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Gres receives Von Schwedler Prize

Work on HIV capsid proteins earns prestigious retrivology award

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Anna Gres studies HIV capsid protein using X-ray crystallography. She recently won the 2016 von Schwedler Prize, which awards her $1,200 and gives her the oppportunity to speak this spring at the Cold Spring Harbor Retrovirus Meeting, one of the largest retroviral research conferences in the world. | photo by Roger Meissen, Bond LSC

Science is all about structure in the work of Anna Gres.

For the past four years, she’s looked closely at one HIV protein to figure out its shape in order to stop the virus.

“Capsid protein is extremely important during the HIV life cycle. About 1,500 copies of it come together to form the protective core around the viral genome,” said Gres, a graduate student in the lab of Bond Life Sciences Center’s Stefan Sarafianos and a Mizzou Ph.D. candidate in chemistry. “So, if you are able to somehow disrupt the interactions between the proteins or make them different, the virus loses its infectivity.”

Gres takes her work on this protein to a national stage next month when she speaks at the Retroviruses meeting at Cold Spring Harbor Laboratory — one of the most prestigious international conference on retroviruses — as the recipient of the 2016 Uta Von Schwedler prize. The prize recognizes the accomplishments of one distinguished graduate student as they complete their thesis.

HIV capsid protein has been studied for almost 30 years, but it’s been tricky to get a precise depiction of what it looks like. Gres uses X-ray crystallography to essentially capture the protein in all its 3-D glory. This method gives scientists the higher resolution picture to study the molecular structure of capsid protein. Her work allows the Sarafianos lab and others to study how it interacts and connects with other capsid proteins and the host protein factors of the cell HIV is trying to take over.

“In the past scientists had been splitting the capsid protein in two halves and crystallizing them separately. Another approach was to introduce several mutations to make it more stable,” Gres said. “You would think that it shouldn’t really matter if we have a few mutations, but the protein behaves in such a way that even slight changes result in subtly different interactions that are enough for the virus to lose its infectivity. We were able to crystallize the native protein without any mutations and that should give us more accurate picture.”

Now that the Sarafianos lab and Gres have a good idea of what that native protein looks like, they’ve moved on to other mutated versions of the protein that impair virus infectivity. This could give them insight into how scientists can stop HIV.

“Many labs reported numerous mutations in the capsid protein over the past 25 years that either increase or decrease the stability of the core, which often results in a noninfectious virus,” she said. “Right now we are interested in seeing what structural changes accompany these mutations and how they can affect the overall stability of the core.”

A Climate Change Recap

It’s been almost a week since the 2016 MU Life Sciences and Society Program Symposium, “Confronting Climate Change” wrapped up. If you missed the event, check out our Flickr gallery to see a little bit of the excitement.

We also had the chance to chat a few minutes with most of the LSSP speakers who graciously shared their insight on climate change in our lives. Check out these conversations on our YouTube page from the link at the top of the page.

Last but not least, we put together a radio piece for KBIA giving some speaker highlights. Visit our SoundCloud to see more.

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.

 

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.

 

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.

Scientists uncover how caterpillars created condiments

The next time you slather mustard on your hotdog or horseradish on your bun, thank caterpillars and brassica for that extra flavor.

While these condiments might be tasty to you, the mustard oils that create their flavors are the result of millions of years of plants playing defense against pests. But at the same time, clever insects like cabbage butterflies worked to counter these defenses, which then started an arms race between the plants and insects.

An international research team led by University of Missouri Bond Life Sciences Center researchers recently gained insight into a genetic basis for this co-evolution between butterflies and plants in Brassicales, an order of plants in the mustard family that includes cabbage, broccoli and kale.

Chris Pires | Image by Roger Meissen, Bond LSC

Chris Pires | Image by Roger Meissen, Bond LSC

The team published these new insights online in Proceedings of the National Academy of Sciences (PNAS) in June.

“We found the genetic evidence for an arms race between plants like mustards, cabbage and broccoli and insects like cabbage butterflies,” said Chris Pires, an MU Bond Life Sciences Center researcher and associate professor of biological sciences in the College of Arts and Sciences. “These plants duplicated their genome and those multiple copies of genes evolved new traits like these chemical defenses and then cabbage butterflies responded by evolving new ways to fight against them.”

A biting taste

While you might like the zing in mustard, insects don’t.

Compounds, called glucosinolates, create these sharp flavors in plants to defend against caterpillars, butterflies and other pests. Brassicales species first evolved glucosinolate defenses around the KT Boundary — when dinosaurs went extinct — and eventually diversified to synthesize more than 120 different types of this compound.

For most insects, these glucosinolates prove toxic, but certain ones like the cabbage butterfly evolved ways to detoxify the compounds.

“Seeing the variation in the detoxification mechanisms among species and their gene copies gave us important evolutionary insights,” said Hanna Heidel-Fischer, a lead author on the study based at the Max Plank Institute for Chemical Ecology in Germany.

To look at these genetic differences, the team used 9 existing Brassicales genomes and also generated transcriptomes — the set of all RNA in a cell — across 14 Brassicales families. This allowed the team to map an evolutionary family tree of sorts over the millennia, seeing where major defense changes occurred. This family tree was compared with the family tree of 9 key species of Pieridae butterflies, which includes the cabbage butterfly.

Pires and his colleagues identified three significant evolutionary waves over 80 million years, where plants developed defenses and insects evolved counter tactics.

Pat Edger | Image by Roger Meissen, Bond LSC

Pat Edger | Image by Roger Meissen, Bond LSC

“We found that the origin of brand-new chemicals in the plant arose through gene duplications that encode novel functions rather than single mutations,” said Pat Edger, a former MU post doc and lead author on the study. “Given sufficient amounts of time the insects repeatedly developed counter defenses and adaptations to these new plant defenses.”

This back-and-forth pressure resulted in the evolution of many more species of plants and butterflies than in other groups without glucosinolate pressures.

Proving an old concept

Co-evolution is not a new idea.

About 50 years ago two now-renowned biologists, Peter Raven and Paul Erhlich, introduced the idea of co-evolution to science. Using cabbage butterflies and Brassica plants as a prime example, the two published a landmark study in 1964 advancing the idea that two species can mutually influence the development and evolution of each other.

To explore the genetics of how this works, Pires’ lab partnered with Chris Wheat, professor of population genetics in the Department of Zoology at Stockholm University.

“Using Ehrlich and Raven’s principles and models, we looked at the evolutionary histories of these plants and butterflies side-by-side and discovered that major advances in the chemical defenses of the plants were followed by butterflies evolving counter-tactics that allowed them to keep eating these plants,” Wheat said.

Chris Pires and colleagues mapped the evolution of Brassicales and butterflies to find how each evolved to combat the defenses of the other. | Courtesy Chris Pires

Chris Pires and colleagues mapped the evolution of Brassicales and butterflies to find how each evolved to combat the defenses of the other. | Courtesy Chris Pires

This research provides striking support for the ideas of Ehrlich and Raven published 50 years ago.

“We looked at the patterns 50 years ago, and found conclusions that proved correct,” said Peter Raven, professor emeritus of the Missouri Botanical Garden and a former University of Missouri Curator. “The wonderful array of molecular and other analytical tools applied now under leadership of people like Chris Pires, provide verification and new insights that couldn’t even have been imagined then.”

Understanding more about how plants and insects co-evolve could one day lead to advances in crops.

“If we can harness the power of genetics and determine what causes these copies of genes, we could produce plants that are more pest-resistant to insects that are co-evolving with them—it could open different avenues for creating plants and food that are more efficiently grown,” said Pires.

Proceedings of the National Academy of Sciences (PNAS) published the study, “The butterfly plant arms-race escalated by gene and genome duplications,” in June. The National Science Foundation (PGRP 1202793), the Knut and Alice Wallenberg Foundation and the Academy of Finland provided the funding for this research.

Poor parenting or BPA?

Endocrine disruptors alter parent behavior in California mice 

California mice exposed to bisphenol A (BPA) or ethinyl estradiol changed their parenting behavior, according to an MU Bond LSC study. | Photo by Roger Meissen, Bond LSC

By Roger Meissen | MU Bond Life Sciences Center

What if a chemical changes the way an animal parents?

That could happen due to endocrine disruptors like bisphenol A (BPA).

A recent study of California mice exposed to BPA showed parents spend less time feeding, grooming and interacting with their babies, according to University of Missouri research. Even mother mice not exposed to the chemical parented differently if their male partner was exposed during development.

Most studies only use laboratory mice and rats — where the mother is the sole parental provider — so how early contact to BPA may affect the father and his partner remained a critical gap in existing research.

Cheryl Rosenfeld

Bond LSC researcher Cheryl Rosenfeld | Photo by Roger Meissen, Bond LSC

“The nature and extent of care received by an infant is important because it can affect social, emotional and cognitive development,” said Cheryl Rosenfeld, a researcher in MU’s Bond Life Sciences Center and associate professor of biomedical sciences in the College of Veterinary Medicine. “We found that females who were exposed early on to BPA spent less time nursing, so the pups likely did not receive the normal health benefits ascribed to nursing. Likewise, we found that developmental exposure of males and females resulted in them spending more time out of the nest and away from their pups, further suggesting that biparental care was reduced.”

BPA and other endocrine disrupting chemicals like ethinyl estradiol (EE) — found in birth control — concern scientists because they build up in the environment and mimic natural hormones produced by animals, including humans. Everyday exposure to these chemicals can impact offspring development and now have been found to alter adult behavior in test animals.

California mice have special significance for studying parental behavior. Unlike most lab mice, Californian mice pair up to mate and care for offspring. This monogamous behavior could give researchers insight into child rearing behavior found in most human societies and other biparental animals that would be impossible to measure in lab mice and rats.

MU graduate student and primary author Sarah Johnson worked with Rosenfeld to design the study to look at both sexes. Female and male mice were fed one of three diets — food supplemented with BPA or ethinyl estradiol or endocrine-free (control) food — two weeks before breeding. The mice were then randomly paired with the same mate for the entire study. The behavior of both sexes was then tracked for activities like time spent grooming pups, time spent in the nest and time mothers spent nursing.

But how do you measure the behavior of parents?

Rosenfeld’s team depended on hundreds of hours of video footage, taken at particular times of day and night for seven days, starting two days prior to birth. By using infrared cameras they tracked all 56 litters of mice, logging the number of and duration of activities mothers and fathers completed. During this time, the body weight and temperature of the F2 pups, who were not directly or fetally exposed to any chemicals, was logged to monitor their development.

While results showed reduced pup attention from BPA/EE exposed mother mice, the most intriguing result showed that unexposed moms mated with exposed fathers reduced the time they groomed and cared for offspring.

“These female mice have not been exposed here, but if you can see they are still reducing parental care when paired with the BPA/EE-exposed males,” Rosenfeld said. “And what’s even more interesting is that if a mother and father are both exposed, that parental care diminishes further, and becomes even more statistically significant.”

Researchers hope these results will spur others to look at long-term effects of endocrine disruptors on parenting behavior from generation to generation in animal models and, more importantly, in humans, to see if these chemicals can disrupt parental behavior of mothers and fathers, and if so, whether these effects can be transmitted to subsequent generations.

The study, “Disruption of Parenting Behaviors in California Mice, a Monogomous Rodent Species, by Endocrine Disrupting Chemicals,” was funded by the National Institutes of Health (Grant: 5R21ES023150-02) and was published in the journal PLoS One.

Move over Arabidopsis, there’s a new model plant in town

Bond LSC researchers showed for the first time ever that a grass, Setaria viridis, can receive 100 percent of its nitrogen needs from bacteria  when associated with plant root surfaces. This grass will now serve as model for research into biological nitrogen fixation that could benefit crop development. | Photo by Roger Meissen, Bond LSC

Bond LSC researchers showed for the first time ever that a grass, Setaria viridis, can receive 100 percent of its nitrogen needs from bacteria when associated with plant root surfaces. This grass will now serve as model for research into biological nitrogen fixation that could benefit crop development. | Photo by Roger Meissen, Bond LSC

By Roger Meissen | MU Bond Life Sciences Center

As farmers spend billions of dollars spreading nitrogen on their fields this spring, researchers at the University of Missouri are working toward less reliance on the fertilizer.

Less dependence on nitrogen could start with a simple type of grass, Setaria viridis, and its relationship with bacteria. The plant promises to lay groundwork for scientists exploring the relationship between crops and the fixing nitrogen bacteria that provide them the nitrogen amount plants need daily.

“In science sometimes you have to believe because we often work with such small microorganisms and DNA that you cannot see,” said Fernanda Amaral, coauthor and MU postdoctoral fellow at Bond Life Sciences Center. “Before this research no one had actually proved such evidence that nitrogen excreted by bacteria could be incorporated into plants like this.”

Fernanda Amaral, coauthor and MU postdoctoral fellow at Bond Life Sciences Center. | Photo by Roger Meissen, Bond LSC

Fernanda Amaral, coauthor and MU postdoctoral fellow at Bond Life Sciences Center. | Photo by Roger Meissen, Bond LSC

Biological Nitrogen fixation — where diazotrophic bacteria fix atmospheric nitrogen and convert it to ammonium — provides a free way for plants to alter and absorb the nutrient. Farmers have long known that legumes like soybean fix nitrogen due to the symbiosis with bacteria in the soil through development of nodules on their roots, but since grasses like corn and rice don’t form this specialized structures that relationship has been trickier to explore.

Yet in fact, this team’s experiments showed the grass Setaria viridis received 100 percent of its nitrogen needs from the bacteria Azospirillum brasilense when associated with plant root surfaces.

“I believed in these bacteria’s ability, but I was really surprised that the amount of nitrogen fixed by the bacteria was 100 percent,” Amaral said. “That’s really cool, and that nitrogen can make so much of a difference in the plant.”

Worldwide farmers used more than 100 million tons of nitrogen on fields in 2011, according to the United Nations Food and Agriculture Organization. In the same year, the U.S. alone produced and imported more than $37 billion in nitrogen.

This grass can serve as a simple model for research, standing in for grass relatives such as corn, rice and sugarcane to explore a similar relationship in those crops. This research, “Robust biological nitrogen fixation in a model grass–bacterial association,” was published in the March 2015 issue of The Plant Journal.

 

A nutrient, a nuclear reactor and a model plant

Proving that this grass actually uses nitrogen excreted from the bacteria took some clever experiments, a global collaboration and a nuclear reactor.

MU researchers in the lab of Gary Stacey, a Bond LSC investigator, partnered with scientists in Brazil and at Brookhaven National Laboratory in New York to find a robust plant model system.

They screened more than 30 genotypes of Setaria viridis grass, looking for a strong nitrogen fixing response when colonized with three different bacteria strains. They germinated the seeds in Petri dishes and inoculated those three days after germination with a bacterial solution. Then plants were transplanted into soil containing no nutrients. By eliminating nitrogen in the soil, the scientists were able to make sure that the bacteria was the only source of nitrogen for plant.

The team settled on Azospirillum brasilense bacteria, which has been used commercially in South America to improve crop plant growth. It colonizes the surface of the roots and showed the greatest amount of plant growth when associated with plant roots.

Proving that the bacteria truly fixed the nitrogen used by the plant, required exposing plants to radioactive isotopes at Brookhaven National Laboratory. That began with Nitrogen 13, an unstable radio isotope that showed exactly where and how quickly this nutrient was taken up from the bacteria.

A radio tracer chamber at Brookhaven National Laboratory was needed to test if Setaria viridis actually used nitrogen produced by the bacteria. The scientists allowed only one leaf to contact the radioactive nitrogen, so they could truly tell if it was being used. | Photo provided by Fernanda Amaral

A radio tracer chamber at Brookhaven National Laboratory was needed to test if Setaria viridis actually used nitrogen produced by the bacteria. The scientists allowed only one leaf to contact the radioactive nitrogen, so they could truly tell if it was being used. | Photo provided by Fernanda Amaral

“Nitrogen 13 is really sensitive matter with a half-life of less than 10 minutes, and we first thought there wouldn’t be that much nitrogen fixed by the plant,” Amaral said. “We administered Nitrogen 13 only on the roots, quickly scanned the samples and calculated how much of the nitrogen the plants assimilated based on the decay analysis of the tracer.”

This experiment, paired with several others, showed that this model grass truly incorporated the nitrogen released by the bacteria and metabolizes it in several components.

 

Model (plant) citizen

But why does a type of grass that doesn’t produce food matter so much?

The answer is time and simplicity.

“Corn is really good at responding to bacterial inoculation, but it’s very big and takes a long time to produce seeds and also the genome is complex,” said Beverly Agtuca, an MU Ph.D. student who worked on the study. “Setaria viridis is a small plant that can produce a lot of seeds faster, has a pretty simple genome and can serve as a model for research.”

That makes it perfect to explore how the plant actually uses its bacterial partners, and labs around the world are already using this plant model for research.

For the Stacey lab, the next step is to pinpoint the gene in the model grass that makes this possible.

“We want to identify the genes responsible for the interaction between plant and bacteria and meanly the ones involved with the nitrogen uptake,” Fernanda said. “We hope that will allow us to improve plant growth based on the gene to further study.” We believe that our findings can stimulate others studies at this area, which seems to be a promise plant friendly way to apply for promoting a sustainable agriculture, especially to crop systems including bioenergy grass.

Amaral and Agtuca work in the lab of Gary Stacey at Bond LSC. Stacey is a Bond LSC investigator and a Curators Professor of Plant Sciences in the College of Agriculture, Food and Natural Resources at the University of Missouri. Collaborators included researchers at Brookhaven National Laboratory, State University of New York, Federal University of Paraná in Brazil and Federal University of Santa Catarina in Brazil.

Funding for this project came from the National Institute of Science and Technology- Biological Nitrogen Fixation, INCT-FBN, the Brazilian Research Council, Ciência Sem Fronteiras Program, The Department of Energy and SUNY School of Environmental Science and Forestry Honors Internship Program.

Forest at your fingertips: smartphones enhance fieldwork

An MU student uses his cell phone while in Costa Rica. | Photo by Jack Schultz, Bond LSC

An MU student uses his cell phone while in Costa Rica. | Photo by Jack Schultz, Bond LSC

By Jack Schultz | Director of MU Bond Life Sciences Center

“Fieldwork” means many things to researchers, but in the past it often meant working without easy access to communication.

Now cell phones allow my students visiting the La Selva Biological Station in the lowland rainforest of Costa Rica to remain connected.

While our science and journalism majors learn to report on biological research, I find that I can be replaced. As an experienced biologist who has taught and worked in the Costa Rican tropics for some time, I normally serve as a biology resource. After all, our journalism students have little or no science background.

Yet, as students interview scientists working in a rainforest, learn about the forest’s biology and write about it daily, they now can go online to find the answer. Everything from ecological theory to species lists for our forest site are accessible to any student with a WiFi connection. Fortunately, the biological station has good WiFi service.

While I need to prompt searches to help students know what to look for, the answer to “what was that animal?” is just a hyperlink away. I’m carrying a bulky field guide to the birds, but most often find myself online, checking my own recollection of animals, plants, and facts and figures.

Students return from the forest with evidence of what they’ve seen, which is much better than a hand-waving verbal description. Group meals are eaten with one hand on the phone and the other on a fork. The day’s plans can be refined at breakfast by checking the weather forecast for our rainforest site.

Any good journalist acquires as much background as possible before an interview. Our students can do that in short order by visiting websites of the people we meet in the field. Over several days, they can refine their knowledge and questions to get the most from conversations with researchers. When a term or concept arises in interviews, clarification is right there on the phone.

Cell phone use goes well beyond fact checking.

Paper maps melt in the rain, but the students took photos of the maps we were given and use their cell phones to find their way on the forest trails. Many actually take notes on their phones, and some compose essays there. The improving quality of cell phone cameras produces excellent pictures to post with blogs and articles. Some of the students are producing photos that rival the quality of photos I take with my bulky DSLR. And the videos they produce are high quality and easy to edit.

While computers and tablets are the instruments of choice for uploading larger essays, cool observations can go direct from a cell phone to Twitter, Instagram or even Facebook. And posting to personal Facebook pages keep family and friends updated on each day’s adventures. Everyone in our group is in close contact with home, even if home is in Saudi Arabia (in one case).

While I will admit to feeling, at first, that cell phones could ruin the fieldwork experience, my perspective has changed to value it as a professional tool and not just a personal toy.

Now I’ll be in line for a cell phone upgrade when I return home.