Kwaku Tawiah, Ph.D. candidate, poses on the fifth floor of Bond LSC. | photo by Mariah Cox, Bond LSC.
By Lauren Hines | Bond LSC
Disease diagnosis takes money, time and technology — something rural communities don’t always possess. Kwaku Tawiah, a fifth-year graduate student, and researchers in the Burke lab are creating a probe that can diagnose and inhibit viral diseases cheaply, in less time and without electricity.
“With some of these infections, the faster you’re able to detect it, the better,” Tawiah said. “So, one of my motivations in grad school was to come up with some of these assays that don’t require all this time for experimentation.”
Some rural communities, like Tawiah’s hometown in Ghana, don’t have the electricity to operate diagnostic machines or the resources to afford them. The probe helps solve this problem.
The probe is a structure made up of a single strand of DNA that can fold into a unique 3D shape, which can recognize and bind to the surface protein of a virus.
“It’s a string of DNA, right?” Tawiah said. “What is unique about these probes is you can easily attach other things to the string of DNA.”
Tawiah and other researchers attach fluorescent molecules to the end of the DNA strand so when it binds to the surface protein, they can detect the virus.
Tawiah’s paper on the probe that can detect and inhibit Marburg, a cousin of the Ebola virus, will be submitted for peer review within a few weeks.
Even though Tawiah is graduating, other students in the lab want to expand the application of the probe.
Since virus surfaces have similar structures, the probe can be modified to detect and inhibit other viruses such as COVID-19.
“The beauty of what I do on the platform that we build is that it can be translated to other viruses,” Tawiah said. “So, with the platform for detection, you can essentially do the same thing for COVID, but you have to have the surrogate COVID virus particles…”
Currently, researchers in the lab are waiting for the particles and know the probe can bind to the virus, but they don’t know where exactly it can bind to.
“The lab is interested in using other techniques to find out where exactly the probe binds to on the surface of the virus,” Tawiah said.
While Tawiah is leaving to start his post doctorate in July to further develop low cost diagnostic methods, the Burke lab is continuing to probe for more answers.
Marc Johnson’s research focus changed suddenly one day this February when he received a shipment. That package of synthesized SARS-COV2 spike genes — the virus that causes COVID-19 — has now taken him down a new path.
“It was unusual, nothing like this has ever happened to me before,” Johnson said, an MU professor of molecular microbiology and immunology and Bond LSC investigator. “I’ve never had to switch directions so abruptly before but, you know, we’re always taking on new projects and shifting, it just usually doesn’t happen as fast.”
Typically, Johnson can be found in his lab studying viral glycoproteins, proteins with sugar attached to them involved in structural functions of the cell wall, and spikes, the knobby proteins on the surface of a cell. His main focus is studying HIV and its interaction with its host. However, his lab got to work right away to apply what they know to find a way to block SARS-COV2, and ultimately, COVID-19.
To do this, they focused on what they know best: glycoproteins and spike.
SARS-COV2 shares both of these features, and, in fact, Johnson has done previous research on other coronaviruses. Glycoproteins and spike allow the virus to attach to other cells. He infects cells with a safe, stripped virus containing the SARS-COV2 Spike and uses trial and error to see what works and what does not.
Early on making the glycoprotein functional was a challenge. Sometimes, when a protein of one virus is taken and stuck on another virus, it does not work. That was the case with SARS-COV2. He decided to cut of the tail of spike to see what happens.
“Most of its [spike] on the outside of the virus. There’s still a little piece on the inside, and if you make the virus smaller they don’t have that inside piece and often work better,” Johnson said, “I couldn’t get anything to work until I made that truncation.”
Without much of the tail, Johnson started looking for ways to block the virus. He has used many methods to find an effective blocking technique.
“We’ve thrown various peptides on and we’ve tested various small molecules,” Johnson said. “We’ve also tried plasma from patients who have recovered to see if they’re producing neutralizing antibodies and, no surprise, they are.”
Antibodies are crucial because they are a sign that someone has an effective defense against the virus. At the very least, antibodies allow the body to keep future infections in check. Taking antibodies from one patient and placing them in another patient passively transfers resistance to a virus and, often, immunity. Researchers are conducting studies to see if this is an effective way of blocking COVID-19.
Having only worked on COVID-19 for three months, there is still a lot Johnson—and the world—does not know.
“80% of people are just like, ‘Yeah, whatever, I’m fine,’ and then others just fall off the deep end. But, we don’t know what’s different. We don’t know why some patients do so poorly and others just shake it off,” he said. “It’s different than anything I’ve ever worked with before. I wake up every morning and it seems like there’s a new discovery every day.”
Working on COVID-19 is much different than working on other viruses, such as HIV. Many scientists are now putting manuscripts of their research online before they are published in hopes of aiding others.
While Johnson is aware that the sooner a vaccine is developed, the better, he knows not to rush things.
“It’s not about having a vaccine, it is about making sure that it’s safe and effective,” Johnson said. “We’re acutely aware that there’s this backlash against vaccines even when they are safe. If you put out one vaccine that wasn’t safe, you would ruin it for all vaccines for generations.”
For now, the search to stop COVID-19 continues and Johnson hopes his work helps come up with a treatment soon.
“We haven’t found a magic bullet yet, but we’ve seen some inhibition with various compounds,” Johnson said, “So it’s a starting point.”
The imaging system helps collect data on the growth of plants. | photo contributed by Samuel McInturf.
By Lauren Hines | Bond LSC
In a corner of the David Mendoza lab, a small machine runs back and forth across a track, taking pictures continuously of plants grown in clear square plates. This machine not only saves time during data collection but also helps the lab track the growth of plants in real time.
After almost five years of development, the Mendoza lab has reached the final stretch in their robotic endeavors. Now, they’re putting the finishing touches on the machine and getting a scientific article together to be submitted in a peer-reviewed journal.
“A few years back, we realized that our phenotyping methods were insufficient to capture subtle changes, things that aren’t just screamingly obvious, and although small, they’re very important,” said Samuel McInturf, a molecular biologist who is part of David Mendoza’s lab. “So, I set out and learned a whole bunch of stuff about robotics.
The project idea came into being in 2015 and transformed itself into a bioengineering senior capstone class where Mendoza, McInturf and many students from bioengineering, computer science and computer engineering worked on creating different prototypes of this idea.
“We are rapidly moving into a phase of a modular revolution, where our older methods, such as phenotyping by eye, aren’t really going to be sufficient,” McInturf said. “So, these machines represent a transition into mechanization or automation of our current methodologies.”
In addition to making the lab more efficient, the machine makes experiments more reliable.
“[The machine] really decreases the bias, decreases human intervention and increases the amount and quality of data that we are gathering,” said David Mendoza, associate professor in plant sciences and Bond LSC investigator.
Another benefit the robot offers to other labs is that it allows undergraduates to do more.
“[Undergrads] did a lot of repetitive tasks, but we are evolving as a society, so now you can teach them to do a lot of different assays and a lot of more complicated, sophisticated things using robotics,” said Ron Mittler, professor of plant sciences and Bond LSC investigator. “So, I look into this as a big advantage, actually.”
“The idea was to make the machines not just for us but for labs across the world,” said Landon Swartz, an undergraduate student researcher who is part of the project. “I’m hoping to see us be able to get machines out to other labs and just see how it affects their research and how much good can come from it.”
Soon to be one of their first users, Mittler needs the robot to help screen through the 1,000-3,000 plates his lab ends up with that need to be imaged, measured and processed to determine root growth and other parameters
“If you can optimize processes like that, then you can screen a lot of mutants,” Mittler said. “You can do a lot of different things that will take a lot of time and energy, and then you will not have to always spend power or time on data collection…That’s something that a lot of labs would have liked to have, including mine.”
Even though there’s a paper underway and labs are eager to get their own robot, the Mendoza lab isn’t yet finished with the project.
“Feels good to reach a benchmark,” McInturf said. “But there’s still so much more that we can expand upon.”
As of now, this particular machine is used to track the growth of roots and shoots of plants. However, the robot has potential to do even more.
“Now that we’ve developed all of this expertise, it’s relatively simple to add a heat sensitive camera, so we can see how active a plant’s metabolism is,” McInturf said. “Perhaps we could find a way to measure photosynthesis on the fly. Those types of expansions would be much more powerful.”
But for now, the Mendoza lab is busy writing manuscripts, doing proof of concept experiments and updating the software and user interface for the paper, which is scheduled to be submitted for review within the next few weeks.
As countries hang their hopes on the drug remdesivir for battling
COVID-19, recent modeling and computer-aided drug evaluation at the University
of Missouri caution to keep an open mind to other drug treatments.
Kamlendra
Singh at MU’s Bond Life Sciences Center assessed remdesivir and several other
drugs for long-term success in treating coronavirus causing the pandemic across
the world. His results were published April 26 in the journal Pathogens.
“Remdesivir
is working against COVID-19, but these other drugs are in no way inferior to
it,” said Singh, the assistant director of the MU Molecular Interactions Core,
Bond LSC investigator and MU associate research professor of Molecular
Microbiology and Immunology in the School of Medicine. “They are all FDA
approved and we believe, based on our research, that they are worth looking
into as a form of treatment.
Working
with colleagues at the Karolinska Institutet in Stockholm, Sweden, where he has
an appointment, Singh and his team started their COVID-19 specific research in
earnest on February 20. Through a combination of bioinformatics, molecular
modeling and computer-aided drug design, they evaluated antiviral medicines
remdesivir, favipiravir, 5-Fluorouracil and ribavirin for their effectiveness against
COVID-19, and possible resistance under pressure of these compounds.
While
Singh agrees that remdesivir works against COVID-19, he sees potential
weaknesses of the drug and believes alternatives could supplement its use.
The
team first assessed at the Gilead pharmaceutical remdesivir to inhibit
SARS-CoV-2 viral polymerase, an antiviral drug originally used to treat Ebola.
Remdesivir is one of the most highly touted drugs at this point in the fight
against COVID-19. But Singh’s findings indicate SARS-CoV-2, — the causative
agent of COVID-19 — is capable of mutating — that is, developing alterations in
its genome — that could help it develop resistance to the drug.
Armed
with this conclusion, Singh’s team looked at three additional compounds to
treat the virus. With T-705, also known as favipiravir, they concluded COVID-19
could also develop resistance, but its past effectiveness as a broad-spectrum
inhibitor of RNA viruses made it worth a look as a potential option to treat
COVID-19. Favipiravir, which has been approved in Japan as an anti-influenza
drug, was recently approved in Italy and China for treatment of COVID-19.
Favipiravir is currently undergoing testing regarding its viability against
COVID-19 and results are pending.
Additionally,
Singh’s lab conducted research on two other broad-spectrum RNA polymerase
inhibitors: 5-Fluorouracil and ribavirin. Singh examined 5-Fluorouracil because
RNA polymerase (the enzyme that copies the virus genome) from SARS-CoV-2 is
structurally similar to the RNA polymerases from human Rhinovirus and
Foot-and-Mouth disease viruses and 5-FU shows effectiveness against these
viruses. Due to this structural similarity, it stood to reason that
5-Fluorouracil may also have some effectiveness in treating COVID-19.
Ribavirin
— one of the most widely used, broad spectrum inhibitors of RNA-viruses — shows
unique properties that make it a prime candidate to bind to the active site of
COVID-19. By binding to the active site, ribavirin theoretically could stop the
replication of the viral genome and slow the spread of COVID-19 within an
individual’s body. Still, doubt remains regarding its effectiveness since the
virus can likely develop resistance to ribavirin through mutations or other
mechanisms.
Singh
has been working on coronaviruses — a broader category of viruses of which
COVID-19 is just one specific manifestation — since his arrival at MU in 2009,
so he is no stranger to this area of research. His broad goal is to ultimately
have his research discoveries “make a difference in the lives of patients.”
He
started his work on COVID-19 with the same goal in mind.
“I
did not start my research on COVID-19 in order to write this paper and have it
published,” he said. “I just wanted to do my part and spend some time
researching compounds that could potentially be effective against COVID-19.”
As
detailed as Singh’s findings are, he is quick to acknowledge that he is part of
a global scientific community and had a team of professionals within his lab,
especially Dr. Kyle Hill (a postdoctoral associate) that helped him to complete
this work.
“This
progress is not made by individuals, but by teams,” he said. “I have a talented
group of individuals working with me and collaborating with me. I am well aware
of their expertise and I know how to
best utilize their skills.”
Ujjwal
Neogi, who works at Karolinska Institutet, spearheaded the bioinformatics
portion of the research. Ujjwal also helped in conceptualizing the idea of
exploring already existing drugs to treat COVID-19. This work involved
analyzing genetic code of the viruses, binding of the drugs with the target,
and analyzing sequences of DNA, RNA, or protein to identify regions of
similarity to find the most unchanging, widespread target for using a drug. This
is important work since the smallest of differences in nucleotide sequence can
result in mutations that would render these proposed COVID-19 treatments
ineffective. Neogi was assisted with these tasks by Kyle Hill at the Bond LSC.
Anoop
Ambikan (at Karolinska Institute) used R, a statistical computing and graphics
program, to help put together the models and graphics behind this research.
Xiao Heng, a member of the faculty at MU, used her expertise in biochemistry to
help Singh conceptualize the research trajectory. Thomas Quinn, director of MU’s
Molecular Interactions Core in the Bond LSC, helped with editing and advising
throughout the process. Siddappa Byrareddy (University of Nebraska Medical
Center, Omaha, Nebraska) brought previous experience with the topic of treating
SARS, a form of coronavirus. Anders Sönnerborg, Singh’s longtime collaborator
at Karolinska, provided funding for this work and is the one who takes the
drugs to the clinic if they demonstrate potential. His involvement was important
from the start of the process. Stefan Sarafianos, a former Bond LSC
investigator and current collaborator had engaged with Singh in similar viral
research in the past, and he imparted a lot of knowledge to Singh that enabled
this research.
Taking
a deeper look at potential drugs in an analytical way like this contributes to the
University of Missouri System’s NextGen Precision Health Initiative. The
NextGen Initiative aims to improve large-scale interdisciplinary collaboration
in pursuit of life-changing precision health advancements and research.
While
Singh concedes that none of his team’s work constitutes a guaranteed effective
form of treatment against the virus, he feels that researchers have an
obligation to continue working on a cure, and should not accept the status quo
of hundreds of thousands of deaths globally. He also makes clear that for now
this is all dry lab work. But plans are already in place to test these drugs in
the wet lab for the validation once the appropriate protocols are
established. Funding from the Karolinska
Institute and the submission of two recent COVID related VA grant applications
with Drs. Deutscher and Whaley-Connell will support compound validation and
additional novel compound discovery and characterization.
“These
treatments, if they turn out to be effective, all have limitations,” he
admitted. “But, in the midst of a global pandemic, they are worth taking a
deeper look at, because we have reason to believe, based on our research, that
all of these drugs could potentially be effective in treating COVID-19.”
Kamlendra
Singh is Assistant Director of the MU Molecular Interactions Core and an MU
Associate Research Professor of Veterinary Pathology in the Collage of Veterinary Medicine (as of
May 1, 2020).
The main halls of Bond LSC are empty due to researchers being told to work from home. | photo by Roger Meissen, Bond LSC.
By Lauren Hines | Bond LSC
On an average day, you can find post doctorate Norman Best surrounded by corn in the greenhouse or at his bench in the McSteen lab doing molecular work. However, since Columbia and state leaders issued a stay-at-home order on March 25 to prevent the spread of COVID-19, this means Bond LSC is mostly empty and researchers like Best are at home writing.
“It’s definitely made me appreciate what I had before,” Best said.
Coronaviruses are a family of viruses that can cause respiratory illness in humans. They’re found circulating among animals, and then passed to humans. While the world has seen dangerous coronavirus outbreaks including severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome coronavirus (MERS) in 2012, the 2019 emergence of COVID-19 has spread much more quickly.
According to the Centers for Disease Control and Prevention, COVID-19 is spread through respiratory droplets produced when an infected person coughs, sneezes or talks. Staying at home and avoiding contact with others will help prevent the spread.
Most researchers are working from a distance, like Best who is writing a paper on how the plant hormones auxin and brassinosteroid affect lateral meristem growth. However, some are deemed essential whether it’s to water plants, finish crucial experiments or study COVID-19 itself.
Marc Johnson, professor of molecular microbiology and immunology, is currently studying glycoproteins which are proteins on the surface of viruses that dictate what cell they’re going to infect. Even though Johnson usually works on HIV, he’s shifting his focus to COVID-19 and its glycoproteins called, spike.
Johnson is taking other viruses and replacing their glycoproteins with COVID-19 spike proteins to basically create a safe version of our current coronavirus. This will allow him to do multiple tests to try to inhibit viral entry.
In addition, these experiments can also test the effectiveness of antibodies. Recovered patients are donating their plasma — blood without red blood cells and just antibodies — to transfer their coronavirus-fighting antibodies to other patients.
Marc Johnson observes cells modified with CRISPR under the microscope. | photo by Jennifer Lu, Bond LSC.
“Of course, if you take plasma from a patient, you want to make sure that there are the antibodies you want in there, so that’s where my [experiment] would come into play to check whether there’s a high level of neutralizing antibodies in their serum,” Johnson said. “If there is, then you know it’s good for injecting. It might be helpful for the patient.”
For those who aren’t working on COVID-19, the interruption has some feeling frustrated.
When Best was in the lab, he was doing molecular work on creating a CRISPR construct. CRISPR is a method of editing genes, essentially splicing DNA into a cell. Now, it’s sitting in the freezer half done.
“I’m working on finishing up a few publications that I’ve had data for that I’ve actually
not been able to analyze before,” Best said. “I had not taken the time to analyze as much as I have now because I am sitting all day on the computer…However, there are still a few things left to do in the lab that have been delayed because of quarantining.”
Jean Camden, senior research associate in the Weisman lab, goes into Bond LSC once a week to check on the mouse breeding colony in addition to working from home.
“For us, the timing was good,” Camden said. “We had just finished some large experiments, and we are now writing, all of us. We have plenty of work to do at home.”
For many researchers, as Camden describes, writing papers is one of the last tasks to do.
“Now there’s no excuse,” Camden said. There’s nothing else to do but write.”
However, even the mounds of data researchers have been sitting on will eventually run out.
“If [the end of social distancing] doesn’t happen within the next two or three weeks, then we will be getting behind on getting experiments done,” Camden said.
So far, MU has moved in-person summer classes online, but is hopeful to re-open campus in the fall under a “new normal.”
“[The end of social distancing] should depend on our preparedness and the resources because one of the reasons why I think we shut down is that we weren’t ready to cater for all the people that were going to be sick, and so the best option was to prevent it,” said Kwaku Tawiah, fifth year graduate student who studies Ebola in the Donald Burke lab. “I don’t know when normal life can return as we knew it before.”
Even though there’s a lot of uncertainty of what’s ahead, Bond LSC researchers are learning to adapt and are continuing their research.
“It is very fortunate that I have been able to work at home and keep my job,” Camden said. “A lot of people here in Columbia have been laid off, and I feel bad for the terrible things that have happened. So, I appreciate the position that I’m in.”
At the moment, Best is still sitting at home with his dog analyzing data and writing. He expects his paper on lateral suppressor1 to be published soon among many others in the works.
“I think we’re all ready to get back to normal life,” Camden said.
With shelter in place orders being extended throughout the country and events being canceled, COVID-19 is a pressing issue, and influenza researchers at MU have been pivoting recently to begin studying the virus.
Henry Wan, an influenza researcher and Bond LSC principal investigator, is planning on expanding his work to start looking at COVID-19 along with a team of epidemiologists, anthropologists, engineers, and more at MU. While influenza and COVID-19 are not the same virus, both are infectious respiratory illnesses transmitted through similar ways.
COVID-19
Wan has spent years investigating the workings of influenza, so as the COVID-19 pandemic has played out he has been keeping an eye on its developments. He explained COVID-19 is “being incorporated into my study, in that human cohort, to study and monitor the disease that is going on here. We have established the diagnostic capacity in the lab for COVID-19 but I haven’t tested any samples yet,” he said as of March 18.
A few weeks ago, there was an online meeting of virus researchers at MU to discuss research efforts to combat COVID-19 and what their labs bring to the table. One idea they came up with is to have a cohort in Columbia, that would provide materials such as blood samples over time to track not only the spread of influenza and COVID-19, but vaccine and medication effectiveness. Jane McElroy, an epidemiologist in the MU school of medicine said, “Right now there’s the flu vaccine, so we want to get serum samples four times a year from community members, around vaccine times, and then a couple of times throughout the year to just see what their antibodies look like.”
“Once we have a COVID-19 vaccine, the participants in a cohort could help us by letting us see how their body’s immune functions are maintained with the vaccination,” she said.
This way, MU researchers would not only be getting information on influenza in Columbia but also COVID-19.
Lisa Sattenspiel, professor and chair of anthropology at MU, is also starting a project that is based on her research on the 1918 influenza epidemic. She is comparing the patterns from 1918 to what is going on with the current pandemic and looking at social, economic, and behavioral data to track the spread. “We’re starting this project now,” she said and a National Science Foundation grant proposal has recently been submitted for funding this idea.
Though a very serious and quick-moving situation, Wan and his collaborators are coming up with ideas to understand COVID-19. Not much is known yet about the virus, but they are doing their best to change that.
MU Collaborators
Wan cannot complete this research on his own. Along with students in his lab, he has already collaborated with more than 15 people throughout MU.
“I really enjoy collaboration, everyone is very passionate,” he said, “I think it is very important.”
Everyone brings a unique perspective and expertise to the team which leads to more breakthroughs.
McElroy has connections in the medical field that help Wan obtain samples from people who have influenza symptoms and, in the future, potentially people with COVID-19 symptoms as well. Working with her colleague, Dr. Shamita Misra, medical director of Mizzou Quick Care Clinics located in Columbia Hy-Vee stores, health care providers gather these samples from community members. Extending this work, McElroy has begun working with Dr. Christopher Sampson, a University of Missouri Health System emergency room physician, to identify and gather blood samples from patients with COVID-19.
By using samples from the community, Wan is able to track trends throughout Columbia and use that information to find ways to make the community healthier.
Another person Wan has collaborated with is Sattenspiel, who has been conducting influenza research for more than 25 years.
Sattenspiel tracks how influenza spreads throughout communities with computerized models of towns that simulate potential outbreaks. Having this knowledge gives Wan and his team a better idea of the rate influenza spreads and how to stop it.
Sattenspiel explained, “We are working on designing models that can deal with the entire life cycle of a virus and what goes on at the community level, how it gets transmitted within communities, how it gets transmitted within households, and what happens inside the human body. Ultimately, the goal is to look at different strategies for controlling it.”
With this model, she can test preventative strategies and see their effects.
“So things like vaccinations, if the vaccination works in a certain way, what’s its impact going to be, not only for the individual who was vaccinated but for the community in which they live?” Sattenspiel said.
Another preventative strategy they are looking at is social distancing, which has become relevant and especially effective recently with the COVID-19 pandemic. Through these collaborations, Wan is able to get a well-rounded view of influenza in order to create effective vaccines. He also hopes the addition of the new Next Gen Precision Medicine Institute will expand his research even further.
Wan’s Influenza Research
Arriving at Bond LSC last summer, Wan hit the ground running. He already has several grants and collaborators working on projects to improve the flu vaccine and figure out how influenza spreads in addition to his other work.
“We mainly focus on two aspects,” Wan said, “On one side I’ve been mainly focused on the influenza vaccine and the other aspect on influenza risk assessment.”
As a result, Wan’s lab looks at many factors such as, why the flu vaccine does not work, how to get a better flu vaccine, and which flu virus is more dangerous to humans, among others.
To test this, Wan gathers swab samples from people who have influenza much like how COVID-19 samples are collected.
“From the swab, we look at the virus whether it matches any flu vaccines,” he said. Wan and his graduate students and other senior lab members then look at this data and interpret it to come up with a solution.
Wan recently received a $3.65 million research project grant from the National Institute of Health (NIH) to determine if the number of vaccines a person has affects the number of times they will be infected with influenza. He said getting a flu vaccine results in “A lot of heterogeneity in our immune profile, affecting the vaccine.”
Wan and his team are curious if these vaccines build off each other and what the yearly effect of getting a flu shot is on the body. By understanding this, he will be able to come up with a way to improve the vaccination program, making it stronger.
In addition to this grant, Wan’s two other grants focus on predicting the influenza virus. Using artificial intelligence (AI), machine learning technology, and sequencing techniques, he looks at how the virus mutates and escapes the immunity in human populations. With this information, scientists are informed on how to make an effective vaccine, especially with new strands of influenza.
The Future
Just because COVID-19 is currently the world’s main focus, does not mean it’s the only virus in town. People still get sick from influenza annually, so Wan and his collaborators plan to continue their research on the spread of influenza and improving vaccines.
“The resources are really focused on coronaviruses because of what’s happening right now, but influenza is not going to go away either,” said McElroy, “Lots of people also die from influenza every year so we know that it is also important.”
Mary Butler, an undergraduate from Truman State University, gains experience working on experiments in the lab of Bond LSC’s Cheryl Rosenfeld. | photo by Roger Meissen, Bond LSC
By Mariah Cox
How did an undergraduate student from Truman State University spend last summer working on a research project with a Bond Life Sciences Center primary investigator and become on track to be published as first author several months thereafter?
A nationwide National Science Foundation (NSF) sponsored program has allowed Mary Butler to jump-start her research career early on.
Butler, a sophomore biochemistry and molecular biology student, wanted to get a head start on research to give her a leg up and figure out what her future might look like. As a freshman, Butler joined the Missouri Louis Stokes Alliance for Minority Participation (MOLSAMP) program and sought out opportunities that aligned with her science interests.
MOLSAMP is a collaborative effort among seven public universities, a private university and a community college. The alliance aims to increase the number of underrepresented minority students throughout the state of Missouri who pursue undergraduate degrees in science, technology, engineering or mathematics (STEM). The Missouri chapter is part of a greater program with the Louis Stokes Alliances for Minority Participation sponsored by the NSF, and Butler qualified because of her Mexican American background.
This past summer, Butler started traveling the 90 miles from Kirksville to Columbia to work with Cheryl Rosenfeld, a primary investigator at Bond LSC. Her research primarily focused on how genistein, a soy-derived phytoestrogen, and BPA, an industrial chemical, affects the behavior of mice.
“BPA and genistein are endocrine-disrupting chemicals, so they can mess with the hormones of an animal and potentially humans. I specifically looked at the different behaviors of California mice under four different diets,” Butler said.
The researchers administered a control, a genistein diet, a low dose BPA and a higher dose BPA diet to pregnant mice. After the baby mice were born, the researchers weened them from the mother to observe if the parents’ diet affected what the pups ate. Additionally, at 30 days, 90 days and 180 days, they put the pups through various social tests to see if their diet and their parents’ diet affected the brain and behavioral patterns.
They used socio-communication testing to determine whether developmental exposure to these chemicals led to deficits in these behaviors, which are reminiscent of those seen in children with autism spectrum disorder (ASD). The results point to less socialization among the mice exposed during the pre- and postnatal period to genistein or a low dosage of BPA.
Afterward, Butler measured gene expression changes in the hippocampus, which regulates learning and memory, and the hypothalamus, which guides socio-sexual behaviors. Besides examining for mRNA that encodes for proteins that can act within or outside of a cell, Butler and Rosenfeld also are the first group to examine whether developmental exposure to BPA and genistein affects the expression of so-called junk RNA within the brain.
Junk RNA strands are very short and do not give rise to proteins. For these reasons, they were historically dismissed as not important in cellular biology. However, it is increasingly apparent these RNA strands, also now called microRNA or miR for short, play vital roles in diverse cells throughout the body.
One way they act is to bind mRNA that would otherwise encode for proteins. In so doing, they help contribute to the demise of these mRNA strands. Essentially, the DNA can be transcribed to mRNA but miR binding prevents them from making a protein, acting as an epigenetic modifier that doesn’t alter the DNA or mRNA. In this sense, miR may serve as a system to help regulate mRNA.
While other research groups have examined how BPA affects miR patterns in the placenta and testes, no previous research group has done so in the brain, even though this organ is vulnerable to early exposure to BPA and genistein.
When Butler started in the Rosenfeld lab, she expressed a desire for challenging and novel work. With assistance from Jiude Mao in the Rosenfeld lab, they set out to test whether developmental exposure to BPA and genistein could alter miR decrease in ASD patients.
Notably, Butler’s work suggests that indeed such chemicals caused down-regulation of the same miR that are reduced in ASD patients. Normal expression of such miR appears to help prevent cell death, oxidative-stress damage, and yield other beneficial effects, and thus, reductions in these miR may leave California mice and, presumably, autism patients more vulnerable to such deleterious effects exposed to such chemicals.
Butler’s studies also suggest that changes in mRNA and miR profiles are associated with socialization deficiencies observed in California mice. Further testing is needed to confirm whether these molecular changes contribute to these behavioral disruptions, but, if so, it may pave way for new prevention and treatment strategies in those at risk of neurobehavioral disorders.
Since high school, Butler knew she wanted to pursue science. But she doesn’t quite know where to go from here with all the different options available; the opportunity at Bond LSC allowed her to look into one of those.
“I want to explore a lot of different types of research. My research with Dr. Rosenfeld is very different than the research I’ve been doing at Truman,” said Butler. “I had been looking at Dr. Rosenfeld’s research for a while and it looked really cool, so I sent her an email and asked if I could work in her lab over the summer. She said ‘yeah’ quickly, and it was really exciting.”
As a sophomore, Butler can say she’s the first author on a paper, and she knows how to do protein expression purification and polymerase chain reaction among other techniques.
“I really enjoy it because I’m more of a hands-on learner. Getting to do it hands-on, I’m able to absorb a lot more information,” Butler said. “In my classes, I’m able to follow along and talk about research things because I already know a lot of the techniques we discuss and practice in the lab.”
Butler’s research was published in the “Journal of Neuroendocrinology” in March 2020 and was funded by the National Institute of Environmental Health Sciences.
Figure B is a colorized radiographic image that shows the path of boron in a five-day-old maize seedling. | photo contributed by Alexandra Housh, Michaela Matthes, Amber Gerheart, Stacy Wilder, Kun-Eek Kil, Michael Schueller, James Guthrie, Paula McSteen, and Richard Ferrieri.
By Lauren Hines | Bond LSC
The element Boron, while extremely low in levels, leaves a trail of green and blue radioactive decay as it travels through the veins of plants.
Due to radiotracer technology, this picture of the element’s movement provides this unique insight to what’s going on inside the leaves, stems and roots of plants for the first time ever.
A new collaboration between Bond LSC’s McSteen lab and the MU Research Reactor’s Ferrieri lab led the two labs to present [18 F]-4-FluoropPhenylboronic Acid (FPBA), a radiotracer they designed that can track and visually report on the movement of boron in plants, in a February journal article in the International Journal of Molecular Sciences. The visual tracking was done inside corn.
“It’s the first time anyone has been able to see boron in plants, which is very exciting,” said Paula McSteen, associate professor and researcher at Bond LSC.
The Fifth Element
Boron is an essential micronutrient for plants, but unfortunately, is deficient in soils worldwide. That leads to defects in the roots and shoots of plants, therefore leading to a reduction in crop yields.
“Up to now, there was no way of imaging boron in the plant, which makes it hard because if you could see it somewhere else, like in the cell wall, you would know it has a role there, right?” said Michaela Matthes, postdoctoral scholar in the McSteen lab. “But no one can see it.”
Now, with the equivalent of a PET Scan (Positron Emission Topography) some might receive in a hospital, researchers are able to image and track the movement of boron in live plants to learn where in the plant it’s important and, eventually, how to manipulate it to result in higher crop yields.
You might not think boron would be a controversial topic in the life sciences community, but a longstanding dispute about its importance has been debated for years. The argument is not whether or not the micronutrient is critical to plant development and growth, but where and how it functions. Since there’s such low levels of boron, it’s undetectable. Imaging boron and being able to pick it out will allow a pathway to more direct evidence of its role.
The research study found that the tracer accumulated in the root tip, root elongation zone, lateral root initiation sites and leaf edges in maize.
“So, the accumulation of the tracer basically means there was a signal of the tracer at the root tip, and at the leaf edges, which means boron is there,” Matthes said.
This presence is exactly the kind of direct, visual evidence that supports the argument of the role boron plays in maize growth.
“The other methodologies we looked at would have measured the natural level of boron in the plant, and because that is too low, it is below detection,” Matthes said. “That is why the radiotracer is superior because we are actually adding something to the plant that we then image.”
In the McSteen lab, Matthes fills falcon tubes that contain seeds with water, so they can germinate with exposed roots. | photo by Lauren Hines, Bond LSC
Tracking Boron
“When we came here, we were keenly aware of Dr. McSteen’s interest in boron and the challenges of imaging boron, so we started a discussion between our groups about what we could bring to the table with our radiotracer technology,” said Richard Ferrieri, research professor at the MU Research Reactor who came to MU from Brookhaven National Laboratory.
A radiotracer is a chemical compound where a radioactive element is added to whatever a researcher wants to track, which acts as a tag as it moves through the body or plant. Scientists take pictures of its radioactive decay, showing where the element travels and ends up in higher levels.
In this case radioactive fluorine was added to phenylboronic acid (PBA), a compound the plant can easily absorb, which turns PBA into FPBA. FPBA moves exactly like boron in the plant, so it provides an accurate representation of where boron goes and possibly its role.
The Ferrieri lab grew maize hydroponically, in a solution of plant nutrients, under normal lighting conditions. Once they grew to a certain size, they were moved into glass beakers where the roots were submerged in water. A formulation of the FPBA tracer was then added so that the roots could natural absorb the radioactivity.
Using another imaging technique similar to getting an X-ray called, autoradiography, the Ferrieri lab was able to obtain snapshots of the radioactivity inside the plant to see where it accumulated.
“The imaging that we do is giving us information on where the radioactive tracer mimicking boron goes. Having that visual feedback gives us insight about boron’s role in plant growth and development,” Ferrieri said. “I would say in the world of plant biology, this is the first example of chemists sitting down and designing a custom molecular probe to answer some hard, biological questions in regard to boron uptake in plants,” Ferrieri said.
McSteen and Matthes work together on discovering the roles of Boron in Maize. | photo by Lauren Hines, Bond LSC
What There’s Left to Discover
“Very little is known so far about boron,” Matthes said. “The very basic research question has not been fully answered, and I think it’s fascinating to contribute to that knowledge.”
Researchers were limited in how they could investigate boron’s role without being able to see where boron was in the plant. Matthes said the biggest gap in knowledge in the field of studying boron is whether or not there is an additional role of boron in the plant cell beyond the cell wall.
While there is so little known about boron, researchers aren’t helping themselves due to the lack of method standardization, according to a review written by Matthes, McSteen and Janlo Robil, a Ph.D. candidate in the McSteen lab. When procedures or measurements aren’t consistent across different studies, it makes it hard for other researchers to duplicate experiments and build on existing knowledge.
They also went into evaluating different methodologies investigating boron and how each of them is limited.
“What I think is the best [methodology] is probably a combination of different approaches depending on the question you are asking,” Matthes said.
However, the radiotracer approach can directly locate boron without destroying the plant.
Even though much has been accomplished already, Matthes will keep working towards filling in those gaps.
Nathan Bivens and Wes Warren. | Photos by Mariah Cox & Erica Overfelt, Bond LSC
By Jerry Duggan | Bond LSC
Behind any breakthrough in science lies a research process full of precise methods and instrumentation essential to moving from hypothesis to discovery.
Some of those genetic breakthroughs just became more possible on UM System campuses, thanks to a new, more efficient genome sequencing instrument at MU’s Genomics Technology Core.
The NovaSeq instrument was first put to use in December, purchased with funds from an UM System tier 1 grant meant to benefit all of campus. MU Genomics Technology Core Director Nathan Bivens said this new instrument has significantly increased efficiency of sequencing operations.
“For most projects, we’re able to bring the cost down about 30% compared to our previous instruments,” he said. “This allows us to work more efficiently and increases productivity.”
The sequences behind the problem
According to Wes Warren, primary investigator at Bond LSC, the use of sequencing technology is a crucial need of many scientific investigations. It allows researchers to determine the specific nucleic acid sequence — that is, the order of nucleotide bases-adenine, thymine, cytosine and guanine — in any living thing. The understanding of this sequence variation has already and can in the future lead to real breakthroughs from a human health application standpoint.
“Any novel base change can often be associated with the presence, or absence of a particular trait,” he said.
For example, in many common diseases, such as cardiovascular, variation in the DNA sequence within the individual that has the disease provides clues to the molecular networks associated with its occurrence. This is especially true in polygenic traits — one whose clinical features is influenced by more than one gene, such as cardiovascular disease. According to Warren, sequencing this genetic variation is crucial for understanding these types of traits.
“In order to make sense of many traits and human health conditions, we need to be able to sequence hundreds of thousands of individuals so that we can see and compare that variation,” he said. “Once we have those numbers, we can start to catalog all those sequence changes and assign them some predictive value in the population where we are trying to understand the disease. The importance is in attributing sequence variation toward disease association.”
Investing in the future
Tier 1 grants were rolled out last year as a significant commitment to research, and usually entailed projects or equipment costing in excess of $1 million.
“This instrument is a significant investment on our part, but we expect to get a lot out of that investment over the next several years,” Bivens said. “This instrument is now available for the whole MU system (including the campuses in Columbia, Kansas City, St. Louis and Rolla), so all in the UM system have access to this new infrastructure and will be able to use it.”
Warren is one of those who will benefit from NovaSeq. Warren helped create the proposal that won the grant, and said the instrument itself is but a vehicle to fuel scientific advances in the future.
“Getting this instrument here in a physical sense was big, but an even bigger deal is what we hope it will enable us to do from a research capability standpoint,” he said.
The path to precision medicine
Although sequencing instruments aren’t new to the Genomics Technology Core, our existing instruments were not state of the art for high-throughput genetic studies. High-throughput is the technical term for being able to sequence a large number of samples in a shortened time period. The increased cost efficiency and data precision (higher throughput) comes in handy with the study of cancer cells, because depth of sequencing and larger sample sizes are crucial for discovering mutations. Take, for example, circulating tumor cells in a given individual’s body. The researcher’s goal is to find the cancer cells, of which there are very few, circulating amongst a vast sea of normal cells in the individual’s bloodstream. Use of the NovaSeq instrument will be a key enabler for special strategies to amplify and capture the individual DNA sequences of those hard-to-find circulating cancerous cells.
The potential benefits of sequencing for making human health breakthroughs are boundless, but according to Warren, just as important is what widespread sequencing could mean on an everyday level for patients everywhere.
“A long-term goal is to get to a system where when a patient comes to the doctor there is a blood sample captured and then the genome of the individual is sequenced shortly thereafter” he said. “That individual’s genome would then enter into the electronic health records, and then in the future once we have a population-wide understanding of how sequence variation affects many aspects of human health, physicians will then be able to have access to and utilize all that information.”
While more efficient sequencing has broad, far-reaching benefits, the NovaSeq has already aided specific projects and discoveries on the MU Campus.
One example is Dr. Gary Johnson’s work at the School of Veterinary Medicine, which involves taking whole genome sequences of dogs to look for rare mutations, or changes, in their DNA that can cause a wide range of diseases. Thanks to this new instrumentation, Johnson is able to sequence cheaper and much more quickly. This allows him to get down to the nitty gritty of analyzing DNA sequences for mutations, and in turn, helps identify ways to prevent such diseases that much quicker.
As practical as the new NovaSeq may be, it also represents MU’s symbolic commitment to cutting edge research and scientific discovery.
“This is central to the mission of the DNA core,” said Bivens. “It makes us up-to-date with the most cutting-edge sequencing instruments out there, from a machinery standpoint. More importantly, though, it allows us to offer that technology at a fair cost, which will allow our researchers to be very competitive in terms of writing grants. This instrument is going to set the stage for the next 5 years of research here on this campus very easily, and its benefits are immeasurable to the MU research community.”
Dong Xu, Bond LSC principal investigator and Shumaker Endowed Professor in the University of Missouri’s College of Engineering. | photo by Roger Meissen, Bond LSC
A Bond Life Sciences Center researcher has been inducted into an elite organization comprised of two percent of all medical and biological engineers.
The American Institution for Medical and Biological Engineering (AIMBE) this week announced the induction of Dong Xu, a Bond LSC principal investigator and Shumaker Endowed Professor in the University of Missouri’s College of Engineering.
“Election to the AIMBE College of Fellows is among the highest professional distinctions accorded to a medical and biological engineer,” said Kamrul Islam, chair of the college’s Electrical Engineering and Computer Science department.
Xu was selected for his “distinguished contributions to bioinformatics and computational biology, and extensive services to University of Missouri and his research community.”
In addition to his endowed faculty position, Xu serves as director of the Information Technology program, whose core facility is housed in Bond LSC.
Membership to AIMBE’s College of Fellows recognizes those who have made outstanding contributions to engineering and medicine research, practice or education, and to those pioneering new and developing fields.
Because of health concerns, AIMBE’s annual meeting and induction ceremony scheduled for this spring was canceled. Under special procedures, the induction was held remotely.
