Becca Wolf

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#IAmScience Jessica Kinkade

November 6, 2020 By Becca Wolf in #IAmScience Tags: #IAmScience, biochemistry, mice

By Becca Wolf | Bond LSC

Growing up in Columbia, Jessica Kinkade never thought she would end up working in town.

“I never expected to come back here, but it’s neat that it worked out that way,” Kinkade said. “It’s nice to be able to see my family and work in a familiar place that I’ve lived my whole life.”

Kinkade went to college about 30 minutes east of MU at Westminster College in Fulton, MO.

“I always had an interest in science, especially natural and biological sciences, ever since I was little,” Kinkade said. “I was always interested in a lot of different aspects of it, and my interests turned into something more so that developed, along with me.”

Now a research lab technician in the Rosenfeld lab at Bond Life Sciences Center, she is happy to be back.

“Mizzou is a really good school for science. There are a lot of job opportunities here because it’s such a big school and they do a lot of interesting research so it’s a good resource for that,” Kinkade said. “Just a good place to find work overall.”

As a research lab technician, Kinkade has several responsibilities. She often helps on research as well as taking care of the animals, coding and collecting data, keeping track of supplies as well as other support work.

A self-proclaimed animal lover, Kinkade especially likes working with the animals. Currently, the lab has California Mice, which is a species of deer mouse.

The dark brown mice with big eyes can be quite a handful.

“They’re really fun to work with, but they’re kind of crazy, because they’re wild and not quite as tame,” Kinkade said.

This can make work quite interesting.

“They jump when they get excited so it’s easy for them to jump out of their cages, we have to chase them around a lot,” Kinkade said. “We’ve had to chase them across the floor and grab them quickly before they can run and hide anywhere.”

For the past two years, Kinkade has been focused on studying the behavioral affects BPA (bisphenol A) and genistein has on the behavior of California mice. Xenoestrogens, BPA and genistein, can be found in plastic and in things such as coffee and soybeans. These are known as “forever chemicals” because they do not easily breakdown in the environment. Knowing the behavioral effects of these xenoestrogens can help researchers better understand how the chemicals may be affecting humans as well.

The past few months Kinkade has been wrapping up this research, “It’s exciting to see the studies all come to a close and get the results,” she said.

Kinkade does not plan on ending her animal research there though.

“Right now, we’re trying to breed a strain of serotonin knockout mice,” Kinkade said. “We’re trying to get a colony started so we can begin a study with them. They’re difficult to breed so we’re still working on that, but hopefully we will get a project started soon.”

Kinkade points to her college years for her passion of animals.

“Back in college I got to work on my own independent research project, which was mapping the territories of Cardinals,” Kinkade said. “It was a lot of being outside, looking around and listening, which I thought was really fun.”

Being excited about your work is important, and Kinkade certainly is. Projects like her independent research project kept her inspired and excited about being in a science field. She carries that energy with her to Bond LSC.

“There’s always a lot to do and maintain here. Sometimes it is a lot of work and routine, but then you remember why you’re doing it and what you’re learning,” Kinkade said. “I remember that excitement I would feel throughout school, and remember that’s what I’m doing right now, which feels good.”

Seed size matters: searching for a gene to make a bigger soybean

November 2, 2020 By Becca Wolf in Research Tags: CRISPR, gene expression, soybean

Bing Stacey | photo by Mariah Cox, Bond LSC

By Becca Wolf | Bond LSC

Patience is a virtue, at least it is for Bing Stacey.

Stacey recently completed a project that took her a total of eight years. It took her five years to develop a fast neutron mutant population and it took an additional three years to screen the population to identify a mutant that showed increase soybean seed size and then identifying the causative gene.

This gene, GmKIX8-1, and the seed size QTL, qSW17-1, can potentially be exploited for increasing yield in soybeans. Being able to alter these to increase seed size is important to improve the economic traits of soybeans such as yield and seed quality.

“The most important thing for farmers is the yield,” said Stacey, assistant professor of plant sciences at Bond Life Sciences Center. “For farmers, they plant soybeans, and the value or the profit they get is based on yield.”

There are two components of yield: seed size and seed quality. Stacey went to work finding what genes contribute to these components.

Mutants are very important for gene study because they have a new DNA sequence for genes. While it sounds like a sci-fi term, mutants are just plants manipulated using chemicals, radiations, or genetic modifications to change their characteristic traits in comparison to the norm. And the DNA changes to the GmKIX8-1 gene Stacey found is exactly the mutation she was looking for.

When she began this research, there were not many mutants for soybeans regarding seed size, so she had to develop her own mutant population using fast neutron irradiation.

After taking several years to develop this population, Stacey eventually found several mutants showing altered seed traits, including one showing increased seed size.

“So, we have the mutant, and then we utilized a fast and cost-effective way to genotype for changes in the DNA sequence of the mutant that is associated with the increased seed size. The genotyping method is called Comparative Genome Hybridization (CGH) which can be completed within one week and specifically detects missing DNA sequences in the mutant genome,” Stacey said.

Next, Stacey used CRISPR/Cas9 mutagenesis, which is a very powerful way to cut out specific DNA sequences in targeted genes in an organism. Before CRISPR, plant scientists could only create random mutations in plants. For example, using processes involving mutagenic chemicals and radiations, which meant one had to look at a very large number of mutants to screen for induced changes in the DNA sequence of specific genes.

Using fast neutron mutagenesis which creates deletions in bases, combined with CRISPR/Cas9, Stacey was able to characterize the role of the GmKIX8-1 gene in controlling seed size in soybeans.

“In a way, we got lucky because there is a QTL overlapping GmKIX8-1,” Stacey said. “And once you know the genetics behind a trait, then it is possible to define a possible mechanism of how the gene works, for example, how it makes soybean plants produce bigger seeds.” GmKIX8-1 controls the ability of cells to multiply. It also increases the size of the organs and the seeds of soybeans, which is exactly what Stacey was looking for.

An additional new and important thing Stacey found in her work was that GmKIX8-1 controls seed size, but not leaf size, in a dosage-dependent manner indicating that this gene is a major player in regulating seed size but not leaf size.

“This gene dosage effect on seed size and the identification of GmKIX8-1 as the causative gene behind a major seed size QTL are the novel aspects of this work,” Stacey said.

These novel aspects have been rewarding for Stacey.

“I’m quite happy, and it’s more fulfilling discovering something novel, something that hasn’t been reported before in other plants, instead of confirming what has been already reported,” Stacey said, “For example, based on what is already well-known in maize, or in rice, or in Arabidopsis, I can ask the question, ‘Does this gene also work in soybean?’ Answering this question is still worthwhile to me because maybe the gene will not work at all, or maybe it works in a different way, and that would also be novel, but to discover new genes or new explanations on how a gene works is more fulfilling.”

For more information on Bing Stacey’s work, check out the September 2020 edition of the New Phytologist Foundation article, “GmKIX8‐1 regulates organ size in soybean and is the causative gene for the major seed weight QTL qSw17‐1”

Using improved technology to create an Immune Cell Atlas

October 28, 2020 By Becca Wolf in Research Tags: agriculture, gene expression, RNA

Ashley Meyer | photo by Mariah Cox, Bond LSC

By Becca Wolf | Bond LSC

When given an opportunity to use the newest technology, one has to take it.

Ashley Meyer, the lab supervisor of the Wes Warren lab at Bond Life Sciences Center, recently started using the improved technology of single cell RNA sequencing to create an Immune Cell Atlas for chickens. This search browser of sorts allows people to search for cells and gene expression in chickens.

“It would be a platform for researchers to have a baseline of what should be normal, and then be able to run comparative experiments to say ‘Okay, if we challenge this chicken with this disease, what biological response are we seeing?’ Which can help you then understand the disease better,” Meyer said. “Like, ‘How is it being transmitted? How is this chicken reacting to it?’”

Poultry is one of the largest agricultural industries in Missouri, so it is important to keep chickens healthy.

“The Immune Cell Atlas will help us analyze tissues and will help us perform the single cell RNA sequencing on them to ask what’s normal and what’s diseased so that we can see what’s happening,” Meyer said. “It will also allow us to see which chickens are more susceptible than others or how the progression diseases happen.”

Meyer and the Warren lab are some of the first researchers to apply this technology to chicken based research.

Single cell RNA sequencing (RNAseq) allows Meyer to sequence the RNA of an individual cell which provides in-depth data. This can be done using single cell RNAseq which uses the entire cell, or it can be done using single nuclei RNAseq which uses just the nucleus of a cell. This profiles gene expression in cells that make up a tissue to get data on a cellular level to understand the composition of cells.

It is important to target the RNA because it is the messenger that brings instructions from the DNA for controlling the synthesis of proteins. RNA is commonly synthesized in the nucleus of a cell.

“We’re some of the first people to get our hands on some avian single cell data, specifically looking at nuclei. My job is a lot of optimizing these protocols to isolate these nuclei,” Meyer said.

Using nuclei allows Meyer to use samples that have been frozen, collected in other locations, etc., which provides more flexibility.

Isolating the nuclei through RNAseq allows Meyer to identify different cell types based on genes being expressed in a cell in an organ such as a spleen or lung. This information can then be added to the Immune Cell Atlas.

Meyer is a part of a team working on this project including two master’s students, a bioinformatics analyst, and principal investigator, Wes Warren, at Bond LSC, as well as a bioinformatics analyst who works remotely from Santa Cruz, California.

While the team is working from around the country, they are all connected by a common goal.

“Things have been pretty hectic, the break with the COVID quarantine kind of paused everything and then when we got back it just kind of took off, we’ve generated a couple data sets just in the last month or so,” Meyer said.

Having started this research about six months ago, they still have a way to go. Meyer and her collaborators are currently focused on analyzing five to six immune system organs, and plan on expanding in the future.

Since single cell RNAseq is so new, it can be challenging being one of the first labs to use it at MU and in the avian community.

“Since it’s still newer, everyone’s still figuring out how it works,” Meyer said. “Everybody’s determining their own way of analyzing the data. We’re working through the hiccups of everything, and not having years and years of research to guide us can be challenging; however, also exciting because then we can find what works and what doesn’t work which is the whole point of science.”

While challenging, it is rewarding. Meyer hopes that in the future once they have created the chicken Immune Cell Atlas, they are able to expand to other animals.

“It’s exciting to be part of something that’s newer and having something that could be beneficial for other researchers,” Meyer said. “I am a huge advocate for sharing science and teaching science so creating something to aid other research is super empowering for me.”

#IAmScience Karl Kerns

October 24, 2020 By Becca Wolf in #IAmScience Tags: animal science, bioinformatics

By Becca Wolf | Bond LSC

It’s all about the journey and Karl Kerns has been places.

Originally from a small town in southwestern Iowa, Kerns did his undergrad years at Iowa State University (ISU), taking internships in Maryland, Texas, southeast Asia, and southern Australia, among other places that focused on animal physiology and fertilization.

“I do like traveling just in the sense of vacation. I like having a purpose while traveling. It’s hard for me to sit back and relax and be in vacation mode,” Kerns said.

This focus and determination eventually brought him to Mizzou where he completed his postdoctoral fellowship in the Dong Xu lab at Bond Life Sciences Center this past spring, co-advised with Peter Sutovsky and Susanta Behura in the Division of Animal Sciences. Kerns was initially attracted to MU for his doctoral training because of the reputation of the animal sciences’ reproductive physiology program, but he then joined Bond LSC to continue his research during his USDA NIFA Postdoctoral Fellowship.

“MU is a world class institution. There are not many places that offer the resources we are fortunate to have here with the combination of animal science, veterinary medicine, and the resources of a research hospital system,” Kerns said. “And it’s not just equipment resources, but also intellect and the people that we [Bond LSC] have, it really provides an atmosphere that lets you dig into science a little bit deeper than you can anywhere else.”

This atmosphere at Bond LSC has aided Kerns in his current position as a newly-minted faculty member at ISU.

“It set me up for success. Especially in the middle of COVID right now, there’s not a lot of faculty positions available, so to get a faculty position when all those other positions were being halted, it speaks volumes to the training I received here,” Kerns said.

Kerns began teaching this fall semester, teaching Anatomy and Physiology labs twice a week.

Going back to his alma mater as a professor was not exactly the homecoming Kerns expected.

“It’s a very odd timeframe coming back during COVID when the buildings are not bustling with students and faculty.”

In the Xu lab, Kerns worked on bioinformatics and artificial intelligence with graduate students. They were able to incorporate machine and deep learning analysis with high throughput, single cell phenotyping, looking at multi-million cell data sets for bioimage analysis.

“There are multiple times that you step back from the microscope or from the computer after doing data analysis and you have so much awe and admiration for how all these intricate biological functions have to come together to support life,” Kerns said. “That is what fuels me.”

Kerns’ first publication at Mizzou was about how zinc, a vital micronutrient, helps regulate fertility in pigs, cattle, and humans.

“Research in the life sciences is extraordinarily rewarding because there is a time between initial discovery and publication that just you and your co-authors know this little piece of how life as we know it functions, previously unknown from the start of time,” Kerns said.

While he enjoys special moments like these in the lab, Kerns has been enjoying his time in the classroom with students during this isolating time of the pandemic as well.

“It’s my duty, being taxpayer funded in the training that I got, to make sure I teach the next generation,” Kerns said, “Let them have a taste of the cutting-edge technologies I learned at Mizzou and cutting-edge research findings that we found. The academia system is all about having people that are experts in a set area and passing it down to the next generation to expand on.”

Along with teaching, Kerns is also doing research at ISU. He is currently working on analyzing the fertility of boars and bulls to see what helps predict their fertility. Significant amounts of research and improvements focus on the female side of reproduction but not male, so by improving male fertility, Kerns will be able to help livestock producers and stakeholders improve their efficiency.

Kerns has big plans for his research in the future.

“Once we optimize male fertility, we can start taking superior genetics to third world countries that need a good protein source that can grow in their particular climate — we know protein is a source of complete amino acids that provides better cognitive function for people — and also provide a source of financial security for their families as well. It’s a win-win,” Kerns said.

Until then, Kerns plans to continue teaching and doing research. His science journey has taken him many places so far, and his is not stopping there.

“Science is always changing,” Kerns said. “When we say, ‘what does the science say?’ The science only says our current interpretation of it. In the grand scheme, we have so much more to learn. Every day we’re learning more about our world and not uncommonly disproving what we used to hold to as true because of new techniques now available to us.”

Finding a Link between Xenoestrogens and Autism

October 12, 2020 By Becca Wolf in Research Tags: Autism Spectrum Disorder, biochemistry, mice

California mice | photo by Roger Meissen, Bond LSC

By Becca Wolf | Bond LSC

It’s hard to see a family member treated differently because of a behavior disorder, but those with relatives diagnosed with Autism Spectrum Disorder (ASD) know its impact. Since it affects how people act, communicate, and learn, people with ASD often get bullied or feel left out because of these behaviors.

Sarabjit Kaur, a former undergraduate researcher in the Cheryl Rosenfeld lab at the Bond Life Sciences Center, witnessed this stigma firsthand growing up with a brother who is autistic. Going to college, she knew she wanted to do something that made a difference in understanding ASD.

In Kaur’s final year at MU, she conducted a correlational study to see the effect of xenoestrogens on the microbiome-gut-brain axis and autistic behavior.

“The dramatic rise in ASD suggests that there’s an environmental component to this because genetics don’t change that fast,” Kaur said, “We know that steroid hormones play an important role in brain development. That’s why we suspect that endocrine disruptors such as the xenoestrogens, BPA [bisphenol A] and genistein, might be an important contributor to this disease.”

Xenoestrogens like BPA are often called “forever chemicals” because they don’t readily break down in the environment. Since we use millions of tons of manufacturing plastics, as food can linings and in things like receipts, our exposure can add up. While genistein is naturally found in plant products such as soybeans and coffee, both are common in our environment and affect the body in some way.

To start, Kaur tested the effects of the chemicals on California mice. She found that mice exposed to genistein were more likely to exhibit repetitive behaviors and socio-communicative disturbances. Mice with a low dose of BPA resulted in more frequent repetitive behaviors compared to those with a higher dose. These social communicative deficits were dose dependent autistic-like behaviors, showing that there is a possible correlation between these xenoestrogens and ASD.

In this study, Kaur also found that there are sex dependent effects of these xenoestrogens. BPA and genistein affected female and male mice differently. Exposed female mice resulted in different types of metabolites relative to control such as amino acids or glucose involved in carbohydrate metabolism, but in males the protein metabolism was affected by BPA and genistein relative to the control group.

These processes occur in the microbiome-gut-brain axis, which is the feedback loop where microbes communicate to the central nervous system through channels involving nervous, endocrine, and immune signaling mechanisms.

Exposure to xenoestrogens may be inducing epigenetic changes through altering gene/protein expression, without changing the DNA sequence itself.

“The results get super complicated because there has been dose dependent effects, both sex dependent effects and treatment dependent effects,” Kaur said.

Scientists use California mice as a model for behavior research because of their relationship similarities to humans. They are social creatures, both parents care for their young, and the male and female mate for life. This gives insight as to how BPA and genistein may impact humans.

“We wanted to do this in an animal model because we could help inform our epidemiology colleagues,” Kaur said, “So those that do work with humans, this could help them see what they are looking for as in terms of a diagnosis tool.”

In the future, doctors could look at the bacterial population or the metabolites in the microbiome-gut-brain axis to determine if a child is autistic.

“I just hope, this research will help my brother and people like him. That’s what research is all about, to help advance. Hopefully, we could give people a fresh, healthy start in the beginning of their lives,” Kaur said.

“It would mean a lot to many if in the future people could help their friends, family members, or their siblings that go through ASD,” Kaur said, “It would really help even if we could alleviate the symptoms or give people a fresh start.”

For more information, check out the December 2020 edition of Scientific Reports article, “Developmental exposure of California mice to endocrine disrupting chemicals and potential effects on the microbiome-gut-brain axis at adulthood.”

#IAmScience Norma Castro-Guerrero

October 9, 2020 By Becca Wolf in #IAmScience Tags: Legume, nutrition

By Becca Wolf | Bond LSC

If you like your work, you won’t ever work a day in your life.

That’s the case for Norma Castro-Guerrero, a research scientist in David Mendoza’s lab at Bond Life Sciences Center. Having a good attitude and making the most of everything is something she strives to do.

“I wake up every day and I am already thinking about what I need to do,” Castro-Guerrero said. “Time flies by during the day. I think that’s a way to know that you love what you do.”

She first witnessed this attitude in action from a principal investigator (PI) in her undergraduate studies at the Universidad Nacional Autónoma de México (UNAM) in her hometown of Mexico City, Mexico. This PI soon became an influential role model in Castro-Guerrero’s life.

“She was such a strong woman and full of enthusiasm for science,” Castro-Guerrero said, “She was so supportive, ‘You’re a woman and you will do great, getting the best grades and publishing papers.’ From there, I think it was very natural to me to keep studying and publishing during my Ph.D. and postdoctorate.”

Castro-Guerrero has implemented this enthusiasm in the Mendoza lab, where she oversees research projects and students. As a senior scientist, she trains the students on how to run a successful lab. She knows firsthand how important an encouraging environment can be for student growth.

“What you want to teach them is how important it is to have a healthy environment to achieve their goals. It’s not only teaching them the techniques or basics, it’s also that they feel comfortable and happy in the lab because they will be more efficient,” she said.

The Mendoza lab gives her the opportunity to express this enthusiasm. Castro-Guerrero enjoys the freedom she has to study her own ideas. Everyone is encouraged to make decisions and solve problems they feel are important.

Castro-Guerrero currently focuses on studying micronutrients in legumes like common bean and soybean, crops used in communities that suffer from nutrient deficiencies. She is looking at what mechanisms the legumes are using compared to other crops that make them accumulate more of these elements. Understanding the key factors will be useful to design better crops.

“Some people’s diet depends on these seeds, so improving them is a perfect vehicle to improve their health,” Castro-Guerrero said.

To further this study, Castro-Guerrero and her team have collaborated with people in South Africa and Mexico to improve the well-being of communities. Helping others motivates her, and this project is rewarding in more ways than one.

Aside from science, Castro-Guerrero’s other passion in life is traveling. And here at Bond LSC, she was able to combine these two passions.

In the past several years, Castro-Guerrero was able to travel to Asia and Africa to discuss research the Mendoza lab is working on at symposiums and other events.

“I really enjoy that people notice what we are doing in Missouri. They know that in the middle of United States we’re doing this great science, and they look for collaboration with us,” Castro-Guerrero said. “So, when you go there and they say start dropping names of people in this building [Bond LSC], we know we are in the right place. Being able to see the recognition not only the university but your own building is getting outside the country, I really enjoy that.”

Castro-Guerrero gets as much as she can out of her international science trips. “I know how people work in Mexico and in the United States. There are differences and it’s very enriching to see how other places work. Their dynamics, their strategies, you learn from them. I think it’s neat to be able to interact with other scientific communities,” Castro-Guerrero said.

“In my lab, the students observe you doing this, talking, writing, discussing, and they want to follow you like I did with my PI in the past,” Castro-Guerrero said, “You see them get excited that they’re working on at Bond LSC. And we are happy and proud of that.”

Science has allowed Castro-Guerrero to wake up every day excited about what it will bring. Whether it is supervising students in the lab, conducting research, or traveling around the world, she is ready for anything.

“I love it, I don’t want to do anything else,” Castro-Guerrero said, then pauses, “Except traveling. But traveling the world while doing science is even better.”

Influenza: How it Transmits from Birds to Pigs

October 5, 2020 By Becca Wolf in Research Tags: influenza, virology

Henry Wan | Photo by Roger Meissen, Bond LSC

By Becca Wolf

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.”

Read more about this study in the September edition of the Journal of Virology titled, “Tissue tropisms of avian influenza A viruses affect their spillovers from wild birds to pigs.”

Four Cell Lines, One Great Discovery

September 21, 2020 By Becca Wolf in Research Tags: copper

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.

For more information on Gudekar’s research, check out the December 2020 edition of Scientific Reports article, “Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess.”

A Big Load: Using Wastewater to Test For COVID-19

September 11, 2020 By Becca Wolf in Research Tags: COVID-19, RNA

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.”

Studying Drug Resistance, One Connection at a Time

June 1, 2020 By Becca Wolf in Research Tags: drugs, virology

Kinjal Majumder | photo by Mariah Cox, Bond LSC

By Becca Wolf | Bond LSC

Where you are creates opportunities.

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.”

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