mizzou

BPA rewires the sex of turtle brains

By Jinghong Chen | Bond Life Sciences Center

Turtle box 2.jpg

Painted turtle eggs were brought from a hatchery in Louisiana, candled to ensure embryo viability and then incubated at male-permissive temperatures in a bed of vermiculite. Those exposed to BPA developed deformities to testes that held female characteristics.Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

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.

DSC_3253.jpg

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.”

DSC_3249.jpg

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.

Building blocks of life in the lab could revolutionize life for us all

NASA, NIH-funded work seeks to understand bio-chemical mechanisms of life on Earth, and among the stars
By Phillip Sitter | Bond LSC

Donald Burke-Agüero

Donald Burke-Agüero stands in his office in Bond LSC, holding a model of an RNA protein structure. Burke-Agüero studies the bio-chemical functions of RNA, and how those functions might be able to be artificially designed or replicated. | Phillip Sitter, Bond LSC

Any child obsessed with Legos knows the fun of creation bound only by imagination and the size or variety of the blocks within their pile.

For some scientists, that spirit extends into adulthood, but instead of plastic parts they think about arranging blocks of nucleic acids.

Scientists may not be able to create dinosaurs, dragons or mythical sea creatures the way kids with Legos can. Through the manipulation of nucleic acid building blocks though, they may be better able to understand how the processes of life on Earth work, as well as out among the stars.

“I have a lot of fun asking what is possible,” said Donald Burke, a Bond Life Sciences Center investigator who spends his time researching the building blocks of life.

Burke said he has been interested in the origins of life for 40 years, and he has been associated with NASA for about 20 years.

NASA’s interest in understanding the origins of life is pretty straightforward. It wants to know what clues to look for on other worlds to figure out if those planets also support life.

Many of Burke’s previous discoveries at Bond LSC are funded by NASA’s exobiology and evolutionary biology program.

“No, I have not thought of an excuse to fly anything up there. I’ve tried to think ‘which of my experiments would make sense to do in a micro-gravity or zero gravity environment?’” he explained of the prospect of sending some of his work into orbit, with a wry smile.

But, there’s even more to understanding the building blocks of life than looking for bio-chemical signatures out among the stars. Knowing how these parts are put together allows scientists like Burke to understand the origins and processes of Earth’s biology, and, conceivably create chemical and biological processes or even organisms not found in nature in the near future.

 

A quadrillion arrangements of blocks, one arrangement at a time

“Many of the molecules of life are built from strings of amino acids, or nucleotides or other building blocks,” Burke explained. He also noted that these buildings blocks are not just strings, but fold up into three dimensional shapes.

RNA, or ribonucleic acid, stands out as an essential building blocks in the bio-chemical processes of life.

Put simply, RNA is a kind of molecular structure of nucleic acids similar to DNA (deoxyribonucleic acid) that comes in many combinations. These combinations are at the core of every cell, and play a role in coding, decoding, regulating and expressing the basic operating instructions for each cell — its genes.

The molecules we’re talking about are almost unimaginably small. In one test tube, Burke said there can be one quadrillion of them — that’s a one with 15 zeroes after it. Put another way, that’s roughly equivalent to the estimated number of ants that live on Earth.

Burke’s work focuses on the end goal of being able to artificially create original RNA combinations. In what’s known as experimental evolution, “the population of molecules in the tube is evolving as a result of us imposing experimental constraints upon it.”

This artificial synthesis of RNA molecules looks to create random sequences or variations on natural RNA to create new ones non-existent in nature. A second route aims to selectively choose molecules with certain properties, and use them to build altogether new combinations.

“Their string-like properties allow us to copy them, and make more copies, and make more copies, and make more copies. Their shape-like properties allow us to observe the bio-chemical behaviors they may have,” Burke explained how he and other scientists interact with RNA’s structure in the lab.

“I don’t think we know what those limitations are yet,” he said of the capabilities of RNA.

The motivation for wanting to be able to intentionally design RNA molecules is so that it “can do the things we want it to do under the conditions where we want it to do those things,” he explained of the process of the process of selecting RNA sequences for specific properties.

“I want the ones that will bind a tumor cell. I want the ones that will bind a viral protein. I want the ones that will catalyze useful chemical reactions.”

 

RNA’s path to the future following in biology’s footsteps

The National Institutes of Health and other organizations recognize that engineered forms of RNA have the potential to fight diseases, and they have funded Burke’s work.

He has studied RNA that instructs human cells on how to defend themselves from HIV and is now looking at other RNA that interferes with the proteins of the Ebola virus.

The expectation is that such therapeutics would work in conjunction with other treatments. In the future, they could be expanded to help fight other viruses, cancers and other diseases.

RNA could also be used to start, or catalyze, chemical reactions. As Burke explained, catalysts remove barriers to chemical reactions — “they don’t make things happen that wouldn’t otherwise happen, but they speed up the process.”

Synthetic RNA could be used to accelerate removal of toxins from soil or to get the bacteria in our guts to recognize cancerous tumor cells and kick-start an immune response.

But, the future of RNA research may soon reveal a few different Holy Grail moments on its horizon.

One such Holy Grail that Burke said will definitely happen will be observations consistent with the presence of life on other worlds, based on evidence like an atmosphere having certain chemical compositions.

Another likelihood could involve construction of a self-replicating, fully-artificial organism, either created from scratch or reverse-engineered from other organisms.

For those of you already anticipating the plot of a low-budget sci-fi thriller, Burke offered to assuage your fears.

“The notion of it escaping out in the world and taking over Los Angeles is [only] good 1950’s B-movie” material, because the conditions under which this artificial organism would survive would probably be difficult to maintain even in the controlled environment of lab, he said.

Instead of B-movie science, Burke explained that “really, I’m thinking about what kinds of chemistries we want to see take place, and then building the enzymes that would make it possible.”

“Biology has had a few billion years to work on this, but we’re just starting to figure it out.”

Donald Burke-Agüero is a professor of molecular microbiology and immunology and joint professor of biochemistry and biological engineering.

Research Core offers new capabilities

Grand opening highlights specialty of large-scale metabolite profiling
By Phillip Sitter | Bond LSC

Dr. Zhentian Lei , assistant director and assistant research professor of the MU Metabolomics Center, provides an overview of an ultra high-pressure liquid chromatograph coupled to mass spectrometry for the large-scale profiling of metabolites at the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

Dr. Zhentian Lei , assistant director and assistant research professor of the MU Metabolomics Center, provides an overview of an ultra high-pressure liquid chromatograph coupled to mass spectrometry for the large-scale profiling of metabolites at the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

You might think you’ve entered the inside of a pinball machine for a moment when you enter lab 243 at the Bond Life Sciences Center.

But the wires and tubes strung around the room, connected to large instruments that produce sounds of whirring fans, humming motors and hissing pumps, are just part of the University of Missouri’s newest core facility, the MU Metabolomics Center.

At its grand opening and open house Friday, August 12, there was even a counter-top half-pipe with metal ball bearings to shoot down it as a demonstration of time of flight mass spectrometry.

This new center will serve as home of high-tech chemical analysis services that scientists in Bond LSC, across campus and the country can use to better understand the organisms they work with on a molecular level.

Lloyd Sumner, director of the MU Metabolomics Center, and Assistant Professor Ruthie Angelovici discuss the use of NMR for metabolite identification during the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

Lloyd Sumner, director of the MU Metabolomics Center, and Assistant Professor Ruthie Angelovici discuss the use of NMR for metabolite identification during the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

“We have a series of experiments that allow us to profile hundreds to thousands of different metabolites, and that gives people a large-scale, high resolution biochemical traits for whatever they’re looking at, whether it be plants, microbes or animals,” explained Lloyd Sumner, director of the center. “That is useful in understanding what is happening in response to stresses, disease, drug treatment or pest/pathogen interactions that occur in nature.”

Metabolites are the building blocks and energy sources that fuel your metabolism. In your body, what you eat and drink is processed and yields small molecules that are ready to become raw chemical material for construction processes and energy to fuel these processes, like energy stored in the form of fats and lipids, amino acids for the construction of proteins and enzymes. Metabolite are essentially the raw materials.

In order to be studied, complex metabolite mixtures are separated and observed as individual, uniquely identifiable molecules.

This separation can be accomplished in a couple different ways.

“We have instruments that couple chromatography with mass spectrometry. We use that for comparative profiling. Some of the instruments utilize gas chromatography, some of the instruments use liquid chromatography. Chromatography is the technology used to separate these complex mixtures into its individual components. Once we have the mixture’s components separated, we weigh them and that gives us an idea of their identification,” Sumner explained.

UMMC_1

These internal components of a triple quadrupole mass spectrometry are used for explaining how the instrument helps identify the metabolites within a sample during the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

Mass spectrometry works by bombarding molecules with electrons. This bombardment process generates charged molecules that can also fragment into smaller, electrically-charged pieces. These charged pieces can then be “weighed,” or separated, according to their mass-to-charge ratio and identified.

“Something that we find a lot of the time is that we see metabolic differences, but we can’t always identify all of the metabolites associated with those differences. In those cases, we also use the gold standard for chemical identification of unknown molecules,” Sumner said of the nuclear magnetic resonance (NMR) spectrometer in the corner of the lab.

A person points at a 600 MHz Nuclear Magnetic Resonance Spectrometer used for metabolite identification during the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

A person points at a 600 MHz Nuclear Magnetic Resonance Spectrometer used for metabolite identification during the University of Missouri Metabolomics Center open house on Aug. 12. | photo by Zivile Raskauskaite, Bond LSC

Placards warn people that when NMR produces a magnetic field 235,000 times stronger than the Earth’s — by comparison, a typical refrigerator magnet’s field is about 83 times as strong as the Earth’s.

Sumner explained that most people at Bond LSC won’t use the equipment directly themselves. The center’s Assistant Director Dr. Zhentian Lei and other staff will perform most analyses and training users to prepare, process and understand their data.

Sumner said “we train our core users to do their own sample preparation, data processing and data interpretation. Most of the equipment we have in here [cost] hundreds of thousands of dollars, and so we actually have staff that will do the data acquisition, and we try to make it more cost-effective for users by training them to prep their own samples and process their own data.”

The training workshop in metabolomics will be August 15 through 19. The training Monday through Thursday will be hands-on, and Friday will be a symposium day highlighting current metabolomics research. We will likely offer another training workshop in the Spring of 2017, and then annually thereafter.

For more information on using the MU Metabolomics Core or future training, email Director Lloyd Sumner at sumnerlw@missouri.edu or Assistant Director Zhentian Lei at leiz@missouri.edu.

Unmasking the unknown

Scientists explore genetic similarities between plants and mice

DSC_5116.jpg

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.

montage.jpg

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.

DSC_5098.jpg

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.”

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.

BPA overrides temperature to decide turtle sex

The environmental build-up of bisphenol A (BPA) can result in a life-changing shift for aquatic animals.

For painted turtles, exposure to this chemical can disrupt sexual differentiation,, according to new research in the  General and Comparative Endocrinology.

Scientists at the University of Missouri have teamed up to show how low levels of certain endocrine disruptors like BPA can cause males to possess female gonadal structures in newly-hatched turtles. This collaboration between MU, Westminster College, the U.S. Geological Survey (USGS) and the Saint Louis Zoo exposed turtle eggs to levels of BPA similar to those currently found in the environment.

“It’s important because this is one of the first times we’ve seen low doses of BPA causing disorganization or reorganization of the male gonad to resemble females,” said Dawn Holliday, adjunct assistant professor of pathology & anatomical sciences at MU’s School of Medicine and assistant professor of biology at Westminster College. “We’re not sure what this means in terms of population-level effects, but certainly it can cause some reproductive dysfunction for turtles.”

Painted turtle eggs were brought from a hatchery in Louisiana, candled to ensure embryo viability and then incubated at male-permissive temperatures in a bed of vermiculite. Those exposed to BPA developed deformities to testes that held female characteristics.  Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center

Painted turtle eggs were brought from a hatchery in Louisiana, candled to ensure embryo viability and then incubated at male-permissive temperatures in a bed of vermiculite. Those exposed to BPA developed deformities to testes that held female characteristics.
Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

Endocrine disruptors leach into rivers and streams and concern scientists because of potential effects on animals and humans. While BPA is used as a hardening agent in plastics, it also is used to line cans and in manufacturing where more than 15 billion tons are produced each year.

In the case of painted turtles, these chemicals have potential to alter sex ratios, which are normally regulated by temperature during incubation. Eggs exposed to cooler temperatures normally produce males and those hatched at warmer temperatures produce females.

Turtle eggs incubated at cooler temperatures result in male hatchlings while warmer temperatures cause females. Researchers are measuring the temperature and weight of this turtle. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center

Turtle eggs incubated at cooler temperatures result in male hatchlings while warmer temperatures cause females. Researchers are measuring the temperature and weight of this turtle. Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

In this experiment, turtle eggs were incubated at temperatures known to rear males and dosed with low, medium and high levels of BPA. BPA-exposed turtles were compared to hatchlings not exposed to chemicals as well as a group exposed to high levels of ethinyl estradiol — an endocrine disruptor found in birth control — at the USGS Columbia Environmental Research Center.

These doses resulted in turtle sex organs that should have been male , but abnormally contained female gonadal elements. The low dose represented BPA concentrations found in fields where turtles can nest while the mid and high doses approximate BPA levels near contaminated sites like landfills.

“We exposed the eggs for a limited amount of time right when they were most vulnerable to the effects,” said Cheryl Rosenfeld, a researcher at MU’s Bond Life Sciences Center and an associate professor of biomedical sciences in the College of Veterinary Medicine. “We found that we got partial feminization in more than 30 percent of turtle eggs exposed to BPA despite being incubated at male-permissive temperatures.”

Dawn Holliday (left), Caitlin Jandegian and Cheryl Rosenfeld examine turtle gonadal tissue to determine if BPA affected proper sexual development. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center

Dawn Holliday (left), Caitlin Jandegian and Cheryl Rosenfeld examine turtle gonadal tissue to determine if BPA affected proper sexual development. Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

These results give the team a look into what real-world exposure levels might mean in the wild and a starting point for comparison.

“Turtles are the most endangered vertebrate taxa and they have all sorts of conservation issues coming at them from people harvesting them to disease, and endocrine disruptors are another potentially big whammy they have against their conservation status,” said Sharon Deem, director of the Saint Louis Zoo’s Institute for Conservation Medicine. “This research is a stepping stone, and we are hoping we can apply these results to populations of turtles throughout the state and use these results as a marker to look at endocrine disruptors in the wild.”

Future studies plan to look at the underlying mechanisms behind sexual disruption and will extend the study to animals including fish and mammals. Rosenfeld’s laboratory is in the process of examining how early exposure of turtles to endocrine disruptors might affect cognitive behaviors, including spatial navigation ability.

Fred vom Saal, Curators Professor of Biological Sciences in the College of Arts and Science at MU, Don Tillitt, an adjunct professor of biological sciences at MU and a research toxicologist with the USGS, Ramji Bhandari, an assistant research professor of biological sciences and a visiting scientist with the USGS at MU and Caitlin Jandegian, a senior research technician at MU, all collaborated on the study.

Candling helps determine whether the painted turtle embryo is viable for the experiment. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center

Candling helps determine whether the painted turtle embryo is viable for the experiment. Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

Funding was provided by Mizzou Advantage, an MU initiative that fosters interdisciplinary collaboration among faculty, staff, students and external partners to solve real-world problems in four areas of strength identified at the University of Missouri. These areas include Food for the Future, Sustainable Energy, Media for the Future and One Health/One Medicine.

“Mutant seeds” blossom in the pollen research field

  • A mutant arabidopsis model nearing pollination.

The thought of pollen dispersed throughout the air might trigger horrific memories of allergies, but the drifting dander is absolutely essential to all life.

Science has long linked this element of reproduction with environmental conditions, but the reasons why and how pollen functions were less understood. Now lingering questions about the nuanced control of plants are being answered.

“Pollen is a very important part of the reproductive process and if we understand how pollen develops and how environmental stresses impinge on this process, we might be able to prevent crop loss due to high temperature or drought stress etc.,” said Shuqun Zhang, a Bond Life Sciences Center investigator.

Zhang has developed a new line of seeds that helped him and his lab identify an influential signaling pathway that triggers a chain reaction associated with normal pollen formation and function.

This research could lead to improvement to a plant’s response to disastrous environmental variables like drought to optimize pollen production and increase the production of food crops.

 

Left: Pollen grains with MAPK3/4 genotypes are illuminated using a fluorescent microscope. RIGHT: Normally developed pollen grains shown by an electronic microscope scan. | Credit: Shuqun Zhang

Left: Pollen grains genotypes MAPK3 and MAPK6 are illuminated by red and yellow dye using a fluorescent microscope. RIGHT: Normally developed pollen grains shown by an electronic microscope scan. | Credit: Shuqun Zhang

Seeds of success

Mutant seeds are the key to this work.

Instead of glowing green in the soil like you might see in a science fiction movie, they are providing important insight on plant reproduction and stress tolerance.

Zhang developed these plants from a mutant strain of Arabidopsis, a model plant used in scientific research. Certain genes were “switched off”to pinpoint where important pollen functions were signaled.

Using this mutant plant and seed system, Zhang found that WRKY34and WRKY2, two proteins that turn on/off genes, are regulated by MPK3and MPK6signaling” enzymes. These enzymes basically transform proteins from a non-functional state to a functional state, turning on specific duties or functions. Zhang, a professor of biochemistry at MU, began tinkering with the MPK3 and MKP6 pathways more than twenty years ago during his post-doc at Rutgers University.

Zhang’s research shows the newly identified MPK3/MPK6-WRKY34/WRKY2 pathway is a key switch in the hierarchy of the signaling system in pollen formation.

The research showed that the plant’s defense/stress response and reproductive process are linked, and the influential proteins MPK3 and MPK6 were part of the bigger WRKY34/WRKY2control pathway, which is activated in early pollen production.

The system is so useful that researchers across the country won’t stop asking for the seeds, Zhang said.

“We have a lot of requests for seeds,” Zhang said. “This is a very nice system to study pollen formation and function.”

 

The cascade of control

The functions of MPK3/MPK6 in plants can be compared to a “mother board” switch. The pathway — MPK3 and MPK6 —are part of a hierarchy of response, turning functions on or off. In other words, it’s a switch that controls a lot of different things. Controlling WRKY34/WRKY2 is one of the many roles played by MPK3 and MPK6.

Shuqun Zhang, University of Missouri Bond Life Sciences investigator.

Shuqun Zhang, University of Missouri Bond Life Sciences investigator.

“Whatever is plugged into it is what comes on,” Zhang said. “We are actually very, very interested in the evolutionarily context, how this came to be.”

This signaling process is just one of many in plants. MPK3 and MPK6 are two out the 20 MPKs, or MAPKs (abbreviated from Mitogen-Activated Protein Kinases) in Arabidopsis. They control plant defense, stress tolerance, growth, and development including pollen formation and functions.

“We determined that this MAPK-WRKY signaling module functions at the early stage of pollen development,” Zhang said.

The “loss of function of this pathway reduces pollen viability, and the surviving pollen has poor germination and reduced pollen tube growth, all of which reduce the transmission rate of the mutant pollen,” according to the research.

Zhang and his lab worked with the MU Division of Biochemistry and Interdisciplinary Plant Group on the research, which published in PLoS Genetics in June of this year.

 

A world without pollen production and defense

Without pollen, plants would not reproduce — there aren’t any Single Bars in the plant world (that we know of) — and if plant generations don’t propagate, there would be no air or food for human life to sustain.

“The factors such as heat and drought stresses cause problems to the plant’s normal developmental process and that’s how pollen fails to develop,” Zhang said. “If we understand the process, and know how environmental factors impact negatively the process, we can then make plants that can handle environmental stress better.”

Zhang and his lab continue to research the complexities of these pathways. Next on the quest is to answer how MPK3/MPK6 are involved in pollen functions such as guiding the pollen tube growth towards ovule to complete the sexual reproduction process in plants.

“It is possible that MPK3 and MPK6 are activated quickly in response to the guidance signals,” he said. “There’s still a long way to go because very few players in this process have been identified, we try to understand the biological process how they work together.” This research is in collaboration with Dr. Bruce McClure, also professor of Division of Biochemistry.

Read more:

1. PLoS Genetics (May 2014): Phosphorylation of a WRKY Transcription Factor by MAPKs is Required for Pollen Development and Function in Arabidopsis — Funded by a Hughes Research Fellowship and grants from the National Science Foundation.

2. Plant Physiology (June 2014): Two Mitogen-Activated Protein Kinases, MPK3 and MPK6, are required for Funicular Guidance of Pollen Tubes in Arabidopsis — Funded by a National Science Foundation grant and a NSF Young Investigator Award.

 

Nerve cell communication mechanisms uncovered, may lead to new therapeutic approaches for neurodegenerative diseases

 

Story by Madison Knapp/ Bond Life Sciences summer intern

Simple actions like walking, swallowing and breathing are the result of a complex communication system between cells. When we touch something hot, our nerve cells tell us to take our hand off the object.

This happens in a matter of milliseconds.

This hyperspeed of communication is instrumental in maintaining proper muscle function. Many degenerative diseases affecting millions of people worldwide result from reduced signaling speed or other cellular miscommunications within this intricate network.

Michael Garcia, investigator at the Christopher S. Bond Life Sciences Center and associate professor of biology at the University of Missouri, conducts basic research to answer fundamental questions of nerve cell mechanics.

“In order to fix something, you need to first understand how it works,” Garcia said.

Garcia’s research illuminates relationships between nerve cells to find factors affecting function.  His goal is to provide insight on fundamental cellular mechanisms that aren’t fully understood.

Garcia’s research has been funded partly by the National Science Foundation and National Institutes of Health.

Technological advancements have made it possible to better understand disease development in the human body to create more effective treatments. Alas, a scientist’s work is never finished— when the answer to one question is found, ten more crop up in its wake.

Garcia’s research, which appeared in several journals including Human Molecular Genetics andthe Journal of Neuroscience Research initially sought to shed light on the neuronal response to myelination, the development of an insulating border around a nerve cell, called a myelin sheath, which is critical in rapid communication between cells.

Eric Villalon, a graduate student in Michael Garcia's lab at the Bond Life Sciences Center, examines results. The Garcia Lab is answering news questions in cell mechanics. | PAIGE BLANKENBUEHLER

Eric Villalon, a graduate student in Michael Garcia’s lab at the Bond Life Sciences Center, examines results. The Garcia Lab is answering news questions in cell mechanics. | PAIGE BLANKENBUEHLER

How it works: Rebuilding cell theory

Garcia’s early research disproved a long-standing hypothesis concerning this cellular feature.

Mammals’ nervous systems are uniquely equipped with myelination, which has been shown to increase conduction velocity, or the speed at which nerve cells pass signals. Low velocity is often associated with neurodegenerative diseases, so research exploring why could later have application in therapeutic technology.

In addition to myelination, cell size makes a big difference in conduction velocity — the bigger the nerve cells, the faster they can pass and receive signals. Garcia’s findings disproved a hypothesis that related myelination to this phenomenon.

The hypothesis, published in a 1992 edition of Cell, claimed that myelination causes a cellular process called phosphorylation which then causes an increase in the axonal diameter (width of the communicating part of a nerve cell), leading to faster nerve cell communication. Garcia found that myelination did cause an increase in axonal diameter, and myelination was required for phosphorylation, but that the two results were independent of one another.

To narrow in on the processes affecting axonal diameter, Garcia identified the protein responsible for growth.

Garcia followed earlier work, showing that one subunit controls whether there is growth at all with myelination, by identifying the domain of this protein that determines how much growth.

After clarifying this part of the process, a question still remains: If not to control myelination, why does phosphorylation happen?

 

Looking forward

Jeffrey Dale, a recent PhD graduate from Garcia’s lab, said current research is in part geared toward finding a connection between phosphorylation and a process called remyelination.

Remyelination could be key to new therapeutic approaches. When a cell is damaged (as in neurodegenerative disease) the myelin sheath can be stripped away. Remyelination is the process a cell goes through to replace the myelin.

Imagine you have a new wooden toy boat, painted and smooth. If you take a knife and whittle away all the paint and then repaint it—even exactly how it was painted before—the boat is not going to be as shiny and smooth as it was before. This is how remyelination works (or rather, doesn’t).  When nerve cells are damaged, the myelin sheath is stripped away and even after the cell rebuilds it, the cell can’t conduct signals at the same speed it was able to before.

“If you can learn what controls myelination, maybe you can improve effectiveness of remyelination,” Dale said.

Garcia said it is possible that revealing the mechanics involved in phosphorylation could lead to better treatments. In context of neurodegenerative diseases, the question why don’t axons function properly might be wrapped up in Garcia’s question: In healthy cells, why do they?

Supervising editor: Paige Blankenbuehler

SoyKB: Leading the convergence of wet and dry science in the era of Big Data

Yaya Cui, an investigator in plant sciences at the Bond Life Sciences Center examines data on fast neuron soybean mutants that are represented on the SoyKB database.

Yaya Cui, an investigator in plant sciences at the Bond Life Sciences Center examines data on fast neuron soybean mutants that are represented on the SoyKB database.

The most puzzling scientific mysteries may be solved at the same machine you’re likely reading this sentence.

In the era of “Big Data” many significant scientific discoveries — the development of new drugs to fight diseases, strategies of agricultural breeding to solve world-hunger problems and figuring out why the world exists — are being made without ever stepping foot in a lab.

Developed by researchers at the Bond Life Sciences Center, SoyKB.org allows international researchers, scientists and farmers to chart the unknown territory of soybean genomics together — sometimes continents away from one another — through that data.

 

Digital solutions to real-world questions

As part of the Obama Administration’s $200 million “Big Data” Initiative, SoyKB (Soy Knowledge Base) was born.

The digital infrastructure changes the way researchers conduct their experiments dramatically, according to plant scientists like Gary Stacey, Bond LSC researcher, endowed professor of soybean biotechnology and professor of plant sciences and biochemistry.

“It’s very powerful,” Stacey said. “Humans can only look at so many lines in an excel spreadsheet — then it just kind of blurs. So we need these kinds of tools to be able to deal with this high-throughput data.”

The website, managed by Trupti Joshi, an assistant research professor in computer science at MU’s College of Engineering, enables researchers to develop important scientific questions and theories.

“There are people that during their entire career, don’t do any bench work or wet science, they just look at the data,” Stacey said.

The Gene Pathway Viewer available on SoyKB, shows different signaling pathways and points to the function of specific genes so that researchers can develop improvements for badly performing soybean lines.

“It’s much easier to grasp this whole data and narrow it down to basically what you want to focus on,” Joshi said.

A 3D-protein modeling tool lends itself especially to drug design. A pharmaceutical company could test the hypothesis and in some situations, the proposed drug turns out to yield the expected results — formulated solely by data analysis.

The Big Data initiative drives a blending of “wet science” — conducting experiments in the lab and gathering original data — and “dry science” — using computational methods.

Testament of the times?

“Oh, absolutely,” Joshi said.

 

Collaboration between the “wet” and “dry” sciences

Before SoyKB, data from numerous experiments would be gathered and disregarded, with only the desired results analyzed. The website makes it easy to dump all of the data gathered to then be repurposed by other researchers.

“With these kinds of databases now, all the data is put there so something that’s not valuable to me may be valuable to somebody else,” Stacey said,

Joshi said infrastructure like SoyKB is becoming more necessary in all realms of scientific discovery.

“(SoyKB) has turned out to be a very good public resource for the soybean community to cross reference that and check the details of their findings,” she said.

Computer science prevents researchers having to reinvent the wheel with their own digital platforms. SoyKB has a translational infrastructure with computational methods and tools that can be used for many disciplines like health sciences, animal sciences, physics and genetic research.

“I think there’s more and more need for these types of collaborations,” Joshi said. “It can be really difficult for biologists to handle the large scope of data by themselves and you really don’t want to spend time just dealing with files — You want to focus more on the biology, so these types of collaborations work really well.

It’s a win-win situation for everyone,” she said.

The success of SoyKB was perhaps catalyzed by Joshi. She adopted the website and the compilation of data in its infant stages as her PhD dissertation.

Joshi is unique because she has both a biology degree and a computer science background. Stacey said Joshi, who has “had a foot in each camp,” serves as an irreplaceable translator.

Most recently, the progress of SoyKB as part of the Big Data Initiative was presented at the International Conference on Bioinformatics and Biomedicine Dec. 2013 in Shanghai. The ongoing project is funded by NSF grants.