Seminal work

How unruly data led MU scientists to discover a new microbiome
By Roger Meissen | MU Bond Life Sciences Center

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This seminal vesicle contains a newly-discovered microbiome in mice. Some of its bacteria, like P. acnes, could lead to higher occurrences of prostate cancer. | contributed by Cheryl Rosenfeld

It’s a strange place to call home, but seminal fluid offers the perfect environment for particular types of bacteria.

Researchers at MU’s Bond Life Sciences Center recently identified new bacteria that thrive here.

Cheryl Rosenfeld1.jpg “It’s a new microbiome that hasn’t been looked at before,” said Cheryl Rosenfeld, a Bond LSC investigator and corresponding author on the study. “Resident bacteria can help us or be harmful, but one we found called P. acnes is a very important from the standpoint of men. It can cause chronic prostatitis that results in prostate cancer. We’re speculating that the seminal vesicles could be a reservoir for this bacteria and when it spreads it can cause disease.”

Experiments published in Scientific Reportsa journal published by Nature — indicate these bacteria may start disease leading to prostate cancer in mice and could pass from father to offspring.


A place to call home

From the gut to the skin and everywhere in between, bacterial colonies can both help and hurt the animals or humans they live in.

Seminal fluid offers an attractive microbiome — a niche environment where specific bacteria flourish and impact their hosts. Not only is this component of semen chockfull of sugars that bacteria eat, it offers a warm, protected atmosphere.

“Imagine a pond where bacteria live — it’s wet it’s warm and there’s food there — that’s what this is, except it’s inside your body,” said Rosenfeld. “Depending on where they live, these bacteria can influence our cells, produce hormones that replicate our own hormones, but can also consume our sugars and metabolize them or even cause disease.”

Rosenfeld’s team wasn’t trying to find the perfect vacation spot for a family of bacteria. They initially wanted to know what bacteria in seminal fluid might mean for offspring of the mice they studied.

“We were looking at the epigenetic effects — the impact the father has on the offspring’s disease risk — but what we saw in the data led us to focus more on the effects this bacterium, P. acnes, has on the male itself,” Rosenfeld said. “We were thinking more about effect on offspring and female reproduction — we weren’t even considering the effect the bacteria that live in this fluid could have on the male — but this could be one of the more fascinating findings.”

But, how do you figure out what might live in this unique ecosystem and whether it’s harmful?

First, her team found a way to extract seminal fluid without contamination from potential bacteria in the urinary tract.

“We gowned up just like for surgery and we had to extract the fluid directly from the seminal vesicles to avoid contamination,” said Angela Javurek, primary author on the study and recent MU graduate. “You only have a certain amount of time to collect the fluid because it hardens like glue.”

Once they obtained these samples, they turned to a DNA approach, sequencing it using MU’s DNA Core.

They compared it to bacteria in fecal samples of the same mice to see if bacteria in seminal fluid were unique. They also compared samples from normal mice and ones where estrogen receptor genes were removed.


The difference in the data

It sounds daunting to sort and compare millions of DNA sequences, right? But, the right approach can make all the difference.

“A lot of it looks pretty boring, but bioinformatics allow us to decipher large amounts of data that can otherwise be almost incomprehensible,” said Scott Givan, the associate director of the Informatics Research Core Facility (IRCF) that specializes in complicated analysis of data. “Here we compared seminal fluid bacterial DNA samples to publicly available databases that come from other large experiments and found a few sequences that no one else has discovered or at least characterized, so we’re in completely new territory.”

The seminal microbiome continued to stand out when compared to mouse poop, revealing 593 unique bacteria.

One of the most important was P. acnes, a bacteria known to cause chronic prostatitis that can lead to prostate cancer in man and mouse. It was abundant in the seminal fluid, and even more so when estrogen receptor genes were present.

“We’re essentially doing a lot of counting, especially across treatments to see if particular bacteria species are more common than others,” said Bill Spollen, a lead bioinformatics analyst at the IRCF. “The premise is that the more abundant a species is, the more often we’ll see its DNA sequence and we can start making some inferences to how it could be influencing its environment.”

Although this discovery excites Rosenfeld, much is unknown about how this new microbiome might affect males and their offspring.

“We do have this bacteria that can affect the male mouse’s health, that of his partner and his offspring,” Rosenfeld said. “But we’ve been studying microbiology for a long time and we still find bacteria within our own bodies that nobody has seen before. That blows my mind.”

The study, “Discovery of a Novel Seminal Fluid Microbiome and Influence of Estrogen Receptor Alpha Genetic Status,” recently was published in Scientific Reports, a journal published by Nature.

 

Rodents of unusual appetites

How food cravings and eating affects the brain
By Jennifer Lu | MU Bond Life Sciences Center

When it comes to cookie dough, we’re not the only ones who can’t control our cravings. Kyle Parker’s rats couldn’t resist, either, thanks to a tweak in their brain chemistry.

Parker studies the neuroscience of food-based rewards.

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Matthew Will, associate professor of psychological sciences at the Bond Life Sciences Center, studies the neuroscience of behaviors such as over-eating and addiction | photo by Jennifer Lu, Bond LSC

“It’s like when I eat dessert after I’ve eaten an entire meal,” said Parker, a former grad student from the lab of Bond LSC’s Matthew Will. “I know that I’m not hungry, but this stuff is so good so I’m going to eat it. We’re looking at what neural circuitry is involved in driving that behavior.”

Behavior scientists view non-homeostatic eating — that’s noshing when you’re not hungry — as a two-step process.

“I always think of the neon sign for Krispy Kreme donuts.” Will said, by way of example.

“The logo and the aroma of warm glazed donuts are the environmental cues that kick-start the craving, or appetitive, phase that gets you into the store. The consummatory phase is when you “have that donut in your hand and you eat it.”

Parker activated a “hotspot” in the brains of rats called the nucleus accumbens, which processes and reinforces messages related to reward and pleasure.

He then fed the rats a tasty diet similar to cookie dough, full of fat and sugar, to exaggerate their feeding behaviors. Rats with activated nucleus accumbens ate twice as much as usual.

But when he simultaneously inactivated another part of the brain called the basolateral amygdala, the rats stopped binge eating. They consumed a normal amount, but kept returning to their baskets in search for more food.

“It looked like they still craved it,” Will said. “I mean, why would a rat keep going back for food but not eat? We thought we found something interesting. We interrupted a circuit that’s specific to the feeding part — the actual eating — but not the craving. We’ve left that craving intact.”

To find out what was happening in the brain during cravings, Parker set up a spin-off experiment. Like before, he switched on the region of the brain associated with reward and pleasure and then inactivated the basolateral amygdala in one group of rats but not the other.

This time, however, he restricted the amount of the tasty, high-fat diet rats had access to so that both groups ate the same amount.

This way, both groups of rats outwardly displayed the same feeding behaviors. They ate similar portions and kept searching for more food.

But inside the brain, Parker saw clear differences. Rats with activated nucleus accumbens showed increased dopamine production in the brain, which is associated with reward, motivation and drug addiction. Whether the basolateral amygdala was on or off had no effect on dopamine levels.

However, in a region of the brain called the hypothalamus, Parker saw elevated levels of orexin-A, a molecule associated with appetite, only when the basolateral amygdala was activated.

“We showed that what could be blocking the consumption behavior is this block of the orexin behavior,” Parker said.

The results reinforced the idea that dopamine is involved in the approach — or the craving phase — and orexin-A in the consumption, Will said.

Their next steps are to see whether this dissociation in neural activity between cravings and consumption exists for other types of diets.

Will also plans to manipulate dopamine and orexin-A signaling in rats to see whether they have direct effects on feeding.

“Right now, we know these behaviors are just associated with these neural circuits, but not if they’re causal.”

 

Behavioral Neuroscience published the study, “Neural Activation Patterns Underlying Basolateral Amygdala Influence on Intra-Accumbens Opioid-Driven Consummatory Versus Appetitive High-Fat Feeding Behaviors in the Rat,” in the December 2015 issue. A grant from the National Institute of Drug Abuse supported this research.

Unmasking the unknown

Scientists explore genetic similarities between plants and mice

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University of Missouri PhD Candidate Daniel L. Leuchtman peers through an Arabidopsis plant. Leuchtman has been experimenting with replacing a gene in the plants immune system with a similar gene from mice. | Photograph by Justin L. Stewart/MU Bond Life Sciences Center

By Justin L. Stewart | MU Bond Life Sciences Center

Almost two-thirds of what makes a human a human and a fly a fly are the same, according to the NIH genome research institute.

If recent research at the University of Missouri’s Bond Life Sciences Center is verified, we’ll soon see that plants and mice aren’t all that different, either.

Dan Leuchtman studies a gene in Arabidopsis plants called SRFR1, or “Surfer One.” SRFR1 regulates plant immune systems and tell them when they are infected with diseases or illnesses. Leuchtman studies this model plant as a Ph.D. candidate at MU, splitting time between the labs of Walter Gassmann and Mannie Liscum.

His research involves breeding Arabidopsis plants missing the SRFR1 gene and then replacing it with the MmSRFR1 gene.

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A series of Arabidopsis plants show the differences between the plants, from left, without SRFR1, with MmSRFR1 and with SRFR1. | Photograph by Justin L. Stewart/MU Bond Life Sciences Center

So, what is MmSRFR1? Leuchtman and company believe it’s the animal equivalent of SRFR1, though they aren’t fully aware of all of its’ functions.

“We’re actually one of the first groups to characterize it,” Leuchtman said.

Arabidopsis plants missing the SRFR1 gene struggle to grow at all, appearing vastly different from normal plants. Leuchtman says that a plant missing the SRFR1 gene is a mangled little ball of leaves curled in on itself. “It’s really strange looking.”

While his experiments haven’t created statuesque plants equal to those with natural SRFR1 genes present, the Arabidopsis plants with MmSRFR1 show a notable difference from those completely lacking SRFR1. Leuchtman says the plants with MmSRFR1 lie somewhere in between a normal plant and one lacking SRFR1.

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University of Missouri PhD Candidate Daniel L. Leuchtman poses for a portrait in a Bond Life Sciences Center greenhouse. Leuchtman has been experimenting with replacing a gene in Arabidopsis plants immune system with a similar gene from mice. | Photograph by Justin L. Stewart/MU Bond Life Sciences Center

“At its’ core, it’s more understanding fundamental biology. How do we work? How do organisms tick? How do you go from DNA in a little bag of salts to a walking, talking organism?” Leuchtman said. “The more you know about how an organism functions, the more opportunities you have to find something that makes an impact.”

What’s in the spit?

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.

 

Bond Life Sciences Center Scientists Named to Thomson Reuters’ 2015 List of Highly Cited Researchers

By Bobby Remis | MU Bond Life Sciences Center

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.

16458632870_36bcd5480e_k-e1427818315742-370x533Chris 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 Shuqun ZhangNatural 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 LiuYidong 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.

You shall not pass: the basic science of blocking HIV

 

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

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

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

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.

Family genes

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.

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 how to apply ear medication to a nervous small dog at Horton Animal Hospital on Forum.

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.

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.

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.

 

Putting down roots

Plant scientist Ruthie Angelovici joins the Bond Life Sciences Center

By Jennifer Lu | MU Bond Life Sciences Center

Ruthie Angelovici

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.”

Maze Runners

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

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.

Five years after the Consortium Linking Academic and Regulatory Insights on BPA Toxicity, or CLARITY-BPA for short, launched, results are beginning to come in. This new information will allow researchers to better compare the effects of fixed doses of BPA on the brain, various cognitive behaviors, reproduction and fertility, accumulation of fat tissue, heart disease, the immune system, and several types of cancers.

“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.

Understanding spit

Scientists find how nematodes use key hormones to take over root cells

Roger Meissen | Bond Life Sciences Center
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. (N=nematode; S=Syncytium). Contributed by Carola De La Torre

This Arabidopsis root shows how the beet cyst nematode activates cytokinin signaling in the 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. (N=nematode; S=Syncytium). Contributed by Carola De La Torre

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.

A team at the University of Missouri Bond Life Sciences Center collaborated with scientists at the University of Bonn in Germany to discover genetic evidence that the parasite uses its own version of a key plant hormone and that of the plants to make root cells vulnerable to feeding. Their research recently appeared in Proceedings of the National Academy of Sciences.

Melissa Mitchum

Melissa Mitchum

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

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.”

Mitchum is a Bond LSC investigator and an associate professor of Plant Sciences in the College of Agriculture, Food and Natural Resources. The study “A Plant Parasitic Nematode Releases Cytokinin that Control Cell Division and Orchestrate Feeding-Site Formation in Host Plants” recently was published by the Proceedings of the National Academy of Sciences and was supported by the National Science Foundation (Grant #IOS-1456047 to Mitchum). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.