Gene-editing is the pinnacle of a biologist’s toolbox, but often left unexplained it seems more magic than science.
Growing rice from a small cluster of cells to 4-foot stalks can take six-months or more of planning and careful nurture. But how do scientists change the intricate genetic material in each cell of the plants?
The CRISPR-Cas9 gene editing tool changes a plant’s DNA. As Ph.D. student Ajay Gupta knows firsthand through work altering plants for the Bing Yang lab.
“CRISPR is relatively new. It’s like 10 years old only and still we are working to modify it and improve it in plants,” Gupta said.
The process starts with a few cells from seeds capable of maturing into any type of cell to form a cell cluster, known as a callus.
But how do scientists insert instructions into a cell?
In the Yang lab, they use particle bombardment to insert DNA, fundamental instructions used to build everything within a cell. These inserted DNA strands tell the cells how to build the CRISPR tools to change their own DNA.
To shoot the DNA molecules into the cells, researchers use high pressure helium gas operated gene gun to bombard the cells with DNA coated gold particles.
Once the DNA instructions are in the cell, they build the CRISPR-Cas9 tool using the plant’s genetic machinery.
CRISPR-Cas9 has two parts: the Cas9 protein and an RNA molecule that only binds to the DNA section scientists want to change. This RNA strand guides the Cas9 to the correct location.
But the parts of the guide RNA do two things. First, the spacer part — usually about 20 base pairs only binds to the section of DNA researchers want it to. Second, a part called the scaffold — nearly four times the size of the spacer is required for attaching to the Cas9.
When the spacer of guide RNA binds to the DNA, the Cas9 enzyme acts like a pair of scissors, cutting that section of DNA.
Now we have DNA broken in two, and the plant’s built-in repair system starts its job. However, this repair system is flawed, and it usually messes up while rebuilding the DNA’s bases, or building blocks. Its errors vary every time, so many different mutations are possible.
“The repair machinery which is employed is not perfect,” Gupta said. “When it repairs those double-strand breaks, it causes some errors. The error could be a single base, and it could be 10 bases or it could be a thousand bases. So that is unpredictable.”
Think of it this way: imagine you have a ten-page instruction manual, and you rip out pages two and three. You could replace them with blank, yellow or red pieces of paper and the book would have ten pages again, but the instruction manual can’t be used again. Just like that, it doesn’t matter which error happens, because any mutated change in the gene results in it not working.
“This is how the normal CRISPR-Cas9 works, when you just want to shut that gene down,” Gupta said.
This puts the gene’s instructions out of commission and is commonly called a knockout gene. That helps scientists determine what the particular gene does. They then compare “normal” plants to plants with the knocked out gene to determine what happens differently.
Now the waiting happens. Researchers grow the plants using different hormones to mature the callus cells into roots and stems.
“Once they have shoots and roots then you transfer them to soil and grow the complete plants out of that,” Gupta said.
When the plants become adults, the researchers check the knocked out changes by extracting its DNA in a process known as genotyping.
While traditional CRISPR-Cas9 is used by researchers to learn more about plant genomes, Gupta and others in the Yang lab also work to edit parts of DNA and replace it with specific mutations. This process is known as prime editing.
“The exciting part about this [prime editing] is that theoretically it can do anything,” Gupta said.
Prime editing takes traditional CRISPR-Cas9 editing a step further, and the Yang lab’s research into it opens the door for future collaborations with labs around the world in applied and basic sciences.
What started as an email correspondence between two aptamer enthusiasts rapidly snowballed into a hat trick of authorships for Donald Burke.
“I was contacted by a student in India asking if I would be an external advisor for her Ph.D. committee,” said Burke, a principal investigator at MU’s Bond Life Sciences Center.
Burke’s extensive research with sticky molecules called aptamers — totaling about 60 publications over 30 years — makes him an expert in the field of aptamer technologies. When Ph.D. student Shringika Soni at Amity University in Noida, India, near New Delhi, began characterizing the use of these molecules for drug testing in a literature review, she turned to Burke for advice on her work. The connection soon turned into a mentorship through regular email correspondence.
“I told her that I was interested in the kinds of things that she does, and I would be happy to interact with her about her science,” Burke said.
That project led Burke to collaborate on a project that attempts to create a rapid test to detect various drugs. This test would provide quicker information than waiting for the results of a blood or urine sample to be returned from a laboratory.
Starting out as a literature review, it soon turned into experimental research at Amity University. Burke’s role from halfway around the world was to ask tons of questions.
“There were a lot of back-and-forth emails around the general idea of defining precisely what her message was, what did she want to say about these systems, and as a particular case study might be mentioned, how much detail should be talked about in the review article,” Burke said.
While the research is not ready to be used commercially, Burke suggests potential applications could be for police officers who suspect drug use or for medical personnel trying to respond quickly to an affected individual.
“Maybe you’re suspicious that [someone is] on a particular drug. Having them spit onto a stick is just a whole lot less invasive and quicker,” Burke said.
Detection of the drugs falls to sticky molecules called aptamers. Essentially a chain of nucleotides folded into a particular shape, aptamers are selective in what they stick to, and each aptamer sticks to a specific compound. The researchers engineered their own aptamers that can stick to particular compounds found in certain drugs.
“The two major layers to this technology are designing the sensor components so that it will bind to the [compounds] really well and specifically,” Burke said. “The second component is to somehow turn that binding event into a detectable signal.”
But, aptamers just sticking to a drug is not enough because the researchers also need a signal to know the aptamer detected it.
To sense the chemical interaction, the researchers focused on electrical currents. With no drugs present, the aptamers do not stick to anything, and the electrical current flows. In contrast, when there are drugs present the aptamers stick to the compounds and block the electrical current from flowing. This allows the researchers to measure the drugs in the solution.
Due to Burke’s help on the project, the research team at Amity University offered to list Burke as a co-corresponding author.
“I just didn’t think that was appropriate, they were the ones leading the charge,” Burke said. “My role was to ask them questions from time to time and to push them to make sure they rounded out their arguments.”
This project adds to Burke’s long list of aptamer research fueled by his fascination with the properties of molecules driving living things.
“Just about every aspect of biology is driven by this choreography of who’s dancing with whom, by which molecules dance with which other molecules,” Burke said. “But why is it that some of the players refuse to dance with certain other players and others are just immediately drawn to certain other players?”
Burke’s research tries to answer these questions and determine why some molecules interact with specific compounds and why others do not.
“When you look at molecules the way they interact, it’s just so fun to watch the dance that they actually do,” Burke said.
Art and science are often considered opposites, but Beatriz Praena Garcia sees overlap.
“I think in this job you need to be very artistic,” Praena said. “I have a basic methodology to do the essays … then I read a little bit online. You can search in another paper and do some research to see how you can apply it to your work. You can be more creative, so it’s not always the same.”
The postdoctoral researcher studies influenza vaccines in the Henry Wan lab, tackling them from three angles. She works to improve vaccine effectiveness by growing it in different types of cell lines and eggs. She also works to improve mouse model systems for her lab and studies the influenza virus receptors.
Praena worked on antivirals for herpes before coming to Mizzou, and the switch to vaccines was a welcome change for her.
“I always wanted to study vaccines. . . there are a lot of antivirals already in the war against viruses. I want to give something [new] to the community,” Praena said.
Praena started in the lab right out of high school in a two-year technical training program where she worked in a biology lab.
“In high school, I was not a very good student, so when I finished high school I didn’t go to the university directly,” Praena said.
Once exposed to the lab, Praena knew that was where she wanted to be.
“I realized this is good for me. My score was very high in the class, and I said, ‘Oh, I will try to go to the university,’” Praena said.
Growing up in Spain, Garcia attended Autonomous University of Madrid for undergrad where she started research on herpes antivirals and its receptors. She stuck with that research for the next eight years, working in the same lab for her masters and Ph.D. studies.
Praena came to MU at the height of the pandemic in 2020. She had to gain a special visa, which she also worked on in the Wan lab during the pandemic. After accepting the position at MU, Praena remembers consulting a large map on her wall to find the landlocked state of Missouri.
“At the beginning it was complicated because when I accepted the position, I didn’t know where Columbia, Missouri was,” Praena said.
For Praena, finding passion for her work is vital to her success.
“You need to be a hard-worker, and you need to have a lot of resilience, because in academia you will never be rich, and you have to work a lot,” Praena said. “So, the first thing you need is to love your job [and] the science.”
Her hard work translates to outside of the lab where she competes in triathlons and bikes trails in Columbia.
“I like to take my bike to the trails. I like the Katy Trail, the MKT Trail and I went to the Ozarks,” Praena said.
Praena and her husband also camp and explore different states. Over winter break, Praena took advantage of having two weeks off and traveled to Arkansas, Texas and Oklahoma.
Praena enjoys Columbia and traveling in the U.S., but she hopes to one day return to Spain and have her own lab. While in the Wan lab she works to improve her research skills and develops project ideas.
“You always have to ask ‘why’ and ‘how,’” Praena said.
Ajay Gupta learned biology basics as a first year undergraduate on the bumpy bus ride from his small hometown to Punjab Agricultural University. Just a few hours’ ride, he made the most of his time before he returned home to help his family’s agricultural goods business.
Working extra hours in the margins of his time has become a habit for Gupta. Now a plant science first year Ph.D. student in the Bing Yang lab and Department of Plant Science and Technology Millikan Endowment recipient, Gupta starts his day around 9 a.m. and works on three plant science projects until night.
“My advisor has to sometimes push me out of the lab to go home,” Gupta said. “I have that fascination with science.”
For his Ph.D. thesis project, he studies how rice plants fight off bacterial leaf blight — a disease caused by the bacterium Xanthomonas oryzae. This disease can devastate rice fields in Asia and other areas that depend on the staple grain. His research focuses on genes that help plants resist the deadly disease.
“The second project, which is very close to my heart, … is the development of CRISPR genome editing toolkit,” Gupta said.
Gupta works to improve the gene-editing tool CRISPR-Cas9 in rice plants. Researchers currently modify chunks of DNA sequence with CRISPR, but Gupta’s goal is to modify the individual building blocks of DNA known as nucleotides. He simplifies the process to make gene editing easier for researchers with less CRISPR experience.
His third project started with self-taught skills rooted in a side passion. Gupta contributes to a global bioinformatics project investigating bacterial resistance genes in rice. By understanding the genetic make-up of plant resistance to disease, the researchers may find innovative solutions to prevent crop infections. Gupta’s input involves assembling the genome of a type of rice that is resistant to the pathogen that causes bacterial blight in rice.
“It’s a global project where everyone across the globe who uses that variety [of rice] can take advantage of that genome,” Gupta said.
Most of Gupta’s time is spent in the lab now, but Gupta started studying biology by accident. As an undergraduate he decided to major in agricultural biotechnology thinking there would be more math involved.
“It mentioned that it will have some math and some biology, and I knew nothing about biology,” Gupta said. “I said, ‘OK, it has math, I should go for it.’”
For the first year Gupta had six biology classes and one math class each semester.
“I said, ‘Oh my god, how am I going to survive,” Gupta said.
Gupta worked through basic biology courses in subjects like genetics, botany and zoology alongside his math courses. Then the math courses dropped off for the following years leaving Gupta’s schedule packed with biology classes.
“And then I started liking it,” Gupta said. “It seems more practical to me than math. I think if I would have gone to math I would have been in engineering, but I’m happy that I chose this path, and right now I’m working with plants which I love.”
Gupta’s small high school in his agricultural hometown left out many biology basics, but without the finances to attend a better school in another city Gupta was left to catch up at the university level while also helping his older brother at home.
“It was very challenging for the first year. Our financial situation was not great, so I had to support myself for my studies,” Gupta said.
Gupta returned every day to work as an accountant at his family’s small agricultural goods store to earn money for his education and to help.
“My father passed away when I was 15, and me and my brother who was 18 at that time, took care of our business without having much experience. So, I had to ride the bus four hours every day to go there and help my brother during my first year in bachelor because we could not afford at that time to hire somebody” Gupta said.
Back on their feet by his second year, Gupta continued working on weekends throughout the rest of his studies, and he only separated from the business when he left for his masters’ studies halfway around the world at South Dakota State University.
Gupta initially stayed with friends in South Dakota to soften the transition to the United States. Although difficult to live an ocean away from family, Gupta maintains relationships with his family.
“I talk with my mom almost every day. That’s a kind of an Indian or Asian thing. We are very close to our parents,” Gupta said.
Gupta started CRISPR research while working on his master’s degree at South Dakota State University. He focused on modifying wheat genes to increase yield and diversity, but on the side he also found a passion for bioinformatics.
“Most of this was self-learning, so I just found things on the internet and found out how they work and then made my own protocols and now have the pipeline,” Gupta said.
After this taste of research, Gupta was hooked. He applied to continue his studies as a Ph.D. student and asked Bing Yang, a collaborator with the lab’s wheat genetics research, for a letter of recommendation.
Instead, Yang asked Gupta to join his lab.
Gupta accepted his offer to continue working on CRISPR alongside expanding to more basic research — a change of pace from his previous work in applied agriculture.
“I want to develop a mindset where I can work on [basic science] well where there is little or no information and then I can think on my own,” Gupta said.
Gupta is the primary bioinformatics researcher in his lab and helps with data analysis for several projects alongside his bioinformatics project. While he spends most of his time hard at work on a research bench — and can often be seen walking across the Bond LSC atrium to the greenhouses late into the evening and over holidays — he also enjoys talking with lab mates.
“Sometimes, post [research] talks, we sit together and then I discuss with them if they have some problem I can try to help, and I do the same as well. If I’m having some problem I talk to them,” Gupta said.
Gupta plans to stay in academia and continue research and teaching others. He enjoys mentoring other students and also works on outreach to high school students.
“I love to do those things and talk to students, and maybe in some ways help this university as well,” Gupta said.
Gupta holds on to the inquisitive spirit that drove him to spend hours studying basic biology on the bus despite now learning in advanced research facilities.
“I love to talk with people [to] find out what they’re doing and if I can learn something out of it,” Gupta said. “Because a lot of the time our knowledge is limited and when talking to people you can get something new.”
Cynthia Tang’s academic career is marked by her propensity to multitask. From earning a major and three minors during her undergrad to making a documentary while getting lab and clinical experience, she makes the most of her time.
Recently Tang received the Excellence in Public Health Award from the United States Public Health Service, and a $181.734 National Institutes of Health grant to be used over four years . . . all while getting an M.D. and Ph.D. simultaneously.
The funding from the grant goes towards Tang’s research on SARS-CoV-2, the virus responsible for the COVID-19 pandemic, and its effect in rural areas. By comparing DNA sequences of the virus from patients in rural and urban areas, Tang looks for differences in variants. Her goal is to understand how the virus has been evolving and why patients in rural areas experience more severe symptoms.
“I’ve always found infectious diseases really interesting,” Tang said. “This pandemic came up and it was exciting to be in the position to help contribute to the knowledge base of it.”
In a family of immigrants, Tang saw the challenges in health literacy and cultural differences affecting patient care in the U.S first-hand, and she also caught a glimpse into a global view of healthcare.
“I had an opportunity to travel to Vietnam and actually see some of the health disparities of different health care structures in different countries,” Tang said. “We were visiting my grandfather who was in the hospital, and it was alarming to see the contrast between the quality of health care and availability of health care there compared to some of the hospitals that we have here.”
Tang recalls noticing lack of supplies, physicians and space for patients. She saw similar issues in the U.S., and shined light on public health disparities here by creating a documentary about the challenges immigrants face in the U.S. healthcare system. She also held competitions across Washington University’s campus to teach students more about public health issues while working as a clinical research coordinator.
“I wanted to help improve health equity,” Tang said.
A bachelor’s in chemistry and minors in philosophy, international development and pre-health professions at the College of Idaho may start to explain Tang’s public health focus and her path to bench and clinical research.
“My minor in international development was really useful for me because that gave me a lot more exposure to the different economies of different countries of the world, their health status and how politics, economics and health care all interrelate in different countries,” she said.
Her research at Washington University led Tang to an interest in its clinical applications.
“I really enjoyed the aspect of working with patients, actually getting to talk to patients and hearing their stories,” Tang said. “I felt like with clinical research, you can see … slightly quicker results from bench to bedside compared to bench research, and that was also when I decided I was interested in becoming a physician.”
Part of an eight-year joint M.D.-Ph.D. program at Mizzou, Tang started with two years of pre-clinical medical school before transitioning to Ph.D. research and then will return to finish the clinical years of medical training.
When she came to Mizzou, Tang continued fighting health disparities for immigrants.
“We organized a group of volunteers that were able to translate COVID-19 information to different non-English speaking communities in Boone County,” Tang said.
Always one to tackle several projects at once, Tang plans to pursue a career as a pediatric physician scientist interfacing with patients while continuing lab research.
“I like the idea of doing both. As a physician, you get that one-on-one contact and you get to make a very direct contribution to someone’s life,” Tang said. “And then with research, you don’t have as much contact with patients, but what you do can affect populations.”
When Samantha Yanders stepped to the front of Monsanto Auditorium, she followed presentations from two researchers with three degrees each.
Yanders only had three years of undergraduate research experience.
Nevertheless, she pinned the microphone to her tie, ran her fingers through her short curly hair, and explained her research with a calm certainty to her voice.
Having just finished her junior year as a plant science undergraduate, Yanders spent the first week of her summer sharing her passion for plants with fellow researchers during the 2022 Interdisciplinary Plant Group Symposium.
“I want to be inclusive in how I talk about my work to be able to educate people,” Yanders said. “Even if you’re talking to molecular biologists, they may have no idea about extracellular ATP.”
A clear communicator and advanced undergraduate researcher, Yanders was selected to present her research in Monsanto Auditorium during the symposium and often helps write and edit manuscripts for her lab.
Yanders began research in the Gary Stacey lab through Freshman Research in Plant Science, a program that places plant science freshmen in MU labs. Yanders’ research focuses on the signaling role of extracellular ATP when a plant experiences high-salt conditions.
Within a cell, ATP is a molecule used for energy. However, research shows that when a plant is in high stress or damaging conditions, ATP outside the cell signals for protective mechanisms.
“It’s really clever because instead of spending energy to make both a signaling molecule and a signal receptor, it already has something that’s really high concentrations in the cell and very low concentrations outside the cell,” Yanders said. “So it’s easy for the plant. It just has to make a signal receptor.”
Under normal conditions there is less ATP outside the cell and a high concentration of ATP inside the cell since it is used as energy. However, under high salt conditions there is an increase in ATP outside the cell, which binds to receptors on the cell surface that communicate with the cell to stunt plant growth. Yanders’ work explores the impact of high salt environments for plants — an increasingly relevant project as climate change raises sea levels and increases salt deposits in soil.
Although she now enthusiastically recounts her work to auditoriums of fellow scientists, biology was not originally Yanders’ first choice.
“In high school, I knew that I wanted to do something scientific because I’ve always liked science,” Yanders said, “But I didn’t think I wanted to go into biology, because I saw biology as mainly oriented towards animals.”
With her geneticist grandfather piquing her interest from a young age and her enthusiasm for environmental science in high school, once she learned Mizzou had a plant science program she was ready to commit.
“So I was like, ‘Okay, I’ll go into biology and focus on plants.’ And then I’m looking on the Mizzou website and they [have] plant science. I didn’t even know that was a thing,” Yanders said. “So I switched to plant science.”
After enjoying high school chemistry labs, Yanders was ready to take the next step into research labs as soon as possible. Freshman Research in Plant Science allowed Yanders the opportunity to join a lab right away — fostering her interest in plant research.
“As humans, we move around. We can adapt to change by moving and changing what we do,” Yanders said. “But a plant has to do all of that management from a stationary position. . . It can’t change the circumstances that it’s in, so it has to adjust itself to be able to adapt.”
Yanders’ love of plants extends beyond the walls of the lab, and she spends her free time in her herb garden where she learns more about plant and human interactions.
“The way that you interact with the plant fundamentally changes how the plant acts,” Yanders said. “So like with basil, if you just let a basil plant grow it just gets leggy and crazy, but if you do what you feel like is harming it by pinching the tops off, then it grows more compact and bushy, which is good for the plant.”
Yanders hopes to explore plant and human interactions in the future, potentially pursuing urban agriculture and ecology.
“I’m really interested in soil health, I feel like we’ve done a lot to deplete soils, and coming up with more renewable ways of doing things [and] producing things for human consumption,” Yanders said.
While uncertain which questions she will ask next, Yanders hopes to continue answering questions and explaining her research to others.
“[I like] being able to offer an explanation, and not just see it, but to create that explanation for other people,” Yanders said.
If the world can be taxing on a person as pressure mounts, just think about how stress must feel to plants.
Humans can add a layer of clothing when cold or get a glass of water when thirsty, but plants do not share this simple luxury and must endure whatever environment they sprout in.
As climate change, pollutants, and extreme weather patterns escalate, this poses a serious global threat to plants and our food supply.
Ron Mittler, a principal researcher at the Bond Life Sciences at the University of Missouri, recently looked at how this piling up of multiple stressors at once can significantly decrease plant survival.
“The principle is that you can have a combination of several different stressors, each by itself has no effect on the plant but when they come together, they’re causing severe effects,” Mittler said.
Studying plant response to stress isn’t a new thing, but Mittler’s focus on the compounded effects of stressors may give us a better idea of the threshold of stress plants can endure in our changing climate. Multifactorial stressors combine three or more stress factors simultaneously impacting plants. Four categories of stress— biotic, climate, anthropogenic, and soil threats — all become worse as climate change and environmental pollution progress, subsequently decreasing plant quality of life.
Biotic threats relate to enemies like pathogenic bacteria and insects. Similarly, soil threats are determined by poor nutrient soil and salinity. Climate threats include extreme temperatures and drought. Anthropogenic threats are man-made as humans use harmful pesticides and create microplastics.
Arabidopsis thaliana seedlings, a model plant used in experiments, were placed side by side on a plate and received a combination of stress conditions such as heat, salt, excess light, acidity, heavy metal, and oxidative stress. Researchers studied the growth, survival, and molecular responses of the seeds. Seedlings were grown on plates rather than in soil to isolate and study the impact of multifactorial stress.
Seedlings grew on separate plates and experienced different individual or combined stresses. Results showed that each individual stressor applied to the seedling had a minimal effect on the plants but with the increase in the complexity and number of stress factors affecting the plants, survival, root growth, and chlorophyll content declined. Similar results were also found for seedlings grown in soil.
Ecosystems are already seeing these impacts in Florida and Germany. Multifactorial stress of heat and pollution prompted algae blooms to grow exponentially. This toxic overgrowth led to thousands of manatees dying. Entire forests in Germany are experiencing massive storms followed by long periods of drought, insect attacks, and fires.
Mittler said things may not look dire now, but we will eventually reach a point of no return where plants die off in mass quantities or even go extinct.
“The harmful effects of stress on the nation can serve as a dire warning for society. We may not see the effects now but 10, 20, 40 years down the road we will be having severe problems with our food chain,” Mittler said.
Since there are multiple factors at stake, predicting negative impacts on agriculture and ecosystems is tricky as researchers are unsure how this domino effect may unravel. What they do know for certain is that there will be severe consequences and we are already seeing them today.
These sporadic weather extremes are weakening the plants, making them more vulnerable to insect predators and other stressors.
The research yielded alarming results, and Mittler highlights that this is a dire problem that people need to take seriously. Once people understand the severity of this problem, he hopes individuals and policymakers will take action before the consequences become irreversible.
The Mittler lab is working on several fronts to address this problem and is trying to find a solution to it. They are currently studying multifactorial stress combination in different crop species, such as soybean, rice, and tomato. In addition, also identifying key plant regulators that are activated during multifactorial stress combination. These will be used in future breeding efforts to increase the tolerance of different crops to multiple stresses.
This study is now an international collaboration between the Mittler and Zandalinas laboratories, as Dr. Sara Zandalinas took on a faculty position in Spain.
Investigators at Bond LSC take steps to apply basic research
By Cara Penquite | Bond LSC
Scribbling in a lab notebook and planning experiments tucked between shelves of equipment, it’s easy to fixate on day-to-day lab operations. But scientists also face the challenge of finding how research can improve the world around us.
“The direction, the vision of the lab, ultimately comes from the principal investigator that bridges the research into applied directions,” said Jay Thelen, biochemistry professor and Bond LSC principal investigator
Despite the focus on basic research within the Bond LSC, many principal investigators choose to take their research to the next level with commercial partnerships.
Thelen’s lab researches ways to increase oil production in seeds and has three patents licensed to Yield 10 Bioscience, a sustainable crop innovation company who applies Thelen’s research to commercial crops. While seed oils like canola and soybean oil are known for their use in cooking, Thelen explains that increased production of these oils could play a larger role in sustainable fuel sources such as biodiesel and sustainable aviation fuel.
“We have to make more oil to balance out our need to eat it [and] our need to wean ourselves off of fossil fuels,” Thelen said. “To do that, we need to either plant more acres of oil seeds, or we have to raise the oil in existing oil seeds.”
Thelen researches enzymes with that potential application in mind. One is acetyl-CoA carboxylase, the enzyme which initializes the production of fatty acid chains found in plant oils.
“We’ve known this is an important enzyme, and we know that any tinkering you do with it has an impact on the oil production,” Thelen said. “In this case we’ve made new discoveries that permitted us to rationally engineer this enzyme to make it more active.”
Thelen suggests thinking of the enzyme as a “gatekeeper” to oil production which initializes the production of fatty acids and increases oil production. Thelen’s lab identified two different gene families that influence the activity of the enzyme in Arabidopsis and camelina plants. Yield 10 then applies these discoveries in other commercial plants.
While Thelen works closely with his commercial partner — having served on their scientific advisory board for three years and now stays in contact with Yield 10’s CEO to develop research projects — some labs stick with short-term arrangements.
Kamlendra Singh — assistant director of the Molecular Interactions Core at Bond LSC and Veterinary Pathobiology research assistant professor — studies HIV treatments. His lab identified a compound licensed by a commercial partner that targets the shell containing the virus’ genetic information.
Singh’s work in HIV started in 1994 with basic research investigating the enzyme that makes the viral DNA.
“I wasn’t into [studying] the drugs when I started working on HIV, I was mostly trying to understand how HIV enzymes works,” Singh said. “Once you know how the enzyme works, then you can target these enzymes for discovering the drugs.”
After years of studying how the enzyme works, Singh switched to HIV treatment. The first step to develop a treatment is to look for structures in the virus that the drug could potentially target to stop the viral replication. Singh targeted the shell around the virus’ genetic information known as the HIV capsid.
Building on previous research, Singh’s lab developed a compound able to bind the HIV capsid and prevent it from releasing the contained genetic information. Even with the licensing of his compound, Singh plans to continue researching ways to improve it.
“There are two reasons to keep working on it. One, well it’s my brainchild,” Singh said. “The second reason is as the company grows, we grow. We get more recognition and more funding. You can use it to [study] different viruses or use the same funding to improve upon it.”
While Singh plans to remain looking towards the applied side of his HIV research, he does not forget his roots in basic research.
“You have to put in time … [to] understand the system first, which is basic science, before you go to applied science,” Singh said.
Michael Roberts, a Chancellor’s Professor Emeritus of animal sciences and biochemistry who has had several patented projects, focuses on improving basic science projects and applies for patents if warranted.
“I don’t deliberately go into anything for commercial purposes,” Roberts said. “If I see something that I think does have commercial application, I’m happy to do it, but that is usually after you do [basic sciences].”
Whether starting a project with applications in mind or focusing on basic research, knowledge gained through research can be building blocks for the future.
“Science is simple. Even the most applied research project has its genesis in basic biology and basic research,” Thelen said.
One step into the Advanced Light Microscopy Core (ALMC) sounds an automated bell prompting Alexander Jurkevich, the core’s assistant director, to step out of his corner office into the open square room. With a friendly smile, Jurkevich coordinates biologists across MU’s campus to reveal the wonders of the microscopic world.
“Our mission is to provide researchers campus-wide with advanced microscopy instrumentation,” Jurkevich said. “We not only provide access to instrumentation, but we also train, advise users and support them during their early research at the core.”
The core hosts an annual image contest celebrates MU researchers’ microscopic imaging throughout the year. After being canceled for the past two years, this year contestants submitted their best images for consideration.
Christie Herd, a postdoctoral fellow in the Alexander Franz Veterinary Pathobiology lab won the Best of Show Award for her image of the La Crosse Virus in mosquito ovaries.
David Porciani, assistant research professor under the supervision of Bond LSC’s Donald Burke, won Director’s Award for Best Technically Challenging Image with his image showing epidermal growth factor receptors in two types of resolution.
Janlo Robil, a graduate student in the Bond LSC’s Paula McSteen lab won Experts’ Choice Award with his image of hormones in a corn leaf.
A wonderful surprise
Herd was “blown away” when she got her colorful image that won the Best of Show Award. Not only had she tried imaging other viruses with less success, she also worked on different La Crosse samples with no luck.
“That image in particular, I was not expecting to see because the day before I was there for three hours and had to quit,” Herd said. “So, when I saw that image, I was surprised because I did not think I would see that much detail.”
Herd’s image shows the La Crosse virus in developing eggs within mosquito ovaries. Herd uses La Crosse, a type of bunyavirus, as a model to study virus transmission from a female mosquito to her larvae to determine how viruses can remain transmitted within generations of mosquito populations. While La Crosse infects a small amount of humans a year, it transmits quickly, making it the perfect model to learn more about other bunyaviruses like Zika and Chikungunya.
“With bunyaviruses, they’re so multifaceted, and I like being able to research different aspects of them,” Herd said. “I just feel like they’re medically important.”
Herd’s image includes different colors to label different parts of the ovaries as well as the virus so she can see where in the ovaries the virus is traveling. Using a technique that allows her to see different focal planes, Herd can see where the virus is in every dimension, but it can be tricky to get high quality images.
“It does require a few hours of playing around, looking at the microscope,” Herd said with a chuckle. “It requires an investment of time, and sometimes there are days where it’s just not what you wanted and it doesn’t work … and you wasted days.”
Even with the challenges, the ability to see multiple dimensions of a sample at once is valuable for research projects like Herd’s.
“You just have to persevere and try again with a new set of samples,” Herd said.
From side project to passion
Although a deviation from his primary research, David Porciani’s project to image cell surface receptors slowly took over his focus.
“The biggest surprise was that I really had fun,” Porciani said. “This was not my primary project, but it became my primary project for a while.”
Porciani, an assistant research professor under the supervision of Donald Burke, studies molecules on the surface of cancer cells which are receptors for growth factors. These receptors act as a lock, with growth factors as a key. When the growth factors and receptors come together, the cells divide and create more cells. In lung cancer, there are more of these receptors, which can lead to uncontrolled tumor growth.
“This receptor, EGFR, has been widely studied,” Porciani said. “But for me there is definitely an interest because it’s one of the markers in lung cancer.”
Porciani tags those receptors with small molecules called fluorophores that glow under the light of the microscope, so he can see where the receptors are and how they move. However, the fluorophores cannot attach to the receptors alone, so he used aptamers — synthetic keys created by researchers that can bind receptor locks with specificity similar to the natural growth factors. Ultimately, the aptamers clip the fluorophores to the receptors.
“If you can follow the motion of the receptors, these receptors are kind of dancing,” Porciani said.
In his image, each dot is a different receptor made visible by the attached fluorophore.
However, fluorophores can become bleached, rendering them invisible after being exposed to the laser from the microscope for a while. If the synthetic keys, or aptamers, are still bound to those receptors, they cannot be imaged any longer. To address this, Porciani developed an aptamer which attaches to the receptor for a shorter time and then detaches so that even if the fluorophore bleaches, another aptamer can replace it and so receptors can be imaged for longer time.
“We engineered an aptamer with lower affinity that could work with this approach,” Porciani said. “By having lower affinity aptamers we can still determine localization of a high number of receptors and their motion.”
For his winning image, Porciani split the image to show the difference between single molecule resolution on the bottom right and a lower resolution image on the top left.
“With [lower] resolution, you don’t have a single molecule solution. If there are two molecules close together you will see them as just one single dot,” Porciani said. “But with the image, after software analysis with the image on the bottom right, then you have single molecule resolution.”
With this technique — made possible with microscopes at ALMC in Bond LSC — Porciani saw his efforts come together.
“At the beginning, I was just focused on the aptamer engineering from a high affinity aptamer to low affinity aptamer, and making them was the fun part to play with the structure,” Porciani said. “But when we started doing the imaging experiments at the Bond Life Sciences Center I realized that it was not just fun, but it was actually meaningful and this approach could have lots of biomedical applications.”
The artist’s touch
Although passionate about biology and microscopy, Janlo Robil decided to submit his image based on aesthetics.
“I chose this one because I am also a graphic artist, and I appreciate the color and composition,” Robil said.
Robil’s image shows a developing corn leaf with different colors labeling different plant hormone response proteins involved in stimulating growth. His unique image of an entire leaf, which is just under a millimeter in length, required piecing together images of sections of the leaf.
“About 0.75 millimeter, that’s still big in the microscope,” Robil said. “This is kind of difficult to make because it means that you need to tile several images [together] and sometimes it takes up to an hour just to [get] an image.”
The experiment requires planning ahead since the plants containing the fluorescent proteins must be crossed with plants with genetic mutations to determine the roles of the hormones in the leaf development.
To tag the plant proteins with fluorescent proteins requires planning ahead since the plants must be grown with genetic mutations.
“The beautiful image is actually a result of the expression of fluorescent proteins that are tagging the hormone response protein and also the hormone transport protein,” Robil said.
Initially from the Philippines where the agricultural staple is rice, Robil came to Mizzou interested in genetic mechanisms to make rice plants more productive. One way to enhance the rice plants is to make the rice leaves more similar to corn leaves, so Robil found interest in the McSteen lab’s project understanding the role of hormones in corn leaves.
“This project is perfect for me because I am studying the leaf and also integrating genetics,” Robil said. “And I love microscopy so much. I had quite a good amount of training starting in 2021 on confocal microscopy, and that’s why I was able to image this.”
Mengran Yang sat perched on a stool too tall for the cart of lush green tobacco plants in front of her. Behind towering shelves of lab equipment, she hunched over the plants and steadily pricked each leaf with a syringe.
Yang works with Arabidopsis and tobacco plants to learn about plant immune systems as a postdoctoral fellow in the Gary Stacey lab. Her research focuses on signals plant cells send to coordinate a fight against pathogens.
“I think it’s very interesting to see how plants can defend against the pathogens,” Yang said.
Just like humans have immune cells that fight germs, plants have a network of cells and signals to deal with harmful microbes. This system is particularly important for plants since they are rooted in place and cannot avoid the attackers in their environment.
The plant immune system protects it both inside and outside its cells. Proteins inside the cell can recognize molecules from invaders and trigger an immune response, and the second — the part Yang’s research focuses on — uses receptors on the surface of the cells to recognize molecular patterns from microbes to signal for the plant to get rid of the pathogen.
“Our lab is focused on the extracellular ATP signaling pathway,” Yang said. “ATP, as maybe everyone knows, is an energy source, but we found it can also be a signal when it is released to the extracellular matrix, where it is referred to as extracellular ATP.”
Yang’s work with the lab started when she moved from China to Columbia for her postdoctoral research at Mizzou. Although originally drawn to the lab because of Stacey’s prominence in the field of plant immunity and signaling, Yang stays because of the community within the lab.
“Our lab is very international — some people are from China, Korea, Brazil, Vietnam and India,” Yang said. “A lot of the same-aged girls will go out for fun.”
This community stems from Stacey’s emphasis on the team’s collective success.
“You try to develop an esprit de corps in the lab where people care about each other’s success,” Stacey said. “They’re taking care of themselves, but at the same time, they care about other people’s success.”
Becoming a good researcher started with strong academics, and Yang remembers being the top of her class in biology, physics and math in middle school and high school. When she started her studies at the university level she followed her growing interest in biology.
Growing up with her twin sister in China, Yang recalls societal pressures to fill any spare time with more study. Her parents thought her teachers gave too much homework and pushed back on this expectation.
“My father thinks this kind of education is not good, he thinks you need to promote your efficiency and not just work hard,” Yang said. “You need to study smart, not just hard, and I think my dad influenced us a lot.”
Yang applies this mentality to her research and is supported by Stacey who encourages her to plan experiments and life goals.
“While I was a Ph.D. student, I spent the most time in the lab for fun,” Yang said. “But now I’m good at scheduling stuff, so I schedule my experiments at least a week ahead.”
Stacey works with Yang to ensure her future impact as a researcher. When picking a project, Stacey guides his researchers towards projects with lots of potential.
“That’s the kind of discussion I have with people, what’s the best project for them where they have the chance to make an impact,” Stacey said.
With her lab mates and other research labs, Yang collaborates to strengthen each other’s weaknesses. If she does not have experience with an experiment, Yang asks other postdoc students with more experience for help with her project. When she publishes her research, she makes sure to give credit to the other students who helped her.
“In Chinese, ‘shuangying’ means ‘good to both sides,” Yang said. “That’s very common. You will read a paper, and you can also see most [papers] have a lot of authors there. That’s the cooperation.”
After finishing her research in the U.S., Yang plans to return to China to continue doing what she loves — research.
“I want to have my own lab in China,” Yang said. “It’s difficult, I know, but it’s my dream.”