminate the doctors visits, lost time at work or school, and costs of only partly helpful drugs that are now necessary for people suffering from this rash. Hopefully, the pain and annoyance that comes with Poison Ivy will soon be a thing of the past.

Image: https://commons.wikimedia.org/wiki/File:Poison_ivy-20141524-038.jpg

A Breath of Fresh Air: Epigenetic Studies Help Asthmatics

Asthma and allergies affect many people worldwide. Up until recently, treatments for both asthma and allergies were administered without an appropriate prediction of responses; However, this is about to change. In a recent study conducted by scientists at Imperial College London, “30 new genes that predispose people to allergies and asthma” were found. The discovery of these genes means that new treatments for allergies are possible and more accurate predictions for current treatment responses will be available.

Photo by Author

Photo by Author

By observing the epigenetic changes, ones that influence gene activity- not genetic code, the scientists were able to identify genes which are linked to triggering allergic responses. Such genes regulate specific antibodies. Genes become inactive through methylation: the attachment of methyl molecules to DNA. The scientists studied white blood cells of families with asthma to see if methylation levels in specific genomic locations were associated with levels of an antibody in the blood. Immunoglobin E (IgE) is the antibody studied in the case. The antibody IgE was known prior to this study, but the genes which activities it regulates were not. After monitoring the IgE levels in the blood, researchers saw a strong correlation between IgE and low methylation at 36 places in 34 genes. These genes are overstimulated in asthmatics, thus the production of IgE is increased, contributing to asthma symptoms. In expanding the investigation, researchers came to believe that IgE-involved genes may activate eosinophils, a type of white blood cell which in asthmatics promotes airway inflammation by gathering and releasing chemicals in airways/lungs. Researchers believed that these genes, and their ability to activate eosinophils, then cause the most damage. In order to test this, researchers isolated eosinophils from the blood of subjects and demonstrated that all 34 genes have high activity levels in asthmatics with high IgE levels. Thanks to the findings of new activation signals, patients can avoid high costs and ineffective “treatment-trials” because we’ll be able to predict responses to treatments with more accuracy. Professors Cookson and Moffatt, the leaders of the investigation, give credit to epigenetics for allowing them to make a breakthrough in discovering new potential drug targets for allergies and asthma and sharpening the accuracy of treatment-response predictions. Professor Cookson explained that, “the genetic code that can influence disease and DNA sequencing can only take you so far. Our study shows that modifications on top of the DNA that control how genes are read may be even more important.”

As someone who suffers from allergic asthma, I find it intriguing how the disease-triggering genes aren’t inactive, thus leading to poor lung function, but rather they are overstimulated. Our genes’ ability to regulate disease-triggering antibody activity is amazing. With new studies like this one, we can see that the solution to proper activity regulation is in epigenetic changes, rather than the broad expectations of “our genes”.  This just goes to show that epigenetics is helping us make strides in the ever-changing world of medicine. It should be interesting to see how epigenetic medical-solutions, the current gold mine of Biological research, evolve in the near future.

 

A Baby’s Immune System Might Be Stronger Than We Think

Lymphocyte_activation_simple

Mothers are often extremely protective of their newborn. Most moms are fearful that everything could potentially make their baby sick. Unfortunately though, there is no absolutely sure way to keep a child from getting sick. The immune system plays a huge role in keeping humans well. Two important parts of the immune system are antibodies and memory cells. Antibodies help kill harmful germs while memory cells help the immune system respond quickly to an infection and prevent disease. In fact, recent studies prove that the Immune system of newborn babies are stronger than people previously believed.

Scientists involved in a study led by King’s College London, are reporting that newborn immune T cells can trigger an inflammatory response to bacteria. Originally, it was believed that babies immune systems were immature and therefore couldn’t trigger the same inflammatory response adults normally demonstrate. The team discovered that whilst T cells in newborn babies manufacture a potent anti-bacterial molecule known as IL8. It activates neutrophils to attack the body’s foreign invaders.

In addition, Dr. Deena Gibbons, Lead author in the Department of Immunobiology at King’s College London believes that this “mechanism by which the baby protects itself in the womb from infections of the mother.” Next, she plans to better understand the reasons that there are many differences between the immune cells in newborns and those in adults.

The T Cell activity demonstrated by newborns could be used for future treatments to boost the immune system or neonates in intensive care (place with major risk of infection).

This article is very interesting and important because it is vital to keep newborn babies as healthy and safe as we possibly can. Sometimes it can be as simple as following common measures such as hand washing, avoiding people who are most likely to be sick, snotty noses or hacking coughs. A mother should try to do anything she can to limit the demands placed and a baby’s immune system in the early months to keep her baby healthy.

I chose this article because I know what it is like to be a patient in a Hospital and the precautions that doctors take to prevent further sickness when the immune system is not fully developed or strong.

Image links: 

Häggström, Mikael. “Medical gallery of Mikael Häggström 2014“. Wikiversity Journal of Medicine 1 (2). DOI:10.15347/wjm/2014.008ISSN 20018762. – Image:Lymphocyte_activation.png

http://en.wikipedia.org/wiki/Immune_system

Article: King’s College London. “Immune system of newborn babies stronger than previously thought.” ScienceDaily. ScienceDaily, 21 September 2014. <www.sciencedaily.com/releases/2014/09/140921145104.htm>.

http://www.sciencedaily.com/releases/2014/09/140921145104.htm

Other Sources:

http://en.wikipedia.org/wiki/Immune_system

http://www.wellness.com/reference/allergies/newborn-immune-system

http://www.nobelprize.org/educational/medicine/immunity/immune-detail.html

A New Drug Can Potentially Cure Radiation Sickness

Radiation from a major nuclear accident can kill thousands of people. It is very dangerous and depending on how much radiation people are exposed, high exposures of radtion make people get radiation  sickness and eventually they die. Radiation exposure levels up to 10 gray would require a bone marrow transplant. After exposure to radiation bone marrow is the first thing that is effected and that is where white blood cells are located and a healthy immune system is needed after exposure to radiation. Radiation can cause bacteria to leak into the blood, causing blood pressure to spike, feder, abnormal blood clotting and organ failure. A bone marrow transplant is almost necessary. However, after a major nuclear accident thousands of people will need bone marrow transplants and that might be very tricky to execute.

But there is a solution! New research has proven that a drug composed of fluoroquinolone antibiotic combined with a microbe fighting compound- a protein called BPI– can protect thousands of people from the effects of radiation. How? This antibiotic and protein can attack the bacteria that is leaked in the blood and causes all these harmful effects. The antibiotic kills the bacteria and the protein latches onto the bacterial endoxtin, a molecule on the coats of bacteria.

Researchers exposed mice to radiation for 24 hours and gave some of the mice the injections of the antibiotic and the protein. The mice who did not get the antibiotic and the protein died 30 days later.  80 percent of the mice who got the antiobiotic and the protein lived almost healthily.

This drug could change the harmful effects of radiation and could be very useful to the world as nuclear sources of energy are used more often. However, it is still in production and is not perfected. Doctor Eva Guinan  of the dana-Farber cancer institute and Harvard medical school believes that “We still have a lot more to explore in detail, but the results are really encouraging.” Do you think this future drug will completely solve the problem of radiation sickness?

 

other links:

 

original radiation sickness article

Information on Quinlone

 

More information on BPI

 

Information on Radiation poising

Information on Bone Marrow transplants

alternate article  on radiation sickness treatment

 

Antibodies to the Rescue!

Photo Credit: RambergMediaImages Flickr

 

 

HIV is an extremely dangerous virus because our own antibodies cannot effectively attack it. HIV uses a coat of sugars to hide itself from our antibodies. Although the body cannot effectively fight HIV, it does its best by making new antibodies to try and attack this powerful virus. These new antibodies attach to different spots on the sugar coating of the virus. It uses the sugar coat to bind to a site on the virus where amino acids are exposed. Then the antibody attacks the virus from that site, disabling it.

 

The discovery of this antibody and the way it binds to the virus is important because it can lead to advances in a cure and a vaccine. It gave scientists key information about binding sites made out of sugars and amino acids. They can use this information, as well as information from other projects and discoveries to make a more effective vaccine. In fact, some recent tests have shown that the antibodies play an important role in the health of someone infected with HIV.

New HIV Prevention Technique

CC licensed by photo Micro World (flickr)

An exciting, cutting-edge approach to HIV prevention is quickly gaining support, as researchers  have been learning about special antibodies that have destroyed HIV in the lab.  Now, biologists at Caltech have taken the next step, as they have discovered a way to insert these antibodies into mice, thus protecting them from HIV infection.

This new approach to HIV prevention — called Vectored ImmunoProphylaxis, or VIP — is outlined in the November 30 advance online publication of the journal Nature.

This new technique in HIV prevention is revolutionary, as supposed to traditional methods that centered on developing a vaccine that would provoke the formation of antibodies or T cells in the body, VIP provides protective antibodies directly.

Mice treated with VIP have been shown to produce high concentrations of the protective antibodies throughout their lives, and remain protected from HIV when it is administered intravenously.

Still, researchers must make the next step and show that the antibodies produced from VIP work to destroy HIV in humans.  According to researchers however, the problem will not be whether the antibodies work, as they are relatively sure of its effectiveness.  Rather, experiments will have to be conducted to see if VIP produces enough of these antibodies.  According to Alejandro Balazs, lead author of the study and a postdoctoral scholar, “In typical vaccine studies, those inoculated usually mount an immune response — you just don’t know if it’s going to work to fight the virus.  In this case, because we already know that the antibodies work, my opinion is that if we can induce production of sufficient antibody in people, then the odds that VIP will be successful are actually pretty high.”

For more information on this revolutionary new technique, visit the page http://intelwars.com/2011/11/30/gene-therapy-turns-muscles-into-hiv-antibody-factories/ 

What do you think?  Will the VIP method be successful in humans, and will HIV and AIDS finally be conquered?

 

 

 

 

 

 

 

 

 

Gamers solve some of biology’s most difficult riddles?

Who is solving some of biology’s most difficult puzzles and riddles? Obviously scientists, right? Think again. It’s the gamers.

An article recently reported that a revolutionary online game called Foldit, allows anyone, from gamers to students, to help predict the foldings and structures of  various proteins by playing competitively online. Protein folding is one of biology’s most difficult and costly problems, and is even a troublesome task for the most capable computers. A game such as Foldit requires much insight and an intuitive understanding to fold the proteins, allowing human intuition to triumph over a computer’s calculations. As we have learned in class, proteins are very prevalent in the human body. Hormones, enzymes, and antibodies are all examples of proteins, but many proteins are also associated with strands of viruses and diseases.

This is where you, as the gamers, come into the picture.

Since proteins play a large role in the functions of viruses and diseases, gamers playing Foldit can help design new proteins to help treat or provide a cure for the condition. The article reported that gamers have most recently solved the structure of an enzyme crucial for the reproduction of the AIDS virus. Knowing the structure, scientists are now able to find certain drugs to neutralize the enzyme and stop the reproduction of AIDS virus.

In class, we have learned that there is basically an infinite amount of combinations of proteins; there are 20 amino acids and can be combined to form chains of various lengths. We have also learned that the structure of a protein is also correlated with its function. The bonds present in the primary, secondary, tertiary, and quaternary structures of proteins are an important part to the shape and folds of a protein, giving the protein certain properties due to its shape. All of the information we have learned about proteins in our AP Biology class, can be seen and easily applied to the game, Foldit.

Now since we know the vital importance of proteins, do you want contribute to the next cure for a virus or disease? Get your game on and try Foldit out and see what you can do to solve some of biology’s most difficult riddles!

 

 

 

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