Undergrad’s passion spurred by mice muscle regeneration research
Rebecca Craigg, an undergraduate biology major, studies muscle regeneration in D Cornelison’s lab. | Photo by Samantha Kummerer, Bond LSC
By Samantha Kummerer | Bond LSC
Uncertainty and curiosity led Rebecca Craigg to work in a lab.
As a first-generation college student with an interest in science but no idea what undergraduate research entailed, her path at the University of Missouri landed her in the Bond Life Sciences Center and the lab of D Cornelison.
“I honestly thought undergraduate research meant you just followed around someone like job shadowing,” she said laughing.
Now, after almost a year of research, the junior biology major is more than familiar with what working in a lab entails.
Craigg started working in Cornelison’s lab as an effort to figure out what kind of science she might be interested in. It was while she waited for her genetically altered mice to grow up when she found something that really sparked her interest.
Rather than spend her time cleaning lab dishes, Craigg began assisting a graduate student on a project studying the role of EphA7 in mice. EphA7 is a receptor on cells that helps mediate important events within the body. Receptors create a change in the body after receiving a signal from outside the cell. The exact hows and whys behind the EphA7 are still relatively unknown.
To unravel EphA7 the team began by breeding genetically altered mice with the gene switched off. Next, specific muscles were isolated, dissected, and preserved once the mice reached different developmental time points. Researchers thinly sliced the muscle and added immunofluorescent staining to track specific elements over a period of time. They marked EphA7 with a green fluorescent and regenerating muscle fibers with red. Imaging revealed every time green came up so did red. This led researchers to understand that EphA7 is connected to how muscles rebuild.
Rebecca Craigg, an undergraduate researcher, works with immunoflourescent staining to mark EphA7 and muscle fibers. | Photo by Samantha Kummerer, Bond LSC
But this isn’t the only role the receptor plays.
Work this summer also discovered the gene is involved in the development of muscles. The team found at the end of development, the mice without EphA7 did not fully recover like they would have if they were not modified. When the team looked at marking for muscle stem cells, both the mice with the receptor and the mice without it had a depleted number of satellite cells, essentially muscle stem cells, that did not recover.
Craigg explained this discovery was puzzling.
In every other case heterozygous mice, the mice with one gene coded with EphA7, were fine. The team predicts the cells and muscles realized they were not adequate so the muscle stem cells began to become muscles. This process would leave fewer stem cells at the end of development.
Fewer muscle fibers really mean the mice are lacking normal muscle strength. Craigg said one reason for this could be EphA7’s relation to motor neuron axons. Motor neurons send signals from the nervous system to muscles to tell them to move. EphA7 is present on all motor neurons. The team hypothesized that the gene may play a role in guiding the motor axon to the muscle, thus without it, muscle size would decrease.
“We kind of know way more than we did, obviously, in that it’s involved in regeneration and all these things and that if a mouse doesn’t have it they have all these decreased numbers all over the board, but we don’t know why per say,” Craigg said.
To better understand why EphA7 functions the way it does, the team will begin to examine the relationship between motor neurons and EphA7. They will also explore if the type of muscle fiber, slow-twitch versus fast twitch, make a difference.
Rebecca Craigg looks through the microscope to closely examine a mouse’s muscle cells.| Photo by Samantha Kummerer, Bond LSC
Craigg said each new piece of knowledge about EphA7 is a step towards better understanding what causes muscle disease in humans.
While the lab faces a lot more work ahead, it doesn’t faze Craigg. For her, the possibility of more discoveries is part of the thrill.
“We kept finding out so many new things about it. I would be counting things and I’d be like, ‘Oh my god, I can’t wait to get this data back, like, I just want to know,’” she exclaimed.
Rebecca Craigg is a junior biology major working in the lab of D Cornelison at Bond LSC.
Melissa Mitchum, D Cornelison and Cheryl Rosenfeld (from left to right) of Bond Life Sciences Center were promoted to full professor on September 1, 2017.
By MJ Rogers, Bond LSC
Scientific success largely hinges on research results, and four recent promotions at Bond Life Sciences Center celebrate that achievement.
Laurie Erb
Cheryl Rosenfeld, D Cornelison and Melissa Mitchum of Bond Life Sciences Center were promoted to full professor as of September 1, while Laurie Erb received a promotion as a non-tenure-track research professor. They are the first female full professors in Bond LSC’s 13-year history.
University of Missouri’s Assistant Vice Chancellor of the Division of Inclusion, Diversity and Equity, Noor Azizan-Gardner, said the promotions made her optimistic.
“Three women all going up to full professor – it’s phenomenal,” she said. “And the fact that they all have labs in Bond LSC makes me deliriously happy. Not just for us and them, but for the women who will be the next generation. The ripple effect is bigger than just the three of them.”
Promotion and tenure at MU follows rigorous guidelines that take teaching, research success and service into account to advance professors through three tiers — from assistant to associate to full professorship — over more than a decade.
But like many technical fields, science lags behind in its proportion of women to men. Growing that diversity is important to the breadth of scientific inquiry. As an advocate of collaboration, the promotion of three women to full professor at Bond LSC hopes to reinforce that diversity.
Cornelison and Mitchum were quick to stress their promotions had nothing to do with their gender, and everything to do with their science.
“It just doesn’t cross my mind,” Mitchum said. “I honestly don’t walk around thinking about gender. I just do the best I can and that’s all I can do.”
Similarly, Cornelison said, “I am not a female scientist. I am a scientist. Period. It should not be a part of the story.”
Rosenfeld, however, is concerned that administrators are not giving women the support necessary to flourish in their careers.
“I work seven days a week and I deserve respect and to be taken seriously on par with my male colleagues,” she said. “I am not doing this as a hobby. This is my passion, and, hopefully in the future, women like myself will be treated equally.”
A Pervasive Problem
A study conducted in 2015 by the Chancellor’s Status of Women Committee and the Status of Women Committee in the College of Arts and Science at MU found that with regard to gender equity on campus, there was no evidence of a systematic pay bias against female faculty. However, it did find that the average salary for female faculty is almost $16,000 (or 15 percent) below the average salary for male faculty and that the colleges with the highest average salaries were predominantly male.
Cornelison, Mitchum and Rosenfeld all believe that female scientists at MU face at least three significant hurdles on their path to full professor: the amount of time it takes compared to their male colleagues, the lack of mentorship, and the high ratio of male full professors compared to female full professors in several departments.
Mitchum stated that there are only two other female full professors — Jeanne Mihail and Michelle Warmund — in the plant sciences department compared to at least 17 males. Rosenfeld and Cornelison had similar ratios in their respective departments.
Recent controversies indicate gender equity is a persistent challenge in the field as a whole.
In 2015, a study published by the American Psychological Association found that when considering requests from prospective students seeking mentoring in the future, the science faculty at research-intensive universities were more likely to hire a male lab manager, mentor him, pay him more and rate him as more competent than a female candidate with the exact same resume. And this year, two senior female scientists sued the prestigious Salk Institute for Biological Studies, alleging pervasive gender discrimination and systematic sexism.
Although female scientists remain underrepresented in many countries, academic journal publisher Elsevier released a report in 2017 that shows improvement. It stated that women’s scholarly authorship increased overall from 30 percent in the late 1990s to 40 percent two decades later. In terms of raw proportions, the percentage of women scientists in the U.S. increased from 31 percent from 1996-2000 to 40 percent from 2011-2015.
Beginning Inspiration
Rosenfeld, Cornelison and Mitchum’s success in the departments of Biomedical Sciences, Biological Sciences and Plant Sciences, respectively, follow several decades of hard work and passion in their fields.
But their interest in science started in unique ways.
“In middle and high school I was always excited about science classes,” said Mitchum. “I liked physics. I liked chemistry. I was lucky to have a science teacher, Patty Gustin, who knew I had an interest in science, saw some potential and encouraged me. She was actually the first person to encourage me to go on to college in science.”
Mitchum went on to get an undergraduate degree in biology at the University of Puget Sound in Tacoma, Washington. She immediately continued her education and received her masters in plant pathology at the University of Nebraska, Lincoln and her Ph.D. in plant pathology and biotechnology at North Carolina State University in Raleigh.
Cheryl Rosenfeld’s high school biology teacher, Patricia Murphy, was also the first person to put her on the science track.
“I can still picture her to this day,” Rosenfeld said, smiling. “She gave me a C on my first lab assignment. My friend received a better grade and we did the same work, so I asked her why I got such a low grade. She told me that I was going to be a scientist, that she expected more of me, and to improve my grade she allowed me to help prep the lab experiments.”
Rosenfeld went on to receive a bachelor of science and DVM (Doctor of Veterinary Medicine) from the University of Illinois at Urbana-Champaign and a Ph.D. in Animal Sciences and Reproductive Biology from MU.
Cornelison’s path was a bit different. Like many undergraduate scientists, she initially thought she would go to medical school. But during an independent study, she was assigned to a lab doing behavior genetics in mice and fell in love with research.
“Unlike my experience in Chemistry classes, I was now in an environment where I was expected to go and do things nobody had ever done before,” Cornelison said. “And I got to tell people about it. And I got to decide what the next unknown thing I wanted to know was. After that, I had to decide whether to apply to medical school or graduate school because I only had enough money to take the GRE or the MCAT, so I took the GRE. And I am still incredibly grateful for the people who took me into their lab and taught me to science.”
Cornelison credits that experience with why she enjoys having undergraduates in her lab. To date, over 20 of them have graduated with departmental honors based on their independent research projects.
“If I can give students a taste of what that experience of discovery feels like, I’m happy. It changes your perspective on many things,” she said.
The concept of mentorship is something Rosenfeld, Cornelison and Mitchum all agree is critical for budding scientists, male or female.
Each shared stories about the vast amount of mentors that inspired them and students they still keep in contact with. Mitchum has an especially meaningful relationship with one of her mentors.
“While I was working in a lab as an undergraduate I had the opportunity to interact with a visiting scientist who would work in our lab, Donald Foard, an older gentleman at the time, and he became my mentor,” Mitchum said fondly. “I don’t think I would be where I am today without his mentorship. As an undergraduate, he encouraged me. He believed in me. He inspired me to go to graduate school. And we still keep in contact today. He is 86 years old now and we still write letters back and forth. I recently had the privilege of sending him my promotion letter. The sheer excitement of sharing that promotion with him was incredibly meaningful.”
“Without him believing in me I don’t think I would be sitting here talking to you about this promotion today,” she added. “He believed in me during a time when I didn’t believe in myself.”
Supporting Women in STEM
In an effort to promote mentorship and address female-specific concerns in the STEM fields, such as wage negotiation and salary differences, MU recently started its first Women in STEM group. The group was spearheaded by Rosenfeld and Azizan-Gardner, and had its first meeting in July.
“The issue of mentoring is something that you see everywhere, not just here,” said Azizan-Gardner. “It is a pervasive problem we need to address. And we can do that here at MU and do something that will really benefit everyone.”
Female mentorship is something that Rosenfeld believes is critical for female scientists and she makes an effort to mentor female undergraduate and graduate students.
“When you’re struggling, you often think that there is no way you can do this,” said Rosenfeld. “But if you see someone that looks like you that has succeeded and is teaching you, all the sudden your goal does not seem impossible.”
Mitchum is another strong proponent of mentorship and undergraduate research. She has mentored 26 undergraduate researchers in her lab, and 12 of them went on to graduate school, while many of the rest went to medical school.
“It’s so important for us as mentors, female or male, to believe in and encourage the younger generation,” she said. “I believe in many cases, you just need someone to believe in you and know you can accomplish things. It’s important to have quality in mentorship — investing in students and giving students your time and direct attention.”
Rosenfeld hopes that the Women in STEM group will empower female scientists to be more assertive. She said the first meeting was “eye opening” because many of the participants had similar experiences and it was powerful to hear their frustrations. About 20 women attended the first meeting, and Rosenfeld is confident that number will increase.
Azizan-Gardner believes that Bond LSC has the potential to be a leader in promoting, recruiting and retaining female scientists. And as a result, will encourage more women to go into STEM fields.
“I hope having a strong Women in STEM group will be great recruitment as well for other general faculty to come to MU,” said Azizan-Gardner. “At least that’s my goal, and that’s the area I’m responsible for. And on top of that, I think it will really entice other undergraduate women to go into STEM.”
“#IAmScience because looking into the unknown and coming up with a plan to take a stab at answering it is so fascinating.”
No one in Emilia Asante’s family works in a science field or attended graduate school.
“As an immigrant from Ghana, my family was unaware of the American educational system,” she said. “So many of my academic journeys were unknown and I had to navigate it by myself. I was always interested in science and with the help of programs such as the Lang Youth Medical program when I was in grades six through 12 and the McNair Program at Earlham College, I was put on the right path. These programs provided the help I need and allowed me to explore science in a way I never knew existed.”
Asante graduated in 2014 from Earlham College in Richmond, Indiana with a degree in biology. She is currently pursuing a Ph. D in biological sciences at MU, and works in Anand Chandrasekhar’s lab at Bond LSC.
“I love being able to walk down the hall and talk to other labs about my project and the getting feedback in real time,” she said. “I love the community that I have built since being here.”
For her dissertation topic, Asante is using the zebrafish model to understand the consequences of abnormal neuronal migration of the facial branchiomotor (FBM) neurons on circuit formation and behavioral function. Her research aims to explore the relationship between behavior and circuit organization due to neuronal migration abnormalities of the FBM neurons.
“Essentially, I’m trying to figure out what happens when the proper connections are not made,” she said. “For example, autism is a neurological disease. How does the defect in the brain connections lead to the complex behaviors such communication and social impairments? I want to understand how the brain rewire itself when there is a misconnection.”
Asante has always been interested in behavioral science and how things work. After graduation, she hopes to take the training from her research and lab experience at Bond LSC to a translational research position. Translational research means she would help people in a more direct way, compared to research that is more removed or abstract. Labs that deal with translational research might discover how certain genes cause diseases or work with translating ALS models to drug therapy.
“The possibility that I might discover something that will add to the knowledge in my field – no matter how small the contribution – that’s very exciting,” Asante said.
Natalie Hickerson stands next to her research poster at MU’s Undergraduate Research Forum. Hickerson, a biochemistry major, spent her summer in Dr. Hannink’s lab.
By: Samantha Kummerer| Bond LSC
It takes a lot of time and patience to be a scientist. This is something that first-time researcher Natalie Hickerson quickly discovered.
“A lot of the time things are so small. I mean you’re using such tiny volumes of DNA that you can’t see anything happening,” said the undergraduate biochemistry major.
For some, this uncertainty pair with long lab hours and multiple trial and errors would be frustrating. Hickerson, however, put in the time and asked the questions, which led her to discover the rewards of research.
“You try something a few times and it wouldn’t work and then you change one thing and suddenly it works and you get results and it was just very exciting, like ‘wow what I’m doing is real,’” she said.
Hickerson attends the University of Miami but worked as a research intern in Mark Hannink’s lab in the Bond Life Sciences Center this summer. She said she chose the University of Missouri because the program was well rounded and fit with her major.
Hannink said while Hickerson entered the lab not understanding everything, she wasn’t afraid to ask questions and that helped her catch on fast.
“What makes a scientist different is that you are an active generator of new knowledge. Instead of being a passive consumer of existing knowledge you have to become an active producer of new knowledge,” Hannink said.
The Missouri native spent two months creating new knowledge on PGAM5, an important protein involved in many mitochondrial processes and cell death.
Hickerson began by intensely studying the protein at its most basic level to determine how and why it works the way it does. Part of this process was comparing different mutations of the protein.
Previous research shows how understanding the sensitivity of PGAM5 and its changes in the cell could help in nerve-degenerate diseases like Parkinson’s and ALS. Individuals suffering from those diseases experience a loss of function or mutation in some proteins.
Part of Hickerson’s initial research aimed at figuring out what signal activates PGAM5 in a cell. A better knowledge of that process will help scientists understand how pathways function and turn off during nerve-degenerative disease.
Hannink explained if a pathway is defective, activating a different pathway may function as a type of therapy.
After months of long hours, Hickerson discovered by changing the pH levels, PGAM5 can be switched from inactive to active.
“We had suspected it to be the case but the data she provided really helped demonstrate that was true,” Hannink said.
While Hickerson is headed back to Florida to continue her pursuit of biochemistry, her findings will continue to be advanced this fall in Hannink’s lab.
“There’s a lot more to be discovered with what we were doing,” Hickerson said. “A lot of the stuff we were doing this summer was new ideas and just developing deeper knowledge on things they’ve discovered in the lab previously. So, I was looking deeper into what they already started and we did find some new things so it was neat.”
She said her experience this summer inspired her to get more involved in undergraduate research.
“It definitely gave me a much better understanding of how to work in a lab and just basic lab techniques. The overall research project gave me a lot of good foundation for that,” she said.
As Hickerson continues to learn about research, Hannink said it’s an ongoing process that he is still a part of.
“I’m doing the same thing all the time that they are learning to do,” he said. “There’s this dynamic and that active learning process also challenges me to becomes a better scientist as well.”
Johanna Morrow collects leaf samples from plants in the Bond Life Sciences Center’s growth chamber. Morrow is a Ph.D. student in the Liscum lab.
By Samantha Kummerer | Bond LSC
“#IAmScience because learning something new is super exciting! I love that by performing research one can contribute to the collection of knowledge.”
Johanna Morrow discovered her love for plant sciences after working in Mannie Liscum’s lab for more than a decade after college.
“I was a biology undergraduate and I didn’t really know what I wanted to do, but I knew I enjoyed research so I pursued a job with Dr. Liscum,” she said. “As time went on, I realized I was really interested in how plants are able to sense light and all the different growth and development processes that hinge on that perception.”
Her love for research brought her into the lab, but once she was there she discovered another interest.
“During the time that I worked in the lab, I found that not only did I enjoy doing bench work, but I found that I also enjoyed mentoring students and watching their passion for plant science and research flourish,” she explained.
This realization led Morrow to go back to school so she could share her love of science with more students.
Now her passion is her reality.
Morrow is working on how plants sense and respond to different types of light. She deals with mutations in the model plant Arabidopsis. She hopes by screening different variations of the plant, she can better understand what happens to a plant after it perceives light.
“Maybe we can identify new players in this mechanism or identify new ways that this could have an agronomic impact,” Morrow said.
New web-based framework helps scientists analyze and integrate data
By Emily Kummerfeld | Bond LSC
Large-scale data analysis on computers is not exactly what comes to mind when thinking about biological research.
But these days, the potential benefit of work done in the lab or the field depends on them. That’s because often research doesn’t focus on a single biological process, but must be viewed within the context of other processes.
Known as multi-omics, this particular field of study seeks to draw a clearer picture of dynamic biological interactions from gigantic amounts of data. But, how exactly can scientists suitably weave multiple streams of information together, especially considering technology limits and other biological variables?
Trupti Joshi and her team are seeking to find a solution to that problem.
Joshi, as part of the Interdisciplinary Plant Group faculty, works on translational bioinformatics to develop a web-based framework that can analyze large multi-omics data sets, appropriately entitled “Knowledge Base Commons” or KBCommons for short. She describes KBCommons as “a universal, comprehensive web resource for studying everything from genomics data including gene and protein expression, all the way to metabolites and phenotypes.”
Her work began about eight years ago with soybeans. Dubbed the Soybean Knowledge Base (SoyKB), her team had developed a lot of their own data analysis tools for soybean research, but they realized the same tools could help research of other organisms. From there sprouted the Knowledge Base Commons, intended for looking at plants, animals, crops or disease datasets without the need to “reinvent the wheel” each time.
Soybean plants used in research that utilizes Soy KB web-based network. | Emily Kummerfeld, Bond LSC
“Our main focus has been in enabling translational genomics research and applications from a biological user’s perspective, and so our development has been providing graphic visualization tools,” Joshi said.
Those tools provide an array of colorful graphics from basic bar graphs to assorted colored pie charts to help the researcher better analyze the data once data has been added to the KBCommons.
Colorful graphs and comparisons lets many researchers look past the lines of text and tables full of numbers that represent genes, plant traits or other experimental results, and making the interpretation of data much more easier and efficient.
One particular tool allows the researcher to look at the differential genes of four different comparisons or samples at the same time. Differential genes are the genes in a cell responding differently between different experimental conditions. For example, a blood cell and a skin cell both have the same DNA, however, some genes are not expressed in the blood cell that are expressed in the skin cell. With this KBCommons tool, a researcher can examine genes to see “what are the common ones, what are the unique ones to that, and at the same time look at the list of the genes and their functions directly on the website, without having to really go and pull these from different websites or be working with Excel sheets,” Joshi explained.
She envisions KBCommons as a tool to enable translational research as well. Users will be able to compare crops, such as legumes and maize for food security studies, or link research between veterinary medicine and human clinical studies for better therapies.
Intended for a wide range of users, Joshi is keenly aware of its potential users right here at MU.
One current user of the Soybean Knowledge Base (SoyKB) system is Gary Stacey, whose lab at Bond Life Sciences Center studies soybean genomics and to date has been the longest user of the SoyKB resource. Like many researchers, Stacey explained the need for a program like SoyKB that can process enormous amounts of data.
“The reason it’s called “Knowledge Base” is the idea that we’re putting information in, and what we hope to get out is knowledge. Because information is different than knowledge,” he said, “we don’t just want to collect stamps, we want to be able to actually make some sense out of it…By having a place to store the data, and then more importantly have a place to analyze it and integrate it, it allows us to ask better questions.”
This is essential, given that one soybean genome is 1.15 GB in size, and one thousand soybean genome sequences could generate 30 to 50 TB of raw sequencing data and tens of millions of genomic variations (SNPs).
But such numbers are modest compared to the program’s true capabilities.
“The KBCommons system is so powerful that it can allow you to run thousands of genomes at the same time using our XSEDE gateway allocations,” Joshi said. “This whole scalability is a unique feature of KBCommons, which a lot of databases do not provide, and we are happy we have been able to bring that to our MU Faculty collaborators on these projects, so that they can really utilize the remote high performance computing (HPC), cloud storage and new evolving techniques in the field.”
KB Commons is a new web-based network for biological data analysis and integration developed by students. | Emily Kummerfeld, Bond LSC
Mass data capability and colorful graphs aside, her favorite part is who exactly is designing the program.
“What I like most about KBCommons is that it serves as a training and development ground and is developed by students, undergraduate and graduate students from computer science and our MUII informatics program.”
KBCommons is still under development, but publication and access for all users is planned for the end of this year or early 2018. Users will not only be able to view public data sets, but add their own private data sets and establish collaborative groups to share data.
Dr. Trupti Joshi is an Assistant Professor and faculty in the Department of Health Management Informatics, the Director for Translational Bioinformatics with the School of Medicine, and Core Faculty of the MU Informatics Institute and Department of Computer Science and the Interdisciplinary Plant Group.
Jay Thelen, a Bond LSC researcher, next to the GC-MS machine in his lab, which tracks seed oil measurements. | photo by Mary Jane Rogers, Bond LSC
By MJ Rogers, Bond LSC
You can’t get blood out of a stone, but Jay Thelen wants to find ways to get more oil from seeds.
“We’re specifically working on the metabolic engineering of oil seeds. Broadly, trying to increase the oil content of crops and raise the value of the seed in the process,” said Thelen, a Bond Life Sciences Center researcher.
Seed biology and metabolic engineering have long been interests for Thelen, and his lab combines biochemistry with cutting-edge proteomics technology to identify new regulatory modules for key metabolic enzymes.
But let’s start with why seed oil is important.
Seed oil is big business, enough so that scientists are trying to maximize the amount of oil that seeds produce. It represents an important, renewable source of food and feedstocks, used for everything from salad dressing to combustible fuel. More than 448 million tons of oilseed crops were consumed in 2015-2016, according to USDA Economic Research Service.
The oil essentially comes from lipids, or fats, in plant seeds or fruits. All plant cells contain lipids, and embryonic cells within young seeds are poised to make an abundance of them, especially the storage lipid triacylglycerol.
While tree nuts can be up to 80 percent oil, most oilseed crops store 15 to 45 percent oil within their seed. Through biotechnology and metabolic engineering scientists want to increased this amount, something Thelen aims to do.
His end goal is to increase the oil in crops such as soybeans and canola, but any biotechnological idea for increasing seed oil starts in the model plant Arabidopsis.
Modified plants that are being grown in Thelen’s lab in Bond LSC. | photo by Mary Jane Rogers, Bond LSC
“Arabidopsis is easy to transform in the lab and has a short life cycle,” he said. “We can use this plant to quickly demonstrate proof-of-principle for enhancing seed oil and then advance successful strategies to soybean, camelina, or canola.”
To increase seed oil content, Thelen’s lab works on a large protein complex called Acetyl-CoA carboxylase or ACCase – an enzyme that catalyzes the first step towards oil production.
“We made a recent breakthrough on the regulation of this complex,” Thelen said.
The proteins critical to this process are called BADC proteins – they are kin to an essential part of ACCase, but are inactive. BADC proteins significantly inhibit the activity of ACCase by mimicking its functional sibling and slowing the complex down.
Basically, BADC is a way for the plant to control and slow down the production of fatty acids. By “turning off” the BADC protein, ACCase is de-regulated and oil content in seeds significantly increases.
“We leveraged this discovery to make higher oil producing plants by simply shutting down expression of this gene family by RNA interference,” said Thelen. “Consequently this increased seed oil content quite a bit. We’re now in the process of studying gene knockouts for this family in soybean and camelina.”
Yajin Ye, a postdoctoral researcher from China, adds water to the plants. | photo by Mary Jane Rogers, Bond LSC
Yajin Ye, a postdoctoral researcher from China in Thelen’s lab, is in the thick of this work. He spends his time modifying seeds to maximize seed oil content and tracking the seed oil measurements in the GC-MS instrument.
The GC-MS machine, which tracks the seed oil measurements. | photo by Mary Jane Rogers, Bond LSC
“If you want to know how much oil is in each seed, you use this instrument,” Ye said, pointing to the GC-MS. “It measures the oil content of the samples we provide it.”
Arabidopsis is a model plant used in Thelen’s lab and the seeds are much smaller than most people would expect. | photo by Mary Jane Rogers, Bond LSC
Arabidopsis seed are much smaller than most people expect, so tiny and light that researchers have to be cautious that they don’t become airborne and cause cross-contamination. In addition, soybean plants are kept upstairs in a fifth floor greenhouse at Bond LSC. While camelina are grown in growth chambers within Schweitzer Hall as part of a collaboration with Dr. Abraham Koo, an assistant professor in the Biochemistry Department.
Thelen’s soybean plants, which are kept in the fifth floor greenhouse of Bond LSC. | photo by Mary Jane Rogers, Bond LSC
“Each of the [soybean] plants are transgenic and were screened for higher oil content as a result of BADC gene silencing. The plants are harvested every three or four months so the seed oil content can be monitored,” said Ye.
Thelen has begun patenting the BADC technology and another strategy for engineering ACCase to “make it a more efficient enzyme complex.” The BADC technology was co-invented with his previous graduate student, Matthew Salie, who now works as a postdoctoral research associate at the Scripps Research Institute in San Diego. The patent examination process can take years, but if the technology is approved it would mean a huge influx of money in the agricultural market.
“The math is quite simple,” Thelen said. “A one percent increase in soybean seed oil translates to hundreds of millions of dollars. The numbers fluctuate depending on the market, but a one percent increase translates to about 200 million dollars for soybean alone. A five percent increase, which again, I think is achievable, when realized across the diversity of oilseed crops, we’re talking billions in added crop value annually.”
Yajin Ye, a postdoctoral researcher from China, with Vanildo Silveria and Caludete Santa Cantarina, two visiting faculty from the State University of Rio De Janerio in Brazil. | photo by Mary Jane Rogers, Bond LSC
His innovative approach has gotten the attention of scientists and researchers all around the world. Vanildo Silveria and Claudete Santa Cantarina, two visiting faculty from the State University of Rio De Janerio in Brazil, came to Bond LSC specifically because of Thelen.
“Jay is an expert in the field and we wanted to work with him,” said Silveria.
At the moment, it seems like his research is on the right track. The initial data from Arabidopsis shows that silencing the entire BADC gene family substantially raises seed oil content, which is the main objective of his study.
“Preliminary, first-generation transgenics show soybean with higher oil. But these are greenhouse grown. Randomized field trials are still be awhile out,” Thelen said. “We’re getting closer, but still a few years away from that goal.
Jay J. Thelen is a professor of biochemistry at MU and a researcher at Bond Life Sciences Center. He received degrees in both biological sciences and biochemistry – a B.S. from the University of Nebraska, Lincoln and a Ph.D from the University of Missouri, Columbia. He was a postdoctoral fellow at Michigan State University and has been at MU since 2002.
Katelynn Koskie, a Ph.D candidate, works in Mannie Liscum’s lab. | Photo by Samantha Kummerer, Bond LSC
By Samantha Kummerer | Bond LSC
“#IAmScience because I want to help unravel the mysteries of nature that will improve our futures and positively impact our planet.”
Katelynn Koskie didn’t always know she loved plants. As an undergraduate, she focused on what was above her rather than what grew below her.
“I was really interested in how galaxies interact and then I started to think, ‘you know I’ve always thought plants were really, really cool,’ and I wanted something that was a little bit more down to earth,” she said.
While she was pursuing a degree in astrophysics, she took one plant biology course and fell in love. From there she signed up for grad school and has been with plants ever since.
Koskie works with a mutated plant called hyper phototrophic hypocotyl, hph. The mutation is a variation of the lab’s model plant Arabidopsis. This variation is special. It produces more seeds, bends more under light and is stronger. It’s up to Koskie to figure out why.
That answer could have a large impact on the agriculture industry. If Koskie’s findings can be applied to crop plants like maize, farmers can grow better crops.
“Maize is more complicated than Arabidopsis, but with new techniques like CRISPR/CAS9 now it might make it a little bit easier,” she said.
She plants genetically modified seeds and then waits and observes and begins again.
It is a lot of time in the growth chamber and in the dark room, hoping the research may reveal a breakthrough.
Beverly Agtuca, a Ph.D. candidate that works in Dr. Gary Stacey’s lab in Bond LSC. | photo by MJ Rogers
Beverly Agtuca was born in New York, but has family in the Philippines, a country that struggles with malnutrition and undernourishment. Her overall goal for her research is to help countries that struggle with undernourishment by increasing the agricultural productivity in those countries.
“When I was little, I went on summer vacation to visit my family, which included my grandmother in the Philippines,” she said. “Everyday my grandmother wanted me to go out to the rice fields from 5 a.m. to 10 p.m. with the other children to get rice for our meals. That was not an easy task and that moment changed my life. That’s when I decided that I wanted to be a plant scientist.”
Agtuca graduated in 2014 with honors in Biotechnology and a minor of Microscopy from the State University of New York College of Environmental Science and Forestry (SUNY ESF) in Syracuse, NY. She’s currently a Ph.D. candidate in plant breeding, genetics, and genomics at MU. She chose to come to Bond LSC because of the community and Dr. Stacey, her supervisor and mentor.
“If you ever need help, there’s always help here,” she said. “Everyone at Bond LSC is so kind, including the staff. I love to make small talk with the custodians and they are always supporting me and say I should never give up when I have a bad day.”
Ever since coming to MU in 2014, Agtuca has been keeping busy. In June, she received a travel award to go to the American Society of Plant Biologists (ASPB) in Hawaii. The International Society for Molecular Plant-Microbe Interactions (IS-MPMI) also awarded her a travel award to attend the 2016 meeting in Portland, Oregon, where she gave an oral and poster presentation. She also has two original research publications under her belt and is currently working in Dr. Gary Stacey’s lab at Bond LSC.
The research for her dissertation is focused on the relationship between rhizobia and soybeans. She collaborates with scientists at George Washington University (GWU) in Washington, D.C. and the Pacific Northwest National Laboratory (PNNL) in Richland, Washington to enhance the capabilities of the 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer (21T FTICR) through application of laser ablation – electrospray ionization mass spectrometry (LAESI-MS) technology that can analyze the contents of single plant cells. This 21T FTICR machine was recently installed at PNNL and represents one of only two such machines in the world.
This is revolutionary because few people do single cell analysis. Usually, scientists deal with the law of averages, which dilutes the final measurements. But this technology gives an in-depth glimpse into a single cell so scientists can obtain a more comprehensive bigger picture.
“After we finish building this technology, we want to spread the technique to different research groups so they can answer these research questions on their own,” said Agtuca. “It can help people outside of plant sciences too, and hopefully will help with cancer treatment and disease prevention.”
What do you do when you have an unknown substance and need to know what it’s made of? Or what if you know what’s in it, just not how much?
Scientists turn to metabolomics to figure out what these pieces are.
Lloyd W. Sumner, Director of the Metabolomics Center at MU’s Bond Life Sciences Center, said analyzing a sample is like going to the doctor and having blood drawn to assess what’s happening inside of your body.
“Plants can’t tell us what’s going on, animals can’t do that either, so we need high resolution biochemical phenotyping to understand how organisms respond to stress and disease. Instead of profiling one, five, 10 or 30 compounds, we’re profiling hundreds to thousands of metabolites, and we use that to assess the biochemical phenotype and how the system is responding,” Dr. Sumner explained.
Lloyd Sumner, biochemistry professor and Director of the Metabolomics Center at Bond LSC. | photo by Morgan McOlash, Bond LSC
This is achieved using methods called chromatography and mass spectrometry. These methods separate and analyze hundreds to thousands of metabolites to better understand the biochemistry of living organisms. Metabolites are the molecules a cell creates that provide the building blocks and energy sources enabling a plant or animal to grow, reproduce and respond to its environment. These small molecules can tell us a lot about an organism.
So how exactly how does mass spectrometry work? It really is the science of weighing molecules. It can identify small molecules, quantify known compounds and reveal structural and chemical properties. Crime shows on TV employ the technique in forensic investigations to analyze the molecular composition of unknown substances found at a crime scene.
Let’s break down the process for you.
The first step is actually chromatography, where the molecules in a mixture are separated based on chemical properties. This helps to see individual metabolites within complex mixtures from plants, animals and microbes. Two of the most common types are gas and liquid chromatography. Sumner’s lab uses both since some molecules cannot be heated during gas chromatography and need to be separated via liquid chromatography.
In gas chromatography, a sample is heated until it vaporizes and then travels through a thin glass tube and interacts with a coating on the tube wall. These interactions vary and help separate all the sample’s parts. The speed at which each separate molecule travels helps us determine the identity of each part.
Here’s where the mass spectrometer takes over. As each small molecule/metabolite exits the chromatography column, it enters through the inlet system, and passes through an ionization system that puts a charge on the molecules, since a mass spectrometer does not weigh actual mass but rather the mass-to-charge ratio of the molecular ions. Once the ions are formed, they go through the mass analyzer, which will tell the ions apart based on their mass to charge ratio. From there, the ions hit a detector where a resultant current is measured. This gives scientists a graph of sorts, showing a series of peaks for each substance, with higher peaks indicating more of the molecule in a sample.
This data from both chromatography and the mass spectrometer is then used to figure out exactly what was in the unknown sample.
Control is the name of the game when it comes to getting accurate results. The system keeps samples in a vacuum so molecules can move around without colliding with other gas molecules, because bad things can happen when they do.
“They can deflect the trajectory of the ions as they’re moving through the instrument, there can be charge loss and neutralization of that, but ultimately all of those affect sensitivity,” Sumner said.
Depending on what Sumner’s team is trying to figure out, other types of mass analyzers can come into play. From quadrapole mass analyzers to magnetic sector instruments, to time-of-flight analyzers, they separate the ions in different ways.
Not all mass analyzers are equal as Dr. Sumner explained, “these different types of mass spectrometers have different performance metrics. And as resolution and sensitivity go up, usually cost does, too. But for the most part, we use mostly TOF (time-of-flight) analyzers because they have good mass resolution and the cost is kind of modest.”
The way a time-of-flight analyzer separates ions is actually quite simple. A burst of ions is emitted and accelerated, and the ions are measured based on their flight time over a specific distance, meaning the smaller and lighter the ion, the faster it will be, with each molecule having a unique time-of-flight.
So how does Dr. Sumner use mass spectrometry in his own research? Currently, his work focuses on plant biochemistry.
“We’re trying to understand how plants synthesize triterpenoids. These are important plant compounds that plants use them to defend themselves. They can’t get up and run away, so they make these defense-related compounds,” Sumner said.
The Metabolomics Center benefits more researchers than just Sumner’s team.
For example, his center is collaborating with other faculty on an animal stress test for milk. That’s because the technology at the center can be applied widely to science not only here on campus but nationally and internationally.
An added bonus for Sumner is that his center feeds his fascination with sophisticated analytical instrumentation and electronics, or “shiny toys,” since once “you understand how they work, you get a greater appreciation for them,” he says.
Lloyd Sumner is a professor of biochemistry and director of the University of Missouri Metabolomics Center at the Bond Life Sciences Center. The Metabolomics Center, which opened in August of 2016, is one of ten research core facilities at the University of Missouri. Learn more about the center at http://metabolomics.missouri.edu.
