Endocrine disruptors impact physical activity and metabolism in mice
By Caleb O’Brien | MU Bond Life Sciences Center
Could your experiences in the womb make you lazy as an adult?
A recent study of California mice suggests that early exposure to environmental chemicals can later impact an animal’s metabolism and level of voluntary physical activity, according to new University of Missouri research.
“We found that if we developmentally exposed California mice to bisphenol A (BPA) or ethinyl estradiol (EE), the estrogen present in birth control pills, it caused later disruptions in voluntary physical activity,” said Cheryl Rosenfeld, a researcher in MU’s Bond Life Sciences Center and associate professor of biomedical sciences in the College of Veterinary Medicine. “What that means is they move around less in their home cage, they’re more likely to sleep, and they engage in less voluntary physical activity.”
Rosenfeld’s lab studies the ways that exposure to environmental chemicals such as BPA can affect other behaviors, including cognition and parenting. Endocrine-disrupting chemicals can accumulate in the environment and act like the hormones naturally produced by many organisms, including humans. To test the chemicals’ impact on metabolism and activity, the lab used California mice. This mouse model is a good model for metabolic diseases. And because these animals are initially derived from the wild, they may better replicate the genetic diversity of most human populations.
The researchers exposed the mice to BPA and EE in the womb and until weaning via the mom’s diet. A third group of mice whose mothers were placed on a phytoestrogen-free control diet was not exposed to either chemical. The scientists then placed all the mice on this same control diet and measured their energy expenditure, body composition and level of voluntary physical activity as adults.
To test those attributes, Rosenfeld’s lab relied on a variety of tools and techniques. They rigged bicycle computers to “hamster wheels” to track how far, fast and for how long the mice ran. Using a device called a “Promethion continuous measurement indirect calorimetry system” the researchers continuously monitored the mice’s energy expenditure by measuring oxygen consumption and carbon dioxide production and by using a three-beam system, tracked the rodents’ movements during the dark and light cycles.
Later, the researcher measured the animals’ body composition using an EchoMRI, a tiny MRI machine the size of a filing cabinet, and finally measured circulating concentrations of glucose and hormones that regulate metabolism.
Female mice exposed to BPA and EE were less active than control mice. They moved around their cages less at night (when the nocturnal California mouse is considered most active), moved more slowly, drank less water, and spent more time sleeping. In addition, BPA-exposed females burned more carbohydrates relative to fats, as compared to control mice. This is similar to the difference between obese and slender humans, and many researchers believe that burning more carbohydrates relative to fats can lead to fats gradually accumulating in the body.
“It’s worrisome that environmental chemicals we are exposed to in utero can override our genes and disrupt our neuro-circuitry,” said Sarah Johnson, a research specialist and graduate student in Rosenfeld’s lab and primary author on the study. “The net effect is that we can have behavioral disruptions into adulthood, including altered physical activity.”
The researchers are currently conducting follow-up studies to determine if the changes caused by exposure to BPA and EE predispose mice to obesity and other metabolic disorders. They also are interested in exploring if exposure could affect the children and grandchildren of these mice and examining the potential underlying neural mechanisms.
“Our findings are significant because decreased voluntary physical activity, or lack of exercise, can predispose animals or humans to cardiovascular diseases, metabolic disorders and even cancer,” Rosenfeld said.
Other authors on the study are Angela Javurek and Michele Painter (MU Biomedical Sciences), Mark Ellersieck (MU Agriculture Experimental Station- Statistics), Charles Wiedmeyer (MU Veterinary Medical Diagnostic Laboratory and Department of Veterinary Pathobiology) and John Thyfault (Kansas University Medical Center, Molecular and Integrative Physiology)
The study, “Sex-Dependent Effects of Developmental Exposure to Bisphenol A and Ethinyl Estradiol on Metabolic Parameters and Voluntary Physical Activity” was supported by NIH Grant 5R21ES023150 (to C.S.R.) and R01DK088940 (JPT) and was published in the Journal of Developmental Origins of Health and Disease.
James Amos-Landgraf, assistant professor of comparative medicine and genetics at the University of Missouri, is helping develop a pig model for colon cancer using CRISPR. //photo by CALEB O’BRIEN/Bond LSC
James Amos-Landgraf needed a pig.
The assistant professor of comparative medicine and genetics at the University of Missouri had joined forces with a startup company developing a tool to detect early colon cancer-causing lesions. They already tried out a rat-sized model, but still needed a full-sized prototype.
Scientists in Europe had an ideal pig model for colon cancer, but importing the animals presented a problem. It would be prohibitively expensive and time consuming, and the method European scientists used to develop the pig took several years and cost a great many Euros, Amos-Landgraf said.
Those obstacles might have been enough to scuttle the project entirely, but CRISPR, a new gene-editing tool discovered in the DNA of a peculiar bacterium, has changed the equation for scientists everywhere.
So when Amos-Landgraf went to the National Swine Resource and Research Center (NSRRC) to ask about importing pigs, they told him, “‘We can just make you the model,’” Amos-Landgraf said. “‘We should be able to do a CRISPR project within a few months.’”
CRISPR is rapidly reshaping the way biologist around the world do their jobs.
At Mizzou, it’s transforming how researchers learn about viruses and mosquitoes, pigs and zebrafish, and the individual genes affecting development, sickness and health. The tool makes research more efficient, cost-effective and vastly more powerful.
Amos-Landgraf knows firsthand just how time-consuming and laborious generating an animal model was pre-CRISPR.
“What was almost a two-year process just to generate an animal now would take us a matter of months,” Amos-Landgraf said. “I think the CRISPR revolution is going to be amazing for all of science. I’m totally intrigued by everything that’s going on with this.”
Borrowing a bacterial relic
CRISPR rolls off the tongue far more readily than its unabbreviated equivalent: “clustered regularly interspaced short palindromic repeats.” The name refers to a strange pattern scientists at the University of California, Berkeley noticed in the genome of a bacterium that lives in acidic, abandoned mines: groups of palindromic bacterial DNA sequences interspersed with segments of viral DNA.
It turned out that the genetic snippets were relics of the bacteria’s prior run-ins with viral invaders, like genetic mug shots on a most-wanted list.
Viruses are tiny packages of genetic material that hijack cells, such as bacteria, in order to reproduce. And when a virus enters a bacterial cell, the host compares the virus’ genetic material to the snapshots preserved in the bacteria’s own DNA. If they match, the bacteria dispatches a bounty-hunter protein called Cas9, which tracks down the virus and slices its DNA in half at the very spot that matched the virus’ genetic fingerprint.
If an unfamiliar virus attacks and the bacterium survives, it will incorporate a segment of the invader’s DNA into its own, adding a new battle scar to its DNA and a new miscreant to the most-wanted list.
When the researchers studying the bacterial immune system figured out how it worked, they realized the process could have implications far beyond the organism’s acidic abode: It could become a powerful, inexpensive, and versatile gene-editing tool.
A ground shift
The journey to better manipulate genes has been a long one.
For decades, scientists relied on various techniques and tricks to tease out the function of genes. The most common tool is forward genetics, where a researcher starts with an interesting characteristic in an organism and then hunts for the gene that caused it. Those characteristics could be traits that occur naturally, such as genetic diseases in purebred dogs or pigmentation in corn kernels, or a scientist could induce defects — essentially altering an organism’s genome by exposing it to a bath of nasty chemicals.
Anand Chandrasekhar, Bond Life Sciences Center biologist and professor in the division of biological sciences, and PhD candidate Suman Gurung take a look at some zebrafish they’ve modified using CRISPR. //photo by CALEB O’BRIEN/Bond LSC
Imagine that an organism is like a car, suggests Anand Chandrasekhar, a Bond Life Sciences Center biologist and professor in the division of biological sciences.
“You take a car that is running nicely and you have some kind of weird mechanic from Hell come in and mess something up — just one thing — and the car doesn’t run. Then you have to figure out why the car doesn’t run by looking carefully for where the defect is.”
Reverse genetics — unsurprisingly — starts on the other end. Researchers pick a gene of interest and try to silence or alter it. If they succeed, then they look for changes in the organism that suggest the altered gene plays a role in the observed characteristic.
This tool shaped how scientists do research and what animals they use in their labs. In fact, model organisms such as mice rose in popularity partly because of how easily reverse genetic techniques like homologous recombination work with them, said Amos-Landgraf. But this approach was time consuming, expensive and didn’t function well on other organisms.
The next step forward were Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). Both act like guided missiles to strike at a gene of interest, targeting a specific region of genetic material and breaking both strands of the organism’s DNA at that spot. Once the DNA is broken, the cell’s natural repair mechanism intervenes and stitches the gene back together.
However, the process is prone to errors — mutations — that can alter or silence a gene. ZFNs and TALENs reliably worked in a broader array of species.
CRISPRs represent the next advancement in this process and is far faster than previous techniques.
“Let’s say if you had 15 or 20 genes that you wanted to study: You could design a CRISPR reagent for each one of them in a couple of afternoons, whereas in the ‘olden days’ (three or four years ago) with TALENs that could have taken you months,” Chandrasekhar said. “And if you were using ZFNS… you would not even imagine doing it, because you would have been crazy.”
Elizabeth Bryda, a professor of veterinary pathobiology at MU who heads the Rat Resource and Research Center, stands by a Genetic Analyzer machine. //photo by CALEB O’BRIEN/Bond LSC
At MU’s NIH-funded Rat Resource and Research Center (RRRC), scientists think CRISPR will help break dependence on default model organisms. The RRRC is the only center of its kind in the US and one of two in the world, serving as a repository and distribution center for rats that model human diseases.
“We’re always preaching, use the species that’s most appropriate for the question you’re asking,” said Elizabeth Bryda, a professor of veterinary pathobiology at MU who heads the RRRC. “If you’re studying human disease, use the species that best recapitulates that disease. I think CRISPR will give people the flexibility to really work in the species they want to be working in.”
For example, the center is using CRISPR to develop rat models of human inflammatory bowel diseases, such as Crohn’s disease. “All of those barriers to making rat models are no longer issues,” Bryda said, “CRISPR is easy and finally allows us to manipulate rats in ways we haven’t before.”
That’s good news for the RRRC: “I do think we’re going to see a huge increase in the number of rat models,” Bryda said, “which would increase our inventory.”
Seeing through the zebrafish
Zebrafish are another model organism that might become even more important thanks to CRISPR.
Originally found in the rice paddies and streams of India and Myanmar, the minnow-like fish is an important model organism. They’re easy to care for, produce abundant offspring and — because their embryos are transparent — make great tools for studying development.
Chandrasekhar uses zebrafish to study cranial motor neurons, the neurons that connect to, and control, muscles in the head. His lab is especially interested in the way those cranial motor neurons are deployed during development: how the neurons know where to go and to which muscles they should link.
“CRISPR is a really big boon for research, because now even small labs can test tens of genes over a short period of time for their effect on a particular biological process,” Chandrasekhar said. “That’s how we use it: We study the process of cell migration within a nervous system, and we want to study a whole slew of associated genes.”
Researchers have identified hundreds of new and potentially important genes using advance genomics, but the old techniques of reverse genetics were too slow and tedious to keep up with the new discoveries.
“CRISPR has removed the bottleneck,” Chandrasekhar said. “We can rapidly go through and, hopefully, find new genes and new signaling pathways that might be playing a very specific role for the migration and the biological process that we study.”
But finding a new gene is just the beginning, Chandrasekhar said.
“We have one student who is testing five genes, and if even one or two of those genes turn out to be important, that will then be sufficient for the lab to continue working on them for two or three years.”
Although scientists primarily use CRISPR as Chandrasekhar does, to silence genes in model organisms, new genes can also be introduced.
Through a process called homologous directed repair, scientists select a location where they want to introduce a gene and design a CRISPR to target that region.
Daniel Davis, a PhD candidate and lab manager for Assistant Professor of Veterinary Pathobiology Catherine Hagan, is developing a technique to screen potential antidepressant drugs by leveraging CRISPR technology and the advantages of the zebrafish. //photo by CALEB O’BRIEN/Bond LSC
Daniel Davis, a PhD candidate and lab manager for Assistant Professor of Veterinary Pathobiology Catherine Hagan, is developing a technique to screen potential antidepressant drugs by leveraging CRISPR technology and the advantages of the zebrafish.
When a zebrafish is stressed, it produces a neurotoxic compound, but when the fish is calm, it produces a different compound, one that is neuro-protective. The difference depends on which key enzyme the fish produces — in a stressful situation, the fish produces more of the enzyme that leads to neurotoxicity.
Davis is using CRISPR to try to link different fluorescent proteins genes to each branch of this stress pathway: If the fish produces more of the stressful compound, it will also produce a red fluorescent protein. If the other pathway is taken, the fish will assemble green fluorescent protein.
“If you take some fish, subject them to a stressor and test a variety of potential therapeutics on them, you could visualize the fluorescent proteins to see which therapeutics are more protective,” Davis said.
Altering the host to understand the virus
Other models present special challenges. In mosquitoes, for instance, it’s hard to knock out genes from its genome using traditional methods.
“The problem is that in mosquitoes such as Aedes aegypti, ‘traditional’ knockouts never really worked, so people tried out new techniques such as ZFNs and TALENs,” said Alexander Franz, assistant professor of veterinary pathobiology at MU. But the other techniques had flaws, too: they were expensive, complicated to assemble and often posed issues of efficiency and specificity.
Franz studies arthropod-borne viruses (arboviruses), specifically dengue virus and chikungunya virus. The life cycle of an arbovirus requires its circulation between arthropods, such as mosquitoes, and vertebrate hosts, such as humans. Because vaccines exist for only a few mosquito-borne viruses — yellow fever and Japanese encephalitis, for example — people usually rely on conventional and often ineffective environmental controls to thwart disease: bed nets, the elimination of breeding areas, insecticides.
Alexander Franz (far right), an assistant professor of veterinary pathobiology at MU who demonstrated that CRISPR is effective in mosquitoes, stands with his lab. From left to right: Velmurugan Balaraman, Asher Kantor, Hannah Gerlt, and Shengzhang Dong. //Photo courtesy of Alexander Franz
Franz is pursuing a different avenue for protection that uses genetic manipulations to interrupt the transmission cycle of a virus in the mosquito.
“If you can stop the virus from taking hold in the mosquito, you can block transmission of the virus to its vertebrate host,” he said. “But to do so, you need an effective way to manipulate the mosquito’s genome.”
This is where CRISPR comes into play. “When people started reporting using the CRISPR system for genome editing in Drosophila or zebrafish, we immediately had the idea to try it out in mosquitoes.” Working with two postdocs, Franz demonstrated for the first time that the CRISPR system was capable of disrupting genes in mosquitoes.
To do so, he started with a line of transgenic mosquitoes that had already been modified to produce red and blue fluorescent proteins in their eyes. The lab designed a CRISPR to silence the gene responsible for the blue fluorescent protein. After trying a few different methods, they found a technique that turned off the target gene when they injected the CRISPR into mosquito embryos.
Because it is a very powerful and easy-to-handle genome editing technique, CRISPR has been recently utilized and further developed by other groups studying mosquito-pathogen interactions.
Other MU researchers focus on the viral interaction with human host cells.
Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies the way a virus puts itself together inside a host cell and fights off the cell’s defenses. //photo by CALEB O’BRIEN/Bond LSC
Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies the way a virus puts itself together inside a host cell and fights off the cell’s defenses.
“We don’t know all the cellular genes, cellular machinery and cellular pathways that viruses are harnessing,” Johnson said. “The best way to say that a virus requires a particular gene would be to knock it out of the cell and see if the virus can still replicate.”
“CRISPR is a real ground shift in how we can do science,” he said. “Things that took 6 months to a year to make one gene before, now we can do half-a-dozen in a week.”
The technique has altered the rate at which Johnson’s research proceeds and expanded the scope of his lab’s work. “It’s allowed me to take a step back and think about the whole genome, as opposed to being totally focused on this one thing or that one thing,” Johnson said. “I’d never really taken a step back to think about the whole genome — every gene, where are they and what families. It’s changed my outlook on the cell, the way I can think about it.”
The CRISPR era
Amos-Landgraf and the researchers at NSRRC are still in the process of validating their pig model: developing primers to identify the mutation and creating the CRISPRs themselves. Once everything is ready, they’ll test out the lesion-detecting colonoscope, and if all goes well, move into human trials — far faster and more economically than would have been possible a few years ago.
But Amos-Landgraf is tantalized by the possibilities the technology offers beyond increased speed and reduced costs: “To be able to tease apart not just a single gene in a pathway, but maybe think about knocking out or altering all the genes in a pathway and looking at combinations of those pathways… You can start thinking about multiple gene knockouts, multiple gene manipulations all within the same experiment,” he said.
“And that is not only cost saving, but it becomes a really powerful tool when you want to interrogate biology. We’ve entered a new era of genetics and genomics.”
Tommy Langdon waits for a bee to land on a flower. // photo by CALEB O’BRIEN/BondLSC
Emily Fulcher came face-to-face with science while dissecting a hackberry gall: “Ewww,” she exclaimed, “it’s peeking out a little bit!”
Fulcher and 12 other high school students were observing plant galls as part of a Summers @ Mizzou camp exploring “The Arts as a Portal to Science Communication.”
The camp, which ran Sunday through Thursday afternoon, was facilitated by three interdisciplinary scholars and artists adept at bringing together science and the arts: Milbre Burch, an award-winning storyteller, poet and artist; Lee Ann Woolery, leader of the MU Extension Community Arts Program; and Suzanne Burgoyne, director of MU’s Center for Applied Theatre and Drama Research.
The goal of the camp was “to strengthen interdisciplinary research,” Woolery said, and to encourage “imaginative, creative minds from different disciplines to work together.”
Heidi Appel and Jack Schultz explain the complicated, contentious relationship between plants and insects during the Summers @ Mizzou camp “The Arts as a Portal to Science Communication.” // photo by CALEB O’BRIEN/BondLSC
On Monday, Heidi Appel, co-director of the Schultz-Appel Chemical Ecology Lab, and Jack Schultz, director of the Bond Life Sciences Center, were invited to give the students a crash-course on the complex realm of plant-insect interactions and the equally nuanced world of scientific research. They delved into the nature and process of science as a knowledge-generating enterprise: How do we know what we know? What is peer review? Why do grants matter?
After the talk, the students split into groups — half dissected galls and scrutinized their contents under the microscope, while the other group ventured out into the Tucker greenhouse and to the tiny prairie patch next door, where they recorded the sounds of insects pollinating and eating plants. And they visited the lab of Rex Cocroft, professor of biological sciences at MU, where they used lasers to record the vibrations produced when caterpillars munch on Arabidopsis leaves.
Drawings and collages, such as this illustration of a gall by Emily Fulcher, lined the entrance to Ellis Auditorium, where students gave presentations on the the final day of the Summers @ Mizzou camp “The Arts as a Portal to Science Communication.” //photo by CALEB O’BRIEN/BondLSC
The rest of the week, the students explored different artistic media and disciplines to communicate science and deepen their own understanding of research. They painted, wrote, created videos and performed.
On the final afternoon of the camp, they gathered in Ellis Auditorium with the other Summers @ Mizzou camp participants, facilitators and family members for a final presentation. The arts and sciences group read brief, poetic explanations of the research they’d encountered earlier in the week, accompanied by amusing theatrical interpretations.
For Appel, the benefits of the camp go both ways: “Explaining my work to non-scientists makes me a better scientist because I have to clarify and distill my work down to a few key points,” Appel said. Illuminating her work for others affords an opportunity to step back and take a fresh look at the broader context of her research.
Cameron Christensen, a fifteen-year-old student from Jefferson City, said the camp offered a unique opportunity to learn how to convey information in different formats. The challenge of their final presentation, for example, was to “take cold data and make it lifelike,” Christensen said.
Appel, who has worked with all three of the camp’s facilitators in the past, said the camp “helps students understand that science is more than just a collection of facts.”
Cameron Christensen listens to the dulcet vibrations produced by a bug on a plant during the Summers @ Mizzou camp “The Arts as a portal to Science Communication.” // photo by CALEB O’BRIEN/BondLSC
Communicating science is increasingly recognized as a key step in the process of conducting and disseminating research, and invoking the arts can be a valuable tool in that process.
But in a way, putting the arts back in the sciences is just a long-overdue return to a rich heritage of creative polymathy, Woolery said. In the days before ubiquitous photography, keen powers of observation and adroit draftsmanship were de rigueur for anyone doing research — witness Audubon’s exquisite watercolors of America’s birds or Santiago Ramón y Cajal’s Miró-esque drawings of neurons.
For her part, Appel sees deep parallels between the arts and sciences.
“The processes themselves are not that different,” Appel said. “We all make observations and construct mental models. Then we either test a model (if we’re scientists), or we figure out a way to depict that model if we’re artists. Hypothesis testing is peculiar to science, but the other components are not that different.”
On the final day of the Summers @ Mizzou camp “The Arts as a Portal to Science Communication,” the students performed theatrical and literary interpretations of the science they studied. //photo by CALEB O’BRIEN/BondLSC
This immunofluorescence picture shows the brain of an Alzheimer’s disease mouse model, also known as the TgCRND8 mouse. In the picture, the amyloid beta plaques are stained green and the microglia, or immune cells of the brain, are stained red. Image courtesy of Luke Woods.
By Caleb O’Brien | MU Bond Life Sciences Center
Jean Camden and Luke Woods have an ant’s-eye view of Alzheimer’s disease.
Both are bench scientists in the laboratory of Gary Weisman, a professor of biochemistry at the Bond Life Sciences Center. Jean has spent the past 12 of her 35 years at the University of Missouri in the Weisman lab, running experiments, managing the lab and working with students. Luke joined the Weisman lab six years ago, doing what he call’s the dirty work of science: “Gary does the writing and the NIH stuff, but down in the trenches — that’s me and Jean.”
Weisman’s lab studies Alzheimer’s and other diseases, so I sat down recently with Jean and Luke to talk about their research for Alzheimer’s & Brain Awareness Month.
Q: What does your lab do, and how does it involve Alzheimer’s?
Luke: We primarily have two projects. One, which has been a very longstanding project, is focused on salivary glands and salivary gland inflammation. The other is the Alzheimer’s project. The link between them is a particular type of cell surface receptor called a nucleotide receptor — more specifically, a P2 nucleotide receptor called P2Y2. These P2 receptors function in a lot of different ways, but the link is with inflammation: We look at P2 receptors in salivary gland inflammation and in Alzheimer’s disease, which has a very large inflammation component that often gets glossed over. In a lot of Alzheimer’s articles that the public reads, you hear about amyloid beta plaques and tau tangles and neurodegeneration, but a large component of that is inflammation, where some of the resident non-neurons in the brain start responding like there’s inflammation in the brain, and it actually kills neurons. That’s been the focus in Gary’s lab for the past 30-plus years.
JEAN: The P2 receptors — especially the P2X7 and P2Y2 which we focus on — Gary during his postdoctoral work started studying these receptors without really knowing that they existed. At the time, he just knew that there was a pore formed in cells caused by the addition of the nucleotide ATP which eventually leads to apoptosis (cell death). Eventually, we cloned the human P2Y2 receptor gene with another group in North Carolina, so we call it “our receptor.” It only appears in cells under inflammatory conditions, such as Alzheimer’s disease, salivary gland autoimmune disease and cardiovascular disease. Any time you have tissue damage, it looks like the P2Y2 receptor is up-regulated. And then once the damage is healed, the receptor goes away.
Inflammation is good — we want inflammation, that’s how we heal — it’s the chronic inflammation that’s bad. But we really don’t know how these receptors work and what their role is during chronic inflammation. Do we want to activate them, or do we want to inhibit them?
LUKE: Scientists have investigated P2X7 receptor antagonists in the treatment of Crohn’s disease and rheumatoid arthritis — there are several clinical trials that have been focused on these receptors, evaluating whether you want to block or activate them. If you block them, you prevent the acute inflammatory responses that are good for wound healing; if you activate them, you may extend those responses past the healing phase into a chronic inflammatory phase that can be quite damaging. So unraveling that fine line of what you want to be doing to these receptors in disease settings is sort of what we do here.
Jean Camden and Luke Woods look at images of a mouse brain with Alzheimer’s disease. // Photo by CALEB O’BRIEN/Bond LSC
Q: When I think of Alzheimer’s, I think of a shriveled, shrunken brain, but I associate inflammation with swelling. Why the difference?
LUKE: I think the distinction is acute versus chronic inflammation. With acute inflammation, you get swelling. The body has different types of immune responses: acute responders like neutrophils and macrophages are immune cells that act quickly. They come in, for example, if you have a scratch, and there can be swelling. Along with macrophages neutrophils can protect cells from bacteria. The macrophages can also clean up damaged tissue and then the repair cells go to work. Cells come in that lay down a new matrix, whereas undamaged cells then migrate onto the matrix and regenerate. Well, what happens after you’re done repairing is that there are signals that tell the inflammation to stop. In chronic inflammation, that’s where you have continued cell death, and the tissue would then shrivel up. The shriveled brain that you’re referring to is during chronic inflammation, and that’s an end-of-life case, after a very long bout with Alzheimer’s.
JEAN: What we think of as inflammation is often a cut or a wound. It’s only been in recent years that Alzheimer’s disease has been considered an inflammatory disease. We have a phenomenal immune system, but when it goes awry, you have problems. In the other disease we look at — an autoimmune disease — your immune system starts to attack your own body. It’s hard to treat and understand the underlying mechanism.
Q: So how are you trying to unravel the role of inflammation in Alzheimer’s?
JEAN: To study Alzheimer’s, we have an Alzheimer’s mouse model. It overexpresses a gene for the amyloid precursor protein that enables the brain to accumulate high amounts of beta-amyloid plaques that you always hear about. So we’re using this mouse model that we’ve crossed with a mouse that does not express any P2Y2 receptor, so it’s called a knockout mouse. The P2Y2 receptor knockout mouse by itself is fine, and the Alzheimer’s mouse does develop Ab plaques, but it lives to approximately 6 months old before it will develop behavioral defects. The interesting thing is that when we cross the P2Y2 receptor knockout mouse with the Alzheimer’s mouse, the offspring that are Alzheimer’s mice without P2Y2 receptors prematurely die. So at least in this Alzheimer’s mouse model, it looks like the presence of the P2Y2 receptor is protective, because without it, the Alzheimer’s mice die much earlier. But we don’t really know which cell type is most important: Is it the P2Y2 receptor up-regulated on neurons that acts to repair them —which we’ve already shown happens — or is it the P2Y2 receptor on microglia (an immune cell of the brain), or is it the P2Y2 receptor on blood vessels in the brain that help recruit immune cells from the cardiovascular system to help with repair?
So we’re using this mouse model to investigate the role of the P2Y2 receptor, plus we also use cell lines because we can easily control the environment for these cell lines in culture. We isolate primary neurons, we can prepare primary microglial cells or we can purchase cell lines that comprise blood vessels. We can then utilize these tools to investigate cell signaling mechanisms for the P2Y2 receptor in individual cell types.
LUKE: One of the findings that we have found interesting in these primary cells is when you take them fresh out of the mouse, put them in a dish and then treat them as you wish. We’ve shown that if you activate the P2Y2 receptor in primary microglia from the mouse, they will actually engulf and chew up beta-amyloid. And so one of the things we think might be happening in this Alzheimer’s mouse model is that P2Y2 receptor activation in these microglial immune cells in the brain is working to break down those beta-amyloid plaques. And when you lose the P2Y2 receptor in that mouse model, those plaques develop quicker because the immune cells are no longer offering protection by chewing up that beta-amyloid. That’s one of the hypotheses we’re exploring right now.
Q: So you’d bet that these receptors are actually protective against Alzheimer’s?
JEAN: Yes. Going back to the human — it’s hard to get human tissues, especially brain tissues, but there is one published study that has shown that in Alzheimer’s patients who have passed away the P2Y2 receptor is down-regulated, meaning there’s not much left. Which would make sense. If it’s down-regulated, the plaques aren’t able to be chewed up, per se, by these microglia. There’s a correlation between low levels of P2Y2 receptors and Alzheimer’s disease that is apparent at the end of life.
LUKE: It’s very difficult to do some of these studies in humans because most of the available Alzheimer’s tissues are from end of life cases where you can only look at the end result of the disease without looking at the progression of the disease. Obviously you can’t take brain tissue from a living person, so the ability to study live cells from Alzheimer’s patients is limited. We rely very heavily on mouse models.
This immunofluorescence picture shows microglia cells that were isolated from the brain of an Alzheimer’s mouse model called TgCRND8 and cultured in a dish for further analysis. Image courtesy of Luke Woods.
Q: What have been the biggest shifts in our understanding of Alzheimer’s in recent years?
LUKE: Maybe one shift — I may not be the best expert to speak about it — is the idea that the beta-amyloid plaques are the cause of disease. It is now being mostly recognized that they’re really the tombstones of the disease. They’re not the initial cause, but rather the end result of the disease. For a long time investigators were focused on trying to prevent the buildup of beta-amyloid because that was one aspect of Alzheimer’s disease that you could see and measure. Now the thinking is that maybe the beta-amyloid does not contribute as much to disease progression as originally thought, and rather is the end result of a complicated mechanism that is actually causing the neurodegeneration.
JEAN: There’s still debate on what causes Alzheimer’s disease. There is a small percentage of patients where it’s actually related to a genetic alteration in the amyloid precursor protein gene.
LUKE: Another link has been with the ApoE (apolipoprotein E) gene, which makes a lipoprotein and cholesterol transporter. We inherit 1 copy of the ApoE gene from each of our parents and it has been shown that individuals who have at least 1 copy of a particular variant of the gene called ApoE4 are at increased risk of developing Alzheimer’s disease.
Q: From the perspective of a lab scientist, why do you care about Alzheimer’s?
JEAN: We care about any disease, really, and if we can show that our receptors have anything to do with any disease, we’d be proud to have a role in that.
LUKE: We don’t do much clinical science here, it’s mostly basic science. We contribute to the basic understanding of the disease so that drug companies and medicinal chemists who develop drugs for clinical use in Alzheimer’s patients can say, “Hey, this group’s research found a new mechanism related to Alzheimer’s disease, so let’s target this pathway to treat the disease.” It’s always nice to contribute to that sort of ground-level science.
JEAN: That would be the ideal, to show that whether you have to activate or inhibit the P2Y2 receptor, it does something to improve the clinical outcome in Alzheimer’s patients. A better understanding of Alzheimer’s and other diseases is what’s needed — we’re just working to provide a piece of the puzzle.
Q: How has being down in the trenches changed your perspective on research and Alzheimer’sin general?
JEAN: We’re the ones who are hoping to clarify the direction for science to go. We do the experiments and we are the first ones to see the data. We collect the data that becomes the cornerstone for deciding the direction our research goes. I think Gary would agree with that — he depends on us a lot to collect the data and we depend on him to help determine which scientific findings to chase and which ones not to chase.
I’ve been doing this for 35 years, and I really do enjoy the science. I’ve seen the science of these nucleotide receptors come a long way. These receptors have in common their use of extracellular nucleotides, particularly ATP (or adenosine triphosphate, more commonly known as the intracellular high energy molecule of all cells). And this ATP, is at a high concentration inside cells, so when it is released by cell damage, it can easily activate nucleotide receptors on nearby cells. It was Dr. Geoffrey Burnstock, now considered to be the grandfather of nucleotide receptors, who claimed a long time ago that there are receptors on the outside of cells that respond to ATP. Everybody kind of laughed at him, “Yeah, sure, right. There’s no way: ATP belongs inside the cell.” So for me personally, to come in on the ground level for these receptors and find a role for them in a variety of diseases has been exciting for me.
LUKE: ATP is the energy currency inside of all cells, so it’s use outside cells would be like tossing money out the window. Why would they want ATP outside the cell? It didn’t make any sense at the time, but looking back I think it does. What happens if you damage or rupture a bunch of cells during an injury? You get the release of a high concentration of ATP that neighboring cells recognize as a danger signal telling them that an injury has occurred. In that sense, ATP makes the perfect signaling molecule to tell other cells that an injury has occurred and they need to start the repair work by recruiting immune cells to the damaged tissue.
Jean Camden has spent 35 years working at the University of Missouri and more than a decade in Gary Weisman’s lab. // Photo by CALEB O’BRIEN/Bond LSC
Q: Where would you like to be in five years with this research?
JEAN: I talked about determining how the P2Y2 receptor in this mouse model was protective. It would be nice to find out which cell type on which the P2Y2 receptor is expressed in contributes most to neuroprotection. Our hypothesis would be that the microglial cells are very important, since they gobble up beta-amyloid, but other cell types including neurons and endothelial cells are likely involved. We’re also anxious to look at other inflammatory diseases to see if the P2Y2 receptor plays a similar role there.
LUKE: From somebody who does a lot of bench work, something I would like to see is a really good tool, a specific agonist or antagonist of the P2Y2 receptor that could be used in the clinic. There are a few suitable compounds available that we use to investigate the P2X7 receptor— I’ve told you that some have been tested in clinical trials — but the P2Y2 receptor has been sort of an enigma, due to the lack of selective inhibitors and agonists that are specific enough for clinical use. I’d like to see the development of a specific agonist or antagonist that could eventually be used to treat inflammatory diseases. There’s no reliable drug that is currently suitable to investigate the P2Y2 receptor in animals or humans, so clearly more work is needed there.
This interview has been edited for length and clarity.
The HIV capsid protein (shown above in an array of hexagons) plays a critical role in the virus life cycle. Bond LSC researchers recently developed the most complete model yet of this vital protein. Image by Karen Kirby and Anna Gres
Seeing the whole picture can mean a lot when it comes to figuring out HIV.
Researchers at the University of Missouri Bond Life Sciences Center are gaining a clearer idea of what a key protein in HIV looks like, which will help explain the flexible protein’s vital role in the virus life cycle.
“The capsid acts as an invisibility cloak that hides the virus’ genetic information, the genome, while it is being copied in a hostile environment for the virus,” said Stefan Sarafianos, a virologist at Bond LSC and lead author of the study. “Fine-tuned capsid stability is critical for successful infection: too stable a capsid shell and the cargo is never delivered properly; not stable enough and the contents are detected by our immune defenses, triggering an antiviral response. Capsid stability is a key to the puzzle, and to solve it you have to understand its structure.”
This is the most complete model yet of an HIV-1 capsid protein. In a virus, the protein combines in groups of five or six — called pentamers and hexamers, respectively — that assemble into a mosaic that forms the capsid shell. Roughly 1,500 copies of the protein, grouped into about 250 hexamers and 12 pentamers, comprise the capsid.
The protein building block of HIV capsid (top left) can assemble to form a hexamer (top middle). Crystals grown using this building block (bottom left and middle) contain an array or lattice of hexamers (right). | Image by Karen Kirby and Anna Gres
HIV, or human immunodeficiency virus, is the retrovirus that leads to AIDS — acquired immunodeficiency syndrome. Roughly 1.2 million people live with HIV in the United States, according to the Centers for Disease Control and Prevention. Globally, about 35 million people were living with HIV in 2013.
A lucky break
Over the years, scientists have employed various techniques and tricks to figure out the structure of the capsid protein. But until now, the clearest image had been made of a mutated version of the protein. It was a compromise: the mutation made the protein stable enough that the scientists could get a good snapshot, but they couldn’t see the detailed interactions between hexamers.
Sarafianos’ lab figured out how to get the full picture: a detailed image of the unmodified proteins that filled all the gaps between hexamers.
The team used a technique called X-ray crystallography to unravel the protein’s secrets. Basically, they took many copies of the protein and coaxed them into forming a patterned, crystalline lattice. Next they shot high-powered X-ray beams at the crystal. By interpreting how the X-rays scattered when they ricocheted off the proteins, the researchers made a 3-D map of the protein.
Karen Kirby
“But it doesn’t make sense until we make an atomic model of the protein to fit in that map,” said Karen Kirby, a research scientist at Bond LSC. “The map is just a grid that you can’t really interpret unless you put a model into it to see ‘Ok, it looks like this part is here, and that part is there, and this is how the protein is put together.’”
The researchers altered, tested and honed their 3-D model until it exactly matched the map produced by the X-ray diffraction pattern. This can be difficult and painstaking, but the researchers’ greatest challenge was creating the protein crystals in the first place: Scientists had been trying to crystallize the unmodified version of the HIV protein for decades without success.
To make a crystal, proteins are suspended in a liquid then slowly precipitated out, just like a “grow your own crystals” kit. But there are a lot of variables that control the process, from salts and additives in the liquid to the amount of protein in the mixture.
“It’s a very delicate balance to grow crystals,” Kirby said. “Many people call it more of an art than a science. It’s frustrating because you can never predict which solution will grow crystals. There are a large number of variables.”
Initially, most arrangements the researchers tried resulted in useless brown junk, Kirby said, caused by the proteins forming solids too quickly. Anna Gres, an MU chemistry grad student who led the project, used a crystallization robot to screen roughly 2,500 conditions.
That was the easy part, Gres said: “The real challenge begins afterwards, as one needs to manually optimize the initial crystallization conditions to find the one that will produce protein crystals of desired quality. This process can take years. In our case, I think we were lucky: It took approximately 500 manual screenings and about 6 month.” But the hard work paid off when she was finally able to produce lovely, hexagonal crystals. Surprisingly, the crystals formed in groups of six proteins, which matched their formation in the viral capsid.
The transition from tiny, useless particulate to invaluable crystals was tremendously exciting, Kirby said. But even to Kirby and Sarafianos, why their attempts succeeded when many others failed remains a little mysterious.
“I still don’t know what are the fine details that made the difference,” Sarafianos said.
“That’s the million dollar question,” said Kirby. “We really don’t have a good answer for that.”
Although solving the enigmatic crystal structure of the native full-length capsid protein was really rewarding, Gres said, she will continue to tinker with her technique: “I am still trying to optimize crystallization conditions, hoping to improve the quality of the crystals and diffraction.”
Water, water everywhere
Once the researchers got a good look at the interactions between hexamers, they were surprised by what they found.
Based on the genetic sequence of the protein, scientists speculated that they would be hydrophobic, or repel water. Instead, they found that “ordered” water molecules at specific sites played a crucial structural role by bridging interactions at the interface between hexamers.
“We thought, ‘How could these lowly waters really be of consequence?’” Sarafianos said. “But if you think about it, there’s 256 of these hexamers in the whole capsid and all kinds of interfaces among them: There’s thousands of water molecules that stabilize the whole structure. We hypothesize that this is an essential part of the stability of the whole capsid molecule.”
To test that hypothesis, they took the crystals, dehydrated them and checked to see if their shape changed. Although the protein lattices may look like sturdy crystals, they’re more like jello, Sarafianos said.
“The protein molecules are precariously touching each other and forming a lattice that is very, very sensitive. It’s held together in this case by water molecules in addition to other interactions.”
The change in shape suggested that water molecules are important in that they allow the capsid to assume different shapes. Moreover, Sarafianos said, the capsid’s malleability and plasticity could be critical to the life cycle of the virus and allow it to act as a multi-functional molecular Swiss army knife.
Onward with research
A clearer image of the capsid protein, could help Sarafianos’ lab gain a better understanding of how the body combats the virus and to discover new ways to disrupt the viral capsid.
“Now we have a system to study effects of capsid-targeting compounds with novel mechanism of action,” Gres said.
Working with a medicinal chemist, Sarafianos’ lab will undertake an iterative process of making compounds, solving their structures, testing them against HIV and then refining the molecules, with the ultimate aim of producing new and effective antiviral drugs.
“You’ve probably never really seen a fat plant before, right?” said Salie, a fourth year MU graduate student in biochemistry. “Humans, we make plenty of extra fat and store that as energy. But plants don’t really need to do that — they make just as much as they need, and that’s about it.”
Salie studies plant metabolism with Bond LSC researcher Jay Thelen, an associate professor of biochemistry. He’s one of 25 winners honored for research presented during Missouri Life Sciences Week 2015.
The Thelen lab looks for ways to increase the amount of vegetable oil that crops such as corn and soybean can produce. Salie focused on an enzyme that is the first step in the pathway to producing fatty acid in plants.
The idea was that if he could reduce metabolic limits at the beginning of the process, then the downstream production of oil would increase.
“I found these new proteins that no one has ever really studied before,” Salie said. “As I started to look into them over the last year or two, it turns out that they actually seem to incorporate themselves into the enzyme and slow down it’s activity.”
Four separate proteins normally combine to form the functional enzyme, but the new proteins Salie identified mimic those components and can take their place, like a cuckoo bird replacing another species’ eggs with its own. The more mimics that replace proteins, the fewer functional enzymes the plant produces, which means less oil.
It’s a simple, nuanced way for the plant to fine-tune the production of fatty acids.
“Instead of being an on-off switch, it’s more like a thermostat,” Salie said. And if he can adjust that thermostat in a plant, it should start packing on the pounds.
Although Salies work was only recently submitted for publication, it’s already receiving recognition. His poster, “The BADC proteins — a novel paradigm for regulation of de novo fatty acid synthesis in plants,” won first place in the Molecular and Cellular Biology category during the Life Sciences Week poster competition in April.
Salie relished the opportunity to share his findings with researchers and non-scientists alike.
“It’s a great experience, because it helps you realize what’s really important about the work that your doing,” he said. “It also really encourages you to work harder. It’s like, ‘Wow, this is actually meaningful stuff!’ which can be hard to see when you’re working 60 or 70 hour weeks at the lab, just sitting there by yourself.”
Salie was among more than 300 students who presented their research during the 31st annual Life Sciences Week poster sessions.
The winners in each of the five categories are:
Molecular and Cellular Biology
Matthew Salie, Matthew Muller, Stephanie Bowers
Organismal Biology
Miqdad Dhariwala, Ryan Sheldon, Carine Collins
Genetics, Evolution and Environment
Julianna Jenkins, Nathan Harness, and a tie for third between Sharon Kuo and Susheel Bhanu Busi
Life Science and Biomedical Engineering Technologies and Informatics
Jamie Hibbard, Hang Xu, Brittany Hagenhoff
Social and Behavioral Sciences
Vaness Cox and Ian George tied for first place
Undergraduate winners are Vincent Farinella, James Mrkvicka, Anette van Swaay, Romanus Hutchins, Dallas Pineda, Kelsey Boschert, Anthony Onuzuruike, Clare Diester, Adam Kidwell and Sean Rogers.
Honorable mention:
Social and Behavioral Sciences
Undergrad Honorable Mention – Kelsey Clark
Undergrad Honorable Mention – Louie Markovits
Genetics, Evolution, and Environment
Grad Honorable Mentions: Megan Murphy (Schul) and Amanda Smolinsky (Holliday)
Undergrad Honorable mention: Anthony Spates (Holliday)
Organismal Biology
Grad Honorable Mention: Kathleen Pennington
Grad Honorable Mention: Kasun Kodippili
Grad Honorable Mention: Christopher Tracy
Undergrad Honorable mention: Chelsie Todd
Undergrad Honorable mention: Holly Doerr
Undergrad Honorable mention: Zeina Zeida
Molecular and Cellular Biology
Grad Honorable mention, Khalid Alam [Burke lab]
Grad Honorable mention, Zhe Li [Sarafianos lab]
Undergrad Honorable mention: Vincent Markovitz [Guo lab]
Additional prizes were awarded for communication prowess and poster design chops.
For photos of some of this year’s winner, check out this Flickr album
A simple virtue lies at the heart of Xuemin (Sam) Wang’s research: thrift.
“A good way to think of it is how to increase output without demanding more inputs,” Wang said.
Wang, the E. Desmond Lee and Family Fund endowed professor at the University of Missouri-St. Louis and a principal investigator at the Donald Danforth Plant Science Center, studies plant membrane lipids. His lab is focused on understanding the relationship between oil production and plant stresses such as drought and nutrient deficiency.
Wang will speak during the 31st annual Missouri Life Sciences Week, a yearly celebration of MU’s research and an exploration of public policy, entrepreneurship and science outreach.
Wang’s lab uses Arabidopsis, the lab mouse of the plant world, as a discovery tool but also works with crops such as soybean and the Camelina species. Camelina was widely grown in Europe before it was supplanted by canola, but Wang and others are working to develop Camelina as a productive oil crop.
The lab studies how lipids — the fatty acids that make up cell membranes — help regulate cell function. For example, they’re trying to figure out how a cell senses water and nutrients and then “determines whether it should grow faster or store more lipid or carbohydrates,” Wang said.
By understanding those processes, future research might develop plants that do more with less. That could mean less water and chemical fertilizer needed for the same or greater yield. Wang pointed to reliance on fertilizers as a major problem.
“Not only does it drive up agriculture production costs, but there can be major environmental consequences.”
Ultimately, Wang’s research could improve plant oil and biomass production while decreasing our dependence on fertilizers and abundant water.
Wang’s presentation on “Lipids as Molecular Switches in plant stress signaling and metabolic integration” constitutes this year’s Charles W. Gehrke distinguished lecture. Gehrke, a MU professor of Biochemistry who died in 2009, was instrumental in advancing the field of chromatography and helped analyze rock samples retrieved from the moon during the Apollo 11 mission. Gehrke grew up in poverty during the great depression and worked in melon fields during his youth before studying at Ohio State University.
Missouri Life Sciences Week is an annual event. In addition to Wang’s talk, this year’s line-up will also focus on HIV and emerging diseases and highlight more than 300 undergraduate and graduate research projects at its poster sessions.
Joya Chandra, associate professor of pediatrics at The University of Texas MD Anderson Cancer Center, explains the epigenetics of pediatric cancers at the 2015 MU LSSP Symposium on epigenetics on Sunday, March 15.//photo by Caleb O’Brien/Bond LSC
The evolving science of epigenetics is shaking up how scientists and doctors think about cancer.
At the 11th annual University of Missouri Life Sciences & Society Program symposium, scientists, historians and philosophers explored the epigenetic theme. On its final day, two researchers spoke about how cancer and epigenetics intersect.
Epigenetics involves changes to how genes work that do not occur within DNA. Epigenetic changes can, in effect, turn genes on or off, and those changes can then be passed from generation to generation. Because cancer involves cells that grow uncontrollably, don’t die and can travel throughout the body, it makes sense to look for epigenetic changes that could account for cancer’s unusual attributes.
Shuk-mei Ho, director of the University of Cincinnati Cancer Center and Chair of the Department of Environmental Health, discussed the developmental origins of health and disease (DOHaD) hypothesis, which suggests that problems early in development or during other key periods such as puberty and pregnancy can have wide-ranging implications later in life.
She centered on whether early exposure to a high fat diet and endocrine disruptors such as bisphenol A can affect an offspring’s risk of developing certain types of cancer.
“These studies are really important because they help us identify some early warning signs that may be related to epigenetics,” she said “They allow us to devise measures that we can use to interfere with and prevent development of cancer.”
A proactive approach to cancer could harness science’s newfound understanding of epigenetics and target cancers before they start.
“It is much more effective to have prevention rather than have the cancer and devise ways to treat it,” she said. “This will minimize a lot of suffering and also reduce the cost of the health care.”
Prevention is especially important because epigenetic effects can arise quickly and persist for multiple generations. For example, breast cancer rates in immigrant populations can increase within one generation of arrival. In mice, scientists have detected the lingering effects of a high fat diet after three generations.
Epigenetics and childhood cancer
Pediatric oncology is especially apt for integration with epigenetics.
Joya Chandra, associate professor of pediatrics at the University of Texas MD Anderson Cancer Center, studies therapies for childhood cancers. She said developments in epigenetics have helped researchers better understand childhood cancers and the ways they differ from adult cancers.
“The field of epigenetics has just exploded in terms of giving us insight into how pediatric cancers are different from adult cancers.” Chandra said. “Just in the past 2-3 years we’ve learned a lot about cancers that are present in adults and children that have really different epigenetic profiles. This presents an opportunity for pediatric cancer oncologists to treat these cancers differently, and increasing knowledge about epigenetics will help serve that goal.”
leukemia, for example, is the most common pediatric cancer but only the sixth most common among adults. And about 70 percent of infants with leukemia exhibit epigenetic changes associated with their cancer.
Children with glioblastoma, a type of brain cancer, exhibit epigenetic traces that are distinct from the same kind of cancer in adults.
A deeper understanding of epigenetics and of how cancer in children differs from adult cancer could allow for the development of new medicines, novel therapies and powerful strategies for proactively protecting patients from certain types of cancers.
Roger Meissen/Bond Life Sciences Center – These soybean roots show some nematode cysts. The small, white circles are the hardened body of the nematodes and form when the nematode attaches itself to the root to create a feeding cell.
Beneath a North Carolina field in 1954, a tiny worm inched its way through the soil and butted against a soybean root. The worm pierced the plant, slipped inside and inserted a needle-like appendage into a cell. It pumped a mixture of proteins into the root cell and waited for the potent blend to take effect on the unsuspecting soybean.
Since the first detection of soybean cyst nematode (SCN) in the US, the worm Heterodera glycines has spread to about 80 percent of American soybean fields. In Missouri, SCN attacks soybeans in almost every county and causes decreased yields even in robust, healthy-looking fields. Nationwide, SCN wreaks havoc to the tune of $1.2 billion per year, making it by far the most costly soybean pest.
Despite the hefty toll, farmers still depend on the same small handful of resistant soybean varieties to combat SCN that they have used for years. But those natural defenses are becoming less effective as nematodes evolve.
“More than 90 percent of the soybean cultivars that farmers plant derive their resistance from a single source,” said Melissa Mitchum, a plant nematologist at the University of Missouri Bond Life Sciences Center and Division of Plant Sciences faculty member in the College of Agriculture, Food and Natural Resources. “Consequently, this has led to widespread virulence in the pathogen population, thereby reducing the effectiveness of those resistant cultivars.”
But in the past 10 years, researchers studying SCN have made numerous breakthroughs, unlocking the secrets of the nematode and exploring how the worm interacts with host plants. Now, scientists are poised to bring that knowledge from the laboratory to the field.
Found in translation
Relatively little was known about SCN a decade ago.
Scientists could determine the type of nematode in a soil sample and had just figured out the cocktail of proteins a nematode pumps into the soy root cell that transform it into a syncytium, or feeding cell.
Working in part with funding from commodity boards and farmer checkoff dollars, researchers around the country made breakthrough after breakthrough, deepening our understanding of SCN and equipping scientists with new tools to fight the pest.
That money helped scientists sequence the soybean genome, draft a SCN genome and pinpoint important soy and SCN genes.
Checkoff investments continued to pay dividends in 2012 when Mitchum and colleagues cloned the first gene linked to natural soybean cyst nematode resistance. This breakthrough is one key step in moving science from the laboratory into the field. With a SCN resistance gene in hand, new avenues for creating soybean varieties that fight off the nematode are opening up.
But other areas of research also hold promise in the struggle against soybean cyst nematode’s parasitic ways.
Mitchum’s group also identified the plant receptors that recognize and respond to the blend of proteins an attacking nematode inserts into a plant. In a recent project published in Plant Biotechnology Journal, Xiaoli Guo, a postdoctoral fellow in Mitchum’s lab demonstrated that silencing those receptors in soybean roots helped the plant resist SCN.
This work has implications for more crops than just soybeans: Working with collaborator Xiaohong Wang at Cornell, Mitchum’s group used their understanding of plant receptors to develop a potato resistant to potato cyst nematode.
A roadmap for discovery
To build on the momentum of recent research, experts drafted a roadmap for the next decade of nematode research. Their goal, Mitchum said, is to address the challenge of translating these research breakthroughs into something tangible for the farmer.
With support from state farmer run organizations such as the Missouri Soybean Merchandising Council, the North Central Soybean Research Program and the United Soybean Board, researchers are formulating teams that “bring together commodity, industry and university funding to develop collaborative, interdisciplinary, multistate projects,” said Mitchum.
And there’s plenty of scientific firepower to advance research: MU’s College of Agriculture, Food and Natural Resources alone has more than 90 faculty studying plant science, plant genetics and other areas of agriculture-related science.
The scientists’ plan for the next 10 years involves a blend of molecular research, plant breeding, population biology and outreach. Researchers will focus on refining the existing draft SCN genome, which will help to develop a quick, inexpensive test for HG type and eventually contribute to understanding of how SCN overcomes a plant’s resistance. They’ll create an “atlas” of SCN genes researchers can use to block the pest. Updating yield loss estimates and mapping SCN distribution will also give scientists a better idea of the nematode’s national impact. Other efforts will allow breeders to incorporate new sources of resistance into commercially-available varieties, refine the use of non-host species to control SCN and develop a pipeline for creating and testing transgenic SCN-resistant soybeans. Finally, videos, webinars and training modules will help scientists, students and producers take advantage of new discoveries and techniques.
Roger Meissen/Bond Life Sciences Center – Michael Gardner, Ph.D. student, Melissa Mitchum, associate professor of Plant Sciences, Xiaoli Guo, post doctoral fellow, conduct research at the University of Missouri. They investigate how soybean cyst nematode overcomes soybean resistance to identify novel approaches for management.
Onward with research
A thorough understanding of SCN resistance and virulence starts with basic research and then moves into the field. “We all need to come together to transfer this knowledge to the breeder,” Mitchum said, “and from there it gets out to the farmer.”
Her lab recently received a National Science Foundation grant to continue their work on soybean protein receptors. Specific targeting of the receptors is just one potential strategy for producing new kinds of SCN-resistant plants. A second grant, from the National Institute of Food and Agriculture, will allow the lab to continue refining their understanding of how SCN proteins overcome a host plant’s defenses. To that end, Mitchum’s graduate student Michael Gardner is identifying the genetic blueprint of the different SCN types present in Missouri fields.
“If we better understand nematode populations and what makes those populations distinct, we can better advise farmers confronted with virulent nematodes,” Gardner said. “We’ll be able to go one step beyond the HG type test and understand how nematodes are able to adapt in the long term, not just the next growing season.”
But these breakthroughs do little good unless they then become useful tools for breeders and ultimately farmers. To that end, Mitchum and other researchers will help breeders use research results to produce soybeans with durable resistance. They‘ll also develop guides so farmers can easily incorporate new technologies and management strategies into their farms.
It’s important for farmers, breeders and researchers to take a unified approach to fighting SCN, Mitchum said, because a tactic that seems successful at first could backfire.
For instance, combining resistance genes in a single soybean variety could actually be harmful. “When we deploy it in the field, we select for nematodes that can overcome multiple types of resistance,” Mitchum said.
A better approach might be to perfect varieties with distinctive resistance mechanisms and insure durable resistance by rotating among the resistant varieties and non-host crops.
“It’s similar to taking antibiotics,” Mitchum said. “Improper use and overuse selects for resistance.” The strategic planning document should help everyone working with soybeans and SCN leverage and build upon new knowledge.
Despite all the research and recent breakthroughs, there remains only one certainty in the ongoing arms race between soybeans and SCN: “It is highly unlikely that we will eradicate it.” Mitchum said, “We’re going to have to find new strategies to protect and bolster soybean yields.”
Thanks to the efforts of researchers such as Mitchum, in the future SCN might be a little easier to get along with.
Roger Meissen/Bond Life Sciences Center – Research specialist and coordinator for the Plant Nematology Lab Amanda Howland processes soil samples for nematodes. Howland replaced Bob Heinz earlier this year.
University of Missouri Plant Nematology Laboratory: An extensive legacy
Bob Heinz spent his last day at work in December surrounded by nematodes. Heinz served as Mitchum’s research specialist and coordinator of the Plant Nematology Laboratory, where he processed soil samples, responded to growers and assisted researchers. After 35 years on the job, he’s retired, and Amanda Howland is now filling his shoes. The scientists and farmers who’ve worked with Bob over the decades thank him for his dedication and wish him luck in his retirement. And Amanda: Welcome aboard.
The Plant Nematology Lab, housed within Mitchum’s lab at MU, represents a successful model for how research, teaching and extension program integration can promote interdisciplinary collaboration. Such an approach helps maintain an effective pipeline that brings research-based information and resources from MU to Missourians. The lab offers an array of tests that help farmers understand and manage nematode populations. The available tests include:
–Vermiform Nematode Identification: Soybean Cyst? Root Knot? Lesion? Find out what kinds of nematodes are in your fields with this test.
–Soybean Cyst Nematode Egg Count: This procedure provides an estimate of the number of SCN eggs in your field.
–Soybean Cyst Nematode HG Type Test: Different types of SCN have overcome various sources of soybean resistance. A HG type test will help you determine the best source of resistance for the particular type of SCN in your field.
Epigenetics involves changes in how your genes work.
In classical genetics, traits pass from generation to generation in DNA, the strands of genetic material that encode your genes. Scientists thought alterations to the DNA itself was the only way changes could pass on to subsequent generations.
So say you lost a thumb to a angry snapping turtle: Because your DNA hasn’t changed, your children won’t be born with smaller thumbs. Classic.
Things get way more complicated with epigenetics. It turns out that some inherited changes pass on even though they are not caused by direct changes to your DNA. When cells divide, epigenetic changes can show up in the new cells.
Getting nibbled on by an irate turtle isn’t likely to epigenetic changes, but other factors such as exposure to chemicals and an unhealthy diet, could cause generation-spanning epigenetic changes.
How does it work?
The main players in epigenetics are histones and methyl groups.
Imagine your genes are like pages in a really long book. Prior to the mid-1800s, books came with uncut edges, so in order to read the book, you’d have to slice apart the uncut pages. That’s sort of what a histone does to DNA: They are proteins that wrap DNA around themselves like thread on a spool. They keep the DNA organized and help regulate genes.
Methyl groups (variations on CH3) attach to the histones and tell them what to do. These molecules are like notes in a book’s margin that say, “These next few pages are boring, so don’t bother cutting them open.” As you read the book, you’ll save time and effort by skipping some sections even though those sections still exist. Or maybe the note will say, “This next section is awesome; you’ll want to read it twice.”
That’s epigenetics. Higher level cues that tell you whether or not to read a gene.
And when a scribe makes a copy of the book, they’ll not only copy all the words in the novel, but all the other stuff, too: the stuck-together pages and the margin notes.
What about my health?
Many areas of health — including cancer, autoimmune disease, mental illness and diabetes — connect with epigenetic change.
For example, scientists link epigenetic changes to neurons to depression, drug addiction and schizophrenia. And environmental toxins — such as some metals and pesticides — can cause multigenerational epigenetic effects, according to research. Once scientists and doctors decipher those processes work, they will be better equipped to treat the sick and be able to take preventative measures to help insure our health and the health of our kids.
Is it epigenetics or epigenomics?
Confusing, I know.
As we learned from the first question, epigenetics is “the study of heritable changes in gene function that do not involve changes in the DNA sequence,” according to my trusty Merriam-Webster.
Epigenomics is the study and analysis of such changes to many genes in a whole cell or organism. It’s comparable to the difference between genetics (dealing with particular pieces of DNA, usually a gene) and genomics (involving the whole genetic shebang).
Where can I learn more?
Start at this year’s Life Sciences and Society Program Symposium, “The Epigenetics Revolution: Nature, Nurture and What Lies Ahead,” on March 13-15, 2015. Speakers from all over the country will delve into the puzzles and possibilities of epigenetics.
For more background, Nature magazine also created this supplement on epigenetics.
