Bond LSC is now producing monthly segments for KBIA, Columbia’s NPR station at 91.3 FM.
This month highlights the work of Melissa Mitchum, a molecular plant nematologist at Bond LSC and an associate professor of Plant Sciences in the College of Agriculture, Food and Natural Resources.
She studies nematodes, a pest that cost soybean farmers billions of dollars each year. Her lab recently helped discover that this tiny parasite produces molecules that mimic plant hormones in order to siphon nutrients from soybean roots.
Tune in at 12:30 to hear her profile or visit the Soundcloud link above to hear the segment.
You can imagine it’s hard to distinguish yourself from the crowd when it comes to scientific papers.
But, publishing quality work in a well-known journal adds value to the whole scientific world by assisting others and inspiring new science. Three Bond LSC researchers recently were recognized for doing just that.
Bond Life Sciences Center scientists Chris Pires, Shuqun Zhang and Yidong Liu are among five University of Missouri System researchers named in the 2015 Thomson Reuters’ Highly Cited Researchers list.
This list spotlights the top 1 percent of papers published from nearly 9 million scientists and scholars. The Highly Cited Researchers 2015 list represents the world’s most influential scientific minds from 21 scientific fields. The rankings are based on how often scientific papers published in the last decade get cited in newly published research, according to Essential Science Indicators (ESI), a component of the Web of Science.
Chris Pires, associate professor of Biological Sciences, studies the evolution of plants by looking at changes in their genetics over millions of years. Pires published work in 2015 looking at how plant defenses evolved in tandem with the defenses of caterpillars that feed on them.
Shuqun Zhang is a Distinguished Researcher from the MU College of Agriculture, Food and Natural Resources and a professor of Biochemistry. His research seeks to improve plants’ response to adverse environmental conditions. By identifying molecular targets that aid in protecting crops from disease, his research aims to help create healthier, more productive agricultural products. In particular, he focuses on a family of enzymes called mitogen-activated protein kinases (MAPKs) that are involved in plant cell-to-cell communication and plant interaction with its environment.
Yidong Liu is a senior research specialist from MU’s Department of Biochemistry that manages Shuqun Zhang’s lab. She also works on MAPKs and their role in plant defense responses such as pathogen-induced ethylene biosynthesis and phytoalexin induction.
The Bond Life Sciences Center is an interdisciplinary research center at the University of Missouri exploring problems in human and animal health, the environment and agriculture since 2004. Learn more about our research by visiting bondlsc.missouri.edu.
Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies viruses such as HIV. | photo by Jennifer Lu, Bond LSC
Nineteen colorful foam flowers decorate the walls of Marc Johnson’s office, a memento from his lab members when they “redecorated” while he was out of town.
Each flower is labeled in bold Sharpie with the names of viruses and viral proteins that his lab studies—MLV, RSV, Gag, Pol, to name a few.
One flower stands out, marked in capital letters: H-I-V.
Johnson, an associate professor of molecular microbiology and immunology, is one of four researchers at Bond LSC who studies HIV, the virus that leads to AIDS. His research focuses on understanding how HIV assembles copies of itself with help from the cells it infects.
Like most viruses, HIV hijacks cellular functions for its own purposes.
“It has this tiny itty bitty little genome and yet it can infect 30 million people,” Johnson said. “It doesn’t do it by itself.”
To understand how viruses reprogram the proteins in our bodies to work against us, he said, you have to understand the cells they infect. If cells were a chamber, then viruses are the keyhole.
For example, cells use a protein called TSG101 to dispose of unwanted surface macromolecules by bending a patch of cellular membrane around the macromolecule until it is surrounded inside a membrane bubble. The process, like trapping a bug inside a sheet of tissue paper, is called budding.
The cell sweeps all the pinched-off bubbles into a larger receptacle, or multivesicular body. These bodies, Johnson said, act as the cell’s garbage collection system. To dispose of the trash, the compartments become acidic enough to disintegrate everything inside or fuse with the cell membrane so that the trash gets dumped outside the cell.
It’s like in the second Star Wars movie, “The Empire Strikes Back,” Johnson said. “They just drop all their garbage before they go into hyperspace, and that’s how the Millennium Falcon got out.”
HIV uses the same housekeeping mechanism to break out of infected cells and infect more cells, but it remains unclear which other host proteins HIV commandeers.
“It’s all part of the puzzle,” Johnson said.
THE GAME CHANGER
On his desk, Johnson keeps a white legal pad with a list of 16 projects written in blue ink.
Marc Johnson observes cells modified with CRISPR under the microscope. | photo by Jennifer Lu, Bond LSC
“Things make it off the list or they’ll get added,” Johnson said. “Or they’ll spend years on the back burner. I have a lot of projects.”
One of the biggest projects involves using CRISPR/Cas9 — a precision gene-editing tool — to identify genes that make a cell resistant to viral infections.
“It’s a game changer. It really is,” Johnson said. “It’s so cool.”
The technology uses a missile-like strand of guide RNA to target specific sites in the genome for deletion. Before CRISPR, scientists had to suppress gene expression using methods that were neither permanent nor absolute.
But because CRISPR manipulates the genome itself, Johnson said, there’s less doubt about what is happening.
Using the CRISPR library, the Johnson lab can scan the effects of 20,000 unique gene deletions in a population of cells. When these cells, each of which contains a different deleted gene, are exposed to HIV, not all of them die. Those that survive can cue researchers in to which genes might be important for blocking HIV infection.
And if another researcher has doubts that a gene is truly knocked out, Johnson said, you can tell them, “I’ll just send you the cell line. You try it and see for yourself.”
A DAY IN THE LIFE
The Johnson lab is a tight-knit group that consists of a lab manager, two grad students, a postdoc and four undergrads.
Dan Cyburt — a third year grad student — studies molecules that interact with proteins that keep HIV from infecting the cell, such as TRIM5α. TRIM5α, a restriction factor, blocks replication of the viral genome.
Graduate student Yuleum Song prepares cells for viral infection in the BL-2 hood. | Image by Jennifer Lu, Bond LSC
Fourth year grad student Yuleum Song focuses on how the viral envelope protein, Env, is packaged into viruses before they break free from cells. While Env isn’t necessary for viral assembly and release, she said, it’s critical for the infection of new cells.
Undergrads work in a tag team, picking up where the other left off, to generate a collection of new viral clones.
And lab manager Terri Lyddon keeps day-to-day experiments on task.
Lyddon, who has been with the Johnson lab for ten years, spends much of her day working with cells inside the biosafety level 2 hood. The area is specifically designated for work with moderately hazardous biological agents such as the measles virus, Samonella bacteria, and a less potent version of HIV.
Normally, HIV contains instructions in its genome for making accessory proteins that help the virus replicate, but the HIV strains used in the Johnson lab lack the genes for some of these proteins. That means the handicapped viruses can infect exactly one round of cells and spread no further.
Lyddon also ensures quality control for the lab by making sure students’ work is reproducible.
As a pet project, Johnson also independently confirms new findings reported in academic journals about HIV. Sometimes, Johnson says, the phenotypes that get published are not wrong, but they tend to represent the best outcomes, which might only exist in very specific scenarios.
“They’re only right by the last light of Durin’s day,” Johnson said, making a Lord of the Rings reference to a phenomenon in The Hobbit that reveals the secret entrance to a dwarven kingdom only once a year.
Because scientists base their work on the research of other scientists, he said, it’s always important to check.
A RECONSIDERED POSITION
According to the World Health Organization, 37 million people worldwide in 2014 have HIV or AIDS. The virus infects approximately two million new individuals every year. Breakthroughs in treatment have turned the autoimmune disease from a highly feared death sentence into a chronic and manageable condition.
For the longest time, HIV researchers scrambled to find better therapies against HIV why trying to develop a vaccine that could prevent AIDS.
But in the past five years, Johnson says he’s noticed a shift: researchers are gaining confidence in the possibility of finding a cure, something he once thought was impossible.
“Now it’s been demonstrated that it’s possible to cure a person,” Johnson said, referring to the Berlin patient. “So it’s only going to get easier.”
However, Johnson pointed out, most people would never undergo the kind of high-risk treatment that Timothy Ray Brown, the Berlin patient, received. Brown underwent a bone marrow transplant to treat his leukemia, and his new bone marrow, which came from an HIV-resistant donor, cured him of AIDS.
A “full blown cure” will be hard to attain, but Johnson believes there may be ways for HIV patients to live their lives without having to constantly take medication.
As an example, he points to certain “elite controllers” who are HIV positive but never progress further to show symptoms of AIDS. If scientists can figure out what’s different about their immune systems, Johnson said, then researchers could train the immune response in AIDS patients to resist HIV or keep it in check.
That’s a project for the immunologists. As a basic scientist, Johnson says he adds to the knowledge of how HIV works.
“I am not thinking about a therapy,” Johnson said, “but I’m also acutely aware that some of the best solutions come from basic science. “
Even though scientists haven’t discovered all the mechanisms behind cellular and viral function yet, Johnson said, the rules do exist.
“The sculpture is already there in the stone,” he said.
Johnson’s job is to chip away at the marble until the rules are found.
MU freshman follows in aunt’s footsteps while exploring career options
Robert Schmidt poses with one of the cats that lives at Horton Animal Hospital, where he works part-time. Schmidt, a freshman studying biochemistry at the University of Missouri, is a member of the Discovery Fellows Program where he is learning about plant genetics by working with biologist Scott Peck in the Bond Life Sciences Center. Photo by Justin L. Stewart | MU Bond Life Sciences Center
By Justin L. Stewart | MU Bond Life Sciences Center
Sometimes it’s socks. Another time, it was a book cover.
Robert Schmidt has retrieved quite a few things from misguided pets’ digestive systems as an assistant at Horton Animal Hospital, where he’s worked with his aunt for the past three years.
While most of the time he helps with simpler things — such as feeding kenneled animals or spaying and neutering pets — he also has amputated a dog’s toe after it had a lawnmower mishap.
Schmidt says he’s kind of following in his aunt’s footsteps. She majored in biochemistry at the University of Missouri and studied at its veterinary school, too. That’s what Robert wants to do with his life.
Schmidt demonstrates to a pet owner how to apply ear medication to a nervous small dog at Horton Animal Hospital on Forum. Schmidt works at the animal hospital part-time with his aunt. Photo by Justin L. Stewart | MU Bond Life Sciences Center
Schmidt likes working with animals, but pets aren’t the only animals he’s interested in. An internship at the MU Animal Sciences Research Center two summers ago had Schmidt elbow deep in fistulated cows’ stomachs as he assisted in research that tested the affects of adding different metals in cows feed to help them better absorb protein.
“My family’s always had a dog, my sister has a cat and I work at a small animal clinic. For a while, I was just like, ‘I’m not going to work with cows. I didn’t grow up on a farm.’ But now, after the internship, I’m interested in getting some more experience.”
As a freshman in the Discovery Fellows Program, Schmidt now finds himself knuckle deep in dirt as he digs into plant genetics.
The Discovery Fellows Program pairs first-semester freshmen and sophomores with scientists in their chosen field, allowing them to get hands-on experience in research on campus while earning a $1,700 stipend.
That led Schmidt, an MU Honors College student, to the lab of biologist Scott Peck in the Bond Life Sciences Center.
Schmidt plants Arabidopsis seeds in a petri dish inside the Bond Life Sciences Center, where his fellowship work is. Photo by Justin L. Stewart | MU Bond Life Sciences Center
Peck studies how plants recognize and respond to infections, specifically focusing on three proteins that begin to chemically modify shortly after a plant is infected.
No one knows what these proteins actually do, Peck said, making the research that much more interesting.
Schmidt has been growing Arabidopsis seeds of three different types, each missing one of the three proteins. Once those seeds have fully grown, Schmidt said they will cross-pollinate the three variations to hopefully create a plant without any of those three proteins.
He hopes to have triple mutants by early next semester, so they can experiment with them to better understand the roles of the absent proteins.
While Schmidt came into Peck’s lab with more of an interest in animals, he sees how the skills translate.
“If I worked with animals, I’d want to do genetics and I’m doing genetics with plants right now. A lot of the lab techniques are transferable.”
Schmidt moves an Arabidopsis seedling from a petri dish to fresh soil with the rest of the grown seedlings. Arabidopsis can grow from seed to seedling with two weeks. It’s a favorite among scientist, according to the National Science Foundation. Photo by Justin L. Stewart | MU Bond Life Sciences Center
Like most freshman, the former Rockbridge Valedictorian is still figuring out his life plans. He’s considering a double major in math, a favorite subject of his, and now wonders whether he might attend graduate school instead of vet school.
“I really like learning, being in a classroom and being a student. I really enjoy that. I think research is almost like a career where you’re almost always a student. You’re just always learning.”
As for now, he’s just waiting for his first batch of Arabidopsis seeds to mature.
Plant scientist Ruthie Angelovici joins the Bond Life Sciences Center
By Jennifer Lu | MU Bond Life Sciences Center
Ruthie Angelovici
Ruthie Angelovici clearly remembers her big eureka moment in science thus far. It didn’t happen in a laboratory. It wasn’t even her experiment.
At the time, Angelovici was in college studying marine biology. She had spent a year going on diving trips to figure out whether two visibly different corals were polymorphs of the same species, or two separate species.
A simple DNA test told her the answer in one afternoon.
“That’s the day I decided that there was a lot to be discovered, just in the lab,” Angelovici said. She switched majors and hasn’t looked back.
Better Nutrition in Crops
Angelovici studies the molecular biology of plants.
As a newly minted assistant professor in biological sciences at the Bond Life Sciences Center, her goal is to increase the nutritional quality of staple crops like corn, rice, and wheat.
Although these crops make up 70 percent of people’s diet across the world, Angelovici said, they aren’t very nourishing.
Corn, rice, and wheat are deficient in several key nutrients called essential amino acids. For example, if a person lived on wheat alone, they would have to eat anywhere from three to 17 pounds of the grain per day to reach the daily recommended amount for these nutrients.
Moreover, harsh growing conditions cause amino acids levels in plants to plummet—an increasingly grave problem as the earth’s climate gets warmer.
“If you think about the future, we’re going to face more droughts, more heat,” Angelovici said. “We need to figure out how we can maintain quality under those circumstances.”
Scientists have been trying to improve the nutritional quality of crops for years, whether through classical breeding or genetic engineering. The latter requires knowing which genes to alter.
Angelovici uses a technique called genome-wide association mapping. This allows her to link the natural variations within a particular trait — say, a special type of amino acids that are branched in structure — with the genes that affect this trait.
In previous studies, Angelovici chose Arabidopsis thaliana, which is popular among plant scientists for its simple genome and short life cycle, as her model plant.
She collected seeds from 313 varieties and burst them open, one seed type at a time, to release their contents. After separating the free amino acids from the rest of the seed pulp, she measured the branched amino acid levels — as a ratio to each other and to other amino acids — to build a nutritional profile that acts like a fingerprint for each plant.
Angelovici used this fingerprint to identify plants that shared similar traits. Then she scanned their DNA for any small genetic variations, or mutations, that plants had in common.
When she tallied up the frequency of each mutation in what is called a Manhattan plot, she found one particular variation that outstripped the others, standing out like a skyscraper over a city: a small section on chromosome 1 close to a gene called bcat2.
Angelovici then switched this gene off. When branched amino acid levels changed, it suggested that this trait was linked to the bcat2 gene.
However, Angelovici warned that often plants resist genetic tinkering. They lose viability, or cannot germinate seeds.
“We get yield penalty,” Angelovici says, “and the question is why?”
Metabolism, she explains, is like a network. “If you pull one way, something else is going to be affected.”
That’s where bioinformatics comes in handy. Angelovici uses an approach called network analysis to look at many pathways within the plant at once. This allows her to see the big picture, as well as the fine detail.
Moving to Missouri
Angelovici has being studying plant metabolism for ten years. Originally from Israel, she earned her PhD in 2009 under Gad Galili at the Weizmann Institute of Science in Rehovot, Israel. Then, she continued her research as a postdoctoral fellow at Michigan State University.
She prefers working with plants to animals because plants are relatively easy to manipulate and breed. Also, she loves animals and at one point wanted to be a veterinarian.
Angelovici says she was immediately drawn to the University of Missouri, and is looking forward to collaborating with researchers at Bond LSC.
“There is a great program here, great plant people here,” she said. “So, Mizzou is spot on.”
Although she has found an undergraduate and a post-doctoral researcher to help her so far, the benchtops in her laboratory remain uncluttered save for some equipment, like glassware and a few gel boxes. Three pristine white lab coats hang neatly from hooks on the wall.
But Angelovici is not fazed by the enormous task of getting her lab up and running.
“I just love doing this. It’s like climbing a mountain,” Angelovici said, about the research process. “You do it slowly and then you feel like you’re going up and you are achieving more and you can see more. It’s really fulfilling.”
As for that big eureka moment, Angelovici says she doesn’t put much stock in it.
Then she laughs. “But maybe I will experience one, and then I’ll change my mind.”
Female rats struggle to find their way in BPA study from MU and the NCTR/FDA
Cheryl Rosenfeld is one of 12 researchers partnering with the NCTR/FDA to study BPA
Despite concerns about bisphenol A (BPA), academic and regulatory scientists have yet to reach a consensus on BPA’s safety.
The National Institute of Environmental Health Sciences (NIEHS), the National Toxicology Program (NTP), the Food and Drug Administration and independent university researchers are working together to change that.
“The idea of this Consortium is to examine the potential systems that have been previously suggested to be affected by BPA,” said Cheryl Rosenfeld, an associate professor of biomedical sciences at the University of Missouri and one of twelve researchers involved in the project.
Rosenfeld’s group looked at spatial navigation learning and memory. They found that prenatal exposure to BPA could potentially hinder the ability of female rats to learn to find their way through a maze. This effect was not seen in male rats.
Approved by the FDA in the early 1960s, BPA can be found in a wide variety of products, including plastic food and drink containers with recycle codes 3 or 7, water and baby bottles, toys, the linings of metal cans and water pipes, even patient blood and urine samples.
BPA has structural similarities to estrogen and can potentially act as a weak estrogen in the body.
In Rosenfeld’s experiment, researchers at the National Center for Toxicology Research gave pregnant rats a fixed dose of BPA every day: a low, medium, or high dose.
After the baby rats were born, researchers continued to dose the babies, both male and female, according to what their mothers had received.
When these rats reached three months old, they were tested in a circular maze with twenty possible exit holes, one of which was designated as the correct escape hole. Every day for seven days, researchers tested the rats’ abilities to solve the maze in five minutes and timed them as they ran.
Rats solve mazes in three ways, Rosenfeld said.
They can run through the labyrinth in a spiral pattern, hugging the outer walls, and work their way in until they find the correct exit hole in what is called a serial search strategy.
Or they might move aimlessly in the maze using an indirect search strategy, Rosenfeld said. “In this case, the rats seemingly find the correct escape hole by random chance.”
Lastly, they can travel directly from the center of the maze to the correct escape hole. The third strategy is considered the most efficient method because the rats find their way swiftly, Rosenfeld said.
Sarah Johnson, a graduate student and first author on the paper, assessed each rat’s performance in the maze using a three-point tracking program that recognizes the rat’s nose, body, and tail.
Using the program, Johnson measured their performances in terms of the total distance traveled, the speed at which the rat ran the maze, how long it took the rats to solve the maze (latency), and how often the rat sniffed at an incorrect hole.
The last two parameters are considered the best gauges of spatial navigation learning and memory.
“What you expect to see is that they should start learning where that correct escape hole is,” Rosenfeld said. “Thus, their latency and sniffing incorrect holes should decrease over time.”
Rosenfeld’s group found that female rats that had been exposed to the highest dose of BPA since fetal development were less likely to find the escape hole than rats that hadn’t been exposed to BPA.
As for how this study may translate to people, Rosenfeld said, “the same brain regions control identical behaviors in rodents and humans.”
She considers it a starting point for setting up future experiments that take into consideration sex differences in cognitive behaviors and neurological responses to BPA.
Immediate next steps for the Rosenfeld group include analyzing tissue collected from the brains of rats that had undergone maze testing. Rosenfeld’s team of researchers will measure DNA methylation and RNA expression in the brain to determine which genes might be involved in navigational learning and memory. Their overarching goal is to determine how changes in observed sex- and dose-dependent behaviors occur on the molecular level.
NIEHS grant U01 ES020929 supported this research. Additional coauthors include Mark Ellersieck and Angela Javurek of the University of Missouri, Thomas H. Welsh Jr. of Texas A&M University, and Sherry Ferguson, Sherry Lewis, and Michelle Vanlandingham of the National Center of Toxicological Research/Food and Drug Administration. Read the full study on the Hormones and Behavior website and browse the supplementary data for this work.
Scientists find how nematodes use key hormones to take over root cells
This Arabidopsis root shows how the beet cyst nematode activates cytokinin signaling in syncytium 10 days after infection. The root fluoresces green when the TCSn gene associated with cytokinin activation is turned on because it is fused with a jellyfish protein that acts as a reporter signal. Contributed by Carola De La Torre
Roger Meissen | Bond Life Sciences Center
This is a story about spit.
Not just any spit, but the saliva of cyst nematodes, a parasite that literally sucks away billions in profits from soybean and other crops every year.
Researchers are working to uncover exactly how these tiny worms trick plant root cells into feeding them for life.
Cytokinin is normally produced in plants, but these researchers determined that this growth hormone is also produced by nematode parasites that use it to take over plant root cells.
“While it’s well-known that certain bacteria and some fungi can produce and secrete cytokinin to cause disease, it’s not normal for an animal to do this,” said Melissa Mitchum, an MU plant scientist and co-author on the study. “This is the first study to demonstrate the ability of an animal to synthesize and secrete cytokinin for parasitism.”
Not Science Fiction
Reprogramming another organism might sound like a far out concept, but it’s a reality for plants susceptible to nematodes.
Cyst nematodes hatch from eggs laid in fields and quickly migrate to the roots of nearby plants. They inject nematode spit into a single host cell of soybean, beet and other crop roots.
Carola De La Torre
“Imagine a hollow needle at the head of the nematode that the parasite uses to penetrate into the plant cell wall and secrete pathogenic proteins and hormone mimics,” said Carola De La Torre, a co-author of the study and plant sciences PhD student with Mitchum’s lab. “Nematodes use the spit to transform the host cell into a nutrient sink from which they feed on during their entire life cycle. This de novo differentiation process greatly depends on nematode–derived plant hormone mimics or manipulation of plant hormonal pathways caused by effector proteins present in the nematode spit.”
These effector proteins and other small molecules in their spit cause the root cell to forego normal processes and create a huge feeding site called a syncytium. In a short period of time, this causes hundreds of root cells to combine into a large nutrient storage unit that the nematode feeds from for its entire life.
Being able to convince a root cell to do the nematode’s bidding starts with a takeover of the plant host cell cycle — which regulates DNA replication and division. This implies that a plant hormone like cytokinin is involved, says Mitchum. Cytokinin normally regulates a plant’s shoot growth, leaf aging, and other cell processes.
Proving the relationship
While Mitchum’s lab had a hunch that cytokinin was key to this takeover, proving it took some creative science.
De La Torre and Demosthenis Chronis, a postdoctoral fellow MU at the Bond LSC depended on mutant Arabidopsis plants to explore the relationship. “One of the great things about using Arabidopsis as our host plant is the vast genetic resources of cytokinin and hormone mutants that are available through the scientific community,” De La Torre said.
She infected Arabidopsis that contained a reporter gene called TCSn/GFP with nematodes. This gene is associated with cytokinin responses within the plant cells and is fused with a jellyfish protein that glows green when turned on. So, De La Torre saw nematodes activated cytokinin responses in the plant early after infection when her plants emitted a green fluorescent glow under the microscope.
Next, she infected plants missing the majority of their cytokinin receptors with nematodes. Then she started counting nematodes present.
“After a careful evaluation of nematode infection, we observed less female nematodes developing in the receptor mutants compared to the wild type” De La Torre said. “The nematodes could not infect well, and that was a clear piece of evidence suggesting that cytokinin plays a main role in plant–nematode interactions.”
Another experiment looked at Arabidopsis containing a reporter gene called GUS that was fused to the regulatory sequences of the cytokinin receptor genes. All three cytokinin receptor genes were activated where the nematode was feeding.
A final experiment used a mutant that created an excess of an enzyme that degrades cytokinin, finding that a base level of plant cytokinin was also necessary for nematode growth.
“The simple statement is that the cytokinin receptors were activated in response to nematode infection and the mutants did not support growth and development of the nematodes,” Mitchum said. “This shows that if you take away the ability of the plant to recognize cytokinin the worms are unable to fully develop.”
An international collaboration
Mitchum’s team did not work alone.
The lab of Florian Grundler at Rheinische Friedrich-Wilhelms-University of Bonn, Germany, was also on a mission to uncover if genes in the nematode controlled cytokinin activation. They had identified a key gene in the beet cyst nematode that makes the cytokinin hormone. When they took away the ability of the nematode to secrete cytokinin certain cell cycle genes were not activated at the feeding site and the nematodes did not develop. Now we know that the nematode is also secreting cytokinin to modulate the pathways.
De La Torre took that information and found the same gene in the soybean cyst nematode.
Now, Mitchum’s team is trying to find how this key gene might work differently in other nematode types, like root-knot nematode as part of a new National Science Foundation grant. They hope this will help lead to better resistance in future crops.
“Understanding how the nematode modulates its host is going to help us exploit new technologies to engineer plants with enhanced resistance to this terribly devastating pathogen,” Mitchum said. “Technology is changing all the time, we’re gaining new tools constantly, so you never know when something new is going to allow us to do something specific at the site of nematode feeding that will lead to a breakthrough.”
Cells that expressed IFITM proteins (bottom row), showed much less spread of HIV-1 compared with cells lacking the protein. | courtesy Jordan Wilkins, Liu Lab
By Jennifer Lu | MU Bond Life Sciences Center
For Shan-Lu Liu, thinking outside the box meant putting an antiviral protein inside HIV-infected cells, rather than into healthy ones.
Liu and his team of researchers studied how interferon-induced transmembrane (IFITM) proteins limit the infection of HIV-1, the primary strain of virus responsible for AIDS. The journal Cell Reports published their results on September 17.
IFITM proteins are biomolecules with broad antiviral properties. Although multiple versions of IFITM have been found in humans, three are known to have antiviral properties: IFITM1, IFITM2 and IFITM3.
In a 2013 paper published in PLoS Pathogens, the Liu laboratory demonstrated that these three IFITM proteins have the ability to thwart a variety of viral infections.
Shan-Lu Liu, Bond Life Sciences scientist and associate professor in the MU School of Medicine department of molecular microbiology and immunology. | Photo by Justin Kelley, University of Missouri Health Care
“They can inhibit influenza virus, Ebolavirus, HIV and SARS coronavirus,” said Liu, an associate professor in the Department of Molecular Microbiology and Immunology at the Bond Life Sciences Center.
Liu wanted to know why IFITM’s inhibition of HIV was uncharacteristically weaker than its inhibition of other viruses.
To study this conundrum, many researchers designed their experiments by expressing IFITM proteins in target or healthy cells. Then they infected these IFITM-bolstered cells with HIV, but saw minimal protection against viral infection.
In a twist, the Liu group put IFITM proteins in HIV-1 producer or infected cells instead of in healthy T-lymphocyte cells, a special kind of immune cell used specifically to study the viral infection by HIV.
They found that IFITM proteins, especially IFITM2 and IFITM3, interacted with the viral envelope protein (Env) that makes up the outer shells of virus particles.
For normal HIV infections to occur, Liu said, envelope proteins need to be cleaved into two parts.
Once processed, the resulting two portions, Env gp120 and gp41, can be incorporated into viral particles. The two processed envelope proteins protrude from the outer surface of the virus like mushroom-shaped pegs that help the virus latch on and fuse to target cells.
But when IFITM binds to envelope proteins they interfere with the viral envelope functions.
“It’s just unexpected,” Liu said, about this finding. In other viruses his group has studied, IFITM inhibited virus’ ability to fuse its outer shell with the membrane of a cell by making the cell membrane rigid during the infection process. He said he assumed IFITM would block HIV the same way.
Instead, they found evidence suggesting that IFITM blocks infection through direct contact with HIV’s envelope proteins.
“It is the first study that shows this kind of interaction,” Liu said. “That’s why this study is so surprising. We did not think about this.”
Liu does not yet know the mechanism behind IFITM and envelope protein interactions, but he said the outcome remains clear. “IFITM proteins inhibit this Env cleavage process and this makes HIV less infectious and less transmissible.”
To visualize IFITM’s inhibitory effects in action, Liu’s group tagged HIV-1 inside infected cells with a green fluorescent dye. Then they colored healthy target cells with a red fluorescent dye. When they mixed the two populations of cells together, they saw two days later a very tiny amount of cells exhibiting green signals within the red cells —a sign of spread of HIV cell-to-cell infection.
By comparison, cells in the control group—healthy red-tagged cells mixed with green HIV-infected cells that do not contain IFITM—showed a higher number of red cells lighting up green inside.
This suggested that having IFITM and HIV-1 inside the virus-producing cells somehow limited the virus’ infectivity and cell-to-cell spread at the same time.
His group also showed through a technique called co-immunoprecipitation that IFITM proteins bound specifically with envelope protein rather than with other proteins, such as Gag. Liu attributed this work to his two talented hardworking graduate students, Jingyou Yu and Minghua Li.
Unfortunately, the benefits of IFITM are short-lived. When the Liu laboratory let HIV-infected cells replicate again and again, they saw that HIV could evolve enough to circumvent the inhibitory effects of IFITM after 30 passages.
Liu said that his research on IFITM was still in its early stages, but that the next step would be to look at the IFITM’s function in HIV patients in order to move the basic research of IFITM from bench to bedside.
“Once we know better how this protein works, we can develop some inhibitors to block HIV, block Ebola, block other viruses,” Liu said. “So that’s our ultimate goal.”
Liu is a Bond Life Sciences Center investigator and an associate professor of molecular microbiology and immunology in the MU School of Medicine. He studies how viruses infect healthy host cells to cause illness and cell response to viral attacks.
The National Institutes of Health and the Canadian Institutes of Health Research partially supported this research. Additional collaborators include Eric Freed, PhD, senior investigator with the National Cancer Institute (NCI) HIV Dynamics and Replication Program; Chen Liang, PhD, at McGill University; and Benjamin K. Chen, PHD, at the Mount Sinai School of Medicine. Read the full study on the Cell Reports website and browse the supplementary data for this work. See more on this research from Mizzou News.
Endocrine disruptors impact physical activity and metabolism in mice
By Caleb O’Brien | MU Bond Life Sciences Center
Could your experiences in the womb make you lazy as an adult?
A recent study of California mice suggests that early exposure to environmental chemicals can later impact an animal’s metabolism and level of voluntary physical activity, according to new University of Missouri research.
“We found that if we developmentally exposed California mice to bisphenol A (BPA) or ethinyl estradiol (EE), the estrogen present in birth control pills, it caused later disruptions in voluntary physical activity,” said Cheryl Rosenfeld, a researcher in MU’s Bond Life Sciences Center and associate professor of biomedical sciences in the College of Veterinary Medicine. “What that means is they move around less in their home cage, they’re more likely to sleep, and they engage in less voluntary physical activity.”
Rosenfeld’s lab studies the ways that exposure to environmental chemicals such as BPA can affect other behaviors, including cognition and parenting. Endocrine-disrupting chemicals can accumulate in the environment and act like the hormones naturally produced by many organisms, including humans. To test the chemicals’ impact on metabolism and activity, the lab used California mice. This mouse model is a good model for metabolic diseases. And because these animals are initially derived from the wild, they may better replicate the genetic diversity of most human populations.
The researchers exposed the mice to BPA and EE in the womb and until weaning via the mom’s diet. A third group of mice whose mothers were placed on a phytoestrogen-free control diet was not exposed to either chemical. The scientists then placed all the mice on this same control diet and measured their energy expenditure, body composition and level of voluntary physical activity as adults.
To test those attributes, Rosenfeld’s lab relied on a variety of tools and techniques. They rigged bicycle computers to “hamster wheels” to track how far, fast and for how long the mice ran. Using a device called a “Promethion continuous measurement indirect calorimetry system” the researchers continuously monitored the mice’s energy expenditure by measuring oxygen consumption and carbon dioxide production and by using a three-beam system, tracked the rodents’ movements during the dark and light cycles.
Later, the researcher measured the animals’ body composition using an EchoMRI, a tiny MRI machine the size of a filing cabinet, and finally measured circulating concentrations of glucose and hormones that regulate metabolism.
Female mice exposed to BPA and EE were less active than control mice. They moved around their cages less at night (when the nocturnal California mouse is considered most active), moved more slowly, drank less water, and spent more time sleeping. In addition, BPA-exposed females burned more carbohydrates relative to fats, as compared to control mice. This is similar to the difference between obese and slender humans, and many researchers believe that burning more carbohydrates relative to fats can lead to fats gradually accumulating in the body.
“It’s worrisome that environmental chemicals we are exposed to in utero can override our genes and disrupt our neuro-circuitry,” said Sarah Johnson, a research specialist and graduate student in Rosenfeld’s lab and primary author on the study. “The net effect is that we can have behavioral disruptions into adulthood, including altered physical activity.”
The researchers are currently conducting follow-up studies to determine if the changes caused by exposure to BPA and EE predispose mice to obesity and other metabolic disorders. They also are interested in exploring if exposure could affect the children and grandchildren of these mice and examining the potential underlying neural mechanisms.
“Our findings are significant because decreased voluntary physical activity, or lack of exercise, can predispose animals or humans to cardiovascular diseases, metabolic disorders and even cancer,” Rosenfeld said.
Other authors on the study are Angela Javurek and Michele Painter (MU Biomedical Sciences), Mark Ellersieck (MU Agriculture Experimental Station- Statistics), Charles Wiedmeyer (MU Veterinary Medical Diagnostic Laboratory and Department of Veterinary Pathobiology) and John Thyfault (Kansas University Medical Center, Molecular and Integrative Physiology)
The study, “Sex-Dependent Effects of Developmental Exposure to Bisphenol A and Ethinyl Estradiol on Metabolic Parameters and Voluntary Physical Activity” was supported by NIH Grant 5R21ES023150 (to C.S.R.) and R01DK088940 (JPT) and was published in the Journal of Developmental Origins of Health and Disease.
James Amos-Landgraf, assistant professor of comparative medicine and genetics at the University of Missouri, is helping develop a pig model for colon cancer using CRISPR. //photo by CALEB O’BRIEN/Bond LSC
James Amos-Landgraf needed a pig.
The assistant professor of comparative medicine and genetics at the University of Missouri had joined forces with a startup company developing a tool to detect early colon cancer-causing lesions. They already tried out a rat-sized model, but still needed a full-sized prototype.
Scientists in Europe had an ideal pig model for colon cancer, but importing the animals presented a problem. It would be prohibitively expensive and time consuming, and the method European scientists used to develop the pig took several years and cost a great many Euros, Amos-Landgraf said.
Those obstacles might have been enough to scuttle the project entirely, but CRISPR, a new gene-editing tool discovered in the DNA of a peculiar bacterium, has changed the equation for scientists everywhere.
So when Amos-Landgraf went to the National Swine Resource and Research Center (NSRRC) to ask about importing pigs, they told him, “‘We can just make you the model,’” Amos-Landgraf said. “‘We should be able to do a CRISPR project within a few months.’”
CRISPR is rapidly reshaping the way biologist around the world do their jobs.
At Mizzou, it’s transforming how researchers learn about viruses and mosquitoes, pigs and zebrafish, and the individual genes affecting development, sickness and health. The tool makes research more efficient, cost-effective and vastly more powerful.
Amos-Landgraf knows firsthand just how time-consuming and laborious generating an animal model was pre-CRISPR.
“What was almost a two-year process just to generate an animal now would take us a matter of months,” Amos-Landgraf said. “I think the CRISPR revolution is going to be amazing for all of science. I’m totally intrigued by everything that’s going on with this.”
Borrowing a bacterial relic
CRISPR rolls off the tongue far more readily than its unabbreviated equivalent: “clustered regularly interspaced short palindromic repeats.” The name refers to a strange pattern scientists at the University of California, Berkeley noticed in the genome of a bacterium that lives in acidic, abandoned mines: groups of palindromic bacterial DNA sequences interspersed with segments of viral DNA.
It turned out that the genetic snippets were relics of the bacteria’s prior run-ins with viral invaders, like genetic mug shots on a most-wanted list.
Viruses are tiny packages of genetic material that hijack cells, such as bacteria, in order to reproduce. And when a virus enters a bacterial cell, the host compares the virus’ genetic material to the snapshots preserved in the bacteria’s own DNA. If they match, the bacteria dispatches a bounty-hunter protein called Cas9, which tracks down the virus and slices its DNA in half at the very spot that matched the virus’ genetic fingerprint.
If an unfamiliar virus attacks and the bacterium survives, it will incorporate a segment of the invader’s DNA into its own, adding a new battle scar to its DNA and a new miscreant to the most-wanted list.
When the researchers studying the bacterial immune system figured out how it worked, they realized the process could have implications far beyond the organism’s acidic abode: It could become a powerful, inexpensive, and versatile gene-editing tool.
A ground shift
The journey to better manipulate genes has been a long one.
For decades, scientists relied on various techniques and tricks to tease out the function of genes. The most common tool is forward genetics, where a researcher starts with an interesting characteristic in an organism and then hunts for the gene that caused it. Those characteristics could be traits that occur naturally, such as genetic diseases in purebred dogs or pigmentation in corn kernels, or a scientist could induce defects — essentially altering an organism’s genome by exposing it to a bath of nasty chemicals.
Anand Chandrasekhar, Bond Life Sciences Center biologist and professor in the division of biological sciences, and PhD candidate Suman Gurung take a look at some zebrafish they’ve modified using CRISPR. //photo by CALEB O’BRIEN/Bond LSC
Imagine that an organism is like a car, suggests Anand Chandrasekhar, a Bond Life Sciences Center biologist and professor in the division of biological sciences.
“You take a car that is running nicely and you have some kind of weird mechanic from Hell come in and mess something up — just one thing — and the car doesn’t run. Then you have to figure out why the car doesn’t run by looking carefully for where the defect is.”
Reverse genetics — unsurprisingly — starts on the other end. Researchers pick a gene of interest and try to silence or alter it. If they succeed, then they look for changes in the organism that suggest the altered gene plays a role in the observed characteristic.
This tool shaped how scientists do research and what animals they use in their labs. In fact, model organisms such as mice rose in popularity partly because of how easily reverse genetic techniques like homologous recombination work with them, said Amos-Landgraf. But this approach was time consuming, expensive and didn’t function well on other organisms.
The next step forward were Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). Both act like guided missiles to strike at a gene of interest, targeting a specific region of genetic material and breaking both strands of the organism’s DNA at that spot. Once the DNA is broken, the cell’s natural repair mechanism intervenes and stitches the gene back together.
However, the process is prone to errors — mutations — that can alter or silence a gene. ZFNs and TALENs reliably worked in a broader array of species.
CRISPRs represent the next advancement in this process and is far faster than previous techniques.
“Let’s say if you had 15 or 20 genes that you wanted to study: You could design a CRISPR reagent for each one of them in a couple of afternoons, whereas in the ‘olden days’ (three or four years ago) with TALENs that could have taken you months,” Chandrasekhar said. “And if you were using ZFNS… you would not even imagine doing it, because you would have been crazy.”
Elizabeth Bryda, a professor of veterinary pathobiology at MU who heads the Rat Resource and Research Center, stands by a Genetic Analyzer machine. //photo by CALEB O’BRIEN/Bond LSC
At MU’s NIH-funded Rat Resource and Research Center (RRRC), scientists think CRISPR will help break dependence on default model organisms. The RRRC is the only center of its kind in the US and one of two in the world, serving as a repository and distribution center for rats that model human diseases.
“We’re always preaching, use the species that’s most appropriate for the question you’re asking,” said Elizabeth Bryda, a professor of veterinary pathobiology at MU who heads the RRRC. “If you’re studying human disease, use the species that best recapitulates that disease. I think CRISPR will give people the flexibility to really work in the species they want to be working in.”
For example, the center is using CRISPR to develop rat models of human inflammatory bowel diseases, such as Crohn’s disease. “All of those barriers to making rat models are no longer issues,” Bryda said, “CRISPR is easy and finally allows us to manipulate rats in ways we haven’t before.”
That’s good news for the RRRC: “I do think we’re going to see a huge increase in the number of rat models,” Bryda said, “which would increase our inventory.”
Seeing through the zebrafish
Zebrafish are another model organism that might become even more important thanks to CRISPR.
Originally found in the rice paddies and streams of India and Myanmar, the minnow-like fish is an important model organism. They’re easy to care for, produce abundant offspring and — because their embryos are transparent — make great tools for studying development.
Chandrasekhar uses zebrafish to study cranial motor neurons, the neurons that connect to, and control, muscles in the head. His lab is especially interested in the way those cranial motor neurons are deployed during development: how the neurons know where to go and to which muscles they should link.
“CRISPR is a really big boon for research, because now even small labs can test tens of genes over a short period of time for their effect on a particular biological process,” Chandrasekhar said. “That’s how we use it: We study the process of cell migration within a nervous system, and we want to study a whole slew of associated genes.”
Researchers have identified hundreds of new and potentially important genes using advance genomics, but the old techniques of reverse genetics were too slow and tedious to keep up with the new discoveries.
“CRISPR has removed the bottleneck,” Chandrasekhar said. “We can rapidly go through and, hopefully, find new genes and new signaling pathways that might be playing a very specific role for the migration and the biological process that we study.”
But finding a new gene is just the beginning, Chandrasekhar said.
“We have one student who is testing five genes, and if even one or two of those genes turn out to be important, that will then be sufficient for the lab to continue working on them for two or three years.”
Although scientists primarily use CRISPR as Chandrasekhar does, to silence genes in model organisms, new genes can also be introduced.
Through a process called homologous directed repair, scientists select a location where they want to introduce a gene and design a CRISPR to target that region.
Daniel Davis, a PhD candidate and lab manager for Assistant Professor of Veterinary Pathobiology Catherine Hagan, is developing a technique to screen potential antidepressant drugs by leveraging CRISPR technology and the advantages of the zebrafish. //photo by CALEB O’BRIEN/Bond LSC
Daniel Davis, a PhD candidate and lab manager for Assistant Professor of Veterinary Pathobiology Catherine Hagan, is developing a technique to screen potential antidepressant drugs by leveraging CRISPR technology and the advantages of the zebrafish.
When a zebrafish is stressed, it produces a neurotoxic compound, but when the fish is calm, it produces a different compound, one that is neuro-protective. The difference depends on which key enzyme the fish produces — in a stressful situation, the fish produces more of the enzyme that leads to neurotoxicity.
Davis is using CRISPR to try to link different fluorescent proteins genes to each branch of this stress pathway: If the fish produces more of the stressful compound, it will also produce a red fluorescent protein. If the other pathway is taken, the fish will assemble green fluorescent protein.
“If you take some fish, subject them to a stressor and test a variety of potential therapeutics on them, you could visualize the fluorescent proteins to see which therapeutics are more protective,” Davis said.
Altering the host to understand the virus
Other models present special challenges. In mosquitoes, for instance, it’s hard to knock out genes from its genome using traditional methods.
“The problem is that in mosquitoes such as Aedes aegypti, ‘traditional’ knockouts never really worked, so people tried out new techniques such as ZFNs and TALENs,” said Alexander Franz, assistant professor of veterinary pathobiology at MU. But the other techniques had flaws, too: they were expensive, complicated to assemble and often posed issues of efficiency and specificity.
Franz studies arthropod-borne viruses (arboviruses), specifically dengue virus and chikungunya virus. The life cycle of an arbovirus requires its circulation between arthropods, such as mosquitoes, and vertebrate hosts, such as humans. Because vaccines exist for only a few mosquito-borne viruses — yellow fever and Japanese encephalitis, for example — people usually rely on conventional and often ineffective environmental controls to thwart disease: bed nets, the elimination of breeding areas, insecticides.
Alexander Franz (far right), an assistant professor of veterinary pathobiology at MU who demonstrated that CRISPR is effective in mosquitoes, stands with his lab. From left to right: Velmurugan Balaraman, Asher Kantor, Hannah Gerlt, and Shengzhang Dong. //Photo courtesy of Alexander Franz
Franz is pursuing a different avenue for protection that uses genetic manipulations to interrupt the transmission cycle of a virus in the mosquito.
“If you can stop the virus from taking hold in the mosquito, you can block transmission of the virus to its vertebrate host,” he said. “But to do so, you need an effective way to manipulate the mosquito’s genome.”
This is where CRISPR comes into play. “When people started reporting using the CRISPR system for genome editing in Drosophila or zebrafish, we immediately had the idea to try it out in mosquitoes.” Working with two postdocs, Franz demonstrated for the first time that the CRISPR system was capable of disrupting genes in mosquitoes.
To do so, he started with a line of transgenic mosquitoes that had already been modified to produce red and blue fluorescent proteins in their eyes. The lab designed a CRISPR to silence the gene responsible for the blue fluorescent protein. After trying a few different methods, they found a technique that turned off the target gene when they injected the CRISPR into mosquito embryos.
Because it is a very powerful and easy-to-handle genome editing technique, CRISPR has been recently utilized and further developed by other groups studying mosquito-pathogen interactions.
Other MU researchers focus on the viral interaction with human host cells.
Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies the way a virus puts itself together inside a host cell and fights off the cell’s defenses. //photo by CALEB O’BRIEN/Bond LSC
Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies the way a virus puts itself together inside a host cell and fights off the cell’s defenses.
“We don’t know all the cellular genes, cellular machinery and cellular pathways that viruses are harnessing,” Johnson said. “The best way to say that a virus requires a particular gene would be to knock it out of the cell and see if the virus can still replicate.”
“CRISPR is a real ground shift in how we can do science,” he said. “Things that took 6 months to a year to make one gene before, now we can do half-a-dozen in a week.”
The technique has altered the rate at which Johnson’s research proceeds and expanded the scope of his lab’s work. “It’s allowed me to take a step back and think about the whole genome, as opposed to being totally focused on this one thing or that one thing,” Johnson said. “I’d never really taken a step back to think about the whole genome — every gene, where are they and what families. It’s changed my outlook on the cell, the way I can think about it.”
The CRISPR era
Amos-Landgraf and the researchers at NSRRC are still in the process of validating their pig model: developing primers to identify the mutation and creating the CRISPRs themselves. Once everything is ready, they’ll test out the lesion-detecting colonoscope, and if all goes well, move into human trials — far faster and more economically than would have been possible a few years ago.
But Amos-Landgraf is tantalized by the possibilities the technology offers beyond increased speed and reduced costs: “To be able to tease apart not just a single gene in a pathway, but maybe think about knocking out or altering all the genes in a pathway and looking at combinations of those pathways… You can start thinking about multiple gene knockouts, multiple gene manipulations all within the same experiment,” he said.
“And that is not only cost saving, but it becomes a really powerful tool when you want to interrogate biology. We’ve entered a new era of genetics and genomics.”
Chris Pires
Yidong Liu is a senior research specialist from MU’s Department of Biochemistry that manages Shuqun Zhang’s lab. She also works on MAPKs and their role in plant defense responses such as pathogen-induced ethylene biosynthesis and phytoalexin induction.
