Plant biologists across the country opened their mailboxes last month to the glowing leaves of Arabidopsis on the cover of the latest issue of Molecular Plant.
That cover taken by post-doctoral researcher Yosef Fichman of Bond Life Sciences Center depicts plants fluorescing in response to reactive oxygen species (ROS) propagation, a technique that allows researchers to track plant response to certain stressors.
The novel approach is the first of its kind to image the response of an entire plant, rather than small samples. To make this technique work, Fichman — who works in the lab of Bond LSC’s Ron Mittler — and his collaborators came up with the idea to fumigate the plant with a dye which enters the plant, reacts with ROS inside the plant and fluoresces under an in vivo imaging system (IVIS). From there, the researchers could watch the stress signals of the plant as they evolve in real-time following the application of different stresses.
ROS are essential regulators of cellular and systemic signals in plants as well as animals. Additionally, they play an important role in hormonal, physiological, and developmental reactions in plants such as growth and development as well as defense and acclimation to environmental stress.
ROS are both beneficial and detrimental to plants. On one hand, they regulate basic biological processes. On the other, if overproduced, they are toxic to cells and can cause cell stress or death. However, recent research by the Mittler lab supports the idea that, at certain levels, ROS can be more beneficial than harmful because they have the power to signal defense mechanisms against stressors in the environment. In essence, it is a way for cells to communicate with each other, creating a domino effect, or wave that sends a message to nearby cells and eventually over long distances within the plant.
Now that they have a technique to detect the fast-moving ROS signal in plants, the researchers can identify the proteins that are involved in the plants’ response to stress. The signal is important because it causes fast adjustments to stress within the environment, which means that other parts of the plant that are not subject to stress can build up protection mechanisms.
“In order to understand plants’ response to stress, we need to know the mechanism of that’s going on in plants,” Fichman said. “Basically, it’s a balance of resources within the plant.”
Currently, the researchers are working with Arabidopsis, but this technique applies to other plant and crop models. Previous methods of tracking biological responses in plants included the arduous process of gene manipulation and expression. The new method allows for a straight-forward approach to tracking and monitoring the biological reactions in plants.
Moving forward the researchers plan to use Arabidopsis mutants and other plants to track the response to different stresses compared to the wild type to see if there are changes in the genes involved. Fichman is particularly interested in the effect of abiotic stresses, such as high-intensity light and leaf destruction by insects.
“Whether you like to call it weather extremes or global climate change, there are changes. Plants are a main food source, so we need to be prepared to create better plants for the future that allows crops to grow in stressful conditions such as high light, less water and heat,” Fichman said. “If plants invest more resources into protecting themselves they will be able to survive these changes and continue to produce food for our future. I’m studying this mechanism in ROS waves and in different pathways to create better plants in the future.”
By understanding the proteins that are involved in plants’ response to stress, researchers can begin to re-engineer plants to survive harsher conditions. Fichman’s technique makes it easier to recognize plants’ responses to stress moving forward.
For Yosef, when it comes to research, imagination is the only limitation.
“In research, we can come up with the craziest ideas and science allows us to check those ideas and discover new things,” Fichman said. “This is why I wake up in the morning.”
Years at MU lands student turned faculty tenure-track position
By Mariah Cox | Bond LSC
Where can passion, hard work and more than a decade worth of experience get you? They landed Maggie Lange-Osborn her own research lab on the University of Missouri campus.
Lange is starting down that path in Bond Life Sciences Center but will move to a permanent space in either the Medical Science Building or Schweitzer Hall eventually. She’s excited to spread her wings and establish herself independently of her past role in the Bond LSC.
In the meantime, she’s working arduously to build her lab from the ground up. From small materials such as plastics for cell culture, pipettes and pipette tips and chemicals to make buffers to large expensive equipment, Lange will eventually need it all.
“Walking into the lab, you don’t realize all of the things you need to do an experiment. When I walk into my lab space, I literally have nothing,” said Lange, a newly appointed assistant professor in the Department of Molecular Microbiology and Immunology (MMI). “You have to think about all of those things, you know it’s not just the equipment it’s the pipettes, the incubators, the hoods and then also the materials to put in those things.”
Luckily with 10 plus years in the building, Lange knows of all of the shared resources available to her, so she doesn’t have to invest in many expensive machines just yet.
“I cannot wait to do my first experiment with my own equipment,” Lange said.
Lange began her career at MU in 2003 as a Ph.D. student in the Molecular Microbiology and Immunology and Veterinary Pathobiology Graduate Program, whether she knew it at the time or not.
When applying for faculty positions, Lange worried that she would be placed in a box, unable to separate herself from her graduate work if she stayed at MU. With time, she’s found that hasn’t been the case.
“Even though I’ve been here for so long, I’ve been able to surround myself with people who know more than me and who have different areas of expertise than I do,” Lange said. “I can still branch off and learn a lot from other researchers, which is what I found really attractive about Mizzou.”
Her segway into research occurred during her first year as a graduate student in an MMI lab in the School of Medicine. Under Michael Misfeldt at the School of Medicine, Lange studied pattern recognition receptors in the innate immune system, which is the body’s first line of defense against a virus or bacteria.
The specific receptor she worked with recognizes a component of viruses. From there, Lange wanted to understand how a host is able to respond.
“After working on that I felt like I had a really good grasp of the host response side and I wanted to get more of the virus side to understand virus replication and what types of replication mechanisms work to signal the host from that perspective,” Lange said.
That led Lange to join Bond LSC in 2008 as a post-doctoral researcher in Donald Burke’s lab. Known for specializing in HIV research and viral biology, the Burke lab gave Lange the opportunity to understand virus side interactions.
Lange wasn’t quite ready to move on at the end of her post-doc, and the success with her research led Burke to invite her to stay on as an assistant research professor.
“During that time, I was more exposed to leading people and mentoring people in the lab. I was able to get some teaching experience in that role as well in the infection, immunity and advanced virology classes,” Lange said. “The position evolved into really enjoying all of the components that would be required for a tenure track position and it grew from there.”
In her sixth year as an assistant researcher, Lange decided she was ready to run her own lab. However, she knew it would be a challenge to secure a faculty position and even more difficult to stay at MU.
But, her experience in the Burke lab and her proven ability to obtain grant funding worked in her favor.
“It’s really nice to stay in Missouri because mine and my husband’s family are here,” Lange said. “When I was growing up, my dad was in the military and we moved around a lot, so I never got to know my grandparents or cousins. It’s really nice now that my kids get to have those relationships with their extended family.”
Her goal with her new lab is to combine her knowledge of viral interaction in the body and hosts’ response to infection.
Three projects currently in the works for grant submission focus on host-virus interactions and how different host factors and viral proteins interact during replication. One specific project looks at the host factors that are involved in HIV induced death caused by different HIV proteins.
“While HIV has been around for a long time, there are still things we don’t know about it. With the research, I’m getting back to my innate immunity roots and looking at exactly how viruses interact with innate immune receptors and signaling pathways and how that interaction dictates pathogenic outcomes,” Lange said.
Understanding the death pathways for HIV can lead to the development of a strategy to preserve T cells and facilitate the death of the virus. Additionally, it can lead to the development of therapies toward eradication.
Her excitement isn’t just for her new lab, it’s also for her newfound opportunity to provide students with lab experience and open up the possibility of research for those who haven’t had access to it.
“I’m from a rural community and I didn’t even know that a Ph.D. existed when I was in high school,” Lange said. “I’d really like to present those opportunities to people like me who have no idea that they’re even available. There are so many things you don’t realize are possible because of the environment that you’re in whether it be in rural or inner-city communities.”
While unknowingly launching her career at the outset of her Ph.D. program, Lange is grateful her path led her here.
“It’s been really fortunate for me, the way the whole process developed. I love this building and the awesome people I’ve met here, but I’ve been here since 2008, so having a fresh perspective elsewhere could be beneficial. I’ve worked a long time with both Marc Johnson and Donald Burke and being away from the building will allow me to meet new investigators and establish new collaborations,” Lange said. “While we still have very productive collaborations and have promising, active projects, it will help demonstrate that I’m separate from them and have my own interests as well.”
It’s a sensitive balance between growth and defense when it comes to plants.
While a built-in, passive immune system helps them survive attackers, this response halts the growth and development of the plant, something that fascinates Ben Spears in the lab of Walter Gassmann at Bond LSC.
“In our lab, we try to pick apart the different signaling pathways governing these processes of growth and maintaining an immune response; we think of them as distinct processes, but, in reality, they are all interconnected,” Spears said. “If we can unlock the ability to manipulate how these signaling pathways work together to allow the plant to decide whether to grow or protect itself, it’ll go a long way toward producing plants that are better adapted to their environments.”
That’s what has led Spears to work with the Arabidopsis TCP Transcription Factor family. Transcription factors are proteins that “turn genes on or off,” as Spears puts it. They selectively activate certain sets of genes by interacting directly with family-specific sequences of DNA.
Spears is a postdoc researching molecular plant genetics such as the plant-specific (TCP) transcription factor family. Classically, the TCPs — named for early family members TEOSINTE BRANCHED1 (maize)/CYCLOIDEA (snapdragon)/PROLIFERATING CELL FACTOR (rice)— have only been known to regulate things such as branching, leaf size, growth, and other external structures of the plant, but, more recently, the Gassmann lab and other groups have identified them as regulators of immunity, making it the perfect subject of research. Spears focused on one of the multiple layers of this function to hone in on immune response in this family of transcription factors.
Detecting an attack
At the surface of a plant cell, there are receptors that will identify the presence of bacteria, and then initiate an immune response in the plant. Spears found that several members of the TCP family seemed to turn on this immunity process. This discovery, along with previously understood processes of growth in TCP showed that these two responses are tied together locally. It is this connection that has sprouted a high level of excitement and interest in the hormone biology research community recently.
“In terms of overall impact, this is a really exciting family of proteins,” Spears said. “They’ve been on the scene for a while, but only recently they’ve started to explode in terms of broad interest in the field. It’s nice to work on something that others are interested in, it makes you feel like you’re doing something that could potentially have an impact.”
Spears said it always comes back to the tradeoff between these two plant processes.
“A plant may be immune to pathogen infection, which is great because agronomically we want our crops to not be affected by pathogens out there, but it can come at the cost of growth, which affects the yield,” he said.
There’s a tight regulatory control system in plants that decides how it will spend its energy resources, and it’s affected by factors such as heat, water, light availability, or pathogen stresses. If researchers can better pinpoint the connection of these growth-immunity processes, they are one step closer to creating crops that can be better suited to their environments.
The nuts and bolts of this work involved a lot of bacterial infection assays and basic genetics, but a new technique presented a challenge for Spears. Chromatin immunoprecipitation (ChIP-Seq) technique helped demonstrate the direct interaction between Spears’ protein, the transcription factor of interest and the DNA. He said it was tricky for the lab because they hadn’t tried this method before, so he had to consult outside resources and push himself to learn new skills. Transcription factors control the rate at which a cell converts the DNA blueprint into the RNA messenger that carries the instructions to make proteins for a cell’s structures, enzymes and signaling, among others.
Collaboration across scientific fields almost always leads to better science, and Spears said conversations with researchers of different expertise helped push him into the next portion of his research. A common theme amongst researchers in Bond LSC, he values the community atmosphere and intellectual diversity.
“This study focuses on the other side of the equation, plant growth, and the role that TCPs may play in the plant response to brassinosteroids, an important group of phytohormones controlling plant growth,” he said.
After two years of work and a successfully published paper, Spears entered this next phase of research, a launchpad into a second experiment on brassinosteroid signaling. He’s trying to figure out how this operates on both sides of growth and defense at the same time.
“There is what we think to be a large regulatory transcription complex that these transcription factors may be a central component of, so the piece that actually does the work turning genes on and off,” he said. “There’s still a lot to learn.”
Ultimately, Spears and others are interested in more clearly identifying how TCPs interact with and/or are controlled by these other pieces to help plants decide whether to defend or to grow.
The tradeoff
Researchers are interested in this because the intricate trade-off affects agriculture in significant ways.
“Yield is important to a world that is desperately going to need to feed people in the future, specifically under increasing amounts of duress,” he said. “Growth environments aren’t getting better; they’re changing, and when things change, host-pathogen interactions change too. In the future, microbes that weren’t necessarily pathogenic might become pathogenic because of shifts in temperature, humidity and light that is available to them.”
Since proteins secreted from a pathogen ‘target’ the TCP-family and biochemically change how the TCP protein works to promote infection, it’s only natural for scientists to try to lessen the hardships crops endure. So, it should be clear that if pathogens are interested in specific transcription factors, then researchers should be, too, according to Spears. If researchers can better understand why and, by extension, tell what the transcription factors are doing, then they can potentially understand how to knockout that weak link that allows for infection and limit an attack within the plant.
Spears said that the interactions between a host and pathogen change faster than researchers can change to combat it, so they must better understand what those pathogens are doing to continually be better in the future. He said this research has the potential to help a world that needs reliable agriculture. Though some parts of the research were stressful, a great team made it a fantastic experience.
Dry erase markers and Styrofoam molecular models are a part of Amanda Paz Herrera’s repertoire when teaching complex scientific processes to the average person.
Teaching the next generation of scientists requires work and discipline, but Paz Herrera is up for the task.
Paz Herrera takes her science on the road with Science on Wheels, a traveling group of graduate students and postdoctoral researchers at MU who aim to make science accessible to rural communities in Columbia’s surrounding counties. Sciences on Wheels visits schools, nursing homes, clubs and public events making it imperative to communicate complex scientific processes and mechanisms to all learning levels.
“We should be able to build a knowledge bridge to communicate what we as scientists do without jargon making people feel uncomfortable,” said Paz Herrera, a third-year biochemistry Ph.D. student in Donald Burke’s lab at Bond LSC. “We’re supported by taxpayer money, so the community has a right to know how that money is being used and how we’re moving forward scientifically.”
Paz Herrera emphasizes the importance of diversity and representation in the field on top of her passion for science education and outreach.
“Scientists look like you and me and there’s way more diversity than is depicted in popular thought,” Paz Herrera said. “One of the reasons I do science on wheels is to show what a scientist can look like and that brings a lot of power to little kids that may look like me. When they see someone that looks like them that can do this, that is life-changing.”
Paz Herrera’s research has been driven by her desire to see and understand things at the tiniest level. When she was in the second grade, she owned a play light microscope and would look at her hair or the fibers from her shirt.
When going through the three rotations at the start of her doctorate program, Paz Herrera visited a nuclear magnetic resonance lab and an X-ray crystallography lab, both of which would provide her expertise in studying biology at the structural level. However, her third rotation in the Burke lab changed her perception of what the rest of her program could look like.
While the Burke lab doesn’t specialize in structural biology, its focus on viruses and cancers offers an avenue for Paz Herrera to apply her interest.
A recent study Paz Herrera collaborated on with her colleagues from the Burke lab and researchers across campus optimized RNA and DNA molecules, called aptamers, to carry cancer diagnostics or therapeutics like backpacks to receptors on cell surfaces.
Now, Paz Herrera is looking to visualize the interaction of those same molecules with a protein on the cell surface of the Ebola virus for her dissertation.
However, working with the actual Ebola virus requires a biosafety level 4, and the Bond Life Sciences Center only has a biosafety level 2. So, the researchers in the Burke lab had to get creative.
To study the virus interaction in a safe manner, collaborative researcher Alex Bukreyev who works in Galveston, Texas, engineered a lab model that combines a cattle virus with the protein present on the Ebola virus cells. Because the virus only infects cattle, researchers won’t be infected but can still study what they’re most interested in – the glycoprotein on Ebola cells.
Paz Herrera wants to visualize the interaction to understand how that happens. Understanding the interaction between the proteins on the surface of cells and the aptamers can help researchers develop drugs or diagnostics further down the line.
Visualizing it isn’t as easy as looking under a microscope. By themselves, aptamers can’t be seen on the surface of a cell, making it impossible to find where they are or see how they are functioning.
Paz Herrera is working on building a ‘plug-and-play’ modular dart made up of an aptamer, an interjecting body and a gold nanoparticle tail. The gold nanoparticle tail allows her to see where the aptamer is and subsequently visualize the interaction happening.
Using a basic electron microscope available in Mizzou’s Electron Microscope Core, Paz Herrera has begun to infer where the interaction is using staining. However, she is looking forward to using cryo-electron microscopy (CryoEM) that will soon be available. Using CryoEM will allow her to see the aptamer interaction embedded in ice, providing more molecular detail.
The interjecting body in her model joins the gold nanoparticle with a compatible aptamer. This technology can expand far past Ebola and will help researchers study the interaction between aptamers and proteins in other applications.
Paz Herrera didn’t have much research experience as an undergrad. Instead, she did a lot of educational outreach with elementary and high school students.
“I would do cheek cell samples with them. They would swab the inside of their cheek, put it on a slide and stain it and see their own cells,” Paz Herrera said. “They would go crazy.”
As she progressed in her biochemistry career, she learned that there was more to science than viewing cells under a microscope. She saw graduate school as an opportunity to solidify herself as a scientist and also to prepare to teach the next generation of scientists.
“I really want to do what some of the best professors did for me – to inspire a thirst for inquiry and asking questions,” Paz Herrera said. “When a professor uses an acronym in class, they expect you to understand what they’re saying, but learning isn’t like that and shouldn’t be like that. Sometimes we have great experts in the field that have the curse of knowledge and don’t have the best tools to communicate that knowledge.”
Paz Herrera is also minoring in college teaching and has been shadowing Margaret Lange’s classes, an assistant professor in the Department of Molecular Microbiology and Immunology, to prepare herself as a future educator. She also has the ambitious goal of being a guest lecturer in all of the biochemistry undergraduate classes to strengthen her skills and receive feedback.
Her goal is to make learning science more relatable and enjoyable.
“As I progress in my education, although I become focused in my field, I will never forget where I started so that when I teach, I can break the complexity barrier,” Paz Herrera said. “I want to make science appealing, understandable and accessible not only to my future students but to the community and public as well.”
Researchers from MU, the University of Maryland and the Pacific Northwest National Laboratory are building a microscope that doesn’t yet exist.
By Mariah Cox | Bond LSC
Tiny neon dots speckle a black backdrop – and no, this isn’t a Hasbro Lite Brite. Rather, these fluorescent dots indicate something about plants that scientists research and help them see the genes, traits and molecules they study amid thousands of possibilities.
To help in seeing that, a new imaging microscope will allow researchers to better pinpoint molecular interactions in plants they have a hard time highlighting to overcome the obstacle plant scientists face with wavelengths of light they can’t necessarily see.
“When you think about imaging, you think about what you can see with your eyes. But, there are a whole variety of other things you can image that aren’t visible to the human eye,” said Gary Stacey, a Bond Life Sciences Center principal investigator who is working to help develop a new microscope technology to view fluorescent quantum dot markers beyond the range of visible light, into the infrared spectrum.
Stacey, along with collaborators from the University of Maryland and the Pacific Northwest National Laboratory (PNNL), was awarded a combined $2.25 million grant from the Department of Energy (DOE) to develop a novel microscope for ‘multiplexed super-resolution fluorescence imaging in plants.’
The call for the development of this new imaging hardware was borne out of the need for a more precise measurement of enzyme function, tracking of metabolic pathways and monitoring the transport of materials and signaling processes within and among cells in plants. Right now, the emission spectrum of plant pigments limits the usefulness of and the number of fluorescent colors that can be detected in a single experiment.
Stacey and his collaborators were one of six groups to be chosen for a total $13.5 million investment from the DOE for new bioimaging approaches. For bioenergy, using quantum dots in combination with other novel technologies could enhance imaging techniques to allow scientists new ways to re-engineer plants and microbes for bioenergy conservation and production.
Quantum dots are small particles that are only a few nanometers in size, one nanometer equals one billionth of a meter, and are used as fluorescent biological labels in cells. These labels can be tagged to particular molecules, cell parts or genes of interest to a researcher.
“Think about [fluorescence] as a black light. If you have a room that’s completely dark with fluorescent paint on the wall and you turn on a black light, then you will be able to see where the paint is on the wall,” Stacey said. “It’s the same concept for quantum dots. One application is localizing where a virus is or label it and watch it move into a cell to try to understand the mechanism by which it moves.”
However, the mechanics behind quantum dots don’t make it simple. When exciting a single molecule, it will fluoresce and emit light but will do so in a diffuse pattern. This makes it difficult to see the molecule itself.
Additionally, plants absorb 490-700nm of light — essentially covers the entire visible range of light — allowing them to photosynthesize. As a result of absorbing these wavelengths of light, they also auto fluoresce, which is natural emission of light by structures inside plants cells such as chloroplasts.
The problem, then, is that viruses labeled with fluorescent probes in leaves are difficult to see because of the natural fluorescent glow coming from the plant.
For Stacey and his collaborators, the idea is to go beyond the visible light spectrum and use infrared light, which is above the visible light spectrum. Infrared light is most commonly known for its use in heat lamps.
“With infrared light, there would be no autofluorescence and so when you shine infrared light on a leaf, it would appear black,” Stacey said.” If you shine an infrared light on a fluorescent molecule, it would emit light and show up against a black background, making it very easy to see.”
The problem, though, is being able to distinguish one fluorescent molecule from another when they are close together. Because the researchers will be using infrared light, which has a longer wavelength, the imaging resolution decreases.
To get around that obstacle, the researchers will be using super-resolution microscopy to compensate for the resolution loss. The use of this technology will allow them to pinpoint the center of the fluorescence.
“It should be a big breakthrough. We would be able to look at single molecules interacting against a black background without any interference from autofluorescence,” Stacey said.
Stacey’s collaborators each contribute to the project in their unique way.
Zeev Rosenzweig from the University of Maryland, who is an expert in quantum dots, will be making the dots and labeling them with probes that absorb infrared light. Galya Orr from the Environmental Molecular Sciences Laboratory (EMSL), PNNL, in Richmond, Washington, has expertise in fluorescent microscopy and she, and her colleagues, will build the microscope.
An attractive part of submitting a proposal for the grant is the microscope’s prospect of being used as part of the EMSL user facility, which will ultimately allow researchers from all over the world to use the microscope when fully developed.
The researchers are excited to begin work on this project because they’re building a microscope that doesn’t yet exist. The microscope will expand the capabilities of researchers all over the world.
Stacey is appreciative that he gets to work with researchers from multiple disciplines. Namely, because he learns more about science from the expertise of others.
“That’s what makes it exciting because you’re constantly learning. The great thing about science is that you’re learning every day. It’s nice to get into these new areas especially where you don’t feel comfortable and learn new stuff,” Stacey said.
This work is funded by the Department of Energy for innovating new bioimaging approaches for bioenergy. The grant is split among the collaborators at the University of Maryland, University of Missouri and the Environmental Molecular Sciences Laboratory at the Pacific Northwest National Laboratory. Specifically, $1.5 mil. is to be used by researchers at the University of Maryland and the University of Missouri and $750,000 by researchers at EMSL.
Hollywood cinema stereotypes leave us with a false vision of voracious piranhas that swim in packs and readily attack beachgoers with their sharp teeth and strong jaws.
This simply isn’t true, but their feeding habits are of particular interest to researchers because they can endure long periods of prey shortages and starvation, and scientists are starting to look at the genes behind that advantage.
Bond Life Science Center primary investigator Wes Warren brought his extensive knowledge of genome analysis to a research project with collaborators from Germany and Canada who looked at the genetic expression of the red-bellied piranha under fasting and well-fed diets.
Most fish are faced with short to long-term periods of starvation throughout their lifetime, which cause changes in behavior and the biochemistry of the fish. While piranhas do swim in schools, their feeding habits are much less horror-movie-like than popular culture suggests.
When feeding, piranhas travel in groups of 20-30 and tend to ambush prey in aquatic vegetation, forage the ocean floor for vegetation or invertebrates and opportunistically prey on sick or injured fish. Only rarely have “feeding frenzy” attacks occurred in which piranhas have fed on larger mammals.
But what is it in their genes that could lead to aggressive feeding behavior? The researchers were keen to discover the association of genes which drive aggressiveness and feeding behaviors.
More specifically, the researchers assigned the genes to biological pathways — interactions among molecules that lead to a change in a cell — to try to correlate the genes to certain evolutionary adaptations.
Biological pathways can be thought about in terms of a control room. Environmental cues have the ability to trigger pathway activation, turn genes on or off in the process, cause a cell to move or lead to biological changes within cells. Further, if a particular animal has a more active pathway, it may take much less to turn it on, so in the case of a piranha, it might be much easier to slow their metabolism or turn on a voracious feeding habit.
“For example, if we a say that piranhas have certain genes that appear to be under natural selection and they’re enriched for pathways in the metabolism, we could speculate the piranha lineage has developed some specific amino acid changes to this particular protein which we know is involved in metabolism that helps them deal with longer periods of starvation,” Warren said.
Before beginning research on piranhas, Warren was awarded a National Institutes of Health (NIH) grant to create genome assemblies of various aquatic species. The sequences of these genomes can even help the scientific community link the traits seen in these fish to human diseases.
Warren’s collaborators in Germany and Canada were particularly interested in studying the red-bellied piranha that inhabits neotropical freshwater rivers of northeastern Brazil and in the Paraguay and Parana basins. Previous research on this fish focused on dietary habits and social feeding behavior, but none had looked at the gene regulatory response during periods of food deprivation.
“This gene expression study is the first of its kind in piranhas and provides new information on changes in the genome under caloric restriction,” Warren said. “In particular, it provides evidence for the upregulation of genes involved in metabolism, suggesting an increased utilization of storage fuels and brain energy. The outcome in the piranha is consistent with that seen in other fish species.”
A database of pathways that represent common properties of a particular signaling module in cells helps the researchers link genes to behavior to see if they are enriched, such as a glucose metabolism pathway. From there, researchers can assess whether there is a similar biological process in humans.
“It’s possible to have these delayed periods of feeding where the liver could be adapting to that type of metabolism, and we’re always interested in trying to find unique angles to understand metabolism,” Warren said. “There are lots of metabolism-related diseases in humans, such as diabetes, so the goal is to understand if there’s any kind of evolutionary conservation of some of these pathways and how they respond to extremes in diet.”
The comparative process of discovery is indirect, but scientists have shown repeatedly that there is an evolutionarily conserved response to various stimuli in the environment. Warren says that it’s hard to make a sound biological inference by associating pathways of biological functions in other species, but scientists can use tactics to see enriched adaptations in species over what you would expect to see by chance.
“With the limited number of piranha samples we had for evaluation, we need to follow up these experiments through other types of validation to see if these genes are really driving the traits in piranhas. It’s always interesting to speculate about the linkage of those genes in the piranha driving the trait we examined, but it’s very challenging to prove without some kind of functional experiment,” Warren said.
This research was published in the journal “Genome Biology Evolution” in July 2019 and was funded by Deutsche Forschungsgemeinschaft, Julius-Maximilians-Universität Würzburg, the M.S.I. Foundation, the National Institutes of Health and the Natural Sciences and Engineering Research Council of Canada.
International flights usually require months of planning to score the best deals and to ensure minimal layovers, so Sara Izquierdo Zandalinas, a post-doc in the Ron Mittler lab, was faced with a challenge as she flew to Spain twice within a month’s span this summer.
But the reason for those flights was a pleasant surprise. Zandalinas recently received the 2019 Sabater award given every two years at the Meeting of the Spanish Society of Plant Physiology held in Pamplona Spain from June 26-28. This award is a tribute to Francisco Sabater, a widely recognized plant physiologist of Spain, and is the most prestigious award given to early-career Spanish researchers in plant science.
“I couldn’t believe it because they told me I won one month before the conference,” said Zandalinas. “At that time, I was in Spain at another conference for 10 days, so I had to come back to Missouri and then I had one-and-a-half weeks to prepare a keynote presentation before I had to go back to Spain.”
Nonetheless, she was thrilled.
Zandalinas was recognized for her research on the role of Reactive Oxygen Species (ROS), a fancy term for reactive chemical species such as peroxides, in regulating plants responses to stresses and combined stresses, such as heat or drought.
“Studying combined stress is very important because with climate change, as temperatures are increasing, plants are facing not only one single stress in the field but also other additional stresses,” said Zandalinas. “Previous reports in this lab have shown that plants response to a single stress is completely different from the response of plants to multiple stresses.”
As a result, Zandalinas has spent the past two-and-a-half years identifying which genes are involved in acclimating plants to combined stresses. By selecting the genes that hold up well against certain stresses, scientists can begin to develop plants that are more tolerant.
When she was completing her undergraduate degree at the Polytechnic University of Valencia, Zandalinas recalls many of her friends and colleagues being drawn to the medical aspect of science. However, not many people were interested in studying plants.
Zandalinas, too, thought she was heading toward a career in the medical field until her final undergraduate project producing human antibodies in tobacco plants changed her mind.
“I remember thinking, ‘how is it possible that we can produce human antibodies in plants and in the future apply it to humans?’ I was fascinated by the powerful tools we have of using plants as bio-factories,” said Zandalinas.
From there, she received two master’s degrees in chromatographic techniques and analytical techniques used in clinical labs and a Ph.D. in plant biotechnology from Jaume I University in Castellon, Spain. Afterward, she began her post-doc with Ron Mittler at the University of North Texas before the lab moved to MU last fall.
While she enjoys having the resources for her research such as MU’s Genomics Technology Core and the Proteomics Center, Zandalinas dreams of the sunny skies and warmth back home in Spain.
Following the completion of her post-doc, Zandalinas hopes to establish her own lab back in Spain. She explains that it won’t be as easy to find a research position in Spain because so many people are completing their post-docs in the U.S. and Europe and are wanting to return to Spain.
“In one to three years I hope to receive a research grant from the Spanish government to begin my own research investigations,” Zandalinas said.
As a result of being awarded the 2019 Sabater Award, Zandalinas is now the Spanish candidate for the award for young European researchers in the biannual conferences of the Federation of European Societies of Plant Biology which will take place in Turin, Italy from June 29-July 2, 2020.
The discoveries from research capture the public’s and other scientist’s attention, but what about the tools, instruments and data management systems that provide more efficient means of getting there?
A new genome sequencing instrument is on its way to the Bond Life Sciences Center thanks to a Tier 1 grant from the UM system’s mission to enhance the ‘well-being for Missouri, the nation and the world through transformative teaching, research, innovation, engagement and inclusion’.
Wes Warren, primary investigator in the Bond LSC, led the effort to speed up the turnaround time for genome sequencing and lower the overall cost. The proposal for NovaSeq instrumentation additionally includes purchasing more data storage to keep up with researchers’ demand for the technology.
“This is an issue that has been noticed in the last three or four years around being able to generate the data in a high throughput fashion. A lot of our genomics researchers were required to seek these services off-campus,” said Nathan Bivens, Director of the Genomics Technology Core. “As part of this strategic initiative, we saw an opportunity for campus to invest in this instrument, make it available and then continue to build our own genomics resources here on this campus.”
With his experience with the McDonnell Genomic Institute at Washington University in Saint Louis for the past 17 years, Warren was the person to lead the charge in bringing this technology to MU.
“I was involved in some of the later stages of curating the human genome and I’ve been involved in many genome sequencing efforts at the McDonnell Genomic Institute, not only that but high-throughput sequencing in populations,” said Warren. “My work mostly revolved around comparative genomics. The experience factor that I brought to this proposal is knowledge of how to do high throughput sequencing and how to curate the data.”
Since the first human draft genome in the 90’s, there have been constant developments in sequencing technology to speed up the process and to do so at a lower cost. Sequencing figures out the order of A’s, C’s,G’s, and T’s that make up the DNA nucleotides, or bases, that define a genome that codes to build an organism.
Genomic and single-cell sequencing machines are ‘disruptive technologies’ which allow scientists to essentially have a blueprint of all of the genes that make up any given organism. These blueprints can be used for a variety of scientific purposes to study issues ranging from cancerous mutations to drought resistance in plants.
The data being generated from such technologies is allowing researchers to better understand scientific complexities such as cancers.
“We have an Illumina instrument that’s a different type of format in terms of its capability. It produces fewer bases per flow cell and as a result of that it’s more expensive,” said Warren. “It’s simply a question of cost here; the technology of the NovaSeq has improved in terms of the base accuracy but the main reason is that with more bases per flow cell, the more experiments you can do.”
The current technology in MU’s Genomics Technology Core costs about 30 percent more to complete similar sequencing and takes longer. The new technology can complete thousands of sequences at once, which means it will generate the data needed for publishing discoveries in a more timely fashion.
This cost-effective high throughput technology will allow researchers across the UM System to increase the number of samples they can test and to find genes of significance and traits of interest at a much faster rate.
“Since 2008, we’ve continued to see an increase in the amount of data that’s being generated on campus and the number of publications that are resulting from genomics research and data and I don’t think that’s going to slow down anytime soon,” said Bivens. “In five to 10 years I think we will still be generating more sequencing data especially with the new NextGen Precision Health Institute here at MU. We’re seeing more and more growth in areas which are going to use this type of technology.”
Inevitably, housing this technology at MU will create a higher demand for sequencing through the Genomics Technology Core Facility. To keep up with the demand for this data, Bivens said the facility is purchasing more data storage, restructuring the lab and potentially looking for an additional hire to help process the workload.
As for when the technology will be installed on campus and the DNACF personnel trained to use it, Bivens hopes to be ready to receive samples at the end of September, beginning of October.
This instrumentation is funded by the UM System’s and all four campuses strategic investment for research and creative works.
Cross-collaborative research team looks to refine delivery of cancer treatments
By Mariah Cox | Bond LSC
“When you want to use a tool to do something in the house, you have to use the right size tool. It does no good to use a large screwdriver to fix the tiny screw on your glasses.”
That’s Donald Burke, Bond Life Sciences Center lead primary investigator, as he begins to explain a project looking to optimize the targeting of cancer cells as part of a large cross-collaborative research team.
And the tools Burke is referring to are aptamers, single-chained synthetic DNA or RNA molecules. Aptamers are tricky molecules. Stemming from the Latin word “aptus,” meaning to fit, and “meros,” meaning part, aptamers must be complementary in size and shape to a certain cell-surface receptor in order to be useful as targeting tools, just like having the right size tool.
Last fall, Burke’s lab was able to identify aptamers as a specialized delivery method that have the potential to carry chemotherapy drugs and imaging agents, or cargo, as little backpacks to diseased sites. An important feature of aptamers is their three-dimensional structures which allow them to bind to target sites with high selectivity. That conceivably means they could deliver a drug to a particular part of the body, like a tumor, and not harm nearby tissues.
Now, the team, comprised of MU experts and researchers who specialize in surgery, radiology, molecular biology and immunology and chemical engineering, is hitting the ground running with a two-year research plan to refine the delivery of cancer treatments. The group was one of 19 innovative research projects across all four UM System campuses to receive a grant from a $20.5 million investment for research and creative works.
“Chemotherapeutics are not very specific and most of them act to block DNA replication or other functions of the cell in both healthy and cancerous cells,” said David Porciani a post-doc who started in the Burke lab in the spring of 2016 after finishing his Ph.D. in molecular biophysics in Italy. “Cells that are dividing are more susceptible to the chemotherapeutic effect, that’s why chemotherapy patients start losing hair.”
In general, therapeutic drugs don’t know to go only to tumors, and they don’t differentiate between cancer cells from healthy cells. The obstacle in only targeting cancer cells is to find specific indicative markers that are unique to tumors. Sometimes researchers have to make do with markers that are overexpressed on tumors cells, but those same markers can also be present in low levels on healthy cells.
The team is working simultaneously towards four major objectives – to identify a comprehensive panel of aptamers that target the majority of tumors, develop molecular tools to enhance the delivery of cargo specifically to cancerous cells, improve imaging for targeted delivery of radiopharmaceuticals, and enhance the efficacy of killing solid tumors through immunotherapy.
Each researcher bringing their unique expertise to the table play an important role in ensuring the project stay on track. Mark Daniels, an associate professor of Molecular Microbiology and Immunology and Surgery from the School of Medicine; Bret Ulery, an assistant professor in the Department of Biomedical, Biological & Chemical Engineering; and Donald Burke, a professor of Molecular Biology and Immunology in the School of Medicine and joint professor of Biochemistry, have been instrumental in the project since its beginning stages.
A collaboration years in the making
“Daniels, Ulery and Burke labs have been collaborating for a number of years, each of us excited about what the other could bring to the collaboration,” said Burke. “[Over the years] we’ve explored several ways of making things move forward, we’ve figured out some productive ways to work together and we’ve identified the key questions that we could pool our respective expertise toward answering,” said Burke.
Daniels’ subgroup of the project has been using flow cytometry, a technique used to detect and measure biochemical and molecular characteristics of tumor cells to see which ones are recognized by a set of different aptamers.
Additionally, Ulery has provided extensive insight into the different ways to package molecules together so that they can move around the body and get to where they need to go.
The grant comes as part of the combined UM system’s and all four campuses mission to supply funding for opportunities that will enhance the ‘well-being for Missouri, the nation and the world through transformative teaching, research, innovation, engagement and inclusion.’
For Burke, the grant couldn’t have come at a better time.
“This team has existed before the announcements were made that there would be these opportunities and so when they announced we said that this was tailor-made for us,” said Burke. “It was just the right time for us. Had they had the same competition three years ago, we weren’t ready for it. Had they had it three years from now hopefully we wouldn’t have needed it anymore and we would’ve already gotten the project to the next level.”
Another key player in the development of this discovery is David Porciani. He has spent three years trying to create ‘smart’ molecules that know exactly where to bind without damaging healthy cells, and he has been working on the project since its beginning.
“There are several ways to target cancer cells. Even on campus, there are different groups that are trying to target cancer cells differently and, in my experience, every strategy has advantages and limitations,” said Porciani. “What I see for this aptamer strategy is that it can provide new molecules that can bind to receptors, and it can also identify new tumor biomarkers.”
Porciani’s portion of the project includes using the advanced microscopy capabilities of the MU Advanced Light Microscopy Core to visualize the kinds of receptors on the surface of cancer cells. This information can be very telling in identifying receptors that are specific only to cancerous cells.
What the future holds
The project now starts down this ambitious road. In the first year, the collaborators are working on discovering their panels of aptamers, developing lung cancer-specific T cells which provide artificial cell receptors for the use of immunotherapy, and beginning testing of the specificity of aptamer-cargo constructs in samples acquired from the American Tissue Cancer Collection. By year two the group hopes to begin testing in biopsy tissues acquired from MU hospital patients acquired under informed consent and in lab mice.
“After we test a specific drug in cell culture samples and see an anti-cancer effect, killing only the cancer cells and leaving the healthy cells, we expect to see the same effect in biopsy tissue samples,” said Porciani. “Having a reduction of the tumor mass and not having side-effects is what we hope to see in biopsy tissues and mice.”
After the end of year two, the team is looking forward to expanding this research to other types of tumors to see if their tumor-targeting method can apply in different cancer types.
“Our team has been trying to piece this together for a long time and it’s been surviving on goodwill up to this point. This is the most substantial funding we’ve had for it to date and we’re really excited that the University of Missouri has chosen to support us on this,” said Burke. “We’re very hopeful that we can use it as the starting point for building a much larger enterprise centered around tumor targeting in general, whether it’s for therapeutics, diagnostics or other purposes.”
Donald Burke is a primary investigator in the Bond Life Science Center and is a professor of Molecular Microbiology & Immunology in the School of Medicine and a joint professor of Biochemistry and Bioengineering. David Porciani is a post-doctoral researcher in the Burke lab. Mark Daniels is an associate professor of Molecular Microbiology and Immunology and Surgery in the School of Medicine. Bret Ulery is an assistant professor in the Department of Biomedical, Biological and Chemical Engineering in the College of Engineering. Other key collaborators on the project include Diego Avella Patino and Jusuf Kaifi in the Department of Surgery and Jeff Smith in the Department of Radiology.
Every year we all tend to pay a visit to the doctor to get ahead of cold and flu season. Nothing could be worse than being in the midst of a hectic time at work or school and being out of commission.
Many don’t think twice about the annual flu shot, it just becomes a part of their autumnal routine. But for Henry Wan, a new primary investigator in the Bond Life Sciences Center, a significant portion of his life revolves around understanding how flu viruses get transmitted from animals to humans and vice versa as well as tracking down more effective influenza vaccination strains.
Flu viruses caused more than 959,000 hospitalizations and 79,400 deaths during the 2017-2018 flu season in the United States, according to the Centers for Disease Control and Prevention. And influenza A virus (IAV), also known as the avian flu, has caused pandemics that resulted in millions of deaths in poultry, and fear of widespread transmission to humans and other mammals.
Wan’s interest in influenza started in 1996 after an outbreak of avian influenza (H5N1 virus) in South China. In 1997, this same virus caused another outbreak in Hong Kong that killed 40 percent of geese infected and crossed over to infect 18 humans, causing six of them to die.
While extremely noteworthy because the strain seemed to jump from poultry to humans, researchers weren’t able to pinpoint how these viruses were circulating in wild birds and predict which avian flu could transmit from wild birds to human and from human to human.
“Over the past decade, we’ve been trying to study how influenza emerged in the animal-human interface,” said Wan. “Influenza transmission among humans and different wild and domestic animals have been well documented for decades, however, the detailed mechanism is far from being understood. We still cannot predict emerging risks and provide precise alerts for influenza emergence for domestic animals and humans.”
Wan grew up in a small town in central China on the Yangtze River. After earning an undergraduate degree at Jiangxi Agricultural University in Nanchang, China, Wan decided to pursue an advanced degree in Avian Medicine at South China Agricultural University in Guangzhou, China. Afterward, he moved to the United States to obtain his Ph.D. in veterinary medicine and a master’s in computer science at Mississippi State University where he studied mycoplasmas in poultry rather than influenza.
While mycoplasmas weren’t the peak of his research interest, he still enjoyed studying them.
“I was a poor student from China and didn’t have the opportunities that are available now. The student assistantship at Mississippi State University provided by mycoplasmas study was a good opportunity,” he said. “While at MSU I also had an opportunity to study computer science while finishing my Ph.D. and that really helped me build a foundation in systems biology.”
Wan spent a short period at Oak Ridge National Laboratory in Tennessee before finishing his post-doc at the University of Missouri in the lab of Dong Xu.
From there, he started his teaching and research career at Miami University of Ohio, however, he didn’t get back to studying influenza until he moved to Atlanta to work at the Centers for Disease Control as a senior scientist fellow in 2007. Wan dived back into academics as a veterinary medicine professor to graduate students at Mississippi State University in 2009 and remained there until coming to MU this fall.
His focus on influenza will be helped by a recent $2.8 million National Institutes of Health (NIH) grant to develop and implement high-throughput technology to study and characterize influenza viruses’ antigenic properties and understand antigenic evolution of influenza A viruses. This technical-sounding purpose truly means his lab will work to advance the technology behind vaccine strain selection, especially for children, elderly and pregnant women, to provide a more universal flu vaccine strain for the general population.
Additionally, the project will explore the mechanisms that cause variation in influenza virus quasi-species and establish a history of prior human exposure to influenza viruses in an effort to improve strain selection.
“The mechanisms that cause variation in quasi-species will help us understand how influenza variants emerge in humans and help develop a better-targeted surveillance and prevention strategy through vaccination,” Wan said.
While Wan has only been at Mizzou since this summer, he’s jumped feet first into research collaborations here. He’s also involved in a cross-campus project funded by a University of Missouri System Tier-2 grant to build more research capacity for data-driven discoveries and a grant from the U.S. Department of Agriculture (USDA) to develop a broadly protective E. coli vaccine, among others.
When not in the lab, Wan enjoys spending time outdoors, running and biking along the Katy trail, as well as playing Ping-Pong with his friends. He believes in exercise as an outlet for brainstorming new ideas, rather than just sitting in a lab or his office.
“I think that’s critical, especially in science and research. A lot of the time I get a sparkling idea from exercise, not from sitting in this office. It’s from when you do something else another idea comes,” Wan said.
This research is funded by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. Wan is a Bond LSC primary investigator and joint professor of Molecular Microbiology and Immunology in the School of Medicine, the Department of Veterinary Pathobiology and Electrical Engineering and Computer Science.