Bond Life Sciences Center

Live long and prosper: healthy mitochondria, healthy motor neurons?

Chris Lorson

Chris Lorson (front) and Mark Hannink (back) collaborate to study the role of mitochondria in motor neuron health, particularly in relation to spinal muscular atrophy, a neuromuscular disorder | photo by Jen Lu, Bond LSC

Chris Lorson, a professor of veterinary pathobiology, and Mark Hannink, a professor of biochemistry, want to find a new way to help motor neurons live a long and healthy life. Their question: what’s the relationship between motor neuron sruvival and a cellular component called mitochondria?

The two researchers at the Bond Life Sciences Center were awarded preliminary funding from the Bond LSC to pursue this question. Their findings could lead to new targets for therapies to treat a type of muscular dystrophy called spinal muscular atrophy, or SMA.

Spinal muscular atrophy, a genetic disease characterized by the death of motor neurons in the spinal cord, is caused by a mutation in the Survival Motor Neuron 1, or SMN1, gene. Patients with SMA develop muscle weakness and deterioration that spread inwards from the hands and feet, which progresses to interfere with mobility and breathing. The severity of symptoms and time of onset depend on how well a related gene is able to compensate for the lack of SMN1. As a result, treatment strategies usually focus on improving the activation of SMN1’s back-up gene.

Hannink and Lorson, however, are interested in a different pathway that is related to mitochondria dsyfunction.

Mitochondria are like the cell’s battery packs. Produced in the cell body, mitochondria migrate to the other end of the motor neuron to provide the energy to send electrochemical signals to recipient muscles and nerves. When mitochondria break down, the cell packs them into vacuoles that return to the cell body for recycling or removal.

“I saw a report that said that in SMA, there’s evidence for dysfunctional mitochondria in spinal motor neuron atrophy,” Hannink said. “My lab knows something about how mitochondria respond to stress.”

“There’s a lot of information out there that hints at it,” Lorson, an expert in SMA, said. “A number of the same responses you see in the stress pathway are also activated in neurodegeneration.”

To test their hypothesis, Hannink and Lorson plan to make motor neurons from pluripotent stem cells taken from people with and without SMA, and compare mitochondrial function and cell survival between the two groups. Then, they will test if a number of different genes that are known to be important for mitochondrial function will affect motor neuron health in both SMA and non-SMA derived cells.

“If you look at the tool chest of SMA therapeutics right now,” Lorson said, “you have a number of very obvious targets.”

Most approaches aim to boost the performance of the SMN or its back-up gene, but there are also options like neuroprotectants and skeletal muscle activators. Molecules that maintain healthy mitochondrial function could be another possibility.

“These are things that don’t worry about the state of the SMN gene and are targeting something in addition to, supplemental to or as an alternative to SMN,” Lorson said. “And that’s where this project would fall.”

This seed funding is one of seven awarded this year at the Bond Life Sciences Center. These awards, which range from $40,000 to $100,000 in funding, foster inter-laboratory collaboration and make possible the development of pilot projects.

One step closer from mice to men

Gene therapy treating the neurodegenerative disease, SMARD1, shows promising results in mice studies.

Shababi uses an instrument to measure grip strength in the forelimbs of mice. Healthy mice are able to cling to the rack with a stronger grip than SMARD1 mice. | photo by Jennifer Lu, Bond LSC .

Shababi uses an instrument to measure grip strength in the forelimbs of mice. Healthy mice are able to cling on with a stronger grip than SMARD1 mice. | photo by Jennifer Lu, Bond LSC

Monir Shababi was confident her experiments treating a rare genetic disease would yield positive results before she even ran them.

Scientists had success with a similar degenerative neuromuscular disease, so she had every expectation their strategy would work just as well in her mice.

Monir Shababi, assistant research professor in the Department of Veterinary Pathobiology, studies SMARD1 in mice. | photo courtesy of the Department of Veterinary Pathobiology

Monir Shababi, an assistant research professor in the Department of Veterinary Pathobiology, studies SMARD1 in mice. | photo courtesy of the Department of Veterinary Pathobiology

“I was expecting to get the same results,” said Shababi, an assistant research profession in Christian Lorson’s lab at the University of Missouri Bond Life Sciences Center. Shababi studies spinal muscular atrophy with respiratory disease type 1, or SMARD1.

The treatment worked, but not without a few surprises.

Her findings, published in Molecular Therapy, a journal by Nature Publishing Group, are one of the first to show how gene therapy can effectively reverse SMARD1 symptoms in mice.

In patients, SMARD1 is considered such a rare genetic disorder by the U.S. National Library of Medicine that no one knows how frequently the disease occurs. It’s only when babies develop the first symptom—trouble breathing–that pediatricians screen for SMARD1.

Shortly after diagnosis, muscle weakness appears in the hands and feet before spreading inwards to the rest of the body. The average life expectancy for a child diagnosed with SMARD1 is 13 months. There is currently no effective treatment.

Since the neuromuscular disease is caused by a recessive gene, SMARD1 comes as a shock to the parents, who are carriers but do not show signs of the illness, Shababi said. This genetic defect prevents cells from making a particular protein that scientists suspect is vital to replication and protein production.

The hereditary nature of the disease has a silver lining, though. Because SMARD1 is a caused by a single pair of faulty genes and not multiple ones, it is a prime candidate for gene therapy that could restore the missing protein and reverse the disease.

To do that, Shababi set up a dose-response study using a tiny virus to carry the genetic instructions for making the missing protein. She injected newborn mice with a low dose of the virus, a high dose, or a placebo with no virus at all.

Injecting at different doses allowed her to ask which dose worked better, Shababi said.

According to the previous research, a higher dose should have resulted in a more effective treatment.

“So I thought a higher dose was going to work better,” Shababi said.

Instead, the high dose had a toxic effect. Mice given more of the virus died sooner than untreated mice. Meanwhile, mice given a low dose of the gene therapy lived longest. They regained muscle function and strength in both the forearms and the hind limbs and became more active.

In fact, some of them survived long enough to mate and produce offspring.

Initially, Shababi housed her SMARD1 mice in the same cage as their mothers so that the moms could intervene if the sick pups become too feeble to feed themselves. When the male pups became well, their moms became pregnant.

“That was another surprise,” Shababi said. “That was when I knew I had to separate them.”

Shababi marks a pup, only a few days old, with permanent marker so she can identify each mouse in her study. | photo by Jennifer Lu, Bond LSC .

Shababi marks a pup, only a few days old, with permanent marker so each mouse in her study can be identified. | photo by Jennifer Lu, Bond LSC .

In another twist, Shababi discovered that the route of injection also mattered.

To get the treatment across the blood-brain barrier and to the spinal cord, Shababi used a special type of injection that passes through the skull and the ventricles of the brain, and into the spine.

This was no easy task.

The newborn mice were no larger than a gummy bear. To perform the delicate work, Shababi — who has written a chapter in a gene delivery textbook about this procedure — had to craft special needles with tips fine enough for this injection. She added food coloring to the injection solution so she could tell when it had reached its intended destination.

“After half an hour, you will see it in the spinal cord,” Shababi said. “The blue line in the spine: that’s how you can monitor the accuracy of the injection.”

Unfortunately, repeated injections in the mice caused hydrocephaly, or swelling in the brain.

“They get a dome-shaped head,” Shababi explained.

The swelling happened in all three treatment groups, but most frequently in the group that received a high dose of viral gene therapy. This reinforced the finding that while a low dose was beneficial, a high dose was even more harmful than no treatment at all. It’s unclear why.

Christian Lorson is a professor of veterinary pathobiology at the Bond LSC. His research focuses on spinal muscular atrophy and more recently, SMARD1. | photo by Hannah Baldwin, Bond LSC .

Christian Lorson is a professor of veterinary pathobiology at the Bond LSC. His research focuses on spinal muscular atrophy and more recently, SMARD1. | photo by Hannah Baldwin, Bond LSC .

The Lorson lab plans to continue studying SMARD1 and this treatment, in particular, how changing the delivery routes for gene therapy can improve outcomes in treating SMARD1.

“It’s not as simple as replacing the gene,” Lorson said. “It comes down to the delivery.”

Injections in the brains of mice are meant to mimic spinal cord injections in humans, but intravenous delivery could be another option. However, intravenous injections, which travel through the blood stream and to the entire body, might cause off-target effects that could interfere with the effectiveness of the treatment.

Once researchers better understand how to optimize dosing and delivery on the cellular and organismal level, the therapy can move closer to clinical trials, Lorson said.

Even though gene therapy for SMARD1 is still in its early stages, he said he was optimistic that developing treatments for rare genetic diseases is no longer the impossible task it seemed even ten years ago.

Spinal muscular atrophy (SMA) is a prime example of a recent success, Lorson pointed out. In the last six years, gene therapy for that disease has moved from the research lab to Phase I clinical trials.

“While it feels like a long time for any patient and their families,” Lorson reassured, “things are moving at a breakneck pace.”

 

The study, “Rescue of a Mouse Model of Spinal Muscular Atrophy With Respiratory Distress Type 1 by AAV9-IGHMBP2 Is Dose Dependent,” was published in Molecular Therapy, a journal published by Nature Publishing Group. This work was supported by a MU Research Board Grant (C.L.L.); MU College of Veterinary Medicine Faculty Research Grant (M.S.); the SMA Foundation (C.P.K.); National Institute of Health/National Institute of Neurological Disorders and Stroke grants; and the Missouri Spinal Cord Injury Research Program (M.L.G.).

Combating Climate Change: Q&A with Naomi Oreskes

Naomi Oreskes is a professor of the history of science at Harvard University and a geologist by training.

At a time when global warming was framed by the media as a debate, her 2004 paper in the journal Science showed that climate change was a settled fact among climate scientists.  Of the 928 papers she sampled in her literature search, not a single author denied the reality of climate change. Digging further, Oreskes explored in her book, Merchants of Doubt, co-authored with Eric Conway, the people, organizations, and motivations behind climate science misinformation. From cigarettes and acid rain to global warming and the ozone hole, Oreskes and Conway uncovered how industries such as Big Tobacco and Big Oil employed a core group of ideologically-motivated scientists to fabricate doubt and stymie government regulations.

Since the publication of Merchants of Doubt, Oreskes has been active in conversations about how we can move beyond debate and towards climate change intervention and action. She and Conway also wrote a sci-fi novel imaging a catastrophic future when society in the past (our present) failed to act on climate, The Collapse of Western Civilization.

Naomi Oreskes speaks on Saturday,3:30 pm as part of the LSSP Symposium, “Combatting Climate Change,” held at the Bond Life Sciences Center.

What has been the response of people who, through reading Merchants of Doubt or watching the documentary, have changed their minds about climate change?

Many people have written to me and Erik Conway to thank us for writing the book.  I’d say the most common response was that the book helped them to understand why there was so much opposition to accepting the scientific evidence.  I can’t say that I know for sure that thousands of people changed their minds after reading the book, but I do know that among those who did, the link to the tobacco industry was most compelling.  Our research showed that the opposition was not rooted in problems with or deficiencies in the science. 

You said in an interview with Mongabay, “In our society, knowledge resides in one place, and for the most part, power resides somewhere else.” How can we hold accountable oil and gas companies which have quietly known since the early 1980s that burning fossil fuels contributes to global warming, but used their power to impede actions that would combat climate change?

I’m not a lawyer, so I cannot answer the legal aspects of this question, but state attorneys around the country are now looking into that question.  As a citizen and a consumer, I can say this:  One way we can hold companies accountable by not investing in them,  and this is why I support the divestment movement. We can also boycott their products. In the current world, that is very difficult to do, but we can make a start. I installed an 8-watt solar PV system in my house, and we are now just about net-zero for electricity.

Is it possible to make up for 30 years of squandered time?

No of course not. Lost time is lost time. But knowing how much time has been lost, we should have a sense of urgency now, try not to lose any more. 

Which strategies are being proposed for immediate climate action? Are environmental scientists and economists in agreement over which courses of action make the most sense?

Yes I think so.  Nearly everyone who has studied the issue agrees that the most effective immediate action that is available to us is to put a price on carbon.  This will immediately make renewables and energy efficiency more economically attractive, and it will send a signal to investors that fossil fuels will no longer be given a free pass for their external costs. This means that future returns will be greater in the non-carbon based energy sector.  Anyone interested in this should read Nicolas Stern’s very informative book, Why are we Waiting?

How might the nomination of Merrick Garland to the Supreme Court and the results of the 2016 presidential elections affect the role that the US will play in combating climate change? Best case and worse case scenarios.

Best case: Republicans in Congress come to their senses, and listen to fellow Republicans like Bob Inglis, Hank Paulson, and George Schultz who have made the conservative case for putting a price on carbon.  They can do this pretty much any way they want— through  a tax, thru tradeable permits, or whatever.  it’s clear Democrats would support either, and we know from experience that either approach can work, so long as the price is real (i.e., not just symbolic.) Right now Alberta is talking about $20—that is probably a bit low. BC  is at $30 

Worst case: read The Collapse of Western Civilization.  You’ll find my answer there.

Of all the important issues out there, what motivates you to devote your time and energy to fighting climate change?

Oh that’s a good question.  I didn’t decide to work on climate change, I fell into it when Erik Conway and I tripped over the merchants of Doubt story.  Then, as I learned more and more about the issue, I came to appreciate scientists’ sense of urgency about it. 

 

Rodents of unusual appetites

How food cravings and eating affects the brain
By Jennifer Lu | MU Bond Life Sciences Center

When it comes to cookie dough, we’re not the only ones who can’t control our cravings. Kyle Parker’s rats couldn’t resist, either, thanks to a tweak in their brain chemistry.

Parker studies the neuroscience of food-based rewards.

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Matthew Will, associate professor of psychological sciences at the Bond Life Sciences Center, studies the neuroscience of behaviors such as over-eating and addiction | photo by Jennifer Lu, Bond LSC

“It’s like when I eat dessert after I’ve eaten an entire meal,” said Parker, a former grad student from the lab of Bond LSC’s Matthew Will. “I know that I’m not hungry, but this stuff is so good so I’m going to eat it. We’re looking at what neural circuitry is involved in driving that behavior.”

Behavior scientists view non-homeostatic eating — that’s noshing when you’re not hungry — as a two-step process.

“I always think of the neon sign for Krispy Kreme donuts.” Will said, by way of example.

“The logo and the aroma of warm glazed donuts are the environmental cues that kick-start the craving, or appetitive, phase that gets you into the store. The consummatory phase is when you “have that donut in your hand and you eat it.”

Parker activated a “hotspot” in the brains of rats called the nucleus accumbens, which processes and reinforces messages related to reward and pleasure.

He then fed the rats a tasty diet similar to cookie dough, full of fat and sugar, to exaggerate their feeding behaviors. Rats with activated nucleus accumbens ate twice as much as usual.

But when he simultaneously inactivated another part of the brain called the basolateral amygdala, the rats stopped binge eating. They consumed a normal amount, but kept returning to their baskets in search for more food.

“It looked like they still craved it,” Will said. “I mean, why would a rat keep going back for food but not eat? We thought we found something interesting. We interrupted a circuit that’s specific to the feeding part — the actual eating — but not the craving. We’ve left that craving intact.”

To find out what was happening in the brain during cravings, Parker set up a spin-off experiment. Like before, he switched on the region of the brain associated with reward and pleasure and then inactivated the basolateral amygdala in one group of rats but not the other.

This time, however, he restricted the amount of the tasty, high-fat diet rats had access to so that both groups ate the same amount.

This way, both groups of rats outwardly displayed the same feeding behaviors. They ate similar portions and kept searching for more food.

But inside the brain, Parker saw clear differences. Rats with activated nucleus accumbens showed increased dopamine production in the brain, which is associated with reward, motivation and drug addiction. Whether the basolateral amygdala was on or off had no effect on dopamine levels.

However, in a region of the brain called the hypothalamus, Parker saw elevated levels of orexin-A, a molecule associated with appetite, only when the basolateral amygdala was activated.

“We showed that what could be blocking the consumption behavior is this block of the orexin behavior,” Parker said.

The results reinforced the idea that dopamine is involved in the approach — or the craving phase — and orexin-A in the consumption, Will said.

Their next steps are to see whether this dissociation in neural activity between cravings and consumption exists for other types of diets.

Will also plans to manipulate dopamine and orexin-A signaling in rats to see whether they have direct effects on feeding.

“Right now, we know these behaviors are just associated with these neural circuits, but not if they’re causal.”

 

Behavioral Neuroscience published the study, “Neural Activation Patterns Underlying Basolateral Amygdala Influence on Intra-Accumbens Opioid-Driven Consummatory Versus Appetitive High-Fat Feeding Behaviors in the Rat,” in the December 2015 issue. A grant from the National Institute of Drug Abuse supported this research.

Unmasking the unknown

Scientists explore genetic similarities between plants and mice

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University of Missouri PhD Candidate Daniel L. Leuchtman peers through an Arabidopsis plant. Leuchtman has been experimenting with replacing a gene in the plants immune system with a similar gene from mice. | Photograph by Justin L. Stewart/MU Bond Life Sciences Center

By Justin L. Stewart | MU Bond Life Sciences Center

Almost two-thirds of what makes a human a human and a fly a fly are the same, according to the NIH genome research institute.

If recent research at the University of Missouri’s Bond Life Sciences Center is verified, we’ll soon see that plants and mice aren’t all that different, either.

Dan Leuchtman studies a gene in Arabidopsis plants called SRFR1, or “Surfer One.” SRFR1 regulates plant immune systems and tell them when they are infected with diseases or illnesses. Leuchtman studies this model plant as a Ph.D. candidate at MU, splitting time between the labs of Walter Gassmann and Mannie Liscum.

His research involves breeding Arabidopsis plants missing the SRFR1 gene and then replacing it with the MmSRFR1 gene.

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A series of Arabidopsis plants show the differences between the plants, from left, without SRFR1, with MmSRFR1 and with SRFR1. | Photograph by Justin L. Stewart/MU Bond Life Sciences Center

So, what is MmSRFR1? Leuchtman and company believe it’s the animal equivalent of SRFR1, though they aren’t fully aware of all of its’ functions.

“We’re actually one of the first groups to characterize it,” Leuchtman said.

Arabidopsis plants missing the SRFR1 gene struggle to grow at all, appearing vastly different from normal plants. Leuchtman says that a plant missing the SRFR1 gene is a mangled little ball of leaves curled in on itself. “It’s really strange looking.”

While his experiments haven’t created statuesque plants equal to those with natural SRFR1 genes present, the Arabidopsis plants with MmSRFR1 show a notable difference from those completely lacking SRFR1. Leuchtman says the plants with MmSRFR1 lie somewhere in between a normal plant and one lacking SRFR1.

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University of Missouri PhD Candidate Daniel L. Leuchtman poses for a portrait in a Bond Life Sciences Center greenhouse. Leuchtman has been experimenting with replacing a gene in Arabidopsis plants immune system with a similar gene from mice. | Photograph by Justin L. Stewart/MU Bond Life Sciences Center

“At its’ core, it’s more understanding fundamental biology. How do we work? How do organisms tick? How do you go from DNA in a little bag of salts to a walking, talking organism?” Leuchtman said. “The more you know about how an organism functions, the more opportunities you have to find something that makes an impact.”

Bond Life Sciences Center Scientists Named to Thomson Reuters’ 2015 List of Highly Cited Researchers

By Bobby Remis | MU Bond Life Sciences Center

You can imagine it’s hard to distinguish yourself from the crowd when it comes to scientific papers.

But, publishing quality work in a well-known journal adds value to the whole scientific world by assisting others and inspiring new science. Three Bond LSC researchers recently were recognized for doing just that.

Bond Life Sciences Center scientists Chris Pires, Shuqun Zhang and Yidong Liu are among five University of Missouri System researchers named in the 2015 Thomson Reuters’ Highly Cited Researchers list.

This list spotlights the top 1 percent of papers published from nearly 9 million scientists and scholars. The Highly Cited Researchers 2015 list represents the world’s most influential scientific minds from 21 scientific fields. The rankings are based on how often scientific papers published in the last decade get cited in newly published research, according to Essential Science Indicators (ESI), a component of the Web of Science.

16458632870_36bcd5480e_k-e1427818315742-370x533Chris Pires, associate professor of Biological Sciences, studies the evolution of plants by looking at changes in their genetics over millions of years.  Pires published work in 2015 looking at how plant defenses evolved in tandem with the defenses of caterpillars that feed on them.

Shuqun Zhang is a Distinguished Researcher from the MU College of Agriculture, Food and Shuqun ZhangNatural Resources and a professor of Biochemistry. His research seeks to improve plants’ response to adverse environmental conditions. By identifying molecular targets that aid in protecting crops from disease, his research aims to help create healthier, more productive agricultural products. In particular, he focuses on a family of enzymes called mitogen-activated protein kinases (MAPKs) that are involved in plant cell-to-cell communication and plant interaction with its environment.

Yidong LiuYidong Liu is a senior research specialist from MU’s Department of Biochemistry that manages Shuqun Zhang’s lab. She also works on MAPKs and their role in plant defense responses such as pathogen-induced ethylene biosynthesis and phytoalexin induction.

The Bond Life Sciences Center is an interdisciplinary research center at the University of Missouri exploring problems in human and animal health, the environment and agriculture since 2004. Learn more about our research by visiting bondlsc.missouri.edu.

You shall not pass: the basic science of blocking HIV

 

Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies viruses such as HIV. | photo by Jennifer Lu, Bond LSC

Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies viruses such as HIV. | photo by Jennifer Lu, Bond LSC

Nineteen colorful foam flowers decorate the walls of Marc Johnson’s office, a memento from his lab members when they “redecorated” while he was out of town.

Each flower is labeled in bold Sharpie with the names of viruses and viral proteins that his lab studies—MLV, RSV, Gag, Pol, to name a few.

One flower stands out, marked in capital letters: H-I-V.

Johnson, an associate professor of molecular microbiology and immunology, is one of four researchers at Bond LSC who studies HIV, the virus that leads to AIDS. His research focuses on understanding how HIV assembles copies of itself with help from the cells it infects.

Like most viruses, HIV hijacks cellular functions for its own purposes.

“It has this tiny itty bitty little genome and yet it can infect 30 million people,” Johnson said. “It doesn’t do it by itself.”

To understand how viruses reprogram the proteins in our bodies to work against us, he said, you have to understand the cells they infect. If cells were a chamber, then viruses are the keyhole.

For example, cells use a protein called TSG101 to dispose of unwanted surface macromolecules by bending a patch of cellular membrane around the macromolecule until it is surrounded inside a membrane bubble. The process, like trapping a bug inside a sheet of tissue paper, is called budding.

The cell sweeps all the pinched-off bubbles into a larger receptacle, or multivesicular body. These bodies, Johnson said, act as the cell’s garbage collection system. To dispose of the trash, the compartments become acidic enough to disintegrate everything inside or fuse with the cell membrane so that the trash gets dumped outside the cell.

It’s like in the second Star Wars movie, “The Empire Strikes Back,” Johnson said. “They just drop all their garbage before they go into hyperspace, and that’s how the Millennium Falcon got out.”

HIV uses the same housekeeping mechanism to break out of infected cells and infect more cells, but it remains unclear which other host proteins HIV commandeers.

“It’s all part of the puzzle,” Johnson said.

THE GAME CHANGER

On his desk, Johnson keeps a white legal pad with a list of 16 projects written in blue ink.

Marc Johnson observes cells modified with CRISPR under the microscope. | photo by Jennifer Lu, Bond LSC

Marc Johnson observes cells modified with CRISPR under the microscope. | photo by Jennifer Lu, Bond LSC

“Things make it off the list or they’ll get added,” Johnson said. “Or they’ll spend years on the back burner. I have a lot of projects.”

One of the biggest projects involves using CRISPR/Cas9 — a precision gene-editing tool — to identify genes that make a cell resistant to viral infections.

“It’s a game changer. It really is,” Johnson said. “It’s so cool.”

The technology uses a missile-like strand of guide RNA to target specific sites in the genome for deletion. Before CRISPR, scientists had to suppress gene expression using methods that were neither permanent nor absolute.

But because CRISPR manipulates the genome itself, Johnson said, there’s less doubt about what is happening.

Using the CRISPR library, the Johnson lab can scan the effects of 20,000 unique gene deletions in a population of cells. When these cells, each of which contains a different deleted gene, are exposed to HIV, not all of them die. Those that survive can cue researchers in to which genes might be important for blocking HIV infection.

And if another researcher has doubts that a gene is truly knocked out, Johnson said, you can tell them, “I’ll just send you the cell line. You try it and see for yourself.”

A DAY IN THE LIFE

The Johnson lab is a tight-knit group that consists of a lab manager, two grad students, a postdoc and four undergrads.

Dan Cyburt — a third year grad student — studies molecules that interact with proteins that keep HIV from infecting the cell, such as TRIM5α. TRIM5α, a restriction factor, blocks replication of the viral genome.

Graduate student Yuleum Song prepares cells for viral infection in the BL-2 hood. | Image by Jennifer Lu, Bond LSC

Graduate student Yuleum Song prepares cells for viral infection in the BL-2 hood. | Image by Jennifer Lu, Bond LSC

Fourth year grad student Yuleum Song focuses on how the viral envelope protein, Env, is packaged into viruses before they break free from cells. While Env isn’t necessary for viral assembly and release, she said, it’s critical for the infection of new cells.

Undergrads work in a tag team, picking up where the other left off, to generate a collection of new viral clones.

And lab manager Terri Lyddon keeps day-to-day experiments on task.

Lyddon, who has been with the Johnson lab for ten years, spends much of her day working with cells inside the biosafety level 2 hood. The area is specifically designated for work with moderately hazardous biological agents such as the measles virus, Samonella bacteria, and a less potent version of HIV.

Normally, HIV contains instructions in its genome for making accessory proteins that help the virus replicate, but the HIV strains used in the Johnson lab lack the genes for some of these proteins. That means the handicapped viruses can infect exactly one round of cells and spread no further.

Lyddon also ensures quality control for the lab by making sure students’ work is reproducible.

As a pet project, Johnson also independently confirms new findings reported in academic journals about HIV. Sometimes, Johnson says, the phenotypes that get published are not wrong, but they tend to represent the best outcomes, which might only exist in very specific scenarios.

“They’re only right by the last light of Durin’s day,” Johnson said, making a Lord of the Rings reference to a phenomenon in The Hobbit that reveals the secret entrance to a dwarven kingdom only once a year.

Because scientists base their work on the research of other scientists, he said, it’s always important to check.

A RECONSIDERED POSITION

According to the World Health Organization, 37 million people worldwide in 2014 have HIV or AIDS. The virus infects approximately two million new individuals every year. Breakthroughs in treatment have turned the autoimmune disease from a highly feared death sentence into a chronic and manageable condition.

For the longest time, HIV researchers scrambled to find better therapies against HIV why trying to develop a vaccine that could prevent AIDS.

But in the past five years, Johnson says he’s noticed a shift: researchers are gaining confidence in the possibility of finding a cure, something he once thought was impossible.

“Now it’s been demonstrated that it’s possible to cure a person,” Johnson said, referring to the Berlin patient. “So it’s only going to get easier.”

However, Johnson pointed out, most people would never undergo the kind of high-risk treatment that Timothy Ray Brown, the Berlin patient, received. Brown underwent a bone marrow transplant to treat his leukemia, and his new bone marrow, which came from an HIV-resistant donor, cured him of AIDS.

A “full blown cure” will be hard to attain, but Johnson believes there may be ways for HIV patients to live their lives without having to constantly take medication.

As an example, he points to certain “elite controllers” who are HIV positive but never progress further to show symptoms of AIDS. If scientists can figure out what’s different about their immune systems, Johnson said, then researchers could train the immune response in AIDS patients to resist HIV or keep it in check.

That’s a project for the immunologists. As a basic scientist, Johnson says he adds to the knowledge of how HIV works.

“I am not thinking about a therapy,” Johnson said, “but I’m also acutely aware that some of the best solutions come from basic science. “

Even though scientists haven’t discovered all the mechanisms behind cellular and viral function yet, Johnson said, the rules do exist.

“The sculpture is already there in the stone,” he said.

Johnson’s job is to chip away at the marble until the rules are found.

Family genes

MU freshman follows in aunt’s footsteps while exploring career options

Robert Schmidt poses with one of the cats that lives at Horton Animal Hospital, where he works part-time. Schmidt, a freshman studying biochemistry at the University of Missouri, is a member of the Discovery Fellows Program.

Robert Schmidt poses with one of the cats that lives at Horton Animal Hospital, where he works part-time. Schmidt, a freshman studying biochemistry at the University of Missouri, is a member of the Discovery Fellows Program where he is learning about plant genetics by working with biologist Scott Peck in the Bond Life Sciences Center. Photo by Justin L. Stewart | MU Bond Life Sciences Center

By Justin L. Stewart | MU Bond Life Sciences Center

Sometimes it’s socks. Another time, it was a book cover.

Robert Schmidt has retrieved quite a few things from misguided pets’ digestive systems as an assistant at Horton Animal Hospital, where he’s worked with his aunt for the past three years.

While most of the time he helps with simpler things — such as feeding kenneled animals or spaying and neutering pets — he also has amputated a dog’s toe after it had a lawnmower mishap.

Schmidt says he’s kind of following in his aunt’s footsteps. She majored in biochemistry at the University of Missouri and studied at its veterinary school, too. That’s what Robert wants to do with his life.

Schmidt demonstrates how to apply ear medication to a nervous small dog at Horton Animal Hospital on Forum.

Schmidt demonstrates to a pet owner how to apply ear medication to a nervous small dog at Horton Animal Hospital on Forum. Schmidt works at the animal hospital part-time with his aunt. Photo by Justin L. Stewart | MU Bond Life Sciences Center

Schmidt likes working with animals, but pets aren’t the only animals he’s interested in. An internship at the MU Animal Sciences Research Center two summers ago had Schmidt elbow deep in fistulated cows’ stomachs as he assisted in research that tested the affects of adding different metals in cows feed to help them better absorb protein.

“My family’s always had a dog, my sister has a cat and I work at a small animal clinic. For a while, I was just like, ‘I’m not going to work with cows. I didn’t grow up on a farm.’ But now, after the internship, I’m interested in getting some more experience.”

As a freshman in the Discovery Fellows Program, Schmidt now finds himself knuckle deep in dirt as he digs into plant genetics.

The Discovery Fellows Program pairs first-semester freshmen and sophomores with scientists in their chosen field, allowing them to get hands-on experience in research on campus while earning a $1,700 stipend.

That led Schmidt, an MU Honors College student, to the lab of biologist Scott Peck in the Bond Life Sciences Center.

Schmidt plants Arabidopsis seeds in a petri dish.

Schmidt plants Arabidopsis seeds in a petri dish inside the Bond Life Sciences Center, where his fellowship work is. Photo by Justin L. Stewart | MU Bond Life Sciences Center

Peck studies how plants recognize and respond to infections, specifically focusing on three proteins that begin to chemically modify shortly after a plant is infected.

No one knows what these proteins actually do, Peck said, making the research that much more interesting.

Schmidt has been growing Arabidopsis seeds of three different types, each missing one of the three proteins. Once those seeds have fully grown, Schmidt said they will cross-pollinate the three variations to hopefully create a plant without any of those three proteins.

He hopes to have triple mutants by early next semester, so they can experiment with them to better understand the roles of the absent proteins.

While Schmidt came into Peck’s lab with more of an interest in animals, he sees how the skills translate.

“If I worked with animals, I’d want to do genetics and I’m doing genetics with plants right now. A lot of the lab techniques are transferable.”

Schmidt moves an Arabidopsis seedling from a petri dish to fresh soil with the rest of the grown seedlings. Arabidopsis can grow from seed to seedling with two weeks. It's a favorite among scientist, according to the National Science Foundation.

Schmidt moves an Arabidopsis seedling from a petri dish to fresh soil with the rest of the grown seedlings. Arabidopsis can grow from seed to seedling with two weeks. It’s a favorite among scientist, according to the National Science Foundation. Photo by Justin L. Stewart | MU Bond Life Sciences Center

Like most freshman, the former Rockbridge Valedictorian is still figuring out his life plans. He’s considering a double major in math, a favorite subject of his, and now wonders whether he might attend graduate school instead of vet school.

“I really like learning, being in a classroom and being a student. I really enjoy that. I think research is almost like a career where you’re almost always a student. You’re just always learning.”

As for now, he’s just waiting for his first batch of Arabidopsis seeds to mature.

 

Putting down roots

Plant scientist Ruthie Angelovici joins the Bond Life Sciences Center

By Jennifer Lu | MU Bond Life Sciences Center

Ruthie Angelovici

Ruthie Angelovici

Ruthie Angelovici clearly remembers her big eureka moment in science thus far. It didn’t happen in a laboratory. It wasn’t even her experiment.

At the time, Angelovici was in college studying marine biology. She had spent a year going on diving trips to figure out whether two visibly different corals were polymorphs of the same species, or two separate species.

A simple DNA test told her the answer in one afternoon.

“That’s the day I decided that there was a lot to be discovered, just in the lab,” Angelovici said. She switched majors and hasn’t looked back.

Better Nutrition in Crops

Angelovici studies the molecular biology of plants.

As a newly minted assistant professor in biological sciences at the Bond Life Sciences Center, her goal is to increase the nutritional quality of staple crops like corn, rice, and wheat.

Although these crops make up 70 percent of people’s diet across the world, Angelovici said, they aren’t very nourishing.

Corn, rice, and wheat are deficient in several key nutrients called essential amino acids. For example, if a person lived on wheat alone, they would have to eat anywhere from three to 17 pounds of the grain per day to reach the daily recommended amount for these nutrients.

Moreover, harsh growing conditions cause amino acids levels in plants to plummet—an increasingly grave problem as the earth’s climate gets warmer.

“If you think about the future, we’re going to face more droughts, more heat,” Angelovici said. “We need to figure out how we can maintain quality under those circumstances.”

Scientists have been trying to improve the nutritional quality of crops for years, whether through classical breeding or genetic engineering. The latter requires knowing which genes to alter.

Angelovici uses a technique called genome-wide association mapping. This allows her to link the natural variations within a particular trait — say, a special type of amino acids that are branched in structure — with the genes that affect this trait.

In previous studies, Angelovici chose Arabidopsis thaliana, which is popular among plant scientists for its simple genome and short life cycle, as her model plant.

She collected seeds from 313 varieties and burst them open, one seed type at a time, to release their contents. After separating the free amino acids from the rest of the seed pulp, she measured the branched amino acid levels — as a ratio to each other and to other amino acids — to build a nutritional profile that acts like a fingerprint for each plant.

Angelovici used this fingerprint to identify plants that shared similar traits. Then she scanned their DNA for any small genetic variations, or mutations, that plants had in common.

When she tallied up the frequency of each mutation in what is called a Manhattan plot, she found one particular variation that outstripped the others, standing out like a skyscraper over a city: a small section on chromosome 1 close to a gene called bcat2.

Angelovici then switched this gene off. When branched amino acid levels changed, it suggested that this trait was linked to the bcat2 gene.

However, Angelovici warned that often plants resist genetic tinkering. They lose viability, or cannot germinate seeds.

“We get yield penalty,” Angelovici says, “and the question is why?”

Metabolism, she explains, is like a network. “If you pull one way, something else is going to be affected.”

That’s where bioinformatics comes in handy. Angelovici uses an approach called network analysis to look at many pathways within the plant at once. This allows her to see the big picture, as well as the fine detail.

Moving to Missouri

Angelovici has being studying plant metabolism for ten years. Originally from Israel, she earned her PhD in 2009 under Gad Galili at the Weizmann Institute of Science in Rehovot, Israel. Then, she continued her research as a postdoctoral fellow at Michigan State University.

She prefers working with plants to animals because plants are relatively easy to manipulate and breed. Also, she loves animals and at one point wanted to be a veterinarian.

Angelovici says she was immediately drawn to the University of Missouri, and is looking forward to collaborating with researchers at Bond LSC.

“There is a great program here, great plant people here,” she said. “So, Mizzou is spot on.”

Although she has found an undergraduate and a post-doctoral researcher to help her so far, the benchtops in her laboratory remain uncluttered save for some equipment, like glassware and a few gel boxes. Three pristine white lab coats hang neatly from hooks on the wall.

But Angelovici is not fazed by the enormous task of getting her lab up and running.

“I just love doing this. It’s like climbing a mountain,” Angelovici said, about the research process. “You do it slowly and then you feel like you’re going up and you are achieving more and you can see more. It’s really fulfilling.”

As for that big eureka moment, Angelovici says she doesn’t put much stock in it.

Then she laughs. “But maybe I will experience one, and then I’ll change my mind.”

Maze Runners

Female rats struggle to find their way in BPA study from MU and the NCTR/FDA

Cheryl Rosenfeld is one of 12 researchers partnering with the NCTR/FDA to study BPA

Cheryl Rosenfeld is one of 12 researchers partnering with the NCTR/FDA to study BPA

Despite concerns about bisphenol A (BPA), academic and regulatory scientists have yet to reach a consensus on BPA’s safety.

The National Institute of Environmental Health Sciences (NIEHS), the National Toxicology Program (NTP), the Food and Drug Administration and independent university researchers are working together to change that.

Five years after the Consortium Linking Academic and Regulatory Insights on BPA Toxicity, or CLARITY-BPA for short, launched, results are beginning to come in. This new information will allow researchers to better compare the effects of fixed doses of BPA on the brain, various cognitive behaviors, reproduction and fertility, accumulation of fat tissue, heart disease, the immune system, and several types of cancers.

“The idea of this Consortium is to examine the potential systems that have been previously suggested to be affected by BPA,” said Cheryl Rosenfeld, an associate professor of biomedical sciences at the University of Missouri and one of twelve researchers involved in the project.

Rosenfeld’s group looked at spatial navigation learning and memory. They found that prenatal exposure to BPA could potentially hinder the ability of female rats to learn to find their way through a maze. This effect was not seen in male rats.

Approved by the FDA in the early 1960s, BPA can be found in a wide variety of products, including plastic food and drink containers with recycle codes 3 or 7, water and baby bottles, toys, the linings of metal cans and water pipes, even patient blood and urine samples.

BPA has structural similarities to estrogen and can potentially act as a weak estrogen in the body.

In Rosenfeld’s experiment, researchers at the National Center for Toxicology Research gave pregnant rats a fixed dose of BPA every day: a low, medium, or high dose.

After the baby rats were born, researchers continued to dose the babies, both male and female, according to what their mothers had received.

When these rats reached three months old, they were tested in a circular maze with twenty possible exit holes, one of which was designated as the correct escape hole. Every day for seven days, researchers tested the rats’ abilities to solve the maze in five minutes and timed them as they ran.

Rats solve mazes in three ways, Rosenfeld said.

They can run through the labyrinth in a spiral pattern, hugging the outer walls, and work their way in until they find the correct exit hole in what is called a serial search strategy.

Or they might move aimlessly in the maze using an indirect search strategy, Rosenfeld said. “In this case, the rats seemingly find the correct escape hole by random chance.”

Lastly, they can travel directly from the center of the maze to the correct escape hole. The third strategy is considered the most efficient method because the rats find their way swiftly, Rosenfeld said.

Sarah Johnson, a graduate student and first author on the paper, assessed each rat’s performance in the maze using a three-point tracking program that recognizes the rat’s nose, body, and tail.

Using the program, Johnson measured their performances in terms of the total distance traveled, the speed at which the rat ran the maze, how long it took the rats to solve the maze (latency), and how often the rat sniffed at an incorrect hole.

The last two parameters are considered the best gauges of spatial navigation learning and memory.

“What you expect to see is that they should start learning where that correct escape hole is,” Rosenfeld said. “Thus, their latency and sniffing incorrect holes should decrease over time.”

Rosenfeld’s group found that female rats that had been exposed to the highest dose of BPA since fetal development were less likely to find the escape hole than rats that hadn’t been exposed to BPA.

As for how this study may translate to people, Rosenfeld said, “the same brain regions control identical behaviors in rodents and humans.”

She considers it a starting point for setting up future experiments that take into consideration sex differences in cognitive behaviors and neurological responses to BPA.

Immediate next steps for the Rosenfeld group include analyzing tissue collected from the brains of rats that had undergone maze testing. Rosenfeld’s team of researchers will measure DNA methylation and RNA expression in the brain to determine which genes might be involved in navigational learning and memory. Their overarching goal is to determine how changes in observed sex- and dose-dependent behaviors occur on the molecular level.

NIEHS grant U01 ES020929 supported this research. Additional coauthors include Mark Ellersieck and Angela Javurek of the University of Missouri, Thomas H. Welsh Jr. of Texas A&M University, and Sherry Ferguson, Sherry Lewis, and Michelle Vanlandingham of the National Center of Toxicological Research/Food and Drug Administration. Read the full study on the Hormones and Behavior website and browse the supplementary data for this work.