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BPA overrides temperature to decide turtle sex

The environmental build-up of bisphenol A (BPA) can result in a life-changing shift for aquatic animals.

For painted turtles, exposure to this chemical can disrupt sexual differentiation,, according to new research in the  General and Comparative Endocrinology.

Scientists at the University of Missouri have teamed up to show how low levels of certain endocrine disruptors like BPA can cause males to possess female gonadal structures in newly-hatched turtles. This collaboration between MU, Westminster College, the U.S. Geological Survey (USGS) and the Saint Louis Zoo exposed turtle eggs to levels of BPA similar to those currently found in the environment.

“It’s important because this is one of the first times we’ve seen low doses of BPA causing disorganization or reorganization of the male gonad to resemble females,” said Dawn Holliday, adjunct assistant professor of pathology & anatomical sciences at MU’s School of Medicine and assistant professor of biology at Westminster College. “We’re not sure what this means in terms of population-level effects, but certainly it can cause some reproductive dysfunction for turtles.”

Painted turtle eggs were brought from a hatchery in Louisiana, candled to ensure embryo viability and then incubated at male-permissive temperatures in a bed of vermiculite. Those exposed to BPA developed deformities to testes that held female characteristics.  Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center

Painted turtle eggs were brought from a hatchery in Louisiana, candled to ensure embryo viability and then incubated at male-permissive temperatures in a bed of vermiculite. Those exposed to BPA developed deformities to testes that held female characteristics.
Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

Endocrine disruptors leach into rivers and streams and concern scientists because of potential effects on animals and humans. While BPA is used as a hardening agent in plastics, it also is used to line cans and in manufacturing where more than 15 billion tons are produced each year.

In the case of painted turtles, these chemicals have potential to alter sex ratios, which are normally regulated by temperature during incubation. Eggs exposed to cooler temperatures normally produce males and those hatched at warmer temperatures produce females.

Turtle eggs incubated at cooler temperatures result in male hatchlings while warmer temperatures cause females. Researchers are measuring the temperature and weight of this turtle. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center

Turtle eggs incubated at cooler temperatures result in male hatchlings while warmer temperatures cause females. Researchers are measuring the temperature and weight of this turtle. Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

In this experiment, turtle eggs were incubated at temperatures known to rear males and dosed with low, medium and high levels of BPA. BPA-exposed turtles were compared to hatchlings not exposed to chemicals as well as a group exposed to high levels of ethinyl estradiol — an endocrine disruptor found in birth control — at the USGS Columbia Environmental Research Center.

These doses resulted in turtle sex organs that should have been male , but abnormally contained female gonadal elements. The low dose represented BPA concentrations found in fields where turtles can nest while the mid and high doses approximate BPA levels near contaminated sites like landfills.

“We exposed the eggs for a limited amount of time right when they were most vulnerable to the effects,” said Cheryl Rosenfeld, a researcher at MU’s Bond Life Sciences Center and an associate professor of biomedical sciences in the College of Veterinary Medicine. “We found that we got partial feminization in more than 30 percent of turtle eggs exposed to BPA despite being incubated at male-permissive temperatures.”

Dawn Holliday (left), Caitlin Jandegian and Cheryl Rosenfeld examine turtle gonadal tissue to determine if BPA affected proper sexual development. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center

Dawn Holliday (left), Caitlin Jandegian and Cheryl Rosenfeld examine turtle gonadal tissue to determine if BPA affected proper sexual development. Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

These results give the team a look into what real-world exposure levels might mean in the wild and a starting point for comparison.

“Turtles are the most endangered vertebrate taxa and they have all sorts of conservation issues coming at them from people harvesting them to disease, and endocrine disruptors are another potentially big whammy they have against their conservation status,” said Sharon Deem, director of the Saint Louis Zoo’s Institute for Conservation Medicine. “This research is a stepping stone, and we are hoping we can apply these results to populations of turtles throughout the state and use these results as a marker to look at endocrine disruptors in the wild.”

Future studies plan to look at the underlying mechanisms behind sexual disruption and will extend the study to animals including fish and mammals. Rosenfeld’s laboratory is in the process of examining how early exposure of turtles to endocrine disruptors might affect cognitive behaviors, including spatial navigation ability.

Fred vom Saal, Curators Professor of Biological Sciences in the College of Arts and Science at MU, Don Tillitt, an adjunct professor of biological sciences at MU and a research toxicologist with the USGS, Ramji Bhandari, an assistant research professor of biological sciences and a visiting scientist with the USGS at MU and Caitlin Jandegian, a senior research technician at MU, all collaborated on the study.

Candling helps determine whether the painted turtle embryo is viable for the experiment. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center

Candling helps determine whether the painted turtle embryo is viable for the experiment. Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

Funding was provided by Mizzou Advantage, an MU initiative that fosters interdisciplinary collaboration among faculty, staff, students and external partners to solve real-world problems in four areas of strength identified at the University of Missouri. These areas include Food for the Future, Sustainable Energy, Media for the Future and One Health/One Medicine.

Harm and response

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Bond LSC’s Jack Schultz and Heidi Appel hold model Arabidopsis plants used in many of their experiments. Roger Meissen/Bond LSC

We often think of damage on a surface level.

But for plants, much of the important response to an insect bite takes place out of sight. Over minutes and hours, particular plant genes are turned on and off to fight back, translating into changes in its defenses.

In one of the broadest studies of its kind, scientists at the University of Missouri Bond Life Sciences Center recently looked at all plant genes and their response to the enemy.

“There are 28,000 genes in the plant, and we detected 2,778 genes responding, depending on the type of insect,” said Jack Schultz, Bond LSC director and study co-author. “Imagine you only look at a few of these genes, you get a very limited picture and possibly one that doesn’t represent what’s going on at all. This is by far the most comprehensive study of its type, allowing scientists to draw conclusions and get it right.”

Their results showed that the model Arabidopsis plant recognizes and responds differently to four insect species. The insects cause changes on a transcriptional level, triggering proteins that switch on and off plant genes to help defend against more attacks.

The difference in the insect

“It was no surprise that the plant responded differently to having its leaves chewed by a caterpillar or pierced by an aphid’s needle-like mouthparts,” said Heidi Appel, Bond LSC Investigator and lead author of the study. “But we were amazed that the plant responded so differently to insects that feed in the same way.”

Plants fed on by caterpillars – cabbage butterfly and beet armyworms – shared less than a quarter of their changes in gene expression. Likewise, plants fed on by the two species of aphids shared less than 10 percent of their changes in gene expression.

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These Venn diagrams show the number of genes expressed due to each treatment and their overlap. Upward pointing arrows indicate upregulated genes, downward pointing arrows indicate downregulated genes. For example, beet armyworm (S. exigua) shared 21 percent of upregulated genes expressed with cabbage butterfly caterpillar (P. rapae). M. persicae and B. brevicoryne are the two types of aphids compared in the study. Courtesy of Heidi Appel

The plant responses to caterpillars were also very different than the plant response to mechanical wounding, sharing only about 10 percent of their gene expression changes. The overlap in plant gene responses between caterpillar and aphid treatments was also only 10 percent.

“The important thing is plants can tell the insects apart and respond in significantly different ways,” Schultz said. “And that’s more than most people give plants credit for.”

A sister study explored this phenomena further, led by former MU doctoral student Erin Rehrig.

It showed feeding of both caterpillars increased jasmonate and ethylene – well-known plant hormones that mediate defense responses. However, plants responded quicker and more strongly when fed on by the beet armyworm than by the cabbage butterfly caterpillar in most cases, indicating again that the plant can tell the two caterpillars apart.

The result is that the plant turns defense genes on earlier for beet armyworm.

In ecological terms, a quick defense response means the caterpillar won’t hang around very long and will move on to a different meal source.

More questions

A study this large has potential to open up a world of questions begging for answers.

“Among the genes changed when insects bite are ones that regulate processes like root growth, water use and other ecologically significant process that plants carefully monitor and control,” Schultz said. “Questions about the cost to the plant if the insect continues to eat would be an interesting follow-up study for doctoral students to explore these deeper genetic interactions.”

Frontiers in Plant Science published the primary study in its November 2014 issue. The sister study can be read here.

 

Holding on: Bond LSC scientist discovers protein prevents release of HIV and other viruses from infected cells

Shan-Lu Liu and Minghua Li, HIV Research at the Bond Life Sciences Center

Shan-Lu Liu, Bond LSC scientist and associate professor in the MU School of Medicine’s Department of Molecular Microbiology and Immunology. Courtesy Justin Kelley, University of Missouri Health System.

Shan-Lu Liu initially thought it was a mistake when a simple experiment kept failing.

But that serendipitous accident led the Bond Life Sciences Center researcher to discover how a protein prevents mature HIV from leaving a cell.

Proceedings of the National Academy of Sciences published this research online Aug. 18.

“It’s a striking phenomena caused by this particular protein,” Liu said. “The HIV is already assembled inside the cell, ready for release, but this protein surprisingly tethers this virus from being released.”

The TIM – T-cell/transmembrane immunoglobulin and mucin – family of proteins hasn’t received much attention from HIV researchers, but recent research shows the protein family plays a critical role in viral infections.  From Ebola and Dengue to Hepatitis A and HIV, these proteins aid in the entry of viruses into host cells.

But its ability to stop the virus from leaving cells remained unknown until now. Liu’s lab stumbled onto this finding in November 2011 when trying to create stable cells for a different experiment. After two months of troubleshooting the HIV lentiviral vector – where genes responsible for creating TIM-1 proteins were inserted into a cell to create a stable cell line that expresses the protein – Liu was confident the vector’s failure was not only interesting but also important.

Shan-Lu Liu and Minghua Li, HIV Research at the Bond Life Sciences Center

Minghua Li, coauthor of the study and an MU Area of Pathobiology graduate student. Courtesy Justin Kelley, University of Missouri Health System.

The lab spent the next two years trying to figure out what was happening. Minghua Li, an MU Area of Pathobiology graduate student, carried out experiments that confirmed the protein’s power to inhibit HIV-1 release from cells, reducing normal viral infection. His experiments showed TIM proteins prevent normal deployment of HIV, created by an infected cell, into the body to propagate.

TIM proteins stand erect like topiary on the outside and inside surfaces of T-cells, epithelial cells and other cells. When a virus initially approaches a cell, the top of each TIM protein binds with fats – called phosphatidylserine (PS) – covering the virus surface. This allows a virus, such as Ebola virus and Dengue virus, to enter the cell, infect and replicate, building up a population inside.

But as the virus creates new copies of itself, the host cell’s machinery also incorporates TIM proteins into new viruses. That causes problems for HIV as it tries to leave the cell. Now these proteins cause the viruses to bind to each other, clumping together and attaching to the cell surface.

“We see this striking phenotype where the virus just accumulates on the cell surface,” said Liu, who is also an associate professor in the MU School of Medicine’s Department of Molecular Microbiology and Immunology. “We consider this an intrinsic property of cellular response to viral infection that holds the virus from release.”

This model shows the  interaction between TIMs and PS among the round HIV virions, as well as that between viral producer cells. This collectively leads to accumulation of HIV virions on the plasma membrane on the outside of the cell. Courtesy Mingua Li.

This model shows the interaction between TIMs and PS among the round HIV virions, as well as that between viral producer cells. This collectively leads to accumulation of HIV virions on the plasma membrane on the outside of the cell. Courtesy Minghua Li.

Further research is needed to determine overall benefit or detriment of this curious characteristic, but this discovery provides insight into the cell-virus interaction.

“This study shows that TIM proteins keep viral particles from being released by the infected cell and instead keep them tethered to the cell surface,” said Gordon Freeman, Ph.D., an associate professor of medicine with Harvard Medical School’s Dana-Farber Cancer Institute, who was not affiliated with the study. “This is true for several important enveloped viruses including HIV and Ebola. We may be able to use this insight to slow the production of these viruses.”

The National Institutes of Health and the University of Missouri partially supported this research. Additional collaborators include Eric Freed, PhD, senior investigator with the National Cancer Institute (NCI) HIV Drug Resistance Program; Sherimay Ablan, biologist with the NCI HIV Drug Resistance Program; Marc Johnson, PhD, Bond LSC researcher and associate professor in the MU Department of Molecular Microbiology and Immunology; Chunhui Miao and Matthew Fuller, graduate students in the MU Department of Molecular Microbiology and Immunology; Yi-Min Zheng, MD, MS, senior research specialist with the Christopher S. Bond Life Sciences Center at MU; Paul Rennert, PhD, founder and principal of SugarCone Biotech LLC in Holliston, Massachusetts; and Wendy Maury, PhD, professor of microbiology at the University of Iowa.

Read the full study on the PNAS website and browse the supplementary data for this work. See more news on this research from the MU School of Medicine.

Critical transport: Bond LSC team finds boron vital for plant stem cells, corn reproduction

Carbon’s next-door neighbor on the periodic table typically receives little attention, but when it comes to corn reproduction boron fills an important role.

According to University of Missouri scientists, tiny amounts of boron play a key part in the development of ears and tassels on every cornstalk. The July 2014 edition of the journal Plant Cell published this research.

“Boron deficiency was already known to cause plants to stop growing, but we showed a lack of boron actually causes a problem in the meristems, the stem cells of the plant,” said Paula McSteen, a Bond Life Sciences Center researcher. “That was completely unknown before, and for plant scientists that’s an important discovery.”

MU post doc Amanda Durkbak, Bond LSC researcher Paula McSteen, research specialist Sharon Pike and Bond LSC researcher Walter Gassmann

Amanda Durkbak, MU Biological Sciences post doc; Paula McSteen, Bond LSC researcher and associate professor of Biological Sciences; research specialist Sharon Pike; and Walter Gassmann, Bond LSC researcher and professor of Plant Sciences.

Meristems are a big deal to a plant. These pools of stem cells are the growing points for each plant, and every organ comes from them. They are how plants can survive for 500 or 5,000 years, continuously making new organs in the form of leaves, flowers, and seeds throughout its life.

“When you mow your grass, it keeps growing because of the meristems,” said Amanda Durbak, first author on the paper and MU biological sciences post doc. “In corn, there are actually hundreds of meristems at the tips and all sides of ears and tassels.”

But without enough boron, these growing points disintegrate, and, in corn, that means vegetation is stunted, tassels fail to develop properly and kernels don’t set on an ear. This leads to reduced yield. Missouri and the eastern half of the U.S. are typically plagued by boron-deficient soil, an essential micronutrient for crops like corn and soybeans, indicating that farmers need to supplement with boron to maximize yield.

 

The tassel-less mutant

The team’s discovery started with a stunted, little corn plant that just couldn’t grow tassels, only created a tiny ear and died within a few weeks. These maligned reproductive organs piqued McSteen’s interest and her team of collaborators set out to figure out which gene was affected in this mutant plant. Graduate student Kim Phillips mapped the mutation to a specific gene in the corn genome involved in transporting molecules across the plant membrane.

But, what was this defect preventing the plant from receiving? Two experiments helped find the answer.

They started by looking at similar genes in other plants and animals. Simon Malcomber of California State University-Long Beach compared the gene – named the tassel-less gene after its mutant appearance – to similar genes in other plants and animals. He found that many were known to make a protein that transports boron and a few other elements.

 

From field to frog

To clinch this hypothesis, McSteen looked to Bond LSC scientist Walter Gassmann and the African clawed frog. Gassmann harvests eggs from these frogs and uses them to “test” the function of genes from both plants and animals.

“What we do is we inject the frog egg with RNA made in a test tube from the corn’s DNA,” Gassmann said. “The egg is a single, living cell that will actually use the message provided by this RNA to make the boron transporting protein and put it in the egg’s membrane.”

Frog eggs don’t naturally have the ability to transport boron, so an uninjected egg in a solution of boron can’t move the element into the cell.

“Corn RNA provides the egg with instructions to make a boron transporter protein, so the boron solution should move from outside to inside the egg,” Durbak said. “The egg should swell, showing this protein moves boron, and, in fact, these eggs swell so much they explode.”

A tank and a bucket guaranteed boron was the culprit. Durbak went back to the cornfield, watering some mutant tassel-less corn with boron fertilizer and other mutant plants with only water.

Only the ones given boron recovered and grew like normal corn plants, showing that the mutant corn has difficulties obtaining enough boron under natural, low boron conditions without this boron transporter. The boron content of the plants were later tested at the MU Research Reactor and the MU Extension Soil and Plant Testing Laboratory, affirming their observations.

 

A closer look

But, what does boron deficiency look like on a cellular level?

To see, the team collaborated with biochemist Malcolm O’Neill at University of Georgia. He looked at the cell walls in the plant and discovered that the pectin was affected. Pectin stabilizes the plant cell wall, and many home canners know pectin for its help in making jelly and jam solidify.  Pectin is strengthened when boron cross-links two carbohydrates together, giving rigidity to the plant cell wall.

“The effect is that it locks in the cellulose, so without it plant cells won’t have nearly the stability,” McSteen said. “What we think is going on is that plant meristems basically disintegrate because they don’t have the support of pectin.”

While McSteen’s team identified the gene that controls the protein for boron transport into a cell, a research team from Rutgers University identified a gene that controls the protein that transports boron out of a cell. See more about both studies in Plant Cell’s  “In Brief” section.

The next step in this research is to look more closely at what happens in these boron-deficient cells early on as they develop to understand the mechanism of boron action in stem cells.

A grant from the National Science Foundation supported this research.

In addition to their Bond LSC appointments, Paula McSteen is an associate professor of biological sciences in the MU College of Arts and Sciences and Walter Gassmann is a professor of plant sciences in the MU College of Agriculture, Food and Natural Resources

A veterinarian abroad: Tanzania

In a second travel log from Bond LSC researcher Cheryl Rosenfeld, learn about the wildlife she encountered in Tanzania this summer. Through the North American Veterinary Community (NAVC), Rosenfeld furthered her veterinary education while encountering wildlife in their natural habitat. See more about the first leg of her trip to Rwanda here

By Cheryl Rosenfeld

In the early morning hours, our group flew from Kigali, Rwanda to the Serengeti in Tanzania. As we began the descent to the dirt runway, we glimpsed our first sight of wildebeest and the awe-inspiring Serengeti plains and I soon boarded “tano,” Swahili for the fifth 4×4 in our convoy. “Tano” soon came to have special meaning, as there are many groupings of five to see in Tanzania: “The Big Five, The Ugly Five, etc.” Emanuel, whose life-long ambition was to attend college to be a guide, led us to see all of these groups of five and then some.

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Cheryl Rosenfeld

From the time we pulled away from the meeting spot, I started photographing animals on one side of the vehicle, but it seemed even more intriguing ones would appear on the other side of the road.

When the NAVC and our veterinary guide, Dr. Carol Walton, organized this trip, it was anticipated that the wildebeest would be in the Western corridor of the Serengeti but she was wrong. It’s no longer the case that the location of the wildebeest migrations can be projected with accuracy. Our lecture the first evening in Tanzania discussed how the wildebeest know to migrate and why past modeling of their migratory pattern is no longer accurate. Theories for the migratory nature of these animals include their ability to smell rain and/or changes in calcium concentrations in the soil. Wildebeest rely heavily on calcium to produce sufficient milk to nurse their calves that expend considerable energy keeping up with the herd.

In the past, the rains, like the wildebeest migration, would occur in similar sites and times throughout the year. Climate change has likely contributed to the rainfall in these sites being less reliable and correspondingly, compromised forecasting where the wildebeest will be located throughout the year. This year the wildebeest decided instead to migrate to the central Serengeti region. Thus, the next day we set off on an over-three-hour journey to this region.

While driving to find wildebeest we came upon new animals and birds including Coke’s Hartbeest, several Masai giraffe, and topis, which seemed to be splattered with oil. Finally, the wildebeest herds starting increasing in size until it reached a crescendo. As far as one could look in all directions there were wildebeest: males, females, and an abundance of baby calves. Eating alongside them were many zebras and their babies, with brown fuzzy fur along their back-end. It was the most amazing spectacle any of us had witnessed.

After seeing the vast wildebeest herds, we then came upon the elephants and lions, along with their babies. In the safety of the vehicle, we watched them engage in their natural behaviors for which no zoo experience can replicate. Both species were incredibly affectionate to the offspring and each other. In the case of the lionesses, each time the females, who were likely related, caught up with another that they had not seen in a short while would lead to emotional bouts of jumping on each other, caressing and licking. It was hardly the acts of a deadly predator. Yet, their existence rests on the wildebeest and other prey. With the wildebeest in this area, it seemed all life was flourishing at this time. A true paradise.

However, even paradise is subject to outside threats. One such threat is the massive poaching of elephants in Tanzania and surrounding African countries with 40,000 being killed last year alone. Prior to 2013, Tanzania had a “shoot to kill” policy for those caught poaching elephants or other endangered species. However, this rule has since been relaxed with the elephant numbers now in severe decline. At this unsustainable rate of poaching, the African elephant may go extinct in the wild in the next few decades.

While we did see some groups of elephants, the size of each matriarch-led group seemed less than those depicted in Sir David Attenborough and National Geographic documentaries. Moreover, one elephant that we came across, which I affectionately referred to as “Stumpy,” bore sad evidence of this brutal practice: He lost part of his trunk in a poacher’s snare. Without a full trunk, this elephant exhibited great difficulty grasping various plant items to place in his mouth.

Our subsequent travel took us to the famous “Olduvai (which should actually be Oldupai) Gorge,” one of the most famous paleoanthropological sites, including the Leakeys’ famous discovery. From there we traveled to the Ngorongoro Crater, a gigantic fracture of the earth’s crust that provides habitat for some 30,000 animals, including the most endangered black rhinoceros that only has 20 to 30 left in this area. The difference between the black and white rhino lies not in their coat color but a structural difference in their lips with black rhino possessing pointed and prehensile upper lip to browse on twigs and leaves. In contrast, white rhino possess a square upper lip to graze on the grasses below.

When Dr. Walton convinced us to set off at first light, around 6 a.m., our goal was to find one of these amazing creatures. Once again, with Emmanuel’s assistance and eagle eyes, what started out as a black dot far off in the horizon began to morph into a recognizable rhinoceros. In utter delight, we strained our necks and photographed as this beautiful animal continued to move along and forage in the distance. It would be inhumane to allow this animal to go extinct. In the case of the rhino, many Asian countries believe that its horn increases male libido, which has never been proved to be the case. This mistaken notion possibly originated from the fact that male rhinos copulate with females for several hours duration. This behavior is, however, not transferrable to men who consume any part of a rhino.

The list of species we saw in Tanzania continued to grow while we drove around the crater with black-backed and common jackals following alongside our vehicle, eland and grants gazelles grazing in the plains, flocks of greater and lesser flamingoes observed in the distance, and more lion prides and spotted hyenas.

The next day, we had our final farewell lunch at the famous Arusha Coffee Lodge, where US presidents have dined. We then set off on our long journey back to the US.

All of us were impacted by the creatures and sites we saw. One male veterinarian, who seemed gruff and quiet for most of the expedition, put it best the night before we left. He unexpectedly stood up after our last lecture and proclaimed that he signed up for the expedition more as an escape from the daily grind of being a veterinarian. However, the trip made such as impression on him to the point that he realized that part of the veterinarian oath on “relieving animal suffering” should include standing up and advocating on their behalf to prevent their brutal murder, as previously occurred in the case of the gorillas and is ongoing for elephants and rhinos. All of us were moved by his emotional comments and the tears welling up in his eyes. Our group was indeed fortunate to see these majestic animals, while they still exist, in their natural environments. I hope there is a wake-up call for nations to come together to prevent their extinction.

A veterinarian abroad: Rwanda

While summer brings a slower pace for many researchers, others use it as an opportunity to learn for their profession and network with others in their field.

Bond LSC researcher Cheryl Rosenfeld recently traveled to Africa to further her learning as a veterinarian. This continuing education gives her the opportunity to learn the newest techniques in the field and network with others to learn what’s current and on the collective minds in veterinary medicine.  Through the North American Veterinary Community (NAVC), Rosenfeld has now gone on three expeditions where participants observe animals in their natural, exotic environments, attend nightly lectures and learn more about the humans near these animals. 

Previous expeditions led Rosenfeld to the Galapagos Islands and the Florida Keys, but her June 2014 trip started in Rwanda and ended in Tanzania. Here’s the first of two entries where Rosenfeld shares here experience. 

By Cheryl Rosenfeld

The fate of animal populations is generally intertwined with the predicament of humans in the area. Nowhere is this truer than in Rwanda. Most people know Rwanda for Dian Fossey’s work with the mountain gorillas and the genocide of more than 1 million Hutus and Tutsis that happened 20 years ago in 1994. In this 100-day period, an average of six individuals were killed per minute. Children that survived were often orphaned and many surviving women suffered being raped and exposed to HIV infection. In all, many still require extensive medical and psychological care. On our flight and checking into our hotel was a medical team from Harvard Medical Center that was there as part of the Clinton Foundation to assist in the medical needs.

We saw the history that continues to shape the country when we first visited a genocide memorial site just outside of Kigali where thousands of individuals were brutally murdered and the Kigali Genocide Museum that was partially funded by an English Jewish Holocaust survivor. The history of the conflict is rooted partially in Western influence that infused a social division. Prior to Europeans colonization, Hutus and Tutsis lived in relative peace and individuals could go back and forth between these two groups. The original difference was that Tutsi individuals owned more than 10 cows. The differential treatment and classification adopted by Europeans began to trigger conflict between the two groups. Prior indicators, including extensive propaganda, were ignored by the United States and United Nations. The museum includes two stained glass windows that depict the evidence that genocide was imminent and failure by other nations to prevent this tragedy. Genocide isn’t unique to Rwanda, though, and the displays describe the commonalities on their sad origins in other countries throughout history.  Outside the museum, there are several mass graves where fresh flowers are placed on a routine basis.

I was originally hesitant about traveling to Rwanda because of this history, but am very glad I took the chance. The Rwandan government has worked hard to turn around and instill pride in the country. Their economy is one of the fastest growing in Africa with construction of new businesses and hotels in Kigali. Moreover, the government has placed a ban on plastic bags and hired teams of individuals to keep the country clean. One Saturday a month, all Rwandans, including the President, are expected to participate in clean-up day, which becomes a convivial social event. While there is still sadness in the eyes of many individuals I met, I also saw hope of something better, which was inspiring to witness.

We were soon off to learn about the mountain gorillas that are now the pride of the country. During Dian Fossey’s time, she battled to prevent poaching of these magnificent and intelligent creatures. The country now realizes the worth of preserving and propagating the mountain gorilla populations. In a reasonable and safe way, they developed a tourist industry to view the various troops of gorillas. It currently costs $750 to spend one hour with the mountain gorillas. The government has restricted access to prevent gorilla habituation and stress from too many tourists.

We spent two days with different troops. While waiting in the morning to find out which troop we were responsible for trekking, we were entertained by local dancers. I regrettably made the mistake of indicating I felt fit to track the one of ten groups that was at the furthest distance.

The group that was involved in trekking the first day was called “Snow” in Kinyarwanda. I believe they received this name because they inevitably reside high in the mountains, which used to have snow. As we set off on our hike, many children came out to say “MooRahHoh”- hello in Kinyarwanda and asked for us to take their picture. We were informed that we should easily return before lunch, and therefore were only provided a package of peanuts. Unfortunately, it took us longer to hike through the forests that transitioned from bamboo to masses of stinging nettles and did not return to the hotel until 6:30 p.m. After more than three hours of hiking and our eyes finally fell upon our first mountain gorilla, the silverback of the group. Even knowing that this was the ultimate goal, we were not prepared for this amazing experience of being so close to a creature in the wild that resembled us.

We had the opportunity to meet the rest of the troop, including several 3 to 4 month old babies that were quite entertaining. The enclosed photos and videos only provide a sliver of the spectacle that we were privileged to be part of these two days of gorilla trekking that made our hunger and continued burning sensation on our face and legs from stinging nettles well worth it.

Hearing danger: predator vibrations trigger plant chemical defenses

Experiments show chewing vibrations, but not wind or insect song, cause response

As the cabbage butterfly caterpillar takes one crescent-shaped bite at a time from the edge of a leaf, it doesn’t go unnoticed.

This tiny Arabidopsis mustard plant hears its predator loud and clear as chewing vibrations reverberate through leaves and stems, and it reacts with chemical defenses. Plants have long been known to detect sound, but why they have this ability has remained a mystery.

University of Missouri experiments mark the first time scientists have shown that a plant responds to an ecologically relevant sound in its environment.

“What is surprising and cool is that these plants only create defense responses to feeding vibrations and not to wind or other vibrations in the same frequency as the chewing caterpillar,” said Heidi Appel, an investigator at MU’s Bond Life Sciences Center and senior research scientist in the Division of Plant Sciences in the College of Agriculture, Food and Natural Resources.

Heidi Appel, investigator at MU’s Bond Life Sciences Center and senior research scientist in the Division of Plant Sciences in the College of Agriculture, Food and Natural Resources, and Rex Cocroft, a professor of Biological Sciences in MU’s College of Arts and Science, found that plants create chemical responses specifically to predator chewing vibrations.

Heidi Appel, investigator at MU’s Bond Life Sciences Center and senior research scientist in the Division of Plant Sciences in the College of Agriculture, Food and Natural Resources, and Rex Cocroft, a professor of Biological Sciences in MU’s College of Arts and Science, found that plants create chemical responses specifically to predator chewing vibrations.

Appel partnered with Rex Cocroft, an MU animal communication expert who studies how plant-feeding insects produce and detect vibrations traveling through their host plants.

“It is an ideal collaboration, that grew out of conversations between two people working in different fields that turned out to have an important area of overlap,” said Cocroft, a professor of Biological Sciences in MU’s College of Arts and Science. “At one point we began to wonder whether plants might be able to monitor the mechanical vibrations produced by their herbivores.”

While Appel focused on quantifying “how plants care and in what ways,” Cocroft worked to capture inaudible caterpillar chewing vibrations, analyze them and play them back to plants in experiments that mimic the acoustic signature of insect feeding, but without any other cues such as leaf damage.

Cocroft used specialized lasers to listen to and record what the plant hears.

“Most methods of detecting vibrations use a contact microphone, but that wasn’t possible with these tiny leaves because the weight of the sensor would change the signal completely,” said Cocroft.

This cabbage butterfly caterpillar munches on an Arabidopsis leaf adjacent to  a leaf where a piece of reflective tape bounces back a laser beam used to detect the vibrations created by its chewing. Roger Meissen/Bond LSC

This cabbage butterfly caterpillar munches on an Arabidopsis leaf adjacent to a leaf where a piece of reflective tape bounces back a laser beam used to detect the vibrations created by its chewing. Roger Meissen/Bond LSC

The laser beam reflects off a small piece of reflective tape on the leaf’s surface to measure its deflection, minimizing contact with the plant. The laser’s output can also be played back through an audio speaker, allowing human ears to hear the vibrations produced by the caterpillar.

Moved by the sound

Recording the sound is just the start.

You can’t put headphones on a leaf, so tiny piezoelectric actuators – essentially a tiny speaker that plays back vibrations instead of airborne sound – is required.

“It’s a delicate process to vibrate leaves the way a caterpillar does while feeding, because the leaf surface is only vibrated up and down by about 1/10,000 of an inch,” Cocroft said. “But we can attach an actuator to the leaf with wax and very precisely play back a segment of caterpillar feeding to recreate a typical 2-hour feeding session.”

Appel and Cocroft tested whether these chewing sounds could create more chemical defenses in the plants and whether these feeding recordings primed defenses when played before an actual caterpillar ate part of a leaf.

“We looked at glucosinolates that make mustards spicy and have anticancer properties and anthocyanins that give red wine its color and provide some of the health benefits to chocolate,” Appel said. “When the levels of these are higher, the insects walk away or just don’t start feeding.”

The researchers played 2 hours of silence to some Arabidopsis plants and 2 hours of caterpillar-chewing noises to others. They then chose three leaves around the plant, and allowed caterpillars to eat about a third of each leaf.  After giving the plants 24 to 48 hours to respond to the caterpillar attack, they harvested the leaves for chemical analysis.

When they found higher levels of glucosinolates in the plants that were exposed to chewing vibrations, they knew they were on the right track.

A similar second experiment went further, testing whether the plants would simply respond to any vibration, or whether their response was specific to chewing vibrations. In this case Appel analyzed anthocyanins, which again were elevated – but only when plants had been exposed to chewing vibrations but not to vibrations created by wind or the sounds of a non-harmful insect.

Past echoes and future promise

While the past is littered with suggestions that people talk to their plants, Appel and Cocroft hope their work is shifting the focus on plant acoustics towards a better understanding of why plants can detect and respond to vibrations.

“The field is somewhat haunted by its history of playing music to plants. That sort of stimulus is so divorced from the natural ecology of plants that it’s very difficult to interpret any plant responses,” Cocroft said. “We’re trying to think about the plant’s acoustical environment and what it might be listening for, then use those vibrational sounds to figure out what makes a difference.”

The National Science Foundation seems to agree with the merit of their endeavor, awarding a grant to extend this project.

The next step includes looking at how other types of plants respond to insect predator sounds and pinpointing precisely what features of the sounds trigger the change in plant defenses.

These questions aim to further basic research understanding of how plants know what’s going on to respond appropriately to their environment. This could one day lead to ways to create better plants.

“Once you understand these things you can mess around with it in plant breeding through conventional methods or biotech approaches to modify plants so they are more responsive in the ways you want to make them more resistant against pests,” Appel said. “That’s the practical application one day.”

This research was published online in the journal Oecologia July 1, 2014 and will appear in print in its August issue.

MU Scientists Successfully Transplant, Grow Stem Cells in Pigs

New line of pigs do not reject transplants, will allow for future research on stem cell therapies

Story by Nathan Hurst/MU News Bureau

COLUMBIA, Mo. – One of the biggest challenges for medical researchers studying the effectiveness of stem cell therapies is that transplants or grafts of cells are often rejected by the hosts. This rejection can render experiments useless, making research into potentially life-saving treatments a long and difficult process. Now, researchers at the University of Missouri have shown that a new line of genetically modified pigs will host transplanted cells without the risk of rejection.

Roberts, Mike

Mike Roberts, courtesy of MU News Bureau

“The rejection of transplants and grafts by host bodies is a huge hurdle for medical researchers,” said R. Michael Roberts, Curators Professor of Animal Science and Biochemistry and a researcher in the Bond Life Sciences Center. “By establishing that these pigs will support transplants without the fear of rejection, we can  move stem cell therapy research forward at a quicker pace.”

In a published study, the team of researchers implanted human pluripotent stem cells in a special line of pigs developed by Randall Prather, an MU Curators Professor of reproductive physiology. Prather specifically created the pigs with immune systems that allow the pigs to accept all transplants or grafts without rejection. Once the scientists implanted the cells, the pigs did not reject the stem cells and the cells thrived. Prather says achieving this success with pigs is notable because pigs are much closer to humans than many other test animals.

Randall Prather, courtesy of MU News Bureau

Randall Prather, courtesy of MU News Bureau

“Many medical researchers prefer conducting studies with pigs because they are more anatomically similar to humans than other animals, such as mice and rats,” Prather said. “Physically, pigs are much closer to the size and scale of humans than other animals, and they respond to health threats similarly. This means that research in pigs is more likely to have results similar to those in humans for many different tests and treatments.”

“Now that we know that human stem cells can thrive in these pigs, a door has been opened for new and exciting research by scientists around the world,” Roberts said. “Hopefully this means that we are one step closer to therapies and treatments for a number of debilitating human diseases.”

Roberts and Prather published their study, “Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency” in the Proceedings of the National Academy of Sciences.

This study was made possible through grants from Konkuk University in South Korea and the National Institutes of Health.

Roberts has appointments in the MU College of Food, Agriculture and Natural Resources (CAFNR) and the MU School of Medicine and is a member of the National Academy of Sciences. Prather has an appointment in CAFNR and is the director of the NIH-funded National Swine Resource and Research Center.

A drug that packs a punch: new compound works better against resistant HIV

Virologist Stefan Sarafianos stands in the atrium of the Bond LSC.

Bond LSC researcher Stefan Sarafianos stands in the LSC atrium. The virologist is an associate professor of molecular microbiology and immunology and Chancellor’s Chair of Excellence in Molecular Virology with appointments in MU’s School of Medicine and the Department of Biochemistry.

Resistance is the price of success when it comes to treating HIV.

Virologists at the Bond Life Sciences Center are helping to test the next generation of anti-AIDS medication to quell that resistance.

Stefan Sarafianos’ lab recently proved that EFdA, a compound that stops HIV from spreading, is 70 times more potent against some HIV that resists Tenofovir – one of the most used HIV drugs.

“HIV in patients treated with Tenofovir eventually develop a K65R RT mutation that causes a failure of this first line of defense,” said Sarafianos, virologist at Bond LSC. “Not only does EFdA work on resistant HIV, but it works 10 times better than on wild-type HIV that hasn’t become Tenofovir resistant.”

Sarafianos and a team of researchers found that EFdA (4′-ethynyl-2-fluoro-2′-deoxyadenosine) is activated by cells more readily and isn’t broken down by the liver and kidneys as quickly as similar existing drugs.

“These two reasons make it more potent than other drugs, and so our task is to look at the structural features that make it such a fantastic drug,” he said.

 

From soy sauce to virus killer

The path from EFdA’s discovery to current research is a bit unorthodox.

A Japanese soy sauce company named Yamasa patented this molecule, which falls into a family of compounds called nucleoside analogues that are very similar to existing drugs for HIV and other viruses. EFdA was designed and synthesized by Hiroshi Ohrui (Chem Rec. 2006; 6 (3), 133-143Org. Lett. 2011; 13, 5264) and shown by Hiroaki Mitsuya, Eiichi Kodama, and Yamasa to have potential usefulness against HIV. Samples sent for further testing confirmed EFdA’s potential usefulness against HIV. This started more than a decade of research to pinpoint what makes the compound special.

EFdA joins a class of compounds called nucleoside reverse transcriptase inhibitors (NRTIs) that includes eight existing HIV drugs. Like all NRTIs, EFdA hijacks the process HIV uses to spread by tricking an enzyme called reverse transcriptase (RT). RT helps build new DNA from the RNA in HIV, assembling nucleoside building blocks into a chain. Since EFdA looks like those building blocks, RT is tricked into using the imposter. When this happens the virus’ code cannot be added to the DNA of white blood cells it attacks.

“NRTIs are called chain terminators because they stop the copying of the DNA chain, and once incorporated it’s like a dead end,” Sarafianos said.

 

A little help from some friends

Sarafianos isn’t alone in studying EFdA.

The virologist’s lab works closely with University of Pittsburgh biochemist Michael Parniak and the National Institutes of Health’s Hiroaki Mitsuya to explore the molecule’s potential. Mitsuya had a hand in discovering the first three drugs to treat HIV and Parniak has spent years evaluating HIV treatments using cultured white blood cells.

Sarafianos’ focus requires him to take a very close look at EFdA to define how it works on a molecular level. He uses virology, crystallography and nuclear magnetic resonance to piece together the exact structure, bonding angles and configuration of the compound.

By looking at subtle differences in EFdA’s sugar-like ring, his lab identified the best structure that looks the most like actual nucleosides, doesn’t break down easily and is activated readily by CD4+ T lymphocyte white blood cells.

“The structure of this compound is very important because it’s a lock and key kind of mechanism that can be recognized by the target,” Sarafianos said. “We’re looking at small changes and the ideal scenario is a compound bound very efficiently by the target and activating enzyme but not efficiently by the degrading enzymes.”

 

Treatment for the future

The research of Sarafianos, Parniak and Mitsuya continue to uncover the magic of EFdA. In 2012, they showed that the drug worked incredibly well to treat the HIV equivalent in monkeys.

“These animals were so lethargic, so ill, that they were scheduled to be euthanized when EFdA was administered,” said Parniak. “Within a month they were bouncing around in their cages, looking very happy and their virus load dropped to undetectable levels. That shows you the activity of the molecule; it’s so active that resistance doesn’t come in as much of a factor with it.”

HIV prevention is the newest focus in their collaboration.

By recruiting formulation expert Lisa Rohan at the University of Pittsburgh, they are now putting EFdA in a vaginal film with a consistency similar to Listerine breath strips.

“The only way we are going to make a difference with HIV is prevention,” Parniak said. “If we can prevent transmission, this approach could make a huge difference in minimizing the continued spread of the disease when combined with existing therapies for people already infected.”

While AIDS in the U.S. occurs mostly in men, the opposite is true in sub-Saharan Africa where more than 70 percent of HIV cases occur. Since a film has a better shelf life than creams or gels, it could benefit those at risk in extreme climates and third-world countries.

“We have nearly 30 drugs approved for treating HIV infected individuals, but only one approved for prevention,” Sarafianos said. “Women in Africa would benefit from a formulation like this as a means to protect themselves.”

Despite this success, Sarafianos and Parniak aren’t slowing down in figuring out how EFdA works so well.

“We want to understand how long EFdA stays in the bloodstream and cells,” Parniak said. “If we understand structurally why this drug is so potent it allows us to maybe develop additional molecules equally potent, and a combination of those molecules could be a blockbuster.”

Grants from the National Institutes of Health fund this research.

In 2013 and 2014, the journals Retrovirology, Antimicrobial Agents and Chemotherapy and The International Journal of Pharmaceutics published this group’s work on EFdA. Sarafianos is an associate professor of molecular microbiology and immunology and Chancellor’s Chair of Excellence in molecular virology with MU’s School of Medicine and a joint associate professor of biochemistry in the MU College of Agriculture, Food and Natural Resources. 

Chemical beacons: LSC scientist discovers how plants beckon bacteria to attack

Scott Peck studies Arabidopsis and how bacteria perceive it before initiating an infection. Roger Meissen/ Bond LSC

Scott Peck, Bond LSC scientist and associate professor of biochemistry, studies Arabidopsis and how bacteria perceive it before initiating an infection. Roger Meissen/ Bond LSC

Sometimes plants inadvertently roll out the red carpet for bacteria.

Researchers at the University of Missouri Bond Life Sciences Center recently discovered how a plant’s own chemicals act as a beacon to bacteria, triggering an infection. Proceedings of the National Academy of Sciences published their study April 21.

“When bacteria recognize these plant chemicals it builds a needle-like syringe that injects 20-30 proteins into its host, shutting down the plant’s immune system,” said Scott Peck, Bond LSC plant scientist and lead investigator on the study. “Without a proper defense response, bacteria can grow and continue to infect the plant. It looks like these chemical signals play a very large role in mediating these initial steps of infection.”

The question of how bacteria actually know they are in the presence of a plant has puzzled scientists for years. Being able to identify the difference between a plant cell and, say, a rock or a piece of dirt, means the bacteria saves energy by only turning on its infection machinery when near a plant cell.

“Our results show the bacteria needs to see both a sugar – which plants produce quite a bit of from photosynthesis – and five particular acids at the same time,” Peck said. “It’s sort of a fail-safe mechanism to be sure it’s around a host before it turns on this infection apparatus.”

Peck’s work started with one mutant plant called Arabidopsis mkp1.

Discovered several years ago by Peck’s lab, this little mustard plant acts differently than others by rebuffing the advances of bacteria. Lab tests confirmed that this mutant didn’t get infected by Pseudomonas syringae pv. tomato DC3000, a bacterial pathogen that causes brown spots on tomatoes and hurts the model plant Arabidopsis. Along with MU biochemistry research scientist Jeffrey Anderson and post doc Ying Wan, they showed that this mutant didn’t trigger the bacteria’s Type III Secretion System, the needle-like syringe and associated proteins that lead to infection.

Pacific Northwest National Laboratory (PNNL) worked with Peck’s team to compare levels of metabolites between the mutant Arabidopsis and normal plants. This comparison helped Peck identify a few of these chemicals – created from regular plant processes – that existed in much lower levels in their special little mutant.

Using the PNNL work as a guide, the team found five acids collectively had the biggest effect in turning on a bacteria’s infection: aspartic, citric, pyroglutamic, 4-hydrobenzoic and shikimic acid.

“The key experiment involved us simply adding these acids back into the mutant,” Peck said. “Suddenly we saw the mutant plant wasn’t resistant anymore and the bacteria were once again capable of injecting proteins to turn off the plant’s immune system.”

First contact and recognition means all the difference, whether bacteria or plant. Just a slight jump out of the starting blocks by one or the other could change who will win a battle of health or infection.

While low concentrations of these five acids trigger the bacteria’s attack, high levels blind it to the plant’s presence, leading Peck to believe it could be used to hinder bacterial growth. If this actually thwarts the bacteria’s head start, it could mean stopping disease in crops and could lead to a different approach in the field.

“A lot of the winning and losing occurs within the first 2-6 hours and it seems to be that if the microbe is too slow to turn off the immune system, the plant can actually fight off the infection,” Peck said. “In the future we could possibly make a new generation of anti-microbial compounds that don’t try to kill the bacteria, but rather just make them no longer virulent by blocking these chemical signals so the natural plant immune system can basically take over.”

Peck’s team believes at least some other bacteria will respond to these chemical signals, and he plans to test other bacterial pathogens to make certain. They also want to test bacteria to see if they are more virulent in humans once primed for attack by these plant chemical signals.

“In the long run the question is how far this extends. A lot of people get salmonella or listeria infections through a food source,” Peck said. “The question is do other bacteria that come in through plant food sources have similar perception systems and end up being more infectious in humans because they are already primed for infection.”

A $500,000 grant from the National Science Foundation supported this research.