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.”
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.”
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.
Mary Butler, an undergraduate from Truman State University, gains experience working on experiments in the lab of Bond LSC’s Cheryl Rosenfeld. | photo by Roger Meissen, Bond LSC
By Mariah Cox
How did an undergraduate student from Truman State University spend last summer working on a research project with a Bond Life Sciences Center primary investigator and become on track to be published as first author several months thereafter?
A nationwide National Science Foundation (NSF) sponsored program has allowed Mary Butler to jump-start her research career early on.
Butler, a sophomore biochemistry and molecular biology student, wanted to get a head start on research to give her a leg up and figure out what her future might look like. As a freshman, Butler joined the Missouri Louis Stokes Alliance for Minority Participation (MOLSAMP) program and sought out opportunities that aligned with her science interests.
MOLSAMP is a collaborative effort among seven public universities, a private university and a community college. The alliance aims to increase the number of underrepresented minority students throughout the state of Missouri who pursue undergraduate degrees in science, technology, engineering or mathematics (STEM). The Missouri chapter is part of a greater program with the Louis Stokes Alliances for Minority Participation sponsored by the NSF, and Butler qualified because of her Mexican American background.
This past summer, Butler started traveling the 90 miles from Kirksville to Columbia to work with Cheryl Rosenfeld, a primary investigator at Bond LSC. Her research primarily focused on how genistein, a soy-derived phytoestrogen, and BPA, an industrial chemical, affects the behavior of mice.
“BPA and genistein are endocrine-disrupting chemicals, so they can mess with the hormones of an animal and potentially humans. I specifically looked at the different behaviors of California mice under four different diets,” Butler said.
The researchers administered a control, a genistein diet, a low dose BPA and a higher dose BPA diet to pregnant mice. After the baby mice were born, the researchers weened them from the mother to observe if the parents’ diet affected what the pups ate. Additionally, at 30 days, 90 days and 180 days, they put the pups through various social tests to see if their diet and their parents’ diet affected the brain and behavioral patterns.
They used socio-communication testing to determine whether developmental exposure to these chemicals led to deficits in these behaviors, which are reminiscent of those seen in children with autism spectrum disorder (ASD). The results point to less socialization among the mice exposed during the pre- and postnatal period to genistein or a low dosage of BPA.
Afterward, Butler measured gene expression changes in the hippocampus, which regulates learning and memory, and the hypothalamus, which guides socio-sexual behaviors. Besides examining for mRNA that encodes for proteins that can act within or outside of a cell, Butler and Rosenfeld also are the first group to examine whether developmental exposure to BPA and genistein affects the expression of so-called junk RNA within the brain.
Junk RNA strands are very short and do not give rise to proteins. For these reasons, they were historically dismissed as not important in cellular biology. However, it is increasingly apparent these RNA strands, also now called microRNA or miR for short, play vital roles in diverse cells throughout the body.
One way they act is to bind mRNA that would otherwise encode for proteins. In so doing, they help contribute to the demise of these mRNA strands. Essentially, the DNA can be transcribed to mRNA but miR binding prevents them from making a protein, acting as an epigenetic modifier that doesn’t alter the DNA or mRNA. In this sense, miR may serve as a system to help regulate mRNA.
While other research groups have examined how BPA affects miR patterns in the placenta and testes, no previous research group has done so in the brain, even though this organ is vulnerable to early exposure to BPA and genistein.
When Butler started in the Rosenfeld lab, she expressed a desire for challenging and novel work. With assistance from Jiude Mao in the Rosenfeld lab, they set out to test whether developmental exposure to BPA and genistein could alter miR decrease in ASD patients.
Notably, Butler’s work suggests that indeed such chemicals caused down-regulation of the same miR that are reduced in ASD patients. Normal expression of such miR appears to help prevent cell death, oxidative-stress damage, and yield other beneficial effects, and thus, reductions in these miR may leave California mice and, presumably, autism patients more vulnerable to such deleterious effects exposed to such chemicals.
Butler’s studies also suggest that changes in mRNA and miR profiles are associated with socialization deficiencies observed in California mice. Further testing is needed to confirm whether these molecular changes contribute to these behavioral disruptions, but, if so, it may pave way for new prevention and treatment strategies in those at risk of neurobehavioral disorders.
Since high school, Butler knew she wanted to pursue science. But she doesn’t quite know where to go from here with all the different options available; the opportunity at Bond LSC allowed her to look into one of those.
“I want to explore a lot of different types of research. My research with Dr. Rosenfeld is very different than the research I’ve been doing at Truman,” said Butler. “I had been looking at Dr. Rosenfeld’s research for a while and it looked really cool, so I sent her an email and asked if I could work in her lab over the summer. She said ‘yeah’ quickly, and it was really exciting.”
As a sophomore, Butler can say she’s the first author on a paper, and she knows how to do protein expression purification and polymerase chain reaction among other techniques.
“I really enjoy it because I’m more of a hands-on learner. Getting to do it hands-on, I’m able to absorb a lot more information,” Butler said. “In my classes, I’m able to follow along and talk about research things because I already know a lot of the techniques we discuss and practice in the lab.”
Butler’s research was published in the “Journal of Neuroendocrinology” in March 2020 and was funded by the National Institute of Environmental Health Sciences.
Bond LSC scientist delves into how domestication alters the fox brainRussian silver foxes that have been tamed show changes in their brains. | photo provided by Anna Kukekova
By Roger Meissen | Bond Life Sciences Center
You might be familiar with the idiom “don’t bite the hand that feeds you,” but when it comes to a certain lineage of tame Russian silver foxes it’s quite literal.
After more than 50 generations of breeding, these tame foxes likely offer insight into how selective breeding leads to domestication, and scientists dove deeper to look at what this trait might mean in terms of changes in the brain and the genes behind them.
“These silver foxes act like domesticated dogs and are assumingly a good model to look at the process of domestication, tracing what happened as the wolves became domesticated into what we recognize today as the domesticated dog. Wouldn’t that be fascinating to know all the things that coincide with that?” said Cheryl Rosenfeld, a Bond Life Sciences Center principal investigator at the University of Missouri and co-lead author on the study. “Presumably, to become domesticated the one biggest organ that would have to change is the brain, because that’s where fearfulness, aggression, and social behaviors emerge from, so my interest is in the brain and how various factors might affect it.”
The path away from aggression
While this story eventually focuses on the brain, the path to a modern model of tameness starts in Soviet Russia in the 1950s.
Russian geneticist Dmitry K. Belyaev bred hundreds of foxes in Siberia, initially selecting them based on their willingness to approach humans in their cage. In its beginning only 10 percent of those tested showed a weakened wild response, and those foxes were bred together to reinforce this docile nature. In later generations, their behavior was further evaluated based on their likelihood to interact with humans approaching their cage, their territorial behavior, response to attempts to pet them and reaction to the experimenter leaving the interaction. Lyudmila Trut, his intern now in her 80s and an author on this study, continues the breeding project.
“Those that showed a muted wild response would get bred together, so with each passing generation, the foxes showed diminished fear response. Once they established this tame fox population, they eventually developed the aggressive ones doing just the opposite in that they paired those foxes showing the most aggressive responses to an experimenter approaching the cage,” Rosenfeld said. “They repeated this over time and, ultimately, what they have now, going out 50 generations, are tame foxes that act and even look in some ways like pet dogs.”
The tame silver foxes love human interaction from attention to belly rubs, and exhibit traits like tail wagging, gazing into the faces of humans and a change in their barks. Physical changes accompanied these behavioral changes, with coat color shifting from silvered-black fur to a more mottled, piebald pattern, and tails becoming curly despite these traits not being intentional selected for by breeders. In contrast, those bred for aggression showed hyper aggressive responses to humans, as indicated by lunging at the cage.
The taming of the brain
A change in behavior as observed in these silver foxes likely comes with unseen shifts in genetics and brain function. Rosenfeld decided to look at these changes in the hypothalamus region of the brain with the help of researchers from the University of Illinois at Urbana-Champaign, Cornell University, the Broad Institute of MIT and Harvard and the Institute of Cytology and Genetics in Novosibirsk, Russia.
“We are interested in changes in gene expression associated with the hypothalamic-pituitary-adrenal axis, the main hormonal system involved in stress-response and it was very exciting and productive to collaborate with Dr. Rosenfeld and her colleagues on the analysis of gene expression in the hypothalamus of tame and aggressive foxes” said Anna Kukekova, the senior author and associate professor in the Department of Animal Sciences at the University of Illinois at Urbana-Champaign.
Rosenfeld compared genes in the hypothalamus between tame foxes and aggressive foxes as well as comparing them to genes in two other brain regions — the prefrontal cortex and the basal forebrain — significant in terms of learning and memory.
“The hypothalamus was a good target organ, because it controls social behaviors like getting along or gregariousness and usually hormones like the pro-social hormone oxytocin tends to increase in animals who are social,” Rosenfeld said. “We basically did a global approach, looking at every gene we could identify and how they differ in terms of expression between the tame and aggressive foxes.”
Among genes with differential hypothalamic expression in tame and aggressive foxes they found seven identical genes with the same pattern of expression between all three brain regions. It suggests that when you select for tameness that some genes will change regardless of brain region.
“It was interesting to us because these genes were involved in processes including cell division and making more neurons, differentiation, adhesion like how the cells contact each other, and the most surprising, carbohydrate processing.”
Rosenfeld commented that some of these involved — such as, carbohydrate processing — suggest potential changes to accommodate domestication between the tame and aggressive foxes. This falls in line with how scientists believe dogs became domesticated 10,000 years ago, growing ever closer to human populations and developing to scavenge their scraps and garbage, although in this study both tame and aggressive foxes were maintained on the same diet.
“As animals get domesticated, they have to also accommodate different diets than what they’ve been eating in the wild because now they’re getting scraps from humans,” Rosenfeld said. “They’re selecting for genes that are involved in carbohydrate process, like metabolism processing, so it’s sort of telling us when you consider domestication that we might have to think about how the animal may have to then turn on a whole set of other genes to accommodate this new diet.”
But there were other brain differences as well.
“We also found genes suppressed in tame foxes associated in inflammation processes we call interleukin signaling, cytokines production and communication between cells to each other, and this gene expression linked to whether they were tame versus aggressive.”
The researchers’ original idea that genes related to hormones like oxytocin ended up not panning out when they saw little change between tame and aggressive foxes. But, she said it was interesting that the prevalent changes they did see involved more genes down-regulated or suppressed in tame than aggressive foxes.
“Most of the genes in the hypothalamus actually we found were suppressed in tame relative to hyper aggressive foxes, you know, squashing a hyperactive response, Rosenfeld said. “So, it’s an interesting question to consider that, as dogs became more domesticated, did they become more prone to disease because they don’t have such a heightened immune response? We can’t really answer that question just yet.”
Kristal Gant, a former MU PREP Scholar and current Ph.D. candidate at the University of Wisconsin – Madison | photo by Roger Meissen, Bond LSC
By Roger Meissen | Bond LSC
Kristal Gant is a long way from the student she was when she donned a lab coat and wielded a pipette in labs at Bond Life Sciences Center nearly four years ago.
As she stood in front of a group of MU students hoping to one day follow their own routes to graduate school, Gant recounted her long and winding path to her Ph.D. program at University of Wisconsin-Madison and how MU’s Post-Baccalaureate Research Education Program (PREP) Scholars helped her get there.
“I’m really passionate about wanting to give back and be kind of like a mentor, but also a motivational, navigational tool for these students,” Gant said. “In undergrad, I didn’t have any idea about research and I feel like it’s my duty to come back and talk about my experience. Most of these students have not seen people that look like them in graduate school, as Ph.D. candidates or doctors. They don’t even know that’s possible for themselves.”
Gant didn’t attend Mizzou for her undergraduate degree, but as a PREP Scholar here she gained the chance to hone her skills. It gives underrepresented groups opportunities for individual academic and professional development that range from mentoring and GRE preparation to mini courses and research experience. The end goal is to prepare them for applying to Ph.D. programs. Those in PREP spend one or two years refining their skills and knowledge.
Gant greatly expanded her experience in research during that time. She spent a year alternating between the labs of Cheryl Rosenfeld and Mike Roberts in Bond LSC, learning the ins and outs of research on reproduction and environmental chemicals like bisphenol A (BPA).
“It gave me a preview of what you go through in your first year of grad school. I knew I wanted to study reproduction, reproductive deficiency and disease, and was also interested in environmental and reproductive toxicology, initially,” Gant said. “When I came here, I had the best of both worlds; I had the BPA aspect with Dr. Rosenfeld looking at mouse models and how that affected their behaviors and I had the reproduction area where Dr. Roberts looks at placental development by converting embryonic stem cells to trophoblasts, the main cells responsible for placental function. The project was essentially perfect in accommodating both of my interests.”
Gant had a unique, sometimes turbulent path to her interest in science. She grew up and went to school on the Westbank of New Orleans, until ninth grade. She remembers first getting an interest in science in junior high when her teacher showed a time-lapse video about pregnancy and the development of a baby.
“I was really interested in like how the woman’s body knew to accommodate the baby and how it adapted to having another human inside of it. That really intrigued me, so I was like, ‘I want to do reproduction,’” Gant said. “That combined with “The Cosby Show” made me want to be a neonatal nurse or obstetrician. I pursued that initially, but then I was like, oh, whoa, I didn’t know you could actually research these things, and I was introduced to research careers and grad school in college and realized this could be a thing. So, yeah, I’m a nerd like that.”
Before she got there, life got a little derailed. When Hurricane Katrina hit, the 15-year-old girl got separated from her mother who was visiting her sister in Virginia, causing her to have to relocate to Texas with a family friend to avoid the storm. What started out as a few days of inconvenience quickly turned into a bigger situation.
“We saw on the news that there was extensive flooding and people were stuck on the roads, and our apartment was flooded out, so we lost everything,” Gant said. “My mom didn’t know if I had made it out or anything because the phone lines were down and we were separated two weeks before the family I was staying with in Texas flew me to Virginia where I ended up finishing high school.”
Gant completed a year of college at Virginia Commonwealth University before she moved to North Carolina to help her mother escape a domestic violence situation. They were left living in a shelter for abused women and children for a year before getting their own apartment, and Gant worked at McDonalds until she started college again at Elizabeth City State University. She graduated in 2014 and followed up with a couple internships before ending up in a customer service job.
“And I hated it because it was, like, I have a whole degree that I’m doing nothing with and I’m in debt from it and I need to do something with,” she said.
A former adviser recommended Gant apply for PREP Scholars to improve her chances of getting into grad school.
Kristal Gant talks with students eager to gain insight into applying to Ph.D. programs at Bond LSC in fall 2019. | photo by Roger Meissen, Bond LSC
Gant said her struggles helped her figure out what she wanted to do and shaped how she saw her future. Understanding it also aided her in writing personal statements for graduate school applications. She related that insight to those current PREP students looking to her for advice.
“It requires a lot of self-reflection to understand how your struggle got you to where you are. It doesn’t have to be family or a natural disaster or relationship troubles or whatever. It could be something within you that you yourself struggle with internally, whether it be confidence or fear or a feeling of not being good enough to do something,” Gant said. “It could be something so small that you struggled with, overcame and still, you pursued something you were deathly afraid to do. That shows that no matter what is supposed to stop you, you’re not gonna allow it.”
But her parting advice was some of the best.
“It really takes a passion, knowing what you’re trying to answer and putting in the work to figure it out. If you’re not passionate then you will be miserable and feel like you’re trapped in a prison,” she said. “But if you want the answers to your research questions and will not rest until you figure it out, then you should be in graduate school.”
Kristal Gant is a fourth-year Ph.D. candidate at the University of Wisconsin-Madison studying Endocrinology and Reproductive Physiology in the Department of Obstetrics and Gynecology. She was an MU PREP Scholar in 2015 and plans to graduate with her doctorate in 2021.
As a budding freshman who began her college career in the School of Journalism, she switched to biological sciences, tackled a double major in music, joined two research labs and kept up with clubs throughout her undergraduate career. Through it all, she has maintained an unwavering ambitious spirit.
Now in her final year, Martin has wrapped up her music degree and enjoys a more relaxed course load than she’s used to. From freshman to junior year, she often took over 18 credit hours at a time and balanced countless hours of studio practice, lessons, ensemble rehearsals, music and aural theory courses, science course labs and the academic rigor of both her degrees.
“My busy scheduled has definitely helped me with my time management skills,” Martin said. “I feel like a time management wizard now. It’s definitely prepared me for eventually going to medical school.”
While Martin is very sure of her future career path, she hasn’t always been this certain.
“When I was in high school, I was a part of my school’s newspaper and I’ve always been good at writing, so I thought I wanted to go into journalism,” Martin said. “I did the Missouri Urban Journalism Workshop and I loved it, but I remember thinking ‘wow, these other students are a lot more gung-ho about it than I am’.”
After she realized the journalism route wasn’t for her, she took some time to critically think about her career options. She recalls being inspired by her mom, who is a nurse, and at that point, decided she wanted to go to medical school.
From there, she looked into research labs her sophomore year and landed in Bing Zhang’s lab and studied Drosophila or small fruit flies. Then she found an opportunity in Cheryl Rosenfeld’s lab which allowed her to study the fetal effects of oxycodone.
“I’ve been helping out with Dr. Rosenfeld’s BPA studies on California mice as well as looking at the maternal-fetal effects of oxycodone on a different strain (CF-1),” Martin said. “[My research partner] is doing placental collections and I’ll be doing the behavioral analysis of the litters. Oxycodone is a relatively new drug that is being prescribed as a painkiller, however, people have been abusing it. There isn’t a whole of research being done into its effects as a drug of abuse.”
In addition to her scientific studies, Martin has been involved in the University Philharmonic Orchestra, the University Wind Ensemble, the Missouri Symphony Orchestra and Sigma Alpha Iota, a music fraternity.
“For the Missouri Symphony Orchestra, we do gigs around the community. Tomorrow we’re doing one for all the third graders in Columbia,” said Martin. “It’s like a get to know music and instruments. They learn about the piece in their music class and then they come hear it live.”
Martin intends to apply to medical school, but in the meantime, she will take a gap year and look into EMT or patient care technician jobs at the hospital. She plans to apply to the University of Missouri School of Medicine as her top choice, but she will also look at other places in the Midwest such as UMKC and a handful in Chicago.
Although she has dreams of becoming a pediatric or obstetrics and gynecology doctor, she plans on carrying music throughout her life.
“I’ve played French horn since middle, so it’s been almost 10 years since I started and it’s something that is stress-relieving to me,” Martin said. “It’s a totally different outlet from the science side and it has helped me with my science and research. Being in an orchestra, you have to be a team player and you have to be able to communicate with your team and step up to the plate and do the work.”
As Rosenfeld’s students graduate, awards and future plans celebrate excellence
Brittney Marshall, a graduating Biological Sciences major, and mentor Cheryl Rosenfeld. Marshall has done undergraduate research in Rosenfeld’s lab for three years. | photo by Roger Meissen, Bond LSC
By Mariah Cox | Bond LSC
Brittney Marshall, a soon-to-be-graduating senior from MU’s College of Arts and Sciences with a degree in biological sciences, received one of 15 University of Missouri Awards for Academic Distinction as well as the 2019 Outstanding Senior in Biological Sciences Award.
Marshall started research three years ago in Bond Life Science Center as a sophomore in Cheryl Rosenfeld’s lab. Rosenfeld, her mentor, nominated her for the award along with Thomas Phillips, one of her professors, that celebrates “evidence of extraordinary intellectual curiosity, actively seeking knowledge beyond the classroom and striving to share that knowledge with public audiences for a broader impact, and significantly contributing to the academic atmosphere at the University of Missouri.”
And Marshall has done just that.
Marshall worked with Rosenfeld to examine how prenatal exposure to bisphenol A (BPA) and genistein, a phytoestrogen found in soy, affect behavior in California mice and the gut microbiome and metabolome.
“We already knew BPA was bad because it was an estrogen mimic that was causing certain effects, so we wanted to see if more natural versions of similar compounds like genistein from soy would compare,” Marshall said. “Some of the behavior that we are seeing with Genistein actually differs from BPA, which we were surprised.”
Working off of the knowledge that BPA can cause autism-like behaviors such as decreased socialization and cognition, the researchers were able to compare those behaviors with mice exposed to genistein.
In order to quantify the results, they measured the mice’s sociability and their willingness to interact with stranger mice. They also utilized the Barnes maze test, to measure their spatial cognition and memory, and the elevated plus maze test, to measure their anxiety when placed on top of a tall platform.
Outside of the lab, Marshall spent her time volunteering at various organizations in Columbia and Boonville, such as a center for at-risk youth, a therapeutic horse riding facility and a hospital.
“Balancing everything can be really challenging between my academics, research and volunteering,” Marshall said. “I know in the end, it’s all going to be worth it because I’m just trying to make the most of my four years here and I feel like I have.”
However, Marshall isn’t quite done with Columbia yet.
In the fall, she will be attending the MU School of Medicine in hopes of one day becoming a primary care physician or an OB-GYN. She enjoys the idea of having long-term relationships with her patients and recognizes the growing need for primary care, especially in rural areas.
“I’ve wanted to be a doctor since I was a little kid, so I’ve been doing everything in my power to get there,” Marshall said. “I just remember learning about cells for the first time in middle school and it just blew my mind that you have all of these teeny tiny structures doing all these super complicated mechanisms in your body and there are millions of cells doing that.”
Marshall has made the most of her time at MU, and she encourages others to do the same.
“College flies by really quickly, so if there’s an opportunity that you think you might want to take advantage of, now is definitely the time to do it,” Marshall said. “I think you’ll be glad that you did. You might be a little busy but I think that the experience and the memories are worth it.”
Madison Ortega, a senior Biological Sciences major, spent three years in the lab of Bond LSC’s Cheryl Rosenfeld. | photo by Roger Meissen, Bond LSC
Another one of Rosenfeld’s undergraduate researchers, Madison Ortega, will also graduate and be moving on to bigger things. The Biological Sciences senior recently was accepted into a two-year National Institutes of Health post-baccalaureate program at Research Triangle Park in North Carolina.
Similar to Marshall, Ortega has been researching in Rosenfeld’s lab for three years now. Unlike Marshall, Ortega is unsure of her future career path. She sees her post-baccalaureate program with the NIH as an opportunity to get more research underneath her belt while she figures out what the future has in store.
“I think it’s going to be nice taking, not a break, but a little time to explore research further, because I’ve been doing research for three-and-a-half years, but it’s always been part-time,” Ortega said. “I’ve never worked 40-hour weeks doing in-depth research, so I think this is going to be a good opportunity.”
When looking back, both students attribute their time in Rosenfeld’s lab as a key experience with their time at MU.
“Cheryl is really ambitious. She really pushes us to push ourselves and try to make the most of our experiences,” Marshall said. “Sometimes that’s obviously challenging but I’ve really appreciated it because I’ve had a lot of good opportunities come out it. She’s been more than willing to help us undergrads get a lot out of our experience.”
Male Amami spiny rats have the rare distinction of not having a Y chromosome. Scientists at MU’s Bond Life Sciences Center are trying to decipher how exactly their sex is determined during development. | photo by Asato Kuroiwa, Hokkaido University
By Roger Meissen | Bond LSC
Joint press release by University of Missouri and Hokkaido University
What causes rats without a Y chromosome to become male?
A look at the brains of an endangered spiny rat off the coast of Japan by University of Missouri (MU) Bond Life Sciences Center scientist Cheryl Rosenfeld could illuminate the subtle genetic influences that stimulate a mammal’s cells to develop as male versus female in the absence of a Y chromosome.
The root of the answer is in the chromosomes of this particular mammal. Males of the Amami spiny rat (Tokudaia osimensis) are not like most therian mammals — a name used to group animals that give live birth including placental mammals and marsupials. Unlike in most mammals, these males have no Y chromosome, which has been shed over eons of evolution. And they only have one X chromosome.
“I’d been interested in these rats for many years now, and it’s unclear how sexual differentiation of the gonads and brain occur in this species since both males and females have a single X chromosome,” said Cheryl Rosenfeld, lead author on the study and an MU researcher.
Since most mammals inherit chromosomes from both parents — an X from our mother and either an X or Y chromosome from our father — the only way to develop as male is to inherit a Y chromosome. That male chromosome contains a sex-determining region Y (SRY) gene that stimulates male sexual differentiation in many mammalian species, including humans. SRY triggers the fetus to become male by producing a protein that binds DNA, which leads to development of testes and subsequent production of testosterone. This steroid hormone then stimulates development of the rest of the male reproductive tract. This surge in testosterone from the testes also programs masculinization of the brain.
Naturally, this got Rosenfeld curious about how the absence of a Y chromosome and SRY might affect gene expression differences in male and female Amami spiny rats. In collaboration with Asato Kuroiwa from Hokkaido University Takamichi Jogahara of the Frontier Science Research Center in Japan, and Scott Givan, associate director of the MU Informatics Research Core Facility, her laboratory received brain samples from both males and females of this species.
They took those brains, isolated the RNA from them and sequenced the samples of the male and females to compare transcripts between the two sexes. Transcripts are the step between a gene and a protein, essentially a piece of RNA that encodes the information to make a particular protein. Subtle variations in the resulting RNA can change the final protein, giving rise to alternative forms that can exhibit increased or decreased potency. Investigators found major differences between males and females.
“Several different transcripts or isoforms encode the same gene, and while there might be, for example, 10 transcripts for a particular gene, some transcripts are more potent than others and have more effect in individual cells,” Rosenfeld said. “When we compared males to females, there were several hundred more transcripts upregulated in males than females. Our thought is that since both have the same sex chromosomes, the resulting differences could be originating from the fact that the males might have more of one of the more potent transcripts.”
Expression differences in such transcripts might also arise due to epigenetic changes, which are alterations that affect the turning on/off of certain DNA regions but without affecting the DNA itself. Potentially, in females, these more effective transcripts are not expressed because of such epigenetic changes.
When the team – including MU student Madison Ortega who is in the MU Maximizing Access to Research Careers/Initiative for Maximizing Student Diversity (MARC/IMSD) program — looked closer, they realized many of the transcripts expressed in males encode for various zinc finger protein genes. In males, these genes can be turned on by SRY and are thought to significantly influence sex development.
“What we think might be happening is the males might be turning on all these other zinc finger transcripts that may compensate for the absence of SRY, so they influence undifferentiated gonad to become a testes and help program the brain to be male. Without these zinc finger protein transcripts, female sexual differentiation of the gonad and brain might result,” Rosenfeld said. “It’s possibly it’s more of a potential gradient within these rodents, where you have to turn on all these other zinc finger protein transcripts in males to stimulate male sexual differentiation and compensate for the absence of SRY.”
With the potential extinction of Amami spiny rats on the horizon, furthering this research has a degree of urgency.
“By elucidating more of the mechanisms in this endangered species, I think it might help us save the species or facilitate them being bred in captivity,” Rosenfeld said.
She hopes this research will continue with her team fully sequencing the genome of the Amami spiny rats to look more closely at what’s going on and how that might lead to a better understanding of the nuances of how animals without a Y chromosome undergoes male sexual differentiation.
“If you move outside the mammals, there are all sorts of sex chromosomes and exceptions like the duckbill platypus who has five sets of X and Y, and they can’t find differences between males and females,” Rosenfeld said. “People are focused on sex chromosomes and the SRY gene that they might be forgetting other contributing factors. By understanding these anomalous species, it opens up the idea that the mechanisms regulating gonadal and brain sexual differentiation are quite complex and not fully understood.”
Cheryl Rosenfeld recently worked with the FDA to study genetic effects of BPA. Her results were published in Epigenetics in July 2018. photo by Roger Meissen | Bond LSC
By Roger Meissen | Bond LSC
After a decade of work, Cheryl Rosenfeld is no stranger to bisphenol A (BPA), and her most recent study challenges the dangers posed by developmental exposure the chemical.
Her results continue to raise concerns about how BPA can potentially turn on or off genes in animals and subsequent effects on that early exposure can have on the development and brains of rats. Their research was published in the journal Epigenetics in July.
Rosenfeld and the University of Missouri joined experts from University of Cincinnati and FDA researchers as part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity, or CLARITY-BPA Consortium project. This collaboration is one of several across the United States meant to judge the chemical’s effect using standardized protocols established by the FDA to determine whether BPA exposure, especially during perinatal life, leads harmful effects.
“This is the first study published since a February 2018 BPA statement that challenges the FDA assertion that there is no concern for BPA,” Rosenfeld said. “We’ve shown using the FDA models and studies done right there at their facility that, indeed, early life exposure to BPA can result in gene expression and epigenetic changes that persist into adulthood.”
The study looked at gene expression changes in two brain regions — the hippocampus and the hypothalamus. The hippocampus is associated with long-term learning and memory and the hypothalamus plays a large role in hormone production that influence both the endocrine and nervous systems and affects diverse behaviors, including socialization, sexual behaviors, and appetite control.
Partners at the FDA/National Center for Toxicological Research fed Sprague-Dawley groups of rats — a standardized animal model in this research — diets of BPA, the synthetic estrogen present in birth control pills, ethinyl estradiol, or a chemical-free diet during a developmental period.
The brains from these animals were sent to Rosenfeld’s laboratory, who took biopsies from specific regions of the brain. They used these samples to evaluate whether a group of 10 genes, shown to be affected by BPA exposure in other studies, was affected by this exposure. They also examined the DNA methylation patterns for the promoters of three of these genes to determine whether prior BPA exposure led to persistent epigenetic changes. Epigenetic modifications do not affect the DNA sequence itself but gene and/or eventual protein expression.
Investigators determined that for several of the genes examined BPA exposure altered the expression pattern relative to animals not exposed to either chemical. Sex differences in gene expression in these two brain regions exists in normal animals, and such differences might thus contribute to masculinization or feminization of the brain manifesting as differences in various behavioral patterns, such as male or female sexual behavior. However, previous exposure to BPA abolished many of these gene expression differences between males and females, suggesting that it could disrupt male- and female-typical behaviors. For a gene, brain derived neural factor (BDNF), involved in learning and memory, BPA exposure led to increased methylation of its promoter, which could affect the expression of this key gene. Hippocampal expression of several genes was associated with prior performance in a test designed to measure learning and memory.
“It has become increasingly apparent that BPA can act as a weak estrogen, but what we’re seeing in these results is that it can elicit other effects in addition to those mirroring estrogen and likely independent of estrogen receptor pathways,” Rosenfeld said.
Initiatives like this and other CLARITY-BPA studies aim to answer questions that may later inform government regulators on how to limit or balance the health effects of manufactured chemicals that end up in the environment and may affect human and animal growth in previously unknown ways. With more than 15 billion pounds of BPA were estimated to be produced in 2013, its ubiquitous use in making plastics, lining cans and other manufacturing is of concern. Rosenfeld hopes a closer look at its epigenetic effects may lead to better regulation of the chemical.
“When people are thinking about the effects of BPA, they need to be thinking about it on a molecular scale,” she said. “These results might be subtle, but they can lead to dramatic consequences with long-standing, irreversible changes. Once BPA exposure resculpts an animal’s brain through DNA methylation and other epigenetic changes, it may be permanent.”
