Chinese Medicine Yields Secrets: Atomic Mechanism of Two-Headed Molecule Derived from Chang Shan, a Traditional Chinese Herb

Chinese Medicine Yields Secrets: Atomic Mechanism of Two-Headed Molecule Derived from Chang Shan, a Traditional Chinese Herb

  9:49:00 AM    Science Lover

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The mysterious inner workings of Chang Shan -- a Chinese herbal medicine used for thousands of years to treat fevers associated with malaria -- have been uncovered thanks to a high-resolution structure solved at The Scripps Research Institute (TSRI).

Scripps Research Institute scientists have determined a molecular 
 structure that helps explain how the Chinese herbal medicine 
Chang Shan works. (Credit: Image courtesy of the Schimmel lab.)

Described in the journal Nature this week, the structure shows in atomic detail how a two-headed compound derived from the active ingredient in Chang Shan works. Scientists have known that this compound, called halofuginone (a derivative of the febrifugine), can suppress parts of the immune system -- but nobody knew exactly how.

The new structure shows that, like a wrench in the works, halofuginone jams the gears of a molecular machine that carries out "aminoacylation," a crucial biological process that allows organisms to synthesize the proteins they need to live. Chang Shan, also known as Dichroa febrifuga Lour, probably helps with malarial fevers because traces of a halofuginone-like chemical in the herb interfere with this same process in malaria parasites, killing them in an infected person's bloodstream.

"Our new results solved a mystery that has puzzled people about the mechanism of action of a medicine that has been used to treat fever from a malaria infection going back probably 2,000 years or more," said Paul Schimmel, PhD, the Ernest and Jean Hahn Professor and Chair of Molecular Biology and Chemistry and member of The Skaggs Institute for Chemical Biology at TSRI. Schimmel led the research with TSRI postdoctoral fellow Huihao Zhou, PhD.

Halofuginone has been in clinical trials for cancer, but the high-resolution picture of the molecule suggests it has a modularity that would make it useful as a template to create new drugs for numerous other diseases.

The Process of Aminoacylation and its Importance to Life

Aminoacylation is a crucial step in the synthesis of proteins, the end products of gene expression. When genes are expressed, their DNA sequence is first read and transcribed into RNA, a similar molecule. The RNA is then translated into proteins, which are chemically very different from DNA and RNA but are composed of chains of amino acid molecules strung together in the order called for in the DNA.

Necessary for this translation process are a set of molecules known as transfer RNAs (tRNAs), which shuttle amino acids to the growing protein chain where they are added like pearls on a string. But before the tRNAs can move the pearls in place, they must first grab hold of them.

Aminoacylation is the biological process whereby the amino acid's pearls are attached to these tRNA shuttles. A class of enzymes known as aminoacyl-tRNA synthetases is responsible for attaching the amino acids to the tRNAs, and Schimmel and his colleagues have been examining the molecular details of this process for years. Their work has given scientists insight into everything from early evolution to possible targets for future drug development.

Over time what has emerged as the picture of this process basically involves three molecular players: a tRNA, an amino acid and the aminoacyl-tRNA synthetase enzyme that brings them together. A fourth molecule called ATP is a microscopic form of fuel that gets consumed in the process.

The new work shows that halofuginone gets its potency by interfering with the tRNA synthetase enzyme that attaches the amino acid proline to the appropriate tRNA. It does this by blocking the active site of the enzyme where both the tRNA and the amino acid come together, with each half of the halofuginone blocking one side or the other.

Interestingly, said Schimmel, ATP is also needed for the halofuginone to bind. Nothing like that has ever been seen in biochemistry before.

"This is a remarkable example where a substrate of an enzyme (ATP) captures an inhibitor of the same enzyme, so that you have an enzyme-substrate-inhibitor complex," said Schimmel.

The article, "ATP-Directed Capture of Bioactive Herbal-Based Medicine on Human tRNA Synthetase," by Huihao Zhou, Litao Sun, Xiang-Lei Yang and Paul Schimmel was published in the journal Nature on Dec. 23, 2012.

This work was supported by the National Institutes of Health through grants #GM15539, #23562 and #88278 and by a fellowship from the National Foundation for Cancer Research.

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Strange Behavior: New Study Exposes Living Cells to Synthetic Protein

Strange Behavior: New Study Exposes Living Cells to Synthetic Protein

  10:22:00 AM    Science Lover

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One approach to understanding components in living organisms is to attempt to create them artificially, using principles of chemistry, engineering and genetics. A suite of powerful techniques -- collectively referred to as synthetic biology -- have been used to produce self-replicating molecules, artificial pathways in living systems and organisms bearing synthetic genomes.

The depletion of ATP in cells of the bacterium Escherichia coli causes them to transition to a filamentous state and form dense lipid structures known as endoliposomes. The structures can be clearly observed in these transmission electron micrographs of increasing magnification. (Credit: Image courtesy of Arizona State University)

In a new twist, John Chaput, a researcher at Arizona State University's Biodesign Institute and colleagues at the Department of Pharmacology, Midwestern University, Glendale, AZ have fabricated an artificial protein in the laboratory and examined the surprising ways living cells respond to it.

"If you take a protein that was created in a test tube and put it inside a cell, does it still function," Chaput asks. "Does the cell recognize it? Does the cell just chew it up and spit it out?" This unexplored area represents a new domain for synthetic biology and may ultimately lead to the development of novel therapeutic agents.

The research results, reported in the advanced online edition of the journal ACS Chemical Biology, describe a peculiar set of adaptations exhibited by Escherichia coli bacterial cells exposed to a synthetic protein, dubbed DX. Inside the cell, DX proteins bind with molecules of ATP, the energy source required by all biological entities.

"ATP is the energy currency of life," Chaput says. The phosphodiester bonds of ATP contain the energy necessary to drive reactions in living systems, giving up their stored energy when these bonds are chemically cleaved. The depletion of available intracellular ATP by DX binding disrupts normal metabolic activity in the cells, preventing them from dividing, (though they continue to grow).

After exposure to DX, the normally spherical E. coli bacteria develop into elongated filaments. Within the filamentous bacteria, dense intracellular lipid structures act to partition the cell at regular intervals along its length. These unusual structures, which the authors call endoliposomes, are an unprecedented phenomenon in such cells.

"Somewhere along the line of this filamentation, other processes begin to happen that we haven't fully understood at the genetic level, but we can see the results phenotypically," Chaput says. "These dense lipid structures are forming at very regular regions along the filamented cell and it looks like it could be a defense mechanism, allowing the cell to compartmentalize itself." This peculiar adaptation has never been observed in bacterial cells and appears unique for a single-celled organism.

Producing a synthetic protein like DX, which can mimic the elaborate folding characteristics of naturally occurring proteins and bind with a key metabolite like ATP is no easy task. As Chaput explains, a clever strategy known as mRNA display was used to produce, fine-tune and amplify synthetic proteins capable of binding ATP with high affinity and specificity, much as a naturally occurring ATP-binding protein would.

First, large libraries of random sequence peptides are formed from the four nucleic acids making up DNA, with each strand measuring around 80 nucleotides in length. These sequences are then transcribed into RNA with the help of an enzyme -- RNA polymerase. If a natural ribosome is then introduced, it attaches to the strand and reads the random sequence RNA as though it was a naturally-occurring RNA, generating a synthetic protein as it migrates along the strand. In this way, synthetic proteins based on random RNA sequences can be generated.

Exposing the batch of synthetic proteins to the target molecule and extracting those that bind can then select for ATP-binding proteins. But as Chaput explains, there's a problem: "The big question is how do you recover that genetic information? You can't reverse transcribe a protein back into DNA. You can't PCR amplify a protein. So we have to do all these molecular biology tricks."

The main trick involves an earlier step in the process. A molecular linker is chemically attached to the RNA templates, such that each RNA strand forms a bond with its newly translated protein. The mRNA-protein hybrids are exposed to selection targets (like ATP) over consecutive rounds of increasing stringency. After each round of selection, those library members that remain bound to the target are reverse-transcribed into cDNA (using their conveniently attached RNA messages), and then PCR amplified.

In the current study, E. coli cells exposed to DX transitioned into a filamentous form, which can occur naturally when such cells are subject to conditions of stress. The cells display low metabolic activity and limited cell division, presumably owing to their ATP-starved condition.

The study also examined the ability of E. coli to recover following DX exposure. The cells were found to enter a quiescent state known as viable but non-culturable (VBNC), meaning that they survived ATP sequestration and returned to their non-filamentous state after 48 hours, but lost their reproductive capacity. Further, this condition was difficult to reverse and seems to involve a fundamental reprogramming of the cell.

In an additional response to DX, the filamentous cells form previously undocumented structures, which the authors refer to as endoliposomes. These dense lipid concentrations, spanning the full width of the filamented E. coli, segment the cells into distinct compartments, giving the cells a stringbean-like appearance under the microscope.

The authors speculate that this adaptation may be an effort to maintain homeostasis in regions of the filamentous cell, which have essentially been walled off from the intrusion of ATP-depleting DX. They liken endoliposomes to the series of water-tight compartments found in submarines which are used to isolate damaged sections of the ship and speculate that DX-exposed cells are partitioning their genetic information into regions where it can be safely quarantined. Such self-compartmentalization is known to occur in some eukaryotic cells, but has not been previously observed in prokaryotes like E. coli.

The research indicates that there is still a great deal to learn about bacterial behavior and the repertoire of responses available when such cells encounter novel situations, such as an unfamiliar, synthetic protein. The study also notes that many infectious agents rely on a dormant state, (similar to the VBNC condition observed in the DX-exposed E. coli), to elude detection by antibiotics. A better understanding of the mechanisms driving this behavior could provide a new approach to targeting such pathogens.

The relative safety of E. coli as a model organism for study may provide a fruitful tool for more in-depth investigation of VBNC states in pathogenic organisms. Further, given ATP's central importance for living organisms, its suppression may provide another avenue for combating disease. One example would be an engineered bacteriophage capable of delivering DX genes to pathogenic organisms.

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Human Evolution Driven By Changing Environment

Human Evolution Driven By Changing Environment

  9:46:00 AM    Science Lover

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A series of rapid environmental changes in East Africa roughly 2 million years ago may be responsible for driving human evolution, according to researchers at Penn State and Rutgers University.

The researchers examined lake sediments from Olduvai Gorge in northern Tanzania, looking for biomarkers -- fossil molecules -- from ancient trees and grasses. (Credit: Gail Ashley)

"The landscape early humans were inhabiting transitioned rapidly back and forth between a closed woodland and an open grassland about five to six times during a period of 200,000 years," said Clayton Magill, graduate student in geosciences at Penn State. "These changes happened very abruptly, with each transition occurring over hundreds to just a few thousand years."

According to Katherine Freeman, professor of geosciences, Penn State, the current leading hypothesis suggests that evolutionary changes among humans during the period the team investigated were related to a long, steady environmental change or even one big change in climate.

"There is a view this time in Africa was the 'Great Drying,' when the environment slowly dried out over 3 million years," she said. "But our data show that it was not a grand progression towards dry; the environment was highly variable."

According to Magill, many anthropologists believe that variability of experience can trigger cognitive development.

"Early humans went from having trees available to having only grasses available in just 10 to 100 generations, and their diets would have had to change in response," he said. "Changes in food availability, food type, or the way you get food can trigger evolutionary mechanisms to deal with those changes. The result can be increased brain size and cognition, changes in locomotion and even social changes -- how you interact with others in a group. Our data are consistent with these hypotheses. We show that the environment changed dramatically over a short time, and this variability coincides with an important period in our human evolution when the genus Homo was first established and when there was first evidence of tool use."

The researchers -- including Gail Ashley, professor of earth and planetary sciences, Rutgers University -- examined lake sediments from Olduvai Gorge in northern Tanzania. They removed the organic matter that had either washed or was blown into the lake from the surrounding vegetation, microbes and other organisms 2 million years ago from the sediments. In particular, they looked at biomarkers -- fossil molecules from ancient organisms -- from the waxy coating on plant leaves.

"We looked at leaf waxes because they're tough, they survive well in the sediment," said Freeman.

The team used gas chromatography and mass spectrometry to determine the relative abundances of different leaf waxes and the abundance of carbon isotopes for different leaf waxes. The data enabled them to reconstruct the types of vegetation present in the Olduvai Gorge area at very specific time intervals.

The results showed that the environment transitioned rapidly back and forth between a closed woodland and an open grassland.

To find out what caused this rapid transitioning, the researchers used statistical and mathematical models to correlate the changes they saw in the environment with other things that may have been happening at the time, including changes in the Earth's movement and changes in sea-surface temperatures.

"The orbit of the Earth around the sun slowly changes with time," said Freeman. "These changes were tied to the local climate at Olduvai Gorge through changes in the monsoon system in Africa. Slight changes in the amount of sunshine changed the intensity of atmospheric circulation and the supply of water. The rain patterns that drive the plant patterns follow this monsoon circulation. We found a correlation between changes in the environment and planetary movement."

The team also found a correlation between changes in the environment and sea-surface temperature in the tropics.

"We find complementary forcing mechanisms: one is the way Earth orbits, and the other is variation in ocean temperatures surrounding Africa," Freeman said. The researchers recently published their results in the Proceedings of the National Academy of Sciences along with another paper in the same issue that builds on these findings. The second paper shows that rainfall was greater when there were trees around and less when there was a grassland.

"The research points to the importance of water in an arid landscape like Africa," said Magill. "The plants are so intimately tied to the water that if you have water shortages, they usually lead to food insecurity.

"Together, these two papers shine light on human evolution because we now have an adaptive perspective. We understand, at least to a first approximation, what kinds of conditions were prevalent in that area and we show that changes in food and water were linked to major evolutionary changes."

The National Science Foundation funded this research.

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Sound Beam Could One Day Be Invisible Scalpel

Sound Beam Could One Day Be Invisible Scalpel

  4:34:00 PM    Science Lover

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A carbon-nanotube-coated lens that converts light to sound can focus high-pressure sound waves to finer points than ever before. The University of Michigan engineering researchers who developed the new therapeutic ultrasound approach say it could lead to an invisible knife for noninvasive surgery.

With a new technique that uses tightly-focused sound waves for micro-surgery, University of Michigan engineering researchers drilled a 150-micrometer hole in a confetti-sized artificial kidney stone. (Credit: Hyoung Won Baac)

Today's ultrasound technology enables far more than glimpses into the womb. Doctors routinely use focused sound waves to blast apart kidney stones and prostate tumors, for example. The tools work primarily by focusing sound waves tightly enough to generate heat, says Jay Guo, a professor of electrical engineering and computer science, mechanical engineering, and macromolecular science and engineering. Guo is a co-author of a paper on the new technique published in the current issue of Nature's journal Scientific Reports.

The beams that today's technology produces can be unwieldy, says Hyoung Won Baac, a research fellow at Harvard Medical School who worked on this project as a doctoral student in Guo's lab.

"A major drawback of current strongly focused ultrasound technology is a bulky focal spot, which is on the order of several millimeters," Baac said. "A few centimeters is typical. Therefore, it can be difficult to treat tissue objects in a high-precision manner, for targeting delicate vasculature, thin tissue layer and cellular texture. We can enhance the focal accuracy 100-fold."

The team was able to concentrate high-amplitude sound waves to a speck just 75 by 400 micrometers (a micrometer is one-thousandth of a millimeter). Their beam can blast and cut with pressure, rather than heat. Guo speculates that it might be able to operate painlessly because its beam is so finely focused it could avoid nerve fibers. The device hasn't been tested in animals or humans yet, though.

"We believe this could be used as an invisible knife for noninvasive surgery," Guo said. "Nothing pokes into your body, just the ultrasound beam. And it is so tightly focused, you can disrupt individual cells."

To achieve this superfine beam, Guo's team took an optoacoustic approach that converts light from a pulsed laser to high-amplitude sound waves through a specially designed lens. The general technique has been around since Thomas Edison's time. It has advanced over the centuries, but for medical applications today, the process doesn't normally generate a sound signal strong enough to be useful.

The U-M researchers' system is unique because it performs three functions: it converts the light to sound, focuses it to a tiny spot and amplifies the sound waves. To achieve the amplification, the researchers coated their lens with a layer of carbon nanotubes and a layer of a rubbery material called polydimethylsiloxane. The carbon nanotube layer absorbs the light and generates heat from it. Then the rubbery layer, which expands when exposed to heat, drastically boosts the signal by the rapid thermal expansion.

The resulting sound waves are 10,000 times higher frequency than humans can hear. They work in tissues by creating shockwaves and microbubbles that exert pressure toward the target, which Guo envisions could be tiny cancerous tumors, artery-clogging plaques or single cells to deliver drugs. The technique might also have applications in cosmetic surgery.

In experiments, the researchers demonstrated micro ultrasonic surgery, accurately detaching a single ovarian cancer cell and blasting a hole less than 150 micrometers in an artificial kidney stone in less than a minute.

"This is just the beginning," Guo said. "This work opens a way to probe cells or tissues in much smaller scale."

The researchers will present the work at the SPIE Photonics West meeting in San Francisco. The research was funded by the National Science Foundation and the National Institutes of Health.

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Maya Scholar Debunks World-Ending Myth

Maya Scholar Debunks World-Ending Myth

  10:58:00 AM    Science Lover

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As we hurtle toward the end of 2012, the conversation about a certain date with roots in an ancient Maya calendar has reached a fever pitch.

David Stuart discusses the new inscriptions with colleagues from Tulane University and Universidad del Valle de Guatemala. Seated left to right: Marcello Canuto (Tulane), Stuart, Tomás Barrientos (UVG), Jocelyn Ponce (UVG). (Credit: Image courtesy of University of Texas at Austin)

Dec. 21, 2012, has taken over popular culture this year: It's been the subject of movies, books and news shows. The date and its supposed prophecy that the world will come to an end has been the subject of water cooler conversations and international media attention.

But the truth regarding the date, according to renowned Maya scholar and University of Texas at Austin art history professor David Stuart, is that the day is indeed meaningful -- but not in the way you might think.

"The Maya never actually predicted the end of times," says Stuart, who recently won a UNESCO medal for his lifetime contributions to the study of ancient Maya culture and archaeological sites, including UNESCO World Heritage Sites. "In the Maya scheme of time, the approaching date was thought to be the turn of an important cycle, or as they put it, the end of 13 bak'tuns. The thing is, there are many more bak'tuns still to come."

Earlier this year, Stuart was working with colleagues at the ruins of La Corona in the Guatemalan jungle, where they excavated many inscribed stones that had been part of a staircase. As the world's leading epigrapher of Maya script, Stuart was brought in to decipher the 56 glyphs carved into the stones. He discovered 200 years of political history and, to his surprise, the second known reference in Maya culture to the so-called end date of Dec. 21, 2012.

But despite the popular misconception, the date doesn't predict the end of times. Rather, it was intended to promote continuity during a time of crisis.

"The hieroglyphs emphasized seventh century history and politics, linking the reign of an ancient king to the turn of the 13th bak'tun many centuries later," Stuart explains. "The point was to associate the divine king's time on the throne to time on a cosmic scale.

"The monument commemorated a royal visit to La Corona in AD 696 by the most powerful Maya ruler of that time, a few months after his defeat by a longstanding rival in AD 695," said Stuart. "This ruler was visiting allies and allaying their fears after his defeat. It was a time of great political turmoil in the Maya region, and this king felt compelled to allude to a larger cycle of time that happens to end in 2012."

Rather than prophesy, the 2012 reference served to place this king's troubled reign and accomplishments into a larger cosmological framework. In times of crisis, the ancient Maya used their calendar to promote continuity and stability.

Assuming 21st century soothsayers are incorrect about the impending end of the world, Stuart's research will continue in 2013, starting in January with the Maya Meetings, an international conference held, alternately, in Austin and Antigua, Guatemala, each year. Stuart has served as director of the event since 2004, and this year it is a family affair. Stuart's father, George E. Stuart, will be the keynote speaker at this year's meeting, which will be in Austin.

The elder Stuart was hired as a cartographer for the National Geographic Society and remained on staff for nearly 40 years working in a variety of capacities, including as editor for archaeology of National Geographic Magazine and chairman of the Committee for Research and Exploration. He founded the Center for Maya Research in 1984.

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Scientists Create Nanoscale Window to Biological World

Scientists Create Nanoscale Window to Biological World

  9:13:00 AM    Science Lover

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If the key to winning battles is knowing both your enemy and yourself, then scientists are now well on their way toward becoming the Sun Tzus of medicine by taking a giant step toward a priceless advantage -- the ability to see the soldiers in action on the battlefield.

A novel microfluidics platform allowed viewing of structural 
details of rotavirus double-layered particles; the 3-D graphic 
of the virus, in purple, was reconstructed from data gathered by 
the new technique. (Credit: Virginia Tech)

Investigators at the Virginia Tech Carilion Research Institute have invented a way to directly image biological structures at their most fundamental level and in their natural habitats. The technique is a major advancement toward the ultimate goal of imaging biological processes in action at the atomic level.

"It's sort of like the difference between seeing Han Solo frozen in carbonite and watching him walk around blasting stormtroopers," said Deborah Kelly, an assistant professor at the VTC Research Institute and a lead author on the paper describing the first successful test of the new technique. "Seeing viruses, for example, in action in their natural environment is invaluable."

The technique involves taking two silicon-nitride microchips with windows etched in their centers and pressing them together until only a 150-nanometer space between them remains. The researchers then fill this pocket with a liquid resembling the natural environment of the biological structure to be imaged, creating a microfluidic chamber.

Then, because free-floating structures yield images with poor resolution, the researchers coat the microchip's interior surface with a layer of natural biological tethers, such as antibodies, which naturally grab onto a virus and hold it in place.

In a recent study in Lab on a Chip, Kelly joined Sarah McDonald, also an assistant professor at the VTC Research Institute, to prove that the technique works. McDonald provided a pure sample of rotavirus double-layered particles for the study.

"What's missing in the field of structural biology right now is dynamics -- how things move in time," said McDonald. "Debbie is developing technologies to bridge that gap, because that's clearly the next big breakthrough that structural biology needs."

Rotavirus is the most common cause of severe diarrhea among infants and children. By the age of 5, nearly every child in the world has been infected at least once. And although the disease tends to be easily managed in the developed world, in developing countries rotavirus kills more than 450,000 children a year.

At the second step in the pathogen's life cycle, rotavirus sheds its outer layer, which allows it to enter a cell, and becomes what is called a double-layered particle. Once its second layer is exposed, the virus is ready to begin using the cell's own infrastructure to produce more viruses. It was the viral structure at this stage that the researchers imaged in the new study.

Kelly and McDonald coated the interior window of the microchip with antibodies to the virus. The antibodies, in turn, latched onto the rotaviruses that were injected into the microfluidic chamber and held them in place. The researchers then used a transmission electron microscope to image the prepared slide.

The technique worked perfectly.

The experiment gave results that resembled those achieved using traditional freezing methods to prepare rotavirus for electron microscopy, proving that the new technique can deliver accurate results.

"It's the first time scientists have imaged anything on this scale in liquid," said Kelly.

The next step is to continue to develop the technique with an eye toward imaging biological structures dynamically in action. Specifically, McDonald is looking to understand how rotavirus assembles, so as to better know and develop tools to combat this particular enemy of children's health.

The researchers said their ongoing collaboration is an example of the cross-disciplinary work that is becoming a hallmark of the VTC Research Institute.

"It's an ideal collaboration because Sarah provides a phenomenal model system by which we can develop new technologies to move the field of microstructural biology forward," said Kelly.

"It's very win-win," McDonald added. "While the virus is a great tool for Debbie to develop her techniques, her technology is critical for allowing me to understand how this deadly virus assembles and changes dynamically over time."

The paper "Visualizing viral assemblies in a nanoscale biosphere" was published online and will appear in a 2013 edition of Lab on a Chip.

The authors are Brian Gilmore, a research associate at the VTC Research Institute; Shannon Showalter, a research assistant at the VTC Research Institute; Madeline Dukes, an applications scientist at Protochips; Justin Tanner, a postdoctoral associate at the VTC Research Institute; Andrew Demmert, a student at the Virginia Tech Carilion School of Medicine; McDonald, in addition to her position at the VTC Research Institute, is an assistant professor of biomedical sciences and pathobiology in the Virginia-Maryland Regional College of Veterinary Medicine; and Kelly, in addition to her position at the VTC Research Institute, is an assistant professor of biological sciences in Virginia Tech's College of Science.

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