The unusual red color of the Lobelias leaves make them stand out among 200 other species that thrive in the 20-foot plant wall at the Bond Life Sciences Center | Paige Blankenbuehler
By Madison Knapp | Bond LSC
A hidden treasure on the University of Missouri’s campus is a living and breathing work of art.
In the Christopher S. Bond Life Sciences Center, a 20-foot plant wall stands as a towering tribute to the diversity of plant life and coexistence of species — a botanical landscape of more than 200 individual plants from 40 to 50 species including many ferns, vines, perennials, orchids and geraniums.
The display continues to be a visual reminder of the building’s interdisciplinary nature – much like the plants within the wall, the Bond Life Sciences Center facilitates the coexistence and collaboration between an array of researchers within its walls.
One plant cascades down the wall and stands out more than most — it looks more like a large insect than any type of plant typically seen in such displays.
Tillandsia is a genus of succulent plant native to Central America that can undergo long dry spells. The wiry plant thrives on the stone beside the plant wall without the help of a pot of soil, and is seemingly absent an essential, anatomical feature of most flora — roots.
With leaves more like Medusa’s hair, Thallandsia are rootless plants mounted on stone alongside the plant wall. | Paige Blankenbuehler
They still have tiny root protrusions, but the mass is minuscule compared to the thick leaves, which take up and store water and nutrients, rather than the root system which handles that work in most plants.
Approximately 60 Tillandsias pepper the plant wall, including a few mounted directly onto the stone wall with a special kind of non-toxic rubber cement manufactured by Davis Farms based out of San Diego.
The strange plant was added to the green tapestry by Jason Fenton, an office support associate at the Bond Life Sciences Center. He first noticed the rootless plants in Belize while on a trip there in 2003 and had been growing them at his home.
“I think they’re really beautiful and distinctive,” Fenton said. “They help give variety to the wall.”
The rootless plants require special treatment — just a little extra attention from facilities manager, Jim Bixby, who waters the Tillandsias with a spray bottle once a week.
The wall started with a vision from Jack Schultz, director of the Bond Life Sciences Center. The execution and maintenance of the feature is credited to Bixby, who has honed the project since 2010.
After trying several watering systems, Bixby found one that worked. He hung rows of pots with modified flat sides and tailored a simple, drip irrigation system made for easy watering along a grid.
Within the diverse landscape and after additions of a variety of plants over the years, Bixby has seen competition between several of the species.
The wall favors the ferns, which have taken over much of the space, crowding out other plants for the eastern sunlight that flows through the windows, Bixby said.
Several long-stemmed species have found ways to cope, however; venturing out between the ferns’ curtain-like fronds to get their fair share of sunlight.
Supervising editor: Paige Blankenbuehler
The 20-foot tall plant wall outside of Monsanto Auditorium in the Bond Life Sciences Center is a nod to coexistence and diversity. | Paige Blankenbuehler
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.
In June 2014 Cheryl Rosenfeld traveled to Rwanda to observe mountain gorillas as part of her veterinary continuing education.
In June 2014 Cheryl Rosenfeld traveled to Rwanda to observe mountain gorillas as part of her veterinary continuing education.
The 1994 Genocide is a omnipresent part of the national psyche of Rwanda.
Sites like this memorialize the millions killed in the 1994 genocide.
Local children from one Rwandan village.
Rosenfeld is pictured in the blue four drums from the left.
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.
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.
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
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.”
Simple actions like walking, swallowing and breathing are the result of a complex communication system between cells. When we touch something hot, our nerve cells tell us to take our hand off the object.
This happens in a matter of milliseconds.
This hyperspeed of communication is instrumental in maintaining proper muscle function. Many degenerative diseases affecting millions of people worldwide result from reduced signaling speed or other cellular miscommunications within this intricate network.
Michael Garcia, investigator at the Christopher S. Bond Life Sciences Center and associate professor of biology at the University of Missouri, conducts basic research to answer fundamental questions of nerve cell mechanics.
“In order to fix something, you need to first understand how it works,” Garcia said.
Garcia’s research illuminates relationships between nerve cells to find factors affecting function. His goal is to provide insight on fundamental cellular mechanisms that aren’t fully understood.
Garcia’s research has been funded partly by the National Science Foundation and National Institutes of Health.
Technological advancements have made it possible to better understand disease development in the human body to create more effective treatments. Alas, a scientist’s work is never finished— when the answer to one question is found, ten more crop up in its wake.
Garcia’s research, which appeared in several journals including Human Molecular Genetics andthe Journal of Neuroscience Research initially sought to shed light on the neuronal response to myelination, the development of an insulating border around a nerve cell, called a myelin sheath, which is critical in rapid communication between cells.
Eric Villalon, a graduate student in Michael Garcia’s lab at the Bond Life Sciences Center, examines results. The Garcia Lab is answering news questions in cell mechanics. | PAIGE BLANKENBUEHLER
How it works: Rebuilding cell theory
Garcia’s early research disproved a long-standing hypothesis concerning this cellular feature.
Mammals’ nervous systems are uniquely equipped with myelination, which has been shown to increase conduction velocity, or the speed at which nerve cells pass signals. Low velocity is often associated with neurodegenerative diseases, so research exploring why could later have application in therapeutic technology.
In addition to myelination, cell size makes a big difference in conduction velocity — the bigger the nerve cells, the faster they can pass and receive signals. Garcia’s findings disproved a hypothesis that related myelination to this phenomenon.
The hypothesis, published in a 1992 edition of Cell, claimed that myelination causes a cellular process called phosphorylation which then causes an increase in the axonal diameter (width of the communicating part of a nerve cell), leading to faster nerve cell communication. Garcia found that myelination did cause an increase in axonal diameter, and myelination was required for phosphorylation, but that the two results were independent of one another.
To narrow in on the processes affecting axonal diameter, Garcia identified the protein responsible for growth.
Garcia followed earlier work, showing that one subunit controls whether there is growth at all with myelination, by identifying the domain of this protein that determines how much growth.
After clarifying this part of the process, a question still remains: If not to control myelination, why does phosphorylation happen?
Looking forward
Jeffrey Dale, a recent PhD graduate from Garcia’s lab, said current research is in part geared toward finding a connection between phosphorylation and a process called remyelination.
Remyelination could be key to new therapeutic approaches. When a cell is damaged (as in neurodegenerative disease) the myelin sheath can be stripped away. Remyelination is the process a cell goes through to replace the myelin.
Imagine you have a new wooden toy boat, painted and smooth. If you take a knife and whittle away all the paint and then repaint it—even exactly how it was painted before—the boat is not going to be as shiny and smooth as it was before. This is how remyelination works (or rather, doesn’t). When nerve cells are damaged, the myelin sheath is stripped away and even after the cell rebuilds it, the cell can’t conduct signals at the same speed it was able to before.
“If you can learn what controls myelination, maybe you can improve effectiveness of remyelination,” Dale said.
Garcia said it is possible that revealing the mechanics involved in phosphorylation could lead to better treatments. In context of neurodegenerative diseases, the question why don’t axons function properly might be wrapped up in Garcia’s question: In healthy cells, why do they?
Yaya Cui, an investigator in plant sciences at the Bond Life Sciences Center examines data on fast neuron soybean mutants that are represented on the SoyKB database.
The most puzzling scientific mysteries may be solved at the same machine you’re likely reading this sentence.
In the era of “Big Data” many significant scientific discoveries — the development of new drugs to fight diseases, strategies of agricultural breeding to solve world-hunger problems and figuring out why the world exists — are being made without ever stepping foot in a lab.
Developed by researchers at the Bond Life Sciences Center, SoyKB.org allows international researchers, scientists and farmers to chart the unknown territory of soybean genomics together — sometimes continents away from one another — through that data.
Digital solutions to real-world questions
As part of the Obama Administration’s $200 million “Big Data” Initiative, SoyKB (Soy Knowledge Base) was born.
The digital infrastructure changes the way researchers conduct their experiments dramatically, according to plant scientists like Gary Stacey, Bond LSC researcher, endowed professor of soybean biotechnology and professor of plant sciences and biochemistry.
“It’s very powerful,” Stacey said. “Humans can only look at so many lines in an excel spreadsheet — then it just kind of blurs. So we need these kinds of tools to be able to deal with this high-throughput data.”
The website, managed by Trupti Joshi, an assistant research professor in computer science at MU’s College of Engineering, enables researchers to develop important scientific questions and theories.
“There are people that during their entire career, don’t do any bench work or wet science, they just look at the data,” Stacey said.
The Gene Pathway Viewer available on SoyKB, shows different signaling pathways and points to the function of specific genes so that researchers can develop improvements for badly performing soybean lines.
“It’s much easier to grasp this whole data and narrow it down to basically what you want to focus on,” Joshi said.
A 3D-protein modeling tool lends itself especially to drug design. A pharmaceutical company could test the hypothesis and in some situations, the proposed drug turns out to yield the expected results — formulated solely by data analysis.
The Big Data initiative drives a blending of “wet science” — conducting experiments in the lab and gathering original data — and “dry science” — using computational methods.
Testament of the times?
“Oh, absolutely,” Joshi said.
Collaboration between the “wet” and “dry” sciences
Before SoyKB, data from numerous experiments would be gathered and disregarded, with only the desired results analyzed. The website makes it easy to dump all of the data gathered to then be repurposed by other researchers.
“With these kinds of databases now, all the data is put there so something that’s not valuable to me may be valuable to somebody else,” Stacey said,
Joshi said infrastructure like SoyKB is becoming more necessary in all realms of scientific discovery.
“(SoyKB) has turned out to be a very good public resource for the soybean community to cross reference that and check the details of their findings,” she said.
Computer science prevents researchers having to reinvent the wheel with their own digital platforms. SoyKB has a translational infrastructure with computational methods and tools that can be used for many disciplines like health sciences, animal sciences, physics and genetic research.
“I think there’s more and more need for these types of collaborations,” Joshi said. “It can be really difficult for biologists to handle the large scope of data by themselves and you really don’t want to spend time just dealing with files — You want to focus more on the biology, so these types of collaborations work really well.
It’s a win-win situation for everyone,” she said.
The success of SoyKB was perhaps catalyzed by Joshi. She adopted the website and the compilation of data in its infant stages as her PhD dissertation.
Joshi is unique because she has both a biology degree and a computer science background. Stacey said Joshi, who has “had a foot in each camp,” serves as an irreplaceable translator.
Most recently, the progress of SoyKB as part of the Big Data Initiative was presented at the International Conference on Bioinformatics and Biomedicine Dec. 2013 in Shanghai. The ongoing project is funded by NSF grants.
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.
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
“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 tangled spool of yarn represents DNA, while the fingers holding the section represent the insulators just added by MU researchers to improve a scientific, screening tool. | Paige Blankenbuehler
Here’s a scenario: You are trying to find a lost section of string in the world’s most massively tangled spool of yarn. Then try cutting that section of yarn that’s deeply embedded in the mess without inadvertently cutting another or losing track of the piece you’re after.
For researchers, this problem is not unlike something they encounter in the study of genetic information in the tangled spool that is DNA.
A new tool will help scientists straighten things out.
The tool, developed by University of Missouri Bond Life Sciences Center investigators helps researchers effectively screen cell behavior by limiting epigenetic silencing, which occurs when a cell packages and stows away important genetic information, much like an accountant puts a client’s information away in a filing cabinet.
The cell can go digging to find that information when it absolutely needs it, but otherwise that information is tucked away and inactive.
Professors of biochemistry Mark Hannink, Tom Mawhinney and research assistant professor Valeri V. Mossine used insulators to develop the piggyBac transposon plus insulators, a better reporter of signaling between cells that makes improved screening possible.
This simple addition to an existing screening tool used in laboratories will help streamline research and contribute to screening products like vitamins and supplements and medicines for authenticity, Hannink said.
This is why the insulator addition to the piggyBac reporter assay by MU researchers is a game changer in the scientific world.
How it works
DNA stretches out to nearly 10 feet when it’s uncoiled. That’s 10 feet of your body’s deepest secrets coiled into a microscopic package and tucked away into each and every one of your cells. The human body, by the way, holds an estimated 10 trillion cells. An inconceivable number, right?
Let’s go back to our yarn analogy. You’re trying to find one specific piece to cut but it’s deeply tangled in the mass of yarn. You need to find the piece that you really care about and clamp your fingers onto the yarn to reduce the slack — straighten it out — so you can cut it easily.
Think of your fingers as the insulators.
The insulators of the new piggyBac transposon tool perform the same task of stretching out the DNA so certain expressions through signaling pathways are held open, enabling the investigation of specific genetic material.
Hannink hypothesizes this new reporter could provide answers to questions like: Does an anti-migraine medicine have the component that will relieve that ailment? Does a multi-vitamin deliver all of the nutrients on its label?
“A lot of botanicals are said to have anti-inflammatory benefits,” Hannink said. “By using an assay like this, we can easily determine if they actually do and if so, what molecules in these complex mixtures are in fact the cause of the punitive inflammatory activity.”
Reproducing results
Replication is a critical part of verifying scientific discovery and epigenetic silencing is a big headache for investigators trying to reproduce results.
Scientists studying genetic material can open certain expressions with other reporter tools but often, the cell will turn expressions off and block signaling pathways, causing an expected result to fail because of epigenetic silencing.
The new assay preserves conditions of an experiment so the same results can be reached. Cell behavior under the same conditions and expressions that were switched on during the experiment will be expressed.
The new version of the reporter assay is being used at the MU Center for Botanical Interaction Studies to understand how botanical compounds affect the immune system and in other research on the central nervous system and on the development of prostate cancer.
This research appeared in the Dec. 20, 2013 edition of PLoS ONE. It was funded by the University of Missouri Agriculture Experiment Station Laboratories and grants from the National Center for Complementary and Alternative Medicines, Office of Dietary Supplements and the National Cancer Institute.
Over the weekend, Bond LSC HIV researchers Stefan Sarafianos, Marc Johnson and Donald Burke-Aguero joined Trail to a Cure, Inc., a Columbia nonprofit organization that helped fund important HIV research.
Since 2008, the organization has raised $74,000 for HIV/AIDS research, with some of that funding going directly to the Bond LSC providing additional hours of lab research. The 2014 online fundraising is still open and donations can be made to Trail to a Cure, Inc. until the end of the month.
The funding from Trail to a Cure helps Bond LSC researchers train future scientists and physicians in labs and in some cases, training them on the development of next generation therapies, Johnson said.
Bond LSC HIV researchers Stefan Sarafianos (left), Marc Johnson (center), and Donald Burke-Aguero (right) at the 7th annual Trail to a Cure along Katy Trail in Rocheport on May 3. | Credit: Trail to a Cure, Inc..Trail to a Cure, Inc., a Columbia-based nonprofit, has raised $74,000 for HIV/AIDS since 2008. Part of the raised funds go to HIV research in the LSC, providing additional hours of lab research. | Credit: Trail to a Cure, Inc.
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), 133-143; Org. 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.
Powdery mildew on a cabernet sauvignon grapevine leaf. | USDA Grape genetics publications and research
A princess kisses a frog and it turns into a prince, but when a scientist uses a frog to find out more information about a grapevine disease, it turns into the perfect tool narrowing in on the cause of crop loss of Vitis vinifera, the world’s favorite connoisseur wine-producing varietal.
MU researchers recently published a study that uncovered a specific gene in the Vitis vinifera varietal Cabernet Sauvingon, that contributes to its susceptibility to a widespread plant disease, powdery mildew. They studied the biological role of the gene by “incubating” it in unfertilized frog eggs.
The study, funded by USDA National Institute of Food and Agriculture grants, was lead by Walter Gassmann, an investigator at the Bond Life Sciences Center and University of Missouri professor in the division of plant sciences.
The findings show one way that Vitis vinifera is genetically unable to combat the pathogen that causes powdery mildew.
Gassmann said isolating the genes that determine susceptibility could lead to developing immunities for different varietals and other crop plants and contribute to general scientific knowledge of grapevine, which has not been studied on the molecular level to the extent of many other plants.
The grapevine genome is largely unknown.
“Not much is known about the way grapevine supports the growth of the powdery mildew disease, but what we’ve provided is a reasonable hypothesis for what’s going on here and why Cabernet Sauvingon could be susceptible to this pathogen,” Gassmann said.
The research opens the door for discussion on genetically modifying grapevine varietals.
Theoretically, Gassmann said, the grapevine could be modified to prevent susceptibility and would keep the character of the wine intact — a benefit of genetic modification over crossbreeding, which increases immunity over a lengthy process but can diminish character and affect taste of the wine.
Grapevine under attack
Gassmann’s recent research found a link between nitrate transporters and susceptibility through a genetic process going on in grapevine infected with the powdery mildew disease.
Infected grapevine expressed an upregulation of a gene that encodes a nitrate transporter, a protein that regulates the makes it possible for the protein to enter the plant cell.
Once the pathogen is attracted to this varietal of grapevine, it tricks grapevine into providing nutrients, allowing the mildew to grow and devastate the plant.
As leaves mature, they go through a transition where they’re no longer taking a lot of nutrients for themselves. Instead, they become “sources” and send nutrients to new “sink” leaves and tissues. The exchange enables plants to grow.
The powdery mildew pathogen, which requires a living host, tricks the grapevine into using its nutrient transfer against itself. Leaves turn into a “sink” for the pathogens, and nutrients that would have gone to new leaves, go instead, to the pathogen, Gassmann said.
“We think that what this fungus has to do is make this leaf a sink for nitrate so that nitrate goes to the pathogen instead of going to the rest of the plant,” Gassmann said.
Walter Gassmann, of the Bond Life Sciences Center at the University of Missouri was the lead investogator on the research. Much of his work has been on grapevine susceptibility to pathogens. | Roger Meissen, Bond Life Sciences Center
According to a report by the USDA, powdery mildew can cause “major yield losses if infection occurs early in the crop cycle and conditions remain favorable for development.”
Powdery mildew appears as white to pale gray “fuzzy” blotches on the upper surfaces of leaves and thrives in “cool, humid and semiarid areas,” according to the report.
Gassmann said powdery mildew affects grapevine leaves, stems and berries and contributes to significant crop loss of the Vitas vinifera, which is cultivated for most commercial wine varietals.
“The leaves that are attacked lose their chlorophyll and they can’t produce much sugar,” Gassmann said. “Plus the grape berries get infected directly, so quality and yield are reduced in multiple ways.”
Pinpointing a cause
Solutions to problems start with finding the reason why something is happening, so Gassmann and his team looked at a list of genes activated by the pathogen to find transporters that allowed compounds like peptides, amino acids, and nitrate to pass.
Genes for nitrate transporters, Gassmann said, pointed to a cause for vulnerability to the mildew pathogen.
Over-fertilization of nitrate increases the severity of mildew in many crop plants, according to previous studies sited in Gassmann’s article in the journal of Plant Cell Physiology.
The testing system for isolating and analyzing the genes began with female frogs.
Gassmann used frog oocytes (unfertilized eggs), to verify the similar functions of nitrate transporters in Arabidopsis thaliana, a plant used as a baseline for comparison.
A nitrate transporter, he hypothesized, would increase the grapevine’s susceptibility to mildew.
“The genes that were upregulated in grapevine showed similarity to genes in Arabidopsis that are known to transport nitrate,” Gassmann said. “We felt the first thing we had to do was verify that what we have in grapevine actually does that.”
The eggs are very large relative to other testing systems and act as “an incubating system” for developing a protein. Gassmann and his team of researchers injected the oocyte with RNA, a messenger molecule that contains the information from a gene to produce a protein. The egg thinks it’s being fertilized and protein reproduces and is studied.
“The oocyte is like a machine to crank out protein,” Gassmann said. “We use that technique to establish what we have is actually a nitrate transporter.”
The system confirmed that the gene isolated from grapevine encodes a nitrate transporter.
“We contributed to the general knowledge of the nitrate transporter family,” Gassmann said. “It turned out to be the first member of one branch of nitrate transporters that, even in Arabidopsis haven’t been characterized before.”
The mounting knowledge of Vitis vinifera genes could make genetically modifying the strain to prevent the susceptibility easier.
“Resistance is determined sometimes by a single gene,” Gassmann said. “Until people are willing to have the conversation of genetic modification, the only way to save your grapevines is to be spraying a lot.”
Sharon Pike, Gassmann, other investigators from the MU Christopher S. Bond Life Sciences Center and post-doctoral student, Min Jung Kim from Daniel Schachtman’s lab at the Donald Danforth Plant Science Center in Saint Louis, Mo. contributed to the report.
The article was accepted November 2013 into the Plant Cell Physiology journal.
