Research

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.).

Learning on Different Levels

New outreach program teaches CAFNR students to make plant science knowledge accessible to a younger audience
Written by Stephen Schmidt | Science Writer in the College of Agriculture, Food and Natural Resources

Although abundant light was shining through the windows, it was the quiet before the storm. Andrew Ludwig, a University of Missouri sophomore majoring in plant sciences, surveyed the small tables and chairs spread out before him in the laboratory of the Benton STEM Elementary School on a recent Monday afternoon. He sifted through his notes. He was ready, even though it was his first time stepping foot in the building — and he was about to talk to a crowd of students spanning from the first to the fifth grade.

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MU graduate student Michael Gardner addresses the students gathered at a recent meeting of the Benton Elementary Science Club. Gardner co-presented a talk called “How Do Flowers Drink?” as part of the “It’s All About Plants” series that was launched earlier this spring. | Photo by Roger Meissen, Bond LSC

“I’m just going to try to stay enthused about everything because I’ve talked to some of my friends who are education majors and the big thing with conveying information to them is just being enthusiastic about it,” Ludwig said about his strategy on the “How Do Flowers Drink?” presentation he was about to give with fellow MU student Michael Gardner.

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MU sophomore Andrew Ludwig talks with Leo Batchelder-Draper during an exercise exploring how plants absorb nutrients as his mother, Miriam Batchelder, looks on. | photo by Justin Stewart, Bond LSC

“I think the key is keeping them moving along, keeping them interested, because as soon as you lose their attention, they’re going to do whatever they want to do, so it’s going to be keeping things moving along at a pace that they’re still getting it, but that they’re not bored out of their minds,” added Gardner, who is a fifth-year graduate student specializing in plant stress biology. “We’re talking about water transport in a plant, which is a concept you can spend a semester on in a college-level course and still not be considered an expert on the topic.”

Both Ludwig and Gardner are a part of the team at the Bond Life Sciences Center lab of Melissa Mitchum, an associate professor in the Division of Plant Sciences. It was Mitchum who was able to launch the new “It’s All About Plants” series this spring with the Benton Elementary Science Club thanks to the science outreach portion of a new National Science Foundation grant that will further delve into a deeper understanding of the interactions between plants and  parasitic nematodes she received in August and a partnership with the MU College of Education’s ReSTEM InstituteMU Office of Undergraduate Research and Columbia Public Schools.

The end result is a program that fosters learning of all varieties: The elementary students learn about plants, while the college students learn how to take complex ideas and break them down to a more accessible level. Furthermore, the program pairs undergraduate students such as Ludwig with graduate students such as Gardner and principal investigator mentors from across campus — involving the existing NSF-funded initiative Freshman Research in Plant Sciences (FRIPS) and the Students for the Advancement of Plant Pathology (SAPP) in the process.

“I think it’s a challenge for all of us who have advanced degrees to really think about where we were long ago and bring the concepts down to a basic level,” Mitchum said, “but I think it’s very important for us to be able to communicate our science at that level to really get the kids excited about research.”

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Melissa Mitchum | photo by Roger Meissen, Bond LSC

She added that the program “really reinforces the concept of engagement and active learning.” In particular, it reinforces hands-on learning through a variety of activities. “I think that makes a big difference in learning,” said Mitchum, who will have sat with the children for five of the 10 presentations that started on Feb. 22. “I think that’s one of the reasons why I’m such a strong advocate of undergraduate research and getting in a lab and learning how to work in a lab. The same thing applies here. When the kids are doing experiments in the science club, they’re going to retain that information a lot more.”

School’s in session

“Class! Class! Class!”

“Yes! Yes! Yes!”

With those loud words reverberating through the room Ludwig had the attention of those before him — for a moment, anyway. The chatter had quieted down as he began to explain the main activity of the day, which would involve carnation flowers and six solutions of household items (sugar, salt, baking soda, vinegar, soda and food coloring) mixed with water in the same-sized cups – and one scenario with just water.

With the help of several volunteer MU students (whose hours are coordinated by Mitchum’s co-PI, Deanna Lankford, a research associate in the College of Education), the assignment was to pour 100 millimeters into each one of the cups.

The cups were then labeled with the name of the solution. This procedure was repeated three times. The white carnations were then cut to the height of the cup and placed in the water as a discussion ensued on what solutions would help the plant (besides plain water) and which ones would hurt.

 

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Deanna Lankford, a research associate from the MU ReSTEM Institute who helped found the Benton Elementary Science Club, helps Adrion Bradshaw open a container of blue food coloring as his twin brother Amahdrion fills writes out some observations with the help of volunteer Alp Kahveci. | photo by Justin Stewart, Bond LSC

Afterwards through the help of a video, the class was introduced to the idea of setting up controls and variables. The one variable, in this case, was the solution. All other factors (such as flower height, flower type, cup size and amount of liquid) were controlled, Ludwig explained. “We use the control to compare our other variables,” he said.

A moment later, Ludwig posed a question to the class: “Raise your hand if you think putting the flower in food coloring is going to change the color of the flower.” A collection of hands sprouted upward.

“We have some flowers that we put in the dye yesterday,” Ludwig continued, as he unveiled carnations that had white petals either tinged in red or blue to pass around to the class.

“Woah!”

“I was right!”

“I knew it!”

“Do we get to keep them?”

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A white carnation begins to show traces of blue food coloring in its petals after sitting in a solution with food coloring and water for a short period of time. | photo by Justin Stewart, Bond LSC

A white carnation begins to show traces of blue food coloring in its petals after sitting in a solution with food coloring and water for a short period of time.

Following a brief video showing a similar experiment relating to celery and food coloring, Gardner explained the phenomenon to the class: “The plant is basically a big straw, so as the water evaporates up the plant, it pulls more water with it and then the food coloring too, so it gets to the very top of the plant, either the leaves of a tree or the top of a flower, and when it gets there the water will be able to evaporate, but the food dye can’t so that’s why your flowers are turning blue or red, OK?”

The food coloring portion of the afternoon turned out to be the top highlight for many of the participants. When asked about all of the projects he had worked on during the spring, Amahdrion Bradshaw, a second grader, proclaimed that “the funnest one was about dying the plants.”

Haily Korn, a fellow second grader, agreed, saying that her favorite part of the program is when you “do fun experiments” and that her favorite experiment was “when you get to dye stuff.”

A perfect fit

Lankford and the ReSTEM Institute formed the Benton Elementary Science Club, which meets one afternoon every week during the school year, seven years ago — the same time Benton officially became a part of the STEM (Science, Technology, Engineering and Mathematics) program.

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Michael Gardner, a doctoral student in Melissa Mitchum’s laboratory, talks with Leo Batchelder-Draper about the water transportation systems in plants while fellow student Beatrice Innocent makes an observation across the table. Also at the table are Leo’s mother, Miriam Batchelder, and volunteer Taylor Mayberry. | photo by Justin Stewart, Bond LSC

“The science club has allowed our students to continue to expand their understanding of a variety of science topics through hands-on experiences after school,” said Heather McCullar, a STEM specialist who works at Benton. “The kids always leave club excited to share what they have learned with their families.”

Over the years, Lankford has helped many principal investigators with the education portion of their grants. When Mitchum asked her about getting involved with an existing partnership, Lankford immediately thought about the science club with its previously established learning format.

“Melissa said ‘I need help with this.’ And I said ‘Great, let’s talk,’” Lankford said, “And we did and we came up with the idea and it has been wonderful. I really like the fact that we have mentors and mentees doing the presentations.”

Under Mitchum’s direction, a series of meetings were set up with mentors and mentees last fall to develop the curriculum and lesson plans that would form the backbone of the course. Given that the grant is funded for a total of three years, the plan is to continue to teach the “It’s All About Plants” program, which has been well-received by students and school administrators alike, at Benton next spring.

Besides Mitchum, the other PI mentors from CAFNR who have taken part in the program are Gary Stacey, Curators Professor of Plant Sciences; Lee Miller, assistant professor of plant sciences; Xi Xiong, assistant professor of plant sciences; Walter Gassmann, professor of plant sciences; John Boyer, Distinguished Research Professor of Plant Sciences; Harley Naumann, assistant professor of plant sciences; Kevin Bradley, associate professor of plant sciences; Heidi Appel, senior research scientist, plant sciences; Jack Schultz, professor of plant sciences and director of the Bond Life Sciences Center; and Scott Peck, associate professor of biochemistry. PI mentors from the College of Arts and Science Division of Biological Sciences included Paula McSteen, Chris Pires, and Mannie Liscum.

The CAFNR students who took part in the science club series planning also recently hosted an outreach booth on t the Science Sleuth event on “Plants and Microbes” the MU campus April 16. In addition, Mitchum gave a talk at the Exploring Life Sciences symposium at the Bond Life Science’s Center recent Missouri Life Sciences Week.

Furthermore, the end goal is to take all of the lessons that are being created and turn them into a booklet that is easily accessible for teachers in Columbia Public Schools, and beyond, by posting the material online.

In the meantime, the lessons are being molded by the interactions of CAFNR students and those at Benton.

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At a recent session of the “It’s All About Plants” series at the Benton Elementary Science Club, students learned about how plants transport water by conducting tests with a variety of solutions made up of household products, including, from left, baking soda, salt and vinegar. | photo by Justin Stewart, Bond LSC

“You have some students who can’t write their names yet, and other people who know everything we’re trying to tell them before we start,” Gardner said, afterwards, referring to the beginning of the class when a student at the front of the room recited a brief and succinct view of a plant’s water transport system. “Apparently his grandma told him all of this stuff already. So yeah that was definitely a challenge that I think we did OK at it.”

“I think it’s really great for everyone involved from the elementary school students who are getting to learn about specific topics from people who are very well informed about it to undergraduate and graduate students who are getting a wider arrange of presentation skills,” Ludwig added. “You can get locked in your head about your research. It’s good to be reminded that the knowledge we gain through research also has a real-world application and that part of the scientific process is sharing.”

It’s a challenge that Mitchum said could serve as a benefit to anyone in the scientific community.

“It’s not easy for us to do many times,” Mitchum said of breaking complex idea down into something easy enough for a child to understand, or even an average adult. “It takes practice. So. It’s something we have to learn. Science communication is very important these days. Especially when we talk about what we’re doing in a lab, it’s very molecular, very cellular, but we have to find a way to make it relevant.”

She added that programs such as this one show children that not all scientists have “crazy hair with the goggles and lab coat. They see ‘Oh, these guys are just like everyday people.’ This is a realistic career for them.”

Would Amahdrion be interested in such a career path?

“Nope,” he said. “I want to be a basketball player.”

Still, when given the choice of attending the science club sessions or just going home after school, he has a quick reply: “Spending extra time at school because you learn more.”

Gres receives Von Schwedler Prize

Work on HIV capsid proteins earns prestigious retrivology award

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Anna Gres studies HIV capsid protein using X-ray crystallography. She recently won the 2016 von Schwedler Prize, which awards her $1,200 and gives her the oppportunity to speak this spring at the Cold Spring Harbor Retrovirus Meeting, one of the largest retroviral research conferences in the world. | photo by Roger Meissen, Bond LSC

Science is all about structure in the work of Anna Gres.

For the past four years, she’s looked closely at one HIV protein to figure out its shape in order to stop the virus.

“Capsid protein is extremely important during the HIV life cycle. About 1,500 copies of it come together to form the protective core around the viral genome,” said Gres, a graduate student in the lab of Bond Life Sciences Center’s Stefan Sarafianos and a Mizzou Ph.D. candidate in chemistry. “So, if you are able to somehow disrupt the interactions between the proteins or make them different, the virus loses its infectivity.”

Gres takes her work on this protein to a national stage next month when she speaks at the Retroviruses meeting at Cold Spring Harbor Laboratory — one of the most prestigious international conference on retroviruses — as the recipient of the 2016 Uta Von Schwedler prize. The prize recognizes the accomplishments of one distinguished graduate student as they complete their thesis.

HIV capsid protein has been studied for almost 30 years, but it’s been tricky to get a precise depiction of what it looks like. Gres uses X-ray crystallography to essentially capture the protein in all its 3-D glory. This method gives scientists the higher resolution picture to study the molecular structure of capsid protein. Her work allows the Sarafianos lab and others to study how it interacts and connects with other capsid proteins and the host protein factors of the cell HIV is trying to take over.

“In the past scientists had been splitting the capsid protein in two halves and crystallizing them separately. Another approach was to introduce several mutations to make it more stable,” Gres said. “You would think that it shouldn’t really matter if we have a few mutations, but the protein behaves in such a way that even slight changes result in subtly different interactions that are enough for the virus to lose its infectivity. We were able to crystallize the native protein without any mutations and that should give us more accurate picture.”

Now that the Sarafianos lab and Gres have a good idea of what that native protein looks like, they’ve moved on to other mutated versions of the protein that impair virus infectivity. This could give them insight into how scientists can stop HIV.

“Many labs reported numerous mutations in the capsid protein over the past 25 years that either increase or decrease the stability of the core, which often results in a noninfectious virus,” she said. “Right now we are interested in seeing what structural changes accompany these mutations and how they can affect the overall stability of the core.”

MU Scientists Fighting World Hunger

By Bobby Remis | MU Bond Life Sciences Center

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Sanborn Field, University of Missouri | photo by Kyle Spradley

In the years to come, climate change and population growth will drastically alter the world around us, impacting farmland and the way we grow food.

Scott Peck

Scott Peck, associate professor of biochemistry, studies how plants perceive and respond to changes in their environment.

New research from an interdisciplinary team at the University of Missouri is hoping to curb the decrease in food production due to climate change by studying the roots of corn and understanding its growth in these intense conditions.

Scott C. Peck — an investigator at the Bond Life Sciences Center — joins an interdisciplinary team that plans to study corn root growth in
drought conditions. The National Science Foundation (NSF) recently awarded them a $4.2 million grant to spend four years developing drought-tolerant corn varieties in an effort to sustain the 9 billion people estimated worldwide by 2050.

The interdisciplinary team is comprised of seven co-primary investigators from four MU colleges as well as the USDA-ARS.

Read the full release from Mizzou News.

Seminal work

How unruly data led MU scientists to discover a new microbiome
By Roger Meissen | MU Bond Life Sciences Center

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This seminal vesicle contains a newly-discovered microbiome in mice. Some of its bacteria, like P. acnes, could lead to higher occurrences of prostate cancer. | contributed by Cheryl Rosenfeld

It’s a strange place to call home, but seminal fluid offers the perfect environment for particular types of bacteria.

Researchers at MU’s Bond Life Sciences Center recently identified new bacteria that thrive here.

Cheryl Rosenfeld1.jpg “It’s a new microbiome that hasn’t been looked at before,” said Cheryl Rosenfeld, a Bond LSC investigator and corresponding author on the study. “Resident bacteria can help us or be harmful, but one we found called P. acnes is a very important from the standpoint of men. It can cause chronic prostatitis that results in prostate cancer. We’re speculating that the seminal vesicles could be a reservoir for this bacteria and when it spreads it can cause disease.”

Experiments published in Scientific Reportsa journal published by Nature — indicate these bacteria may start disease leading to prostate cancer in mice and could pass from father to offspring.


A place to call home

From the gut to the skin and everywhere in between, bacterial colonies can both help and hurt the animals or humans they live in.

Seminal fluid offers an attractive microbiome — a niche environment where specific bacteria flourish and impact their hosts. Not only is this component of semen chockfull of sugars that bacteria eat, it offers a warm, protected atmosphere.

“Imagine a pond where bacteria live — it’s wet it’s warm and there’s food there — that’s what this is, except it’s inside your body,” said Rosenfeld. “Depending on where they live, these bacteria can influence our cells, produce hormones that replicate our own hormones, but can also consume our sugars and metabolize them or even cause disease.”

Rosenfeld’s team wasn’t trying to find the perfect vacation spot for a family of bacteria. They initially wanted to know what bacteria in seminal fluid might mean for offspring of the mice they studied.

“We were looking at the epigenetic effects — the impact the father has on the offspring’s disease risk — but what we saw in the data led us to focus more on the effects this bacterium, P. acnes, has on the male itself,” Rosenfeld said. “We were thinking more about effect on offspring and female reproduction — we weren’t even considering the effect the bacteria that live in this fluid could have on the male — but this could be one of the more fascinating findings.”

But, how do you figure out what might live in this unique ecosystem and whether it’s harmful?

First, her team found a way to extract seminal fluid without contamination from potential bacteria in the urinary tract.

“We gowned up just like for surgery and we had to extract the fluid directly from the seminal vesicles to avoid contamination,” said Angela Javurek, primary author on the study and recent MU graduate. “You only have a certain amount of time to collect the fluid because it hardens like glue.”

Once they obtained these samples, they turned to a DNA approach, sequencing it using MU’s DNA Core.

They compared it to bacteria in fecal samples of the same mice to see if bacteria in seminal fluid were unique. They also compared samples from normal mice and ones where estrogen receptor genes were removed.


The difference in the data

It sounds daunting to sort and compare millions of DNA sequences, right? But, the right approach can make all the difference.

“A lot of it looks pretty boring, but bioinformatics allow us to decipher large amounts of data that can otherwise be almost incomprehensible,” said Scott Givan, the associate director of the Informatics Research Core Facility (IRCF) that specializes in complicated analysis of data. “Here we compared seminal fluid bacterial DNA samples to publicly available databases that come from other large experiments and found a few sequences that no one else has discovered or at least characterized, so we’re in completely new territory.”

The seminal microbiome continued to stand out when compared to mouse poop, revealing 593 unique bacteria.

One of the most important was P. acnes, a bacteria known to cause chronic prostatitis that can lead to prostate cancer in man and mouse. It was abundant in the seminal fluid, and even more so when estrogen receptor genes were present.

“We’re essentially doing a lot of counting, especially across treatments to see if particular bacteria species are more common than others,” said Bill Spollen, a lead bioinformatics analyst at the IRCF. “The premise is that the more abundant a species is, the more often we’ll see its DNA sequence and we can start making some inferences to how it could be influencing its environment.”

Although this discovery excites Rosenfeld, much is unknown about how this new microbiome might affect males and their offspring.

“We do have this bacteria that can affect the male mouse’s health, that of his partner and his offspring,” Rosenfeld said. “But we’ve been studying microbiology for a long time and we still find bacteria within our own bodies that nobody has seen before. That blows my mind.”

The study, “Discovery of a Novel Seminal Fluid Microbiome and Influence of Estrogen Receptor Alpha Genetic Status,” recently was published in Scientific Reports, a journal published by Nature.

 

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.”

What’s in the spit?

Bond LSC is now producing monthly segments for KBIA, Columbia’s NPR station at 91.3 FM.

This month highlights the work of Melissa Mitchum, a molecular plant nematologist at Bond LSC and an associate professor of Plant Sciences in the College of Agriculture, Food and Natural Resources.

She studies nematodes, a pest that cost soybean farmers billions of dollars each year. Her lab recently helped discover that this tiny parasite produces molecules that mimic plant hormones in order to siphon nutrients from soybean roots.

Tune in at 12:30 to hear her profile or visit the Soundcloud link above to hear the segment.

 

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.