Jacqueline Ihnat, one of the 12 Cherng Summer Scholars, outside Dr. Cornelison’s lab at the Bond Life Sciences Center.
Sometimes the most learning occurs outside of the classroom.
For Jacqueline Ihnat, an opportunity to pursue research at the Bond Life Sciences Center this summer will give her that chance. She recently became one of 12 Cherng Summer Scholars, a full-time, ten-week program within the Honors College at MU.
“Doing research helps keep me focused on the bigger picture,” Ihnat said. “Sometimes in class we learn things that don’t seem entirely relevant or useful, but being part of a research lab allows me to apply some of the knowledge that I gain in the classroom. It’s a daily reminder of why I’m learning what I am.”
Jacqueline Ihnat’s passion for science started in high school. Her high school biology teacher ignited that love by teaching her how to struggle through difficult problems and concepts. Now, Ihnat is an MU pre-med student with a major in business management and a minor in Spanish.
Since Ihnat is fascinated with cells and how our bodies function, she’ll be studying the role of specialized stem cells in muscle regeneration and how they interact with muscle fibers — specifically the role of Eph-A3, a type of cell-surface receptor. This project will take place in the lab of Dawn Cornelison, a Bond LSC biologist who will be mentoring Ihnat this summer.
Jacqueline Ihnat as she pipettes samples in Dr. Cornelison’s lab in Bond LSC. | photo by Mary Jane Rogers, Bond LSC
Each muscle in the body is unique in its length, fiber organization and fiber type patterning, so Ihnat hopes to explore why two types of muscle — fast and slow twitch muscle — develop and regenerate to maintain each specific muscle fiber-type composition.
When a muscle is damaged from exercise or injury, a muscle’s stem cells, or “satellite cells,” will multiply, move towards the injury and form new muscle, replacing the damaged fiber. There is no research that determines if “fast” satellite cells create fast fibers and if “slow” satellite cells create slow fibers, and Ihnat hopes to tackle that question this summer. This kind of research gives scientists a deeper understanding of degenerative muscle diseases such as ALS, which could lead to more effective treatments and therapies.
The Cherng Summer Scholars program is supported by a gift from Andrew and Peggy Cherng and the Panda Charitable Foundation. The Cherng’s are the founders of Panda Express, a well-known restaurant chain. These scholarships support individually designed theoretical research, applied research or artistry projects under the mentorship of an MU faculty member.
For young scientists who are just starting to conduct research and struggling to feel successful, Ihnat has a few words of motivation.
“My high school biology teacher always said that research is 30 years of frustration and disappointment followed by 30 seconds of elation when you finally make a breakthrough,” she said. “Patience is key.”
Bond LSC Biologist Paula McSteen | photo by Jennifer Lu, Bond LSC
When developmental plant geneticist Paula McSteen thinks about the specimens she studies, one word comes to mind: potential.
She thought it as she stood in the midst of the first corn field she ever planted as a post-doctoral fellow in corn genetics.
She thinks it as she counts kernels from corn crosses that will be sent to Hawaii, a hotspot for corn geneticists looking to add a second harvest to their research year.
And she sees it in the students she mentors as a professor of biological sciences at MU and a researcher at the Bond Life Sciences Center.
Embracing the corn
McSteen entered the field of corn genetics 21 years ago, as a post-doctoral fellow in Berkeley.
“What’s really amazing is that when you plant a field of corn, the field is just bare,” McSteen says.
“A few weeks later, you come back and your plants are this high,” she says, gesturing with her hands. “And a few weeks later, they’re this high. And a few weeks later, they’re massive and it’s all just coming from nothing. The instructions are there in the seeds, but otherwise the plant is taking nutrients and water from the soil and using the air and sun to generate sugar for growing. It’s amazing. You come back and you’ve got this whole field of corn.”
During her first corn season, McSteen remembers being surrounded as far as the eye can see by corn as tall as people. “I feel like that’s one reason why people get into corn. You’re not staring down into a microscope, you’re embracing it. It’s right there in front of you.”
The feeling hasn’t gone away.
“When I see my plants,” McSteen says, “I’m excited about what’s going on with them. What could be happening here? What’s the meaning of these results?”
McSteen, who is Irish, grew up far removed from the sunny cornfields she works in. As a child in Dublin, she wasn’t particular outdoorsy. When her family went camping by the sea over the summer holidays, McSteen spent most of her time reading books. Her favorite subject in school was science. By the time she sat for her high school leaving exam, her classes were mainly in geography, science or math.
“I’ve always been fascinated with genetics,” McSteen says. Ever since she learned about Punnett squares in high school, its puzzle-like quality has appealed to her. “I just loved that you could figure out what the prediction could be from a certain cross.”
She applied and was accepted to her top choice university, Trinity College Dublin, where she studied genetics. She went on to pursue a PhD in Norwich, England studying how snapdragons make flowers.
When it came time for her to do her post-doc, she had the choice to work on Arabidopsis, a popular plant model, or maize, a crop with many opportunities for research funding. She chose the latter. The decision changed the course of her career, from her research focus to her country of residence.
Corn brought her to California, Pennsylvania and then the University of Missouri, which has a long history of corn genetics.
Researchers cross-pollinate corn by pouring pollen collected from the tassel over the ear. | Photograph by Jennifer Lu, Bond LSC
Everything we do starts with mutants
McSteen studies a part of corn plants called the meristem, which is filled with stem cells that become the reproductive organs of the plant: the tassel and the ears.
The tassel, where pollen is produced, is found at the top of the cornstalk, while the ears, which are the female reproductive organs, jut out from the sides. When and how they form depend on a growth hormone called auxin.
To understand auxin regulation, McSteen begins every summer with a field full of mutants. Each kernel contains a mutation, but it’s impossible to tell at first what is causing the mutation.
“To me, every single mutant is just potential. You can’t wait to find out what is mutated. You never know what you’re going to end up with.”
McSteen is interested in mutations that affect the tassels or ears. These plants produce ears with fewer kernels or tassels with fewer branches. Or they fail to make ears or tassels altogether. The defects are outward signs of problems in meristem development, and hint at disruptions to genes that are involved in how auxin is made, transported or perceived by the plant.
Once a mutant is identified, McSteen works backwards to find out which gene is causing the mutation, and where it is located on the chromosome. To date, her group has identified multiple genes related to auxin-mediated development, as well as two genes that affect the uptake or synthesis of essential nutrients.
A third project revolves around a strain of corn that produces half as many kernels as regular corn, causing it to look like grains such as rice or wheat. McSteen thinks that if they can understand what’s causing the shortfall in kernel development, it may be possible to engineer grains like rice and wheat to “double kernel” the way that corn does.
Ultimately, studying these genes help corn researchers to better understand plant development and improve yield.
Graduate student Eden Johnson photographs a mutant that has produced half as many rows of kernels. | Photograph by Jennifer Lu, Bond LSC
You always love the organism
To keep all their experiments going, the McSteen lab plants three acres of corn every summer. Each acre contains 750 rows; each row holds 30 plants.
With the aid of a hand held planter, they drop 67,500 kernels into the soil. Then they do what McSteen calls a lopsided “planter’s shuffle” to stamp the soil down so that it covers every kernel.
“You can do a whole acre of corn in a few hours,” she said. “It’s hard work. You’re sore afterwards.”
During Missouri summers, temperatures can reach over 100 degrees Fahrenheit. It feels even hotter when it rains. The ground is either hard as a rock before it rains or so muddy after it rains that researchers have to take care not to wrench their ankles in the thick muck.
McSteen has worked on corn for so long that she’s developed severe allergies to corn pollen. It’s not uncommon among corn researchers, but her allergies prevent her from taking part in the pollination step that takes place at the height of summer.
“You’re out there in 100-degree heat getting the job done,” she says. “It’s a real bonding experience for the lab.”
To pollinate the corn, they slip paper bags over the ears and tassel of their plants. Covering the ears prevents accidental fertilization of the ears from stray pollen blown about by the wind. The bag over the tassel allows researchers to collect the yellow powder that will be used for controlled pollination.
“The next day, you bang the tassel and the pollen falls out into the bag,” McSteen says. “Then you gather it all up and you pour it on the ear.” It’s possible to pollinate about 100 plants in an hour, but you have to start early and work quickly, McSteen says. Otherwise, all the pollen is dead by noon.
McSteen’s allergies prevent her from shelling corn as well, but she’s on deck for planting and harvest time and all the other stages in between.
“If you’re a corn geneticist, you’re out there working with the plants. You always love the organism.”
Paula McSteen labels the envelope of kernels from a corn cross that will be grown in Hawaii. | Photograph by Jennifer Lu, Bond LSC
A Collaborative Community
“To be a corn geneticist, you have to be very organized and plan ahead,” McSteen says. Because it takes a long time to grow several generations of corn, she’s only beginning to see the results of experiments she started years ago.
As a way to increase productivity, corn researchers send their seeds to warm places such as Mexico, Chile or Hawaii that can accommodate a winter harvest.
In the lab, McSteen chooses kernels from carefully selected mutants to ship to an island in Hawaii. There, a company will plant and harvest the corn for her, but she usually sends two of her researchers down to take care of the pollinations themselves.
McSteen counts out thirteen yellow kernels that are shiny and mold-free. “Potential,” she says, as she slips the seeds in an envelope with the cross information labeled on the front.
A three week trip to Hawaii in the winter isn’t as exotic as it sounds, says Eden Johnson, a third year graduate student in McSteen’s lab. On the island, the beaches are rocky and full of riptides. “It is literally cornfields, one diner and a stop sign. The whole island exists for corn.”
“When you’re down there in Hawaii, you hang out with the other researchers,” McSteen says. “If their field is peaking and your field is not, then you’ll go help.”
In her experience, the corn community tends to be collaborative rather than competitive. She suspects it’s because everyone recognizes that corn takes a long time to grow.
“If you find out you’re working on similar things, you’ll work together, divide the work and do it together,” McSteen says. Researchers don’t race each other to be the first one to publish. “They won’t do that because they have respect for how long it takes to grow the corn.”
Katy Gurthrie removes a bag used to collect pollen from the tassel. | Photograph by Jennifer Lu, Bond LSC
Growing careers alongside corn
McSteen is as serious about mentorship as she is about corn. “It’s part of the job of being a professor”.
According to Katy Guthrie, a second year graduate student in the lab, McSteen takes a Goldilocks approach to managing her students.
Neither too hands on or too hands off, “it’s exactly what I need,” Guthrie says. “She’s kind of like my academic parent for the next five years.”
Back at Penn State, McSteen supervised a PhD student who was talented writer. “I noticed this and gave her opportunities to write.” McSteen introduced her to a science writer and encouraged her to spend a summer writing for a science publication. “Now she works for the National Academy of Sciences. I’m really proud of her, and I feel like she’ll have a big impact communicating science to the public.”
A post-doc wanted to do go into teaching, so McSteen invited her to co-teach her class. Her post-doc went on to become a teaching professor at Mizzou. Another student turned her research experience in mapping mutants in corn into a successful career at a corn company.
“I want to enable the people in my lab to reach their full potential,” McSteen says. “I always try to figure out what they want to do in the future and try and facilitate that.”
Researchers find evidence of a genetic modifier that can improve symptoms of Spinal Muscular Atrophy
Chris Lorson examines axons through a microscope. Lorson’s lab recently published results that showed evidence that the protein plastin 3 affects the severity of SMA. | Photo by Eleanor Hasenbeck, Bond LSC
Eleanor Hasenbeck | Bond Life Sciences Center
Two new potential treatments might improve the lives of patients living with Spinal Muscular Atrophy.
Researchers in the Lorson lab at Bond Life Sciences Center recently produced a new drug that increases the lifespans of mice with SMA, and they found evidence that an increased level of the protein plastin 3 lengthened life span and improved the animal’s nerve function.
Two genes impact Spinal Muscular Atrophy, SMN1 and SMN2. In a healthy body, SMN1 creates a protein called SMN that helps maintain motor neurons controlling muscle movement. If someone is born without SMN1, their body relies on SMN2 to produce this protein, but a small change in the SMN2 gene causes it to make much less of the protein than needed. This leads to SMA, a disorder where an individual loses motor and nervous function, often starting in childhood, over a number of years.
Although rare, sometimes siblings develop SMA, providing an unusual insight into SMA development. Discordant siblings — or siblings that both have SMA but have different severities of the disorder— suggest that other factors could contribute to SMA.
Researchers are investigating why this happens. One theory is that a “genetic modifier,” another gene or protein elsewhere in the DNA, impacts the severity of SMA. The protein plastin 3 could be this modifier.
Plastin 3 doesn’t improve the severe SMA mice, but extended the lives of mice with more mild cases of the disorder. The Lorson lab created its own SMA drug, an antisense oligonucleotide that allows SMN2 to produce a functional protein. The drug is capable of extending survival of SMA mice from approximately 13 days up to 150 days from a single treatment. The typical lifespan of a lab mouse is 1.3 to 3 years.
Kevin Kaifer, a graduate student in the Lorson lab, gave the SMA mice a low dose injection of the drug, increasing their lifespan to about 30 days. Then, they modified a gene in the mice to increase the level of plastin 3. Mice that received the drug and the plastin 3 therapy lived about 40% longer than mice that received only the drug.
Lorson said the results provide proof of concept that plastin 3 does not make more SMN, but actually decreased disease severity. The SMA mice showed improved neuromuscular junctions, the sites where nerve cells fire electrical impulses to the muscles in the body.
“That’s really where plastin 3 is designed to function, at the neuromuscular junction,” Lorson said. “So that brings the idea of plastin 3 full circle; it does not increase SMN, but it does improve the function of the nerve which is where plastin 3 is supposed to function normally.”
This discovery shows promise for a future treatment to some with the disease.
“SMA is a very broad clinical spectrum disease, so there are patients who have an incredibly severe form, and patients that don’t develop disease until adulthood,” said Chris Lorson, a Bond LSC scientist. “Perhaps one therapy is not going to address that very broad clinical spectrum, and you’re going to need to address different parts of the disease with different therapeutics.”
Despite it’s relative rarity as a disease, new treatments for SMA are hitting the market. In December, the Food and Drug Administration approved Spinraza, an antisense oligonucleotide similar to the drug the Lorson lab. But the infrequency of SMA means treatment comes at a cost: $750,000 for the first year of Spinraza and $375,000 for subsequent years. Spinraza is an FDA-designated orphan drug, meaning it’s a treatment for a disease that affects less than 200,000 people in the U.S. To incentivize research into rare diseases, The Orphan Drug Act allows pharmaceutical companies longer exclusive patent rights. Drugs that treat rare diseases that impact children, including Spinraza, can be allowed priority review, basically putting these drugs on a faster track from lab to market. Though the act has led to more research in certain diseases, it has sparked controversy as patients with no other treatment options are burdened with the resulting drugs’ high cost.
Still, it’s the first FDA approved treatment available to the 9,000 Americans living with SMA.
“I think collectively this is a very exciting time in the SMA field, whether we’re talking about SMN targeting compounds or drugs that are capable of augmenting function,” Lorson said. “To have a rare disease that has so many shots on goals, so to speak is really exciting.”
“The SMA community is really a model for how foundations, families, patients and government agencies can come together,” Lorson said. He said families and government agencies are often in the same room as academics, biotechnology and pharmaceutical companies during meetings.
“The amount of support from the patients, the families and the non-profit world has really helped drive SMN research… I think that’s really helped push SMA from an unknown 20 years ago, to an approved drug.”
Christian Lorson is a professor of veterinary pathobiology at the Bond LSC. His research focuses on spinal muscular atrophy. The results of this study were published in an article in JCI Insight, “Plastin-3 extends survival and reduces severity in mouse models of spinal muscular atrophy.” This work is partially funded by grants from the Muscular Dystrophy Association, FightSMA, the Gwendolyn Strong Foundation, and the Missouri Spinal Cord Injury/Disease Research Program. CureSMA provided the initial support for the development of the drug/antisense oligonucleotide used in these studies.
Cool dudes, hot mommas. This is the underlying concept behind sex development in painted turtles, a species that lacks sex chromosomes.
A painted turtle’s sex is determined by temperature at which the eggs are incubated at critical stages during early development. Eggs incubated at lower temperatures produce male turtles, while those incubated at higher temperatures results in females.
However, early exposure to certain environmental chemicals that mimic hormones naturally produced in individuals can override incubation temperature. Scientists at the Bond Life Sciences Center have teamed up to study how endocrine disrupting chemicals (EDCs), namely bisphenol A (BPA) and ethinyl estradiol (EE), result in irreversible sexual programming of the brain in painted turtles.
“Turtles do not have sex chromosomes. Instead, they demonstrate temperature sex determination. But if they are exposed to EDCs prior to when certain organs form, such chemicals can cause partial to full sex reversal to female both in terms of the gonad and brain,” said Cheryl Rosenfeld, a Bond Life Sciences Center investigator and an associate professor of biomedical sciences at the University of Missouri. “The males will essentially act like females in terms of their behavioral responses.”
The hormones Rosenfeld refers to are BPA and EE, two widely used EDCs. BPA is present in many commonly used household, such as plastic food storage containers, store receipts, and dental fillings. The EE is present in birth control pills and can accumulate in many aquatic environments. These chemicals have been identified in all aquatic environments tested to date, including rivers and streams. Thus, exposure of turtles and other species that inhabit such environments can potentially lead to irreversible effects.
Previously, Rosenfeld and colleagues had studied how these chemicals change behavior of painted turtles after treating the eggs with BPA and EE under male-promoting temperatures. They discovered that male turtles that are early exposed to chemicals exhibit greater spatial navigational ability and improved memory, which are considered female-typical behaviors.
Rosenfeld and colleagues postulated that if the behavioral patterns differ between those exposed to BPA and those who were not, the different behaviors may be due to underlying and persistent differences in the neural circuitry between these two groups.
Cheryl Rosenfeld is a Bond Life Sciences Center investigator and an associate professor of biomedical sciences at the University of Missouri. | photo by Jinghong Chen, Bond LSC
A gene map for turtle
In order to address this possibility, Rosenfeld teamed up with Scott Givan, associate director of the Informatics Research Core Facility, to study the gene expression profiles of the turtles and potentially identify patterns of gene expression associated with the altered behaviors.
After Rosenfeld’s team tested the behavior of the turtles, they collected RNA from the turtle brains to perform a technique called RNAseq that isolates all of the transcripts expressed in this organ. RNA is a nucleic acid that carries genetic information and is indicative of the expression level of every gene in the turtle genome. Once these sequencing results were obtained, Givan had to align the results to the painted turtle genome that has been previously sequenced and annotated. He then determined the transcripts that were differentially expressed in turtles developmentally exposed to BPA or EE versus those unexposed individuals.
There are no existing turtle gene pathway profiles. Therefore, Givan had to analyze the turtle gene expression profiles based on those previously identified in human samples.
“[One] of the most important things in this paper is the linkage of the gene expression profile to behavior difference,” Givan said. “But the gene and metabolic pathway data don’t exist for turtles. We had to basically infer pathway modeling based on the human metabolic pathway maps.”
Scott Givan is the associate director of Informatics Research Core Facility and research assistant professor of molecular microbiology and immunology. | photo by Jinghong Chen, Bond LSC
The results suggest that BPA and, to a much lesser extent, EE exposure overridden incubation temperature and altered the gene expression profile in the brain to potentially reprogram brain to the female rather than male pathway. Specifically, BPA exposure was associated with metabolic pathway alterations involving mitochondria, such as oxidative phosphorylation and influenced ribosomal function.
Mitochondrial activity provides energy. Up-regulation of metabolic pathways in mitochondria can lead to more energy in brain cells, which may have permitted BPA-exposed turtles to demonstrate faster responses and greater cognitive flexibility, including enhanced spatial navigational ability that was previously identified in this group.
The other changes — oxidative phosphorylation and ribosomal function — play key roles in protein synthesis. Specifically, oxidative phosphorylation generates is one of main pathways involved in generating ATP, which is considered an energy source. Ribosome functions to assemble amino acids together for the synthesis of proteins, including enzymes that may facilitate metabolic reactions.
Less appealing males
The possibility for shifting brain sex has a real impact in the wild.
In the beginning, a turtle’s brain is neutral, as it is requires hormones to sculpt and direct it to be male or female. However EDCs, such as BPA and EE, can usurp these normal pathways and cause the brain of otherwise male turtles to develop more feminine characteristics.
“There are certain programmed behaviors [male turtles] have to do to entice the female to select them as their reproductive partner, but if he is not demonstrating these male-typical behaviors, she will likely reject him,” Rosenfeld said. “Even if such chemicals reprogram the brain and subsequent adult behaviors without affecting the gonad, it could have individual and population consequences by reducing a male’s likelihood of breeding.”
Combined with possible shrinking and already inbred population, declines in male turtles or skewing of sex ratio to females that could originate due to EDC-exposure, could push turtle species that exhibit temperature-dependent sex determination (TSD) to the brink of extinction.
“The concern also with turtle species that exhibit TSD, climate change and exposure to EDCs can lead to detrimental and irreversible imbalances in sex ratio favoring females over males, and thereby compromising genetic diversity of the population as a whole,” Rosenfeld said.
Future studies in Rosenfeld’s lab plan to extend the research to female turtles to learn whether the chemicals have any effects on females derived based on TSD rather than those due to exposure to environmental chemicals that are similar to estrogen. She also hopes to look into individual brain regions, such as the forebrain and hippocampus that are essential for cognitive abilities.
Cheryl Rosenfeld is a Bond LSC investigator,associate professor of biomedical sciences, and research faculty member in the Thompson Center for Autism and Neurobehavioral Disorders. Scott Givan is the associate director of Informatics Research Core Facility and research assistant professor of molecular microbiology and immunology.
This research was funded by the Mizzou Advantage Program, the Bond Life Sciences Center and the University of Missouri Office of Research.
Researchers in the Mendoza-Cozatl lab grow beans in a soil that simulates Martian soil
By Eleanor C. Hasenbeck | Bond Life Sciences
As NASA works to send people to Mars, researchers at the Mendoza-Cozatl lab at Bond Life Sciences Center are exploring the possibility of sending beans to the red planet. The journey from Earth to Mars alone would take somewhere between 100 to 300 days. To feed astronauts on these longer missions, scientists are studying space horticulture.
Norma Castro, a research associate in the lab, studies how common beans grow in a soil that simulates Mars’ red soil. The common bean is a good candidate for interstellar cultivation. Beans are a very nutritious crop, and their affinity for nitrogen-fixing bacteria can improve soil health while requiring less fertilizer. Castro is trying to understand how different varieties of beans could grow in the soil.
“This kind of research not only will tell us the right plants to take to Mars, but also which kind of technology needs to be developed,” Castro said.
Mahmoud Khalafalla, a Ph.D. student at Weisman’s lab, is isolating RNA from salivary glands of Sjögren’s syndrome mouse model to look for the expression of pro-inflammatory genes. | photo by Jinghong Chen, Bond LSC
By Jinghong Chen | Bond Life Sciences Center
Our immune system is often the key to our health. Everyday, it works to protect us from foreign invaders such as bacteria and virus, but what happens when it attacks our own tissues?
Gary Weisman, a Curator’s Distinguished Professor of Biochemistry at the Bond Life Sciences Center, is working to advance our understanding of the mechanisms behind immune system function and autoimmune diseases such as Sjögren’s syndrome.
In our immune system, B cells are responsible for producing antibodies to recognize foreign invaders. However, in many autoimmune diseases, B cells produce autoantibodies that recognize our own proteins, causing inflammation and tissue damage. In Sjögren’s syndrome (SS), they attack the glands that produce saliva and tears.
Patients with SS often suffer chronic dry eyes and dry mouth, which might lead to bacterial infection, difficulties in swallowing and speech.
“The symptoms decrease the quality of life rather than the length of life,” said Lucas Woods, research lab manager in Weisman’s lab.
Although SS patients are at higher risk of developing lymphoma cancers and other concurrent autoimmune diseases that may increase mortality, Woods further explained.
According to the Sjögren’s Syndrome Foundation, there are an estimated four million people living with the disease in the U.S. For unknown reasons, 90 percent of them are female.
Yet, current clinical treatments only reduce symptoms by using artificial saliva and tears or cholinergic agents to promote fluid secretion, but there is no approved treatment to reduce the inflammation of the glands themselves. This is the focus of Weisman’s lab.
Sensor of danger
There are 15 different types of nucleotide receptors in humans that regulate numerous cell processes from inflammatory responses to tissue regeneration. Those receptors are stimulated by nucleotides such as ATP. In the past three and a half years, Mahmoud Khalafalla, a Ph.D. student in Weisman’s lab, has focused on one of them in particular – the P2X7 receptor.
Previous studies show increased P2X7 expression in salivary glands from SS patients, as compared to healthy individuals. To understand the reasons behind this, Weisman’s lab used genetically modified mice that develop disease traits similar to SS patients.
In this mouse model, Sjögren’s-like disease occurs when the immune cells invade salivary glands and damage the tissue, leading to decreased saliva production. The invasion of immune cells is triggered by proinflammatory cytokines, a type of signaling molecule that promotes the recruitment of immune cells to the inflamed areas.
But what induces those cytokines?
Weisman’s lab tries to piece together the answer. For the first time, they found that the P2X7 receptor is responsible for the release of these proinflammatory molecules from salivary gland epithelial cells.
To function, most cell-surface receptors require ligands that bind to the receptor to induce cellular responses. The ligand for the P2X7 receptor is ATP – the “energy currency inside of cells.” P2X7 receptors are activated when high concentrations of ATP are released to the outside of the cells, which typically occurs when the cells are injured during inflammation.
“P2X7 receptors [act like] the sensor of danger,” Khalafalla said.
After identifying the role of the P2X7 receptor, the lab then asked: if we stop its activation, what would happen?
Using a drug that inhibits P2X7 receptor activation, they blocked the receptor in their SS mouse model to determine its effect on the development of autoimmune disease. Interestingly, saliva secretion was restored when the P2X7 receptor is blocked while the levels of invading immune cells in salivary glands were dramatically reduced.
“This gives us the thought that [blockade of the] P2X7 receptor is really a promising strategy to reduce salivary inflammation. This may not only relate to Sjögren’s syndrome, but to other autoimmune diseases as well,” Khalafalla said.
Our receptor
Another similar receptor that plays a role in autoimmune diseases is the P2Y2 receptor, which has been referred to as “our receptor” by Weisman’s lab.
As one of the researchers who proved the existence of this receptor, Weisman has spent most of his career studying it.
One of his research projects investigating P2Y2 receptors in human disease recently gained a grant extension for another five years from the National Institutes of Health. The lab found that in a mouse model of SS, similar to the P2X7 receptor, the expression of P2Y2 receptors was increased in both the salivary gland epithelial cells and immune cells.
Furthermore, after they knocked out the P2Y2 receptor in the SS mouse model by breeding them with genetically-modified P2Y2 receptor knockout mice, the inflammation of salivary glands was dramatically reduced.
“The very next step is that we are going to isolate these immune cells out of the diseased mouse salivary glands, and characterize what kinds of cells they are. We want to know exactly which ones are controlling the development of autoimmune diseases, and how P2Y2 receptors and nucleotides like ATP in general are contributing to the diseases,” Woods said.
Gary Weisman is a Curator’s Distinguished Professor of Biochemistry at the Bond Life Sciences Center. His research focuses on the relationship between inflammatory diseases and nucleotide receptors. He currently works on a collaborative research project with Dr. Carisa Petris, an eye surgeon at the MU Hospital, to understand the mechanism of how Sjögren’s syndrome damages the tear-secreting lacrimal glands in mice.
Plants on the left grow with rhizobia bacteria, one type of fixing nitrogen bacteria, in the greenhouse, while the plants on the right grow without the bacteria. | photo by Jinghong Chen, Bond LSC
Jinghong Chen | Bond Life Sciences Center
Since eight years old, Beverly Agtuca knew she wanted to be a scientist.
A trip to Philippines changed Agtuca, an American-born Filipino, and inspired her passion on plants.
“My grandma always told me to work in the field all day so that they can have enough food for us to eat,” Agtuca said. “The life [in Philippines] is so different from here…I want to not just provide food but be that scientist trying to figuring something out, and hopefully saving the world.”
Agtuca is on her way to her dream. She is now a third year doctoral student in Gary Stacey’s lab at Bond Life Sciences Center with a focus on nitrogen-fixing bacteria.
Although she has been involved in research since high school, Agtuca recently faced a new challenge of telling people about her work. The Preparing Tomorrow’s Leaders of Science class tasked her with making a 90-second video to explain her two-year study to the general public.
Her team, “The A Team,” chose to go with the benefits of having nitrogen-fixing bacteria.
For decades, people have been adding nitrogen fertilizers to plants to improve yields, but this can lead to pollution in water systems and ecosystems. Scientists need to enhance plant productivity to meet a huge food demand by the year of 2050.
One little bacteria might make this possible and save the world. Rhizobia, a type of natural bacteria in soil, are able to fix nitrogen via biological nitrogen fixation. These bacteria can convert nitrogen gas into ammonia as a plant nutrient source, while the plants give all the carbon sources back to the bacteria.
“It is like a walky-talky,” Agtuca said. “They are communicating with each other.”
Yet before speaking to the public, Agtuca needs to explain the plant-bacteria interaction to her teammates. Students less well versed in science like Jessica Kaiser, a strategic communication student, thinks of science differently.
“The biggest issue we ran into is jargon, like basic science words that [my teammates] are so comfortable with,” Kaiser said. “We need to focus on what people care about instead of the technical sides, to focus on why it matters to anybody rather than just to a science person.”
Within two weeks, they produced the video “Good Microbes: reducing pollution one farm at a time.” Along with two other teams, their videos will be commented and judged by representatives from Monsanto.
“The A Team” stands together at Bond Life Sciences Center. From left to right: Jessica Kaiser, Sven Nelson, Anna Glowinski, Eleni Galata and Beverly Agtuca. | photo by Jinghong Chen, Bond LSC
The 90-second video is just a glimpse of Agtuca’s study. In the last two years she has been focusing on the use of a new technique — laser ablation electrospray ionization mass spectrometry (LAESI-MS) — that does in situ metabolic profiling of tissues. The lab is using LAESI-MS to investigate the metabolites in a well-characterized model plant-rhizobium system, specifically nitrogen-fixing soybean nodules resulting from root infection by the symbiotic bacterium Bradyrhizobium japonicum.
This work includes a huge collaboration that was developed through a Department of Energy (DOE) grant involving the George Washington University, Washington D.C. and the Environmental Molecular Science Laboratory (EMSL), Pacific Northwest National Laboratory, Richland, WA.
LAESI-MS works like a superhero’s laser-like beams. You first aim the laser on the sample, which then heats it and causes neutral particles to be released into the air. This plume of neutrals is then captured and ionized by the electrospray, and finally analyzed by the spectrometer to figure out the exactly what metabolites in nodules are involved in biological nitrogen fixation.
“It takes about three seconds to analyze one sample using this LAESI-MS technique,” Agtuca said. Other metabolic techniques require extensive pre-treatment of the sample before analysis.
By analyzing the data collected via LAESI-MS, the lab is able to confirm that future plant studies could apply this new approach to understand the interactions between plant and bacteria.
Agtuca’s research is a long way from her first experiences with plants. She still remembers the moment she found her plants in her own garden died. She was less than 10 years old, yet devoted to taking care of her plants with water and fertilizers.
“I was really sad. I could not get my tomatoes, peppers and eggplants to live.…That makes me think that I want to answer why they didn’t grow,” Agtuca said.
More than ever, her future is helping her answer those question for herself.
Gary Stacey is a Bond LSC investigator and MU curators’ professor of plant science and MSMC endowed professor of soybean biotechnology. Read more here about Stacey lab.
Sven Nelson is a USDA/ARS postdoctoral research scientist at the University of Missouri. Anna Glowinski is a Ph.D. student in the USDA/ARS lab. Jessica Kaiser is a graduate student in strategic communication. Eleni Galata works as the team mentor and she is a Ph.D. student in agricultural and applied economics at MU.
Vinit Shanbhag mixes the CRISPR plasmid DNA with cells. The lab will test whether the gene of interest has been knocked out of the cells later. | photo by Jinghong Chen, Bond LSC
Jinghong Chen | Bond Life Sciences Center
It might be strange to say, but in a way the Australian soil led scientist Michael Petris to where he is now.
In certain areas of Australia, soils suffer from extremely low level of copper bioavailability, resulting in poor growth and neurological problems on sheep.
Petris, a Bond LSC investigator and professor of biochemistry who was born in Australia, now spends his time studying how copper, an essential mineral in human body, works in cells to build and maintain essential functions.
Recently published work from his lab focuses on how the ATP7A protein, one of the major proteins, cycles within the cell.
“Copper is solely acquired from diet. The absorption of copper from the intestine in the blood needs ATP7A,” Vinit Shanbhag, a Ph.D. biochemistry student at Petris’ lab and an author of the study, said. “It transports copper to different copper dependent enzymes and exports free copper from the cell to the outside.”
After exporting copper at the cell membrane, ATP7A needs to come back to its steady-state location within the Golgi apparatus of cells – via a process called retrograde trafficking. But one question baffled scientists: what are the key elements that lead ATP7A coming back?
Back in the late 90s, Petris discovered the importance of one single di-leucine in retrograde trafficking of ATP7A. For those of you wondering, leucine is an amino acid that forms the building blocks of proteins like ATP7A, while di-leucine consists of two of them connected via a peptide bond.
His team wished to identify other signals for retrograde trafficking, but one technical hurdle stood in the way— the ATP7A gene is unstable when grown in bacterial plasmids, the traditional way of amplifying genes in the lab.
Commercial DNA synthesis was the answer. This method could create artificial genes in the laboratory.
“We reasoned that if we introduced enough silent mutations into a DNA sequence, we could avoid or change the region of instability in the native sequence without affecting the encoded protein,” Petris said.
To stabilize the gene, they changed more than 1,000 nucleotides within a 3,000 nucleotides segment, and thus solving the problem of instability of the ATP7A gene. In doing so, they subsequently found that in fact multiple di-leucines that are required for retrograde trafficking of ATP7A. This approach could be used by other laboratories whose gene of interest is also unstable.
An overlooked mineral
“If you ask [people], is it important to understand iron nutrition? Is it important to understand calcium nutrition? Most people would say of course! … But, perhaps you would not get the same answer for copper, despite the fact there is a little dispute that copper is important,” Petris said.
As an essential micronutrient, copper performs central functions to develop and maintain human skin, bones, brains and other organs.
“If you don’t have enough copper in your body, you cannot use oxygen to make energy,” Petris said. “If you don’t have copper, you would not survive.”
Pregnant women who carry a mutated ATP7A gene on their X chromosome can pass it on to their children in the form of Menkes disease.
Menkes disease is a genetic disorder that results in poor uptake and distribution of copper to cells. The incidence of this disease is estimated to be one in 100,000 newborns, according to U.S. National Library of Medicine.
Infants with Menkes disease typically begin to develop symptoms during infancy and rarely live past the first few years of life. Abnormally high accumulation of copper in kidneys and low-level accumulation in the liver and brain, cause visible symptoms like sparse hair, loose skin and failure to grow.
Despite copper’s importance, it also can be a potentially toxic nutrient.
“Copper deficiency can be a problem but too much copper is also a problem. There should be a balance,” Shanbhag explained.
The liver normally stores excessive copper and excretes it into bile to release it out of the body. Yet people with genetic disorders that preventing copper excretion might suffer Wilson’s disease, leading to life-threatening organ damage.
Shanbhag said people with Wilson’s disease accumulate toxic amounts of copper in liver and other organs, causing Kayser–Fleischer rings that encircle the pigmented regions of the eye, a hue caused by copper deposits in the cornea.
Its clinical consequences differ from chornic liver failure to neurological sysmptoms like tremors, dystonia, ataxia and cognitive deteriortation.
About one in 30,000 people have Wilson disease, according to National Institute of Diabetes and Digestive and Kidney Diseases.
Starving tumors
Vinit Shanbhag mixes the CRISPR DNA with mammalian cells to specifically delete a gene in these cells in lab hood. | photo by Jinghong Chen, Bond LSC
In 2013, Petris’ lab published the first direct evidence suggesting ATP7A is essential for the dietary absorption of copper. Since then they have dug deeper into this copper transporter, and his lab now sets their sights on a greater enemy of human health — cancer.
Tumor growth requires access to large amounts of nutrients. Without an adequate supply of oxygen and nutrients, tumors fail to grow and survive. Scientists have identified that by preventing access to nutrients—for example by blocking the growth of new blood vessels—they could starve the tumor of nutrients.
Copper is a key nutrient for tumor growth. With the new-introduced system CRISPR-Cas9 — a genome editing tool to knock out specfic genes — his lab has explored how to exploit understanding of copper metabolic pathways to withhold copper from cancer cells.
“Copper starvation might be a good approach as an anti-cancer strategy,” Petris said.
Weapon of the immune system
Michael Petris, a professor of biochemistry at MU, stands with his lab. From left to right: Vinit Shanbhag, Nikita Gudekar, Michael Petris, Kimberly Jasmer-McDonald, Aslam Khan. | photo by Jinghong Chen, Bond LSC
Currently, four members study in Petris’ lab to tackle the relationship between copper and various diseases. Petris plans to expand his research to another area: the role of copper in innate immunity against bacterial pathogens.
This is the topic of Petris’ next grant. Nutritional immunity, which describes how the mammalian host withholds nutrients from the invading bacteria during infection, is very well-described for iron and zinc.
Yet copper performs differently.
During infection, the level of copper in blood actually goes up instead of going down. The immune system concentrates copper at sites of infection and within regions where the bacteria are engulfed.
“We speculate that copper is being used as weapon by the host to kill the bacteria,” Petris said. “That is the area we are trying to develop further.”
What if you could have pork without the pig? Nicholas Genovese’s cultured meat could provide a more environmentally friendly meat
This screenshot of a supplemental video included in Genovese’s study shows cells contracting in response to a neurotransmitter. | photo courtesy of the Nicholas Genovese
By Eleanor C. Hasenbeck | MU Bond Life Sciences Center
Scientists are one step closer to that reality. For the first time, researchers in the Roberts’ lab at Bond Life Sciences Center at MU were able to create a framework to make pig skeletal muscle cells from cell cultures.
In vitro meat, also known as cultured meat or cell-cultured meat, is made up of muscle cells created from cultured stem cells.
As a visiting scholar at the University of Missouri, Nicholas Genovese mapped out pathways to successfully create the first batch of in vitro pork. Genovese also said it was the first time it was done without an animal serum, a growth agent made from animal blood.
According to Genovese, his research in the Roberts Lab was also the first time the field of in vitro meats was studied at an American university.
“I feel it’s a very meaningful way to create more environmentally sustainable meats, which is going to use fewer resources, with fewer environmental impacts and reduce need for animal suffering and slaughter while providing meats for everyone who loves meat,” Genovese said.
The research could have environmental impacts. According to the United Nation’s Food and Agriculture Organization, livestock produce 14.5 percent of all human-produced greenhouse gas emissions. Livestock grazing and feed production takes up 59 percent of the earth’s un-iced landscape. Cultured meat takes up only as much land as the laboratory or kitchen (or carnery, the term some members of the industry have coined for their facilities) it is produced in. It uses energy more efficiently. According to Genovese, three calories of energy can produce one calorie of consumable meat. The conversion factor in meat produced by an animal is much higher. According to the FAO, a cow must consume 11 calories to produce one calorie of beef for human consumption.
And while Michael Roberts, the lab’s principal investigator, is skeptical of how successful in vitro meat will be, he said the results could yield other benefits. Researchers might be able to use a similar technique as they used to create skeletal muscle tissue to make cardiac muscle tissue. Pork muscles are anatomically similar to a human’s and can be used to model treatments for regenerative muscle therapies, like replacing tissue damaged by injury or heart attacks.
“I was interested in using these cells to show that we could differentiate them into a tissue. It’d been done with human and mouse, but we’re not going to eat human and mouse,” Roberts said. “The pig is so similar in many respects to humans, that if you’re going to test out technology and regenerative medicine, the pig is really an ideal animal for doing this, particularly for heart muscle,” he added.
While you won’t find in vitro meat in the supermarket just yet, Genovese and others are working toward making cultured meats a reality for the masses. Right now, producing in vitro meat is too costly to make it economically viable. Meat is produced in small batches, and the technology needed to mass-produce it just isn’t there yet. Genovese recently co-founded the company Memphis Meats, where he now serves as Chief Scientific Officer. The company premiered the first in vitro meatball last year, at the hefty price tag of $18,000.
“We are rapidly accelerating our process towards developments of technology that we hope will make cultured meats accessible to everyone within the not-so-distant future,” Genovese said.
Nicholas Genovese was a member of the Roberts lab in Bond LSC from 2012 to 2016. The study “Enhanced Development of Skeletal Myotubes from Porcine Induced Pluripotent Stem Cells” was recently published by the journal Scientific Reports in February 2017.
Scientists prove parasite mimics key plant peptide to feed off roots By Roger Meissen | Bond LSC
A nematode (the oblong object on the left) activates the vascular stem cell pathway in the developing nematode feeding site (syncytium) on a plant root. | photo by Xiaoli Guo, MU post-doctoral research associate
When it comes to nematodes, unraveling the root of the issue is complicated.
These tiny parasites siphon off the nutrients from the roots of important crops like soybeans, and scientists keep uncovering more about how they accomplish this task.
Research from the lab of Bond LSC’s Melissa Mitchum recently pinpointed a new way nematodes take over root cells.
Melissa Mitchum | photo by Roger Meissen, Bond LSC
“In a normal plant, the plant sends different chemical signals to form different types of structures for a plant. One of those structures is the xylem for nutrient flow,” said Mitchum, an associate professor in the Division of Plant Sciences at MU. “Plant researchers discovered a peptide signal for vascular stem cells several years ago, but this is the first time anyone has proven that a nematode is also secreting chemical mimics to keep these stem cells from changing into the plant structures they normally would.”
Stem cells? Xylem? Chemical mimics?
Let’s unpack what’s going on.
First, all plants contain stem cells. These are cells with unbridled potential and are at the growth centers in a plant. Think the tips of shoots and roots. With the right urging, plant stem cells can turn into many different types of cells.
That influence often comes in the form of chemicals. These chemicals are typically made inside the plant and when stem cells are exposed to them at the right time, they turn certain genes either on or off that in turn start a transformation of these cells into more specialized organs.
Want a leaf? Expose a stem cell to a particular combination of chemicals. Need a root? Flood it with a different concoction of peptides. The xylem — the dead cells that pipe water and nutrients up and down the plant — requires a particular type of peptide that connects with just the right receptor to start the process.
But for a nematode, the plan is to hijack the plant’s plan and make plant cells feed it. This microscopic worm attaches itself to a root and uses a needle-like mouthpiece to inject spit into a single root cell. That spit contains chemical signals of its own engineered to look like plant signals. In this case, these chemicals — B-type CLE peptides — and their purpose are just being discovered by Mitchum’s lab.
“Now a nematode doesn’t want to turn its feeding site into xylem because these are dead cells it can’t use, so they may be tapping into part of the pathway required to maintain the stems cells while suppressing xylem differentiation to form a structure that serves as a nutrient sink,” Mitchum said. “To me that’s really cool.”
This means these cells are free to serve the nematode. Many of their cell walls dissolve to create a large nutrient storage container for the nematode and some create finger-like cell wall ingrowths that increase the take up of food being piped through the roots. For a nematode, that’s a lifetime of meals for it while it sits immobile, just eating.
But how did scientists figure out and test that this nematode’s chemical was the cause?
Using next generation sequencing technologies that were previously unavailable, Michael Gardner, a graduate research assistant, and Jianying Wang, a senior research associate in Mitchum’s lab, compared the pieces of the plant and nematode genome and found nearly identical peptides in both — B-type CLE peptides.
“Everything is faster, more sensitive and we can detect things that had gone undetected through these technological advances that didn’t exist 10 years ago,” Mitchum said.
To test their theory, Xiaoli Guo, postdoctoral researcher and first author of the study in Mitchum’s lab synthesized the B-type CLE nematode peptide and applied it to vascular stem cells of the model plant Arabidopsis. They found that the nematode peptides triggered a growth response in much the same way as the plants own peptides affected development.
They used mutant Arabidopsis plants engineered to not be affected as much by this peptide to confirm their findings.
“We knocked out genes in the plant to turn off this pathway, and that caused the nematode’s feeding cell to be compromised. That’s why you see reduced development of the nematode on the plants.”
This all matters because these tiny nematodes cost U.S. farmers billions every year in lost yields from soybeans, and similar nematodes affect sugar beets, potatoes, corn and other crops.
While this discovery is just a piece of a puzzle, these pieces hopefully will come together to build better crops.
“You have to know what is happening before you can intervene,” Mitchum said. “Now our biggest hurdle is to figure out how to not compromise plant growth while blocking only the nematode’s version of this peptide.”
