Geese will soon fill the skies as they migrate south in V-formation as the weather gets colder and the leaves start changing color. For a month or so, migrating birds take over, crossing roads, sitting in parks and stopping to eat leftover seeds in farm fields or swim in ponds as they travel south for the winter.
What people may not realize is that some of these birds are carrying something harmful, yet invisible to the naked eye. That something is influenza A viruses that can transmit from birds to pigs and then to humans.
Henry Wan, influenza researcher in Bond Life Sciences Center, and his collaborators recently identified which influenza A viruses pose a risk for the pigs and are studying how the viruses transmit from pig to pig.
“We hope to identify risk which is very important,” Wan said. “I feel good about the study because we believe this would be the foundation for people be able to do the risk assessment on how the bird flu can go to pig and go to humans.”
By identifying which influenza A viruses are a risk for pigs, it helps to determine which viruses can ultimately be a risk for the human population, a more typical concern for the researcher’s usual work to improve influenza vaccines.
“It’s difficult to make a universal vaccine,” Wan said. “If I know which viruses are a risk then I can tell which ones to protect against.”
Influenza A can have a massive impact on people — causing outbreaks like the 1918 influenza pandemic — and more rapidly mutating genetically and antigenically, compared to other viruses. Understanding how it spreads is crucial in pandemic preparedness and creating vaccines.
Pigs, typically by direct contact or breathing in droplets in the air, contract an influenza A virus in the environment contaminated by birds migrating through the area. Some viruses go to the lungs, which means it cannot escape the pig and cannot be transmitted. Other viruses however, go to the upper respiratory tract of the pig where it can shed and transmit to other pigs, quickly infecting other pigs. How this happens on a molecular level is more of a mystery.
Wild birds serve as a reservoir for diverse viruses across many bird species. While not all of the viruses are a risk to pigs, Wan’s lab wanted to identify which ones are a risk to pigs and how it is transmitted to other pigs.
“Many viruses in nature are not likely to go to humans, only a small portion can. How you detect them is key,” Wan said.
Through analyzing characteristics, Wan found that cells and tissues that support influenza A viruses affect their transmission from birds to pigs. This phenotype — a set of characteristics resulting from the interaction of being with their environment — was determined by markers across the structures of genomes. For example, those in an RNA complex.
To do this, Wan collaborated with researchers from the National Wildlife Research Center and the National Veterinary Services Laboratory at the United States Department of Agriculture, Mississippi State, The Ohio State University, and The United States Department of Agriculture, among other labs. Wan’s lab initiated this study which led to collaborations across labs.
“Different people have different expertise. I really enjoy collaborations and different ideas,” Wan said.
Wan also found only a small portion of the virus can grow and adapt to different pigs and then transmit to other pigs. He then mapped the distribution of these risky viruses across multiple wild bird species.
Now that Wan and his team know how influenza A virus is transmitted into the pig, they are working on the next step, predicting a pig has a risky virus using artificial intelligence (AI).
“I think we have reached one stone and are ready to move to the next one,” Wan said, “Of course, everybody’s pretty excited.”
He plans on furthering his research of pandemic preparedness by looking at if the virus adapts to the pig and gains a receptor or an additional feature in order for it to easily transmit from pig to pig.
“That’s how this virus goes to endemic or even pandemic.”
Lucas Woods from the Weisman lab watches lung cancer cells and oral epithelial cells grow. | photo by Lauren Hines, Bond LSC
By Lauren Hines | Bond LSC
Vaccine development remains a central goal to get the current COVID-19 pandemic under control. While vaccines are highly vital in the fight against the current pandemic, what if scientists could prevent the virus from entering cells altogether? Researchers at Bond Life Sciences Center are working to do just that and, so far, they’re the only ones at Mizzou on the case.
For the Gary Weisman lab, that starts with their study of a family of purinergic receptors which are found on the plasma membrane of cells. Molecules outside the cell bind to receptors and serve as signals to trigger an action, whether it’s transporting something into a cell, alerting its defenses or initiating programmed cell death. The specific receptors they study sound off one of the first alarms to alert host immune cells when a cell is damaged.
Weisman, Bond LSC principal investigator and Curators’ Distinguished Professor of biochemistry, noticed that the spike protein in the virus that causes COVID-19, SARS-CoV-2, shares an amino acid sequence — arginylglycylaspartic acid or RGD — with one of the receptors his lab studies named, P2Y2.
“So, we had this one niche that no one I think has followed,” Weisman said. “Even though people understand RGD sequences are important for protein to protein interactions, and possibly viral entry, no one has looked at this with respect to coronaviruses.”
While ACE2, a plasma membrane receptor, serves as the primary cell-surface receptor for SARS-CoV-2 binding, a potential role for the RGD sequence in the spike protein is to bind to alternate receptors and subsequently enhance viral infectivity.
“We postulated that if there was a way to use our receptor, it might be to block the entry of the virus,” Weisman said. “The virus can’t replicate on its own. It has to get inside the cell to be replicated, so if we prevent the entry, we can potentially stop the infections.”
They found plasma membrane receptors like ACE2 and P2Y2 all share this sequence that binds to other cell-surface receptors called, integrins. The lab is currently exploring ways to block these pathways without blocking other functions.
Few other labs have made this connection because RGD is not known to be present on many of the membrane receptors. In addition, the Weisman lab already has the supplies and connections to study these receptors.
“We, as the lab, have been studying this receptor and this family of receptors for so long that if there is a lab that has the tools and the knowledge to answer this question, I definitely think that is us,” said Kevin Muñoz Forti, Ph.D. candidate for the department of biochemistry.
Kevin Muñoz Forti is doing cell culture work in “the hood,” which is a space used to prevent researchers and other outside factors from contaminating the cells. | photo by Lauren Hines, Bond LSC
The lab partners with Kamal Singh, assistant director of the Molecular Interactions Core at Mizzou and associate research professor in the department of veterinary pathobiology, who works with viruses in a way that won’t infect workers.
Singh achieves this by using look-alikes, or pseudovrises, of the spike protein on SARS-CoV-2. To the cell, this mimics the outer shell of SARS-CoV-2, but is not contagious.
Even though the Centers for Disease Control and Prevention and Dr. Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases, expect a vaccine soon, this work is for the long run.
“I can guarantee you this isn’t the last coronavirus that will come up,” Singh said. “There will be many because it has been kind of periodic lately. Coronaviruses have been emerging, so the research done in collaboration with Gary’s lab will definitely be available for… future coronaviruses.”
Coronaviruses are a family of viruses which may cause respiratory illness in humans. The most recent diseases, before COVID-19, caused by previous coronaviruses were Severe Acute Respiratory Syndrome (SARS) from 2002 and Middle East Respiratory Syndrome (MERS) from 2012. Severe Acute Respiratory Syndrome Coronavirus 2 is the virus that causes COVID-19.
“This is the virus that causes COVID-19 or SARS-CoV2,” said Lucas Woods, Weisman research lab manager. “If this type of research were to have been investigated during the first coronavirus epidemic, there might have been some type of lead on the type of work we’re doing… so, absolutely this work will be useful after a vaccine or treatment is available.”
Looking ahead, the study has two years of funding from the American Lung Association in addition to grants from the National Institutes of Health. The Weisman lab hopes to eventually make it to clinical trials, but the road from basic research to clinical trials is long and arduous.
“What’s on our side, at least for the future, is that with some of these pathways we’re looking at, there are already available drugs that target these particular pathways,” Woods said.
These pathway-blocking drugs aren’t approved for humans yet, but this research could facilitate clinical scientists to test the safety of these or related drugs in humans.
The Weisman lab’s research of P2 receptors can be applied to many systems in the body and other diseases. This also allows for more ways to receive grants to fund studies longer. Along with the COVID-19 studies, they study P2 receptors in diseases that affect salivary glands.
Cheikh Seye, Mizzou professor of biochemistry, studies vascular diseases that relate to the receptors studied by the Weisman lab. Seye understands that one of the complications of COVID-19 in humans is vascular related.
“I mean there’s a lot of interaction between the vasculature and other systems, so it doesn’t stay with the salivary gland,” Seye said. “It’s all interconnected, so that’s why we need to have a multi-dimensional approach.”
In addition, the lab has found commonalities between oral and lung epithelial tissue that may contribute to COVID-19.
While this study has quite a bit of potential, it’s a long way away from seeing practical results.
“All the people in the lab have confidence because they have been successful on their own doing this before,” Weisman said. “So, it’s just another challenge, but we know how to get the answers, we have the tools and we’re optimistic that we’ll find out something.”
Researchers working on this study include Gary Weisman, Kamal Singh, Lucas Woods, Cheikh Seye, Jean Camden and Kevin Muñoz Forti.
Using cell lines to better understand copper homeostasis
Nikita Gudekar working at her lab table. | photo by Becca Wolf, Bond LSC
By Becca Wolf | Bond LSC
When one thinks of copper, they often think of a shiny new penny. A striking engravement of Abraham Lincoln’s side profile with the words, ‘In God we trust’ engraved over his head. This, however, is not the case for Nikita Gudekar.
Gudekar, a genetics area program Ph.D. candidate in the Petris lab, thinks of copper and gets inspired.
Copper is an essential element in the body that has many roles such as energy production, the formation of blood vessels, protection against reactive oxygen species, and so on. However, the body must maintain a balance of this metal because too much or too little could cause disorders such as Wilson’s Disease and Menkes Disease. Both neurological diseases can be deadly without treatment.
Two proteins that help balance copper are ATP7A, a copper exporter, and Metallotionein (MT), a copper binding protein. Knowing these play a crucial role in copper homeostasis, Gudekar created four cell lines to test just how much of a role ATP7A and MTs play in regulating the balance of copper.
Gudekar created a cell line with solely ATP7A present, one with solely MT present, one with both present, and one with neither present.
Comparing the four, Gudekar realized the significance of each protein in copper homeostasis.
Nikita Gudekar looking at the cell lines through a microscope. | photo by Becca Wolf, Bond LSC
“As I was doing the experiment, I realized that these cells lines could not only be used to study copper homeostasis but also to discover novel genes in copper metabolism and diseases,” Gudekar said, “So we knew that this has got a lot of potential. And it might take some time, but we will get something out of it.”
In the cell lines where neither ATP7A nor MTs were present, there was a hyperaccumulation of toxic levels of copper which prevented the growth of the cells in the cell culture medium. However, when one or both proteins are present, the cells can survive, even in excess copper concentrations. This suggests that ATP7A and MTs may function to safeguard against copper toxicity. With this knowledge, there is potential for better understanding copper metabolism during pathological copper deficiency in humans.
A major observation Gudekar saw was in the cell line with only ATP7A present. She noticed that the cells were able to grow at a higher copper concentration compared to the cell line with only MTs present. This signified that, “ATP7A is kind of an important one when it comes to saving the cells from excess copper concentration,” Gudekar said.
However, the lack of MT results in trafficking ATP7A from its normal steady state localization in the trans Golgi complex to the plasma membrane, which only occurs when there is an excess amount of copper in the cell. With MTs present, ATP7A is stabilized and this does not happen. The discovery of this novel regulatory mechanism suggest that both proteins contribute to cellular copper homeostasis.
The most important aspect of this research was the discovery of the cell line with neither ATP7A nor MT present. This cell line is extremely sensitive which can be used to isolate novel copper metabolism genes in the future.
“The extreme sensitivity of ATP7A and MT knockout cells to copper represents an opportunity of using these cells to identify novel copper tolerance genes,” Gudekar said, which is her current thesis project.
“It has become more exciting are this point,” Gudekar said, “We have isolated a novel copper metabolism gene that allows the sensitive cells to now live in copper. Our current focus is to understand the molecular mechanism of how this gene regulates copper homeostasis.”
Using the momentum of her new discovery, Gudekar hopes to find more genes that are involved in copper homeostasis.
“Knowing that something you’ve created is now being put to use to discover novel genes that could be potential drug targets in a disease is very fascinating, it’s really satisfying to see that,” Gudekar said.
Marc Johnson collecting pellets in his lab. | photo by Becca Wolf, Bond LSC
By Becca Wolf | Bond LSC
There is not much thought that goes into using the bathroom. You do your business, flush, and wash your hands. It is just a part of the daily routine.
Recently though, human waste has become a golden nugget to researchers. In fact, waste from toilets throughout the community are contributing to figuring out where the next COVID-19 outbreak could happen.
And Marc Johnson, Bond Life Sciences Center principal investigator and MU professor of molecular microbiology and immunology, is the person figuring out how this human waste can be an asset as he processes samples from the sewer systems of more than 70 communities across the state.
A map of the sewersheds Marc Johnson’s lab receives samples from. | map contributed by Marc Johnson
These samples may be able to serve as the canary in the coal mine before large outbreaks strike in a community, allowing health officials to prepare for and potentially prevent the spread of COVID-19.
SARS-CoV-2, the virus that causes COVID-19, appears in wastewater several days before an infected person shows symptoms, so Johnson wants to find a faster way to test for the virus.
“We’re very good at predicting what’s happening right now based on samples we collected five days ago,” Johnson said, “We are predicting the future, we just don’t know what our prediction is until the future is actually here.”
This data may not seem helpful, however, it proves that the COVID-19 case numbers are accurate and, if acted upon quickly, can prevent an outbreak in a concentrated population such as prisons or schools.
“With the students, when you’re talking about patients that don’t necessarily know they’re infected, it can be really helpful to have a heads up that this dorm has some infections even if they don’t know it,” he said.
Testing wastewater for SARS-CoV-2—an RNA virus—consists of two phases. Phase one is obtaining the sample and phase two is RNA extraction.
Starting phase one, Johnson receives a 24 hour composite from a wastewater testing facility, “So that no matter when, it doesn’t matter whenever people go, as long as they go once a day we’ll catch them,” he said. This gives his lab a representative sample of the population.
Once the sample is taken from a wastewater treatment facility, it is put into a courier system that brings it to the state lab in Jefferson City, Mo. After it arrives there, the Missouri Department of Natural Resources brings them to Johnson’s lab at Bond LSC so he can begin phase two, RNA extraction.
In Johnson’s lab, the samples are put through a filter that nothing bigger than a virus can get through. This eliminates the solids and other bacteria in the wastewater. Next, a chemical is added that allows the viral particles to stick together so they can be pelleted.
This is then put in a centrifuge and is spun for a few hours to get a small, invisible pellet. RNA is then extracted from this pellet in a QIAcube, a robot Johnson’s lab purchased for this project. The QIAcube is the same robot used by the Missouri Department of Health Services for COVID-19 testing in their own facilities.
Marc Johnson placing the samples in the QIAcube. | photo by Becca Wolf, Bond LSC
After Johnson extracts the RNA, the samples are sent to Chung-ho Lin, MU research associate professor at the College of Agriculture, Food and Natural Resources, who continues testing.
This whole process takes about five days.
Johnson hopes that test results will soon go public so people have an idea of where outbreaks are, although reading the data can be confusing.
Said Johnson, “I think we all agree we’ll be better if we put the data out the way we want it to be seen, because it’s just when you look at the raw numbers, without understanding how to interpret it, you can make any conclusion you want.”
It will be a learning curve for the public to be able to understand the data. Johnson even struggled with it at first, “We learned this lesson the hard way,” he said, “I freaked out several times where I thought I had just discovered Armageddon going to our state, but you have to put it in perspective. When you figure out how much it actually takes to get a signal and how big the sewer shed sample is, it’s like, ‘No okay, that’s normal.’”
Along with other testing sites, Johnson’s lab has been testing several universities and colleges in the state, including MU. With time and improvements, it is realistic that his lab will be able to predict the number of infected patients in an area.
“It’s a crap ton of work,” Johnson said, “But it’s kind of interesting. It worked far better than I would have expected.”
While research on COVID-19 was not on Johnson’s agenda earlier this year, he has been trying to have fun with it.
“The puns are unending,” he said, “There are just so many great puns I’ve gotten out of it.”
“It’s serious research but it’s nice that the topic has gotten a little bit of levity.”
These maps show the receptors’ locations, density and count in a cancer cell. | photo contributed by Alexander Jurkevich, Donald Burke-Agüero, David Porciani and Skyler Kramer.
By Lauren Hines | Bond LSC
With two laptops in front of him and a supercomputer on the edge of campus, graduate student Skyler Kramer runs through code daily in the Dong Xu lab with a purpose far beyond deciphering lines of data. He helps his colleagues defeat cancer.
Working in Bond LSC with senior post doctorate David Porciani from the Donald Burke lab, Kramer and the Dong Xu lab are part of an effort to target cancer tissue and develop more precise delivery of treatment.
In addition, they’re trying to learn more about the biology that drives cancer growth and what makes cancer tissue resistant to treatment after a certain point in time.
“[Kramer] has been a tremendous asset for the project,” Porciani said. “His contribution is so critical at many levels.”
Porciani set out four years ago to find a way to easily differentiate between cancer and healthy tissue. By developing small pieces of RNA and DNA called aptamers that can bind to receptors on cancer cells, he slowly tried to determine the pieces that would only interact with cancer — not bothering the healthy cells — that could deliver a cancer-killing drug. But the process meant Porciani would amass mounds of data.
“There is a lot of statistics in there, which is very important,” Porciani said. “So, that brings in the collaboration with Skyler.”
About a year ago, Porciani was presenting his work on aptamers at a talk in Bond LSC. Xu later inquired if Porciani was open to any collaboration, which led to discussion on what the two labs could do together.
“It was just really really fascinating,” Kramer said. “So, I was really interested in it.”
Now, the two are working together to turn microscopy images into something more meaningful.
Through super resolution microscopy images of the receptors on the cancer cells, Porciani took a short video showing the thousands of receptors shaking.
“Most of the receptors like to shake hands with each other, and when that happens it is critical for the growth of the cells,” Porciani said. “So, the handshakes are sort of a signal that says, ‘Okay, let’s move this house to the next stage. Yes, let’s make the cell grow even more.’”
Porciani could analyze only a small part of these shakes with his naked eye but using a bioinformatics approach —using software and statistics to understand biological data — Kramer can take that data from the thousands of receptors and turn it into digestible and meaningful information.
“[I’m] trying to take the massive amounts of data that we’re able to generate and get some sort of useful information out of it,” Kramer said. “So, a lot of times it’s useful not only to get some sort of numeric output but some sort of visualization also.”
This means turning an image of a cancer cell into a receptor density map or a receptor count map. By doing so, researchers can investigate why receptors clump into one area, what that means and why cancer cells become resistant to treatment over time.
“It’s our job to understand why there is this difference,” Porciani said. “This type of information can generate new hypotheses that we need to test.”
With the help of bioinformatics, Porciani and other researchers can find the answers to their questions.
“I think [bioinformatics] is hugely impactful,” Kramer said. “A lot of times, the life sciences will give you the actual experiments and a rough idea of what happened in the experiment, but I think that when you apply the bioinformatics you get more to why specific things are happening.”
The collaboration brings more than just new results, but also can help expand science.
“I think I’m learning a lot from him,” Porciani said. “I think, hopefully, he is learning a lot from me as well.”
To learn more about Porciani and Burke lab’s findings regarding aptamers, the research article can be found here.
Clement Essien poses outside Bond LSC near the building’s garden. | photo by Lauren Hines, Bond LSC.
By Lauren Hines | Bond LSC
Artificial intelligence (AI) can do more than just write a book given a few words. It can help make cancer treatments more effective and predict the presence of disease in cells, which doctoral student Clement Essien did through his recent project.
“It’s exciting because for several years, I was a software engineer, and then I felt like I wanted to do something more with that,” Essien said. “I want to make some contribution to understanding disease and also in the diagnosis and treatment of diseases. So, I had to look for ways that I could apply computing to understand and possibly solve many biological problems.”
Essien — who works with Bond LSC principal investigator Dong Xu — is trying to predict the binding sites of metal-binding proteins called metalloproteins using AI technology, specifically deep learning.
“Metal binding can play a very functional role,” said Dong Xu, Shumaker Endowed Professor in Bioinformatics, Director of Information Technology Program. “That is why it’s important to know whether a protein binds to a particular metal, and also if you really could, you’d want more details on where it binds.”
Even though Essien’s work is still underway, it has big implications.
“[My work] helps to advance the research geared towards improving the prediction capability of machine learning modules that work on this problem and also provide an important step towards understanding protein functions, and their implications for gene product characterization, drug design for certain diseases and enzyme engineering.
Not only could predicting the binding sites of metal proteins help create drug targets and advance other research, but it could also possibly help identify the presence of disease.
By predicting the binding site, researchers can figure out the protein’s structure and therefore infer the function.
“[The function is] what tells you what role the protein plays in the body,” Essien said. “Also, the presence or absence or mutation of a particular protein sequence can cause diseases.”
Essien’s previous project had the same goal of predicting zinc binding sites, but now he is expanding his research to the study of many other metals found in the human body. Essien is also using an AI technique called Natural Language Processing (NLP).
One use for NLP would be to give an AI all the words in the dictionary and then ask it to write a book. In Essien’s work, he is trying to model the protein sequences as a text because they consist of a sequence of letters, and then get the AI to learn representations from it.
“So, we are trying to model the problem as a natural language problem in the sense that those series of letters you see in the protein sequences, they may be represented as words,” Essien said. “So, if you’re able to break that code you might be able to learn some important things.”
Essien published one paper in a conference and expects to have another one out in a few months. Although, he has much more to discover until then.
“It’s one thing to see I’m able to predict these to a certain accuracy, but it’s also important to learn what is going on inside,” Essien said. “Is there any new significance to learn?”
Jiude Mao works on BPA testing in the lab of Cheryl Rosenfeld.
By Mariah Cox | Bond LSC
Bisphenol A, more commonly known as BPA, has been a source of scientific dispute for the past decade. With a lack of consensus among scientists, consumers are left unaware of the potential harms of the chemicals in plastic.
In response to a recent report by the Food and Drug Administration (FDA) that claims BPA is safe at the current levels occurring in foods, Bond Life Sciences Center principal investigator Cheryl Rosenfeld and a group of researchers across the country have teamed up to release a secondary analysis of the existing data, which disputes this claim.
The industrial chemical is used in manufacture of plastics and resins, and it is commonly found in plastic food containers, water bottles, food can linings and other consumer products. BPA can leach out into water supplies and food where humans and wildlife may be exposed to this ‘persistent chemical’ by ingestion or inhalation.
All of the researchers on the second report were a part of the original team put together by the FDA to study the effects of BPA. However, many researchers on that team disagree with the FDA’s re-analysis and interpretation of their individual findings.
By using the publicly available data published on the National Toxicology Program’s website, these scientists reevaluated the information originally compiled by Rosenfeld and dozens of colleagues as part of a Consortium Linking Academic and Regulatory Insights on Bisphenol A Toxicity (CLARITY-BPA).
Cheryl Rosenfeld had concerns of this Consortium project from the beginning.
“The idea at the outset was that individual investigators and FDA scientists partner together to address the question as to the safety of BPA, but even at the initial meetings, several concerns were raised,” Rosenfeld said.
The major source of disagreement boiled down to lab procedures, statistical analysis and a lack of regard for the inter-related effects of BPA on possibly multiple target organs and bodily functions. Going into it, the researchers had minimal input into the general experimental design, including a rat model that may be less sensitive to the effects of this chemical, the dosages of BPA that were tested, the fact that BPA was administered by what many consider a stressful procedure, oral gavage, and the period of administration.
One problem that was not thoroughly considered is the potential for nonmonotonic effects of BPA. That essentially means BPA shows adverse effects on the body at low and high doses, but not in between or middle-of-the-road doses.
On top of discrepancies over the research procedures, the researchers criticize the FDA for using stringent statistical analysis that may filter out important differences between groups.
“It’s like a metaphor about dropping your keys in a parking lot and looking over by the curb for them because there’s better light there,” said Gail Prins, a professor at the University of Illinois – Chicago and a collaborator on the original and secondary research project. “They’re concluding that BPA is not significant, but they’re not looking in the right places for significant results.”
In statistics, there are type one and type two errors. A type one error concludes that the results of the study were statistically significant when they’re not. Vice versa, a type two error concludes that the results are not statistically significant, but they are. Also, margin of error comes into play. P-value — a measure of deviation that determines which results are noteworthy — sets the stage for what is considered significant. Based on the method of a study, researchers can have stringent requirements for assessing the significance of a result (p≤.01), but most research uses p≤.05.
In simpler terms, p≤.05 allows researchers to be 95 percent certain that a result is meaningful. While the FDA used a p-value of <0.05, the researchers in the secondary study believe that the FDA failed to look at the statistical significance of the inter-related effects of BPA on multiple parts of the body, including the mammary glands, ovaries, kidneys, the prostate gland and cognitive-behavioral function.
Additionally, the statistical approaches the FDA sought to use would require hundreds of research replicates to be statistically valid. The FDA only had a budget to repeat the experiments up to 12 times per group, which some investigators questioned whether findings on these alone, especially with the methods the FDA sought to use, would provide meaningful results.
In 2012, the FDA banned the use of BPA in baby products, although that decision was largely due to public concern. However, the primary route of exposure to the effects of BPA are before babies are born. Since BPA is present in products used by pregnant mothers, it can lead to the development of health problems in babies including cancer later on in life.
The original statistical analysis for Rosenfeld’s portion of the project was done by Mark Ellersieck of MU, who has 30 years of experience, and a statistician with the FDA. When the analyses disagreed with each other, a neutral third-party was brought in to review the approaches used by Ellersieck and corroborated they were appropriate for the study design.
Now, Jiude Mao, a research scientist from the Division of Animal Sciences in Rosenfeld’s lab at Bond LSC, is working with Rosenfeld to reanalyze the results of the original study.
“I downloaded the raw data package online,” Mao said. “If you look at the effects of BPA on individual organs versus combining them and looking at its effects on multiple organs, the picture is very different.”
By using special informatics approaches, Mao found that the lowest dose of BPA tested simultaneously led to multiple effects on various target organs in females including the ovaries, uterus, mammary glands, heart, and fat tissue. In males, the prostate gland, along with the heart and adipose tissue showed inter-related changes due to BPA exposure.
Mao and Rosenfeld have also linked multi-organ effects of BPA at two other doses, with all doses tested currently considered safe by the FDA. They examined these inter-relationships at three age ranges: 21 days of age, 90-120 days of age, and 180 days of age. To the investigators’ knowledge this is the first type of toxicological study that has linked such data obtained in multiple investigators’ laboratories and shown such complex relationships.
The data from these three doses of BPA and three age ranges clearly indicate that BPA affects on a single organ can radiate out to affect many other organs throughout the body. By tugging on one organ, BPA can damage intricate webs that connects organs to each other. Such inter-relationships between individual CLARITY-BPA investigator data have not been considered by the FDA.
While a consensus hasn’t been met between the two parties, a potential solution for the data analysis discrepancy could be looking to machine learning or ‘deep learning’ to avoid human error or bias. This would include inputting both data sets into a program that can assess what the similarities and differences are and why the two groups are achieving different conclusions. This approach would ensure more confidence in the accuracy of the results instead of choosing a side to believe based on human calculations.
For the researchers, reevaluating the data means providing the full scope of the effects of BPA on multiple parts of the body. It also means giving consumers the correct information so that they can make well-informed decisions about their health.
“I am concerned that government agencies are not providing the public the fully story as to how BPA exposure might affect various organs, especially in infants exposed to this chemical during pre- and post-natal development when they do not have the full capacity to metabolize BPA and their organs are developing at this time,” Rosenfeld said.
Rosenfeld was joined by Jerrold Heindel, Scott Belcher, Jodi Flaws, Gail Prins, Shuk-Mei Ho, Juide Mao, Heather Patisaul, Ana Soto, Fred vom Saal and Thomas Zoeller from the Healthy Environmental and Endocrine Disruptor Strategies Commonweal, North Carolina State University, University of Illinois at Urbana-Champaign, University of Illinois at Chicago, University of Cincinnati College of Medicine, University of Missouri and University of Massachusetts at Amherst in this data reevaluation. Read more of their secondary results at the Journal of Reproductive Toxicology and see the original FDA CLARITY-BPA publication at FDA.gov.
Mizzou was always near the top of Olivia Warner’s list for Ph.D. programs.
Its renowned psychological sciences program, sound training in Warner’s specialty of addiction and supportive, collaborative atmosphere that she didn’t see at other places made it a top choice on paper.
But she was not introduced to her most formative program in terms of professional development until she had already moved across the country from Arizona to mid-Missouri. Within her first month, Warner learned of the Maximizing Access to Research Careers/Initiative for Maximizing Student Diversity (MARC/IMSD) program.
“I found out about IMSD right away, and I’m glad I did,” Warner said. “Without this program, I would have had a tougher time networking on campus, and wouldn’t have picked up a lot of the skills that I have today.”
MARC/IMSD is a federal program that partners with universities to identify and train the next generation of researchers in biomedical and behavioral sciences. It provides research, mentoring, academic and social support, and professional development to help them along the way to doctoral degrees that will lead them to diversify the workforce. These underserved students include minorities from different ethnic and racial backgrounds, those with disabilities, students from low socioeconomic backgrounds and first-generation college students. The program gives them a helping hand in graduate school.
“Evidence says teams from diverse backgrounds approach problems differently and, ultimately, better in terms of solutions than teams of individuals from similar backgrounds,” said Dr. Mark Hannink, director of MARC/IMSD, Bond Life Sciences Center principal investigator and Biochemistry faculty member. “Data drives the National Institutes of Health’s recognition that entire research enterprises benefit from different perspectives and approaches.”
For 20 years, Mizzou was the recipient of an MARC/IMSD grant from the National Institutes of Health (NIH), that provided mentored research training and professional development to both undergraduate and graduate students. Hannink said that while the mentoring and professional development will continue, there will be changes in MARC/IMSD’s structure with the new grant. The most significant change is that the undergraduate and graduate training programs are now separated into two different grant-funded programs. The graduate MARC/IMSD program, which will become a T32 training program, has recently received $2.2M in funding from NIH to support training of 25 students over five years. A number of units at MU, including the School of Medicine, College of Veterinary Medicine, College of Engineering, College of Food, Agriculture and Natural Resources, the Division of Biological Sciences and the Graduate School, have provided funding to support training of at least 10 additional students. The MARC/IMSD program is truly a partnership between NIH and MU that will have a significant impact on diversity in biomedical research.
“This is more than just a scholarship,” Hannink said. “It’s a training program, in which students develop three different areas of expertise that our students need to develop to become successful scientists: professional, technical and operational skills.”
These objectives include increasing the percentage of underrepresented minorities in participating biomedical predoctoral students to 20% from 16%, to improve the Ph.D. completion rate for such students to 90% from its current 84%. An end goal is that 30% of trainees obtain external fellowships during their time in the program.
While MARC/IMSD provides two years of financial support to participants, its key focus is to foster professional development programs, workshops and support mechanisms to ensure that students in the program have whatever they need to succeed.
Coming out of Arizona State University with degrees in Psychology and Human Development, Warner worked full time for three years and volunteered in a lab, researching the ways in which contextual and interpersonal factors influence alcohol use motives and subsequent problematic use.
Despite significant research experience, Warner’s transition into the six-year clinical psych sciences Ph.D. program would have been much more difficult if not for MARC/IMSD.
“I was connected with Dr. Hannink pretty much immediately after I arrived on campus, and surrounded with people in the program that had a lot of shared experiences with me,” she said. “The program provided extra support and a sense of togetherness for us, which is really important since a lot of us are underrepresented in Ph.D. programs. It really helps to be around a group of people you can relate to and connect with.”
Under the new grant, much of the mentoring aspect of the program will remain the same, but the new format will be much more explicit in terms of the specific training activities used to develop said technical, operational and professional skills.
In addition to moral support and togetherness, MARC/IMSD aims to equip participants with academic skills. For Warner, who just completed her second year of the program, this meant taking a professional development class taught by Hannink. The class included a segment on public speaking which was especially beneficial for Warner.
“Having public speaking and presentation skills is a large part of any successful researcher’s skill set, so it was really helpful for me to be able to develop those skills early on in the program,” Warner said.
Participants also complete rotations in other labs besides their own as part of the program in order to broaden their knowledge about all areas of research, and are given opportunities to network with experts in the field. Many cycle through labs within Bond LSC.
“The next generation of researchers need to have exposure to disciplines outside their degree, to be able to talk with people doing research that’s very different than their own and collaborate effectively,” Hannink said.
Warner wants to be able to continue her research in some capacity for the rest of her career, so being a well-rounded graduate able to face any challenges thrown at her is invaluable.
“Everything I’ve been a part of in this program, from the professional development class, to being exposed to areas of research I might not have otherwise known anything about, has been very beneficial,” she said.
The science community as a whole reaps benefits from this development.
“Any research effort is more effective if it’s inclusive and brings different perspectives and approaches to the problem, and that’s how the skills IMSD teaches really translate long term into a better biomedical workforce.”
MU received a National Institutes of Health five-year grant for $2,228,008 to continue its focus on minority graduate student development in biomedical and behavioral sciences. This grant started February 1, 2020.
The structure and placement of labs encourages researchers to collaborate and talk to each other and often, connections and friendships are formed.
For Kinjal Majumder, a virologist and postdoctoral fellow in the David Pintel lab at Bond Life Sciences Center, that has meant bouncing many ideas off friends and colleagues from neighboring labs. Little did he know that one connection would lead him to interesting findings in a field outside of virology.
Those results may help make progress toward understanding the machinery behind drug resistance and toxicity in the body.
“The big picture is that it will help future studies on how drugs are metabolized by our body,” Majumder said.
The connection started with Andrew Huber, the first author of the paper, who completed his Ph.D. while in the lab of Dr. Stefan Sarafianos at Bond LSC. He graduated two years ago, moved to the lab of Dr. Taosheng Chen at St. Jude Children’s Research Hospital (SJCRH) in Memphis, Tenn. and began his postdoctoral work.
Dr. Chen’s lab focuses on mechanisms of cellular drug metabolism, one of which functions through the pregnane X receptor (PXR) protein.
PXR is found in the liver and is activated by drugs and other substances in the blood. Once activated, it binds to DNA and activates genes responsible for detoxifying the system. However, this system often leads to metabolism and reduced efficacy of administered drugs such as chemotherapies and antibiotics.
“When Andrew was in the Sarafianos lab, we’d talk science often and we collaborated on how different viral proteins bind to host and virus DNA, and how this can aid viral life cycles,” Majumder said, “When he moved on to his postdoctoral work, he saw that he can start applying similar techniques to study how the liver metabolizes toxins. So that’s when we set up this collaboration to pursue these things together.”
Majumder studies how viral DNA and proteins interact in virus-infected cells. One technique he uses is called chromatin immunoprecipitation assay (ChIP) which determines where proteins of interest can bind to DNA molecules. Majumder freezes protein and DNA that are interacting together in the process and he pulls that complex down with an antibody. “It’s like going fishing,” he said.
In order to apply ChIP and other techniques to study PXR signaling, Majumder drove down to SJCRH in Memphis to work with Huber on these assays.
Once in Memphis, Majumder and his collaborators worked and conducted experiments around the clock, but they also found time to explore the city and have fun.
“We go into lab and try to get done as many experiments as we can,” Majumder said, “And whenever we have a long break we go out on the town. It’s usually a really fun way to go and experience the life and scientific culture of a new place.”
It is a lot to pack into one week, but Majumder and his collaborators make the most of it, finding a way to effectively balance their studies on PXR with soaking up life in Memphis.
Majumder compares PXR to a light dimmer switch. When activated, it “brightens” by increasing drug metabolism, leading to decreased drug efficacy. PXR inhibitors work to “dim” this effect. At St. Jude, Dr. Chen’s group found that of the 434 amino acids that make up PXR, only one mutation is needed to “turn a dimmer into a brightener.”
“We sort of got lucky, we thought that you’d have to make a lot of changes in order to make PXR do something different,” Majumder said, “Without making too many changes — Dr. Chen’s group just switched one thing — and that was sufficient enough to turn it into a brightener. So that was a happy and unexpected outcome.”
Once these results were reviewed, the Cellular and Molecular Life Sciences journal accepted their results and published them this March in an article titled “Mutation of a single amino acid of pregnane X receptor switches an antagonist to agonist by altering AF-2 helix positioning.”
Looking ahead, Majumder and his collaborators hope to make more progress in understanding how drugs are metabolized in the body.
All of this would not have been possible if it wasn’t for the connections Majumder made at Bond LSC a few years ago.
“It was so cool to have this opportunity to collaborate with someone from the LSC that I have collaborated with before and apply our experimental techniques to study a new system,” Majumder said, “It feels very encouraging that now the work can be expanded upon and applied to other systems and settings.”
Haval Shirwan and Esma Yolcu arrive at Bond LSC as accomplished researchers.
While having different expertise within the field of immunology, the married couple collaborates extensively on research and together developed ProtEx technology, an alternative to traditional methods of gene therapy for immunomodulation with applications to autoimmune diseases, transplantation, cancer immunoprevention and immunotherapy, and vaccines against infections
With work published in numerous peer-reviewed journals and 19 patents to Shirwan’s name, they both arrive at Mizzou ready to build on their research background and contribute to the new precision medicine focus for the UM System.
But success didn’t always come easily for the couple.
Shirwan grew up in the shadows of Mount Ararat in eastern Turkey, and was always fascinated by how the beautiful nature in the area came to life during spring after a harsh winter. From then, he knew he wanted to spend his life studying something in a science-related field.
After completing his undergraduate education in Turkey, Shirwan received a scholarship via NATO to complete his Ph.D. in the United States at UC-Santa Barbara. From there, he obtained a postdoctoral fellowship at California Institute of Technology, where he worked under an esteemed group of researchers, spent a few years in Philadelphia and arrived at the University of Louisville where he has spent over 20 years in the Department of Microbiology and Immunology.
Yolcu also grew up in Turkey, but did not meet the man who is now her husband until much later in life.
After completing her undergrad and Ph.D. in her homeland, she faced a difficult decision, between continuing in her field of training — cancer biology — or taking a leap of faith to pursue her emerging passion of immunology. That decision would require her to move to the United States for a fellowship at the University of Louisville, which she did. She originally planned to return to Turkey after the fellowship, but after meeting Shirwan, decided to settle in the US and make her career here.
Shirwan and Yolcu bonded over their passion for immunology worked together to develop their esteemed ProtEx technology. But, their initial desire to do so came not out of inspiration but rather out of frustration at the shortcomings of traditional gene therapy methods.
“DNA-based gene therapy is a complicated, exhaustive and expensive technology,” Shirwan said. “We wanted to see if there was a more efficient way to do immunomodulation.”
In traditional methods of gene therapy, DNA must first be introduced into the cell, but ProtEx technology bypasses that step, instead sticking the protein directly on the cell surface for immunotherapy.
The advantages of this method are twofold: it is much more efficient than gene therapy and allows proteins which may be harmful to the cell to “get out of the way” quicker once the process is completed.
Although this technology has received much acclaim from the global scientific community, the breakthroughs took years of work. While Shirwan took charge of the more technical details of ProtEx, it was Yolcu who urged him to be patient and keep trying new strategies when he was ready to give up on the technology altogether.
“In the mid 90s was when we had our breakthrough with ProtEx,” he said. “By the time we reached that point, I was about ready to give up, but (Yolcu) urged me to keep trying.”
Their breakthrough came when a group of animals treated with ProtEx techniques experienced unprecedented recession of cancerous tumors on their body. At first skeptical, Shirwan and Yolcu repeated the experiment several times. The result was the same; the control groups saw enlarged tumors while the group treated with ProtEx technology saw their tumors recede greatly. Their innovative immunomodulation technique of placing protein directly on the cell surface had worked. The couple published their ProtEx findings initially in 2002, but continued to fine-tune ProtEx and discover new things along the way.
While they have already accomplished so much together, they believe they still have much left to learn.
“What’s so fascinating about immunology is that we often think we’ve made so much progress and know so much about the immune system, but in reality, we still know very little,” Yolcu said. “Studying immunology gives me a sense of peace, and I’m excited to continue our work at Bond LSC.”
To that end, the couple aims to establish themselves at Mizzou as soon as it’s safe to do so (they are currently in the midst of a multi-phased moving process due to COVID-19).
This type of research contributes to the University of Missouri System’s NextGen Precision Health focus. The NextGen Initiative unites scientists, government and industry leaders with innovators from across the system’s four research universities in pursuit of life-changing precision health advancements.
Shirwan and Yolcu will establish a Center for Immunomodulation and Translational Research and aim to collaborate with clinical colleagues and scientists from all different backgrounds to continue to make headway in their work with the immune system.
“We want to build a powerhouse here,” Shirwan said.
