virology

Studying Drug Resistance, One Connection at a Time

June 1, 2020 By Becca Wolf in Research Tags: drugs, virology

Kinjal Majumder | photo by Mariah Cox, Bond LSC

By Becca Wolf | Bond LSC

Where you are creates opportunities.

The structure and placement of labs encourages researchers to collaborate and talk to each other and often, connections and friendships are formed.

For Kinjal Majumder, a virologist and postdoctoral fellow in the David Pintel lab at Bond Life Sciences Center, that has meant bouncing many ideas off friends and colleagues from neighboring labs. Little did he know that one connection would lead him to interesting findings in a field outside of virology.

Those results may help make progress toward understanding the machinery behind drug resistance and toxicity in the body.

“The big picture is that it will help future studies on how drugs are metabolized by our body,” Majumder said.

The connection started with Andrew Huber, the first author of the paper, who completed his Ph.D. while in the lab of Dr. Stefan Sarafianos at Bond LSC. He graduated two years ago, moved to the lab of Dr. Taosheng Chen at St. Jude Children’s Research Hospital (SJCRH) in Memphis, Tenn. and began his postdoctoral work.

Dr. Chen’s lab focuses on mechanisms of cellular drug metabolism, one of which functions through the pregnane X receptor (PXR) protein.

PXR is found in the liver and is activated by drugs and other substances in the blood. Once activated, it binds to DNA and activates genes responsible for detoxifying the system. However, this system often leads to metabolism and reduced efficacy of administered drugs such as chemotherapies and antibiotics.

“When Andrew was in the Sarafianos lab, we’d talk science often and we collaborated on how different viral proteins bind to host and virus DNA, and how this can aid viral life cycles,” Majumder said, “When he moved on to his postdoctoral work, he saw that he can start applying similar techniques to study how the liver metabolizes toxins. So that’s when we set up this collaboration to pursue these things together.”

Majumder studies how viral DNA and proteins interact in virus-infected cells. One technique he uses is called chromatin immunoprecipitation assay (ChIP) which determines where proteins of interest can bind to DNA molecules. Majumder freezes protein and DNA that are interacting together in the process and he pulls that complex down with an antibody. “It’s like going fishing,” he said.

In order to apply ChIP and other techniques to study PXR signaling, Majumder drove down to SJCRH in Memphis to work with Huber on these assays.

Once in Memphis, Majumder and his collaborators worked and conducted experiments around the clock, but they also found time to explore the city and have fun.

“We go into lab and try to get done as many experiments as we can,” Majumder said, “And whenever we have a long break we go out on the town. It’s usually a really fun way to go and experience the life and scientific culture of a new place.”

It is a lot to pack into one week, but Majumder and his collaborators make the most of it, finding a way to effectively balance their studies on PXR with soaking up life in Memphis.

Majumder compares PXR to a light dimmer switch. When activated, it “brightens” by increasing drug metabolism, leading to decreased drug efficacy. PXR inhibitors work to “dim” this effect. At St. Jude, Dr. Chen’s group found that of the 434 amino acids that make up PXR, only one mutation is needed to “turn a dimmer into a brightener.”

“We sort of got lucky, we thought that you’d have to make a lot of changes in order to make PXR do something different,” Majumder said, “Without making too many changes — Dr. Chen’s group just switched one thing — and that was sufficient enough to turn it into a brightener. So that was a happy and unexpected outcome.”

Once these results were reviewed, the Cellular and Molecular Life Sciences journal accepted their results and published them this March in an article titled “Mutation of a single amino acid of pregnane X receptor switches an antagonist to agonist by altering AF-2 helix positioning.”

Looking ahead, Majumder and his collaborators hope to make more progress in understanding how drugs are metabolized in the body.

All of this would not have been possible if it wasn’t for the connections Majumder made at Bond LSC a few years ago.

“It was so cool to have this opportunity to collaborate with someone from the LSC that I have collaborated with before and apply our experimental techniques to study a new system,” Majumder said, “It feels very encouraging that now the work can be expanded upon and applied to other systems and settings.”

#IAmScience: Kamlendra Singh

February 27, 2020 By Jerome Duggan in #IAmScience Tags: HIV, virology

By Jerry Duggan | Bond LSC

Kamal Singh was in the town of Allahabad in his native India, preparing for competitive exams to become a government official. As he craned his head to the left, he saw a highly respected official getting berated by an arrogant and disrespectful political leader. While Singh always knew that, in these positions, one was under the government’s thumb to an extent, that incident was what sealed the deal for him and made him realize he did not want to spend his life simply taking orders and being subject to verbal abuse by corrupt politicians.

It was at that moment — roughly 30 years ago — he decided he wanted to dedicate his life to something more personally fulfilling: research.

Singh was raised in a small village in rural India. While his primary and high school education came from a military school, he obtained a Master’s degree in physics from Agra University, in Agra, the city where Taj Mahal is located. Still, Singh thought he wanted to work as a public official until that incident served as the last straw. After that, he decided to complete his graduate degree in quantum physics, and committed fully to a life in academia.

That led him to cross the Atlantic in 1994 when he moved to the U.S. and joined Rutgers New Jersey Medical School. He realized that now was his chance to focus on a different area of science that he had always been fascinated with: biochemistry.

“I taught myself everything from scratch in New Jersey,” he said. “I had always wanted to work with DNA polymerase, and I felt like now was my chance to really get involved with that.”

Over the span of 15 years in New Jersey, he occupied a number of positions including postdoctoral fellow, instructor and assistant professor. His work transitioned from research related to DNA polymerase to his current focus on Human Immunodeficiency Virus (HIV) and other viruses including Foot-and-Mouth Disease Virus, SARS and MERS coronaviruses.

That’s where Singh’s story came to Mizzou where he arrived in Jan. 2009. While he has always been highly motivated and driven by collaborations, the bread and butter of his work for more than a decade has come at the Bond LSC focused on HIV.

The goal of Singh’s HIV-focused research, in a nutshell, is to find drugs that bind to the active site of the enzymes that replicate HIV, thereby blocking the spread of the disease. According to Kyle James Hill, a postdoctoral student working in Dr. Singh’s lab, the function of his research is twofold: to better inform human understanding of the viral life cycle and to help identify novel targets of drug identification to potential treatments.

Kyle James Hill works on a bacterial transformation in Dr. Singh’s lab.

More precisely, Singh’s work is focused on enzymes/proteins that serve particular functions for the virus after the virus enters human body. Most enzymes have a suffix of -ase, and what comes before the suffix generally states the function of a given enzyme. For example, protease has to do with proteins, specifically the breakdown of proteins into smaller proteins or other subunits. Enzymes can have a positive function such as promoting growth and generation of muscle tissue, or a negative function, such as what happens when HIV highjacks their function for its own benefits. The HIV infection, in turn, weakens a person’s immune system and lowers his/her her ability to fight disease, in this case AIDS. Each enzyme has an active site, or a region where its specific function is carried out. All research that deals with enzymes, therefore, must center on what goes on at the active site.

Singh’s HIV-focused research is centered specifically on three HIV enzymes: reverse transcriptase, integrase and protease. Reverse transcriptase catalyzes reverse transcription: that is, the formation of DNA from an RNA genome of HIV. Reverse transcriptase converts RNA genome of HIV into DNA.

HIV then uses another enzyme called integrase to insert viral DNA into the genetic framework of the host cell. This allows the virus to further embed itself in what was once a healthy cell. A third enzyme called protease breaks down long proteins into smaller functional proteins, and these smaller proteins then combine with the genetic material of HIV to infect another cell.

The practical application of Singh’s work is that it is focused on moving research toward tangible results that physicians, in turn, can use to better diagnose and treatment plans for patients. Singh’s current lab work focuses most of all on finding drugs that are able to work to treat HIV patients from different parts of the world.

Singh became interested in investigating why certain drugs designed to treat HIV in the developed world, have less effect on the particular strains of HIV that exist in low- and middle-income countries. To help him with this process, Singh developed a collaboration with scientists at the renowned Karolinska Institutet in Stockholm, Sweden. Singh is also an Associate Faculty member at Karolinska.

While HIV infects people all over the world, there are actually many different strains of the virus, and some strains are more abundant in particular regions of the world. According to Singh, there are 10 subtypes of the virus, and more than 100 hybrid or combined types of it. Some drugs treat a particular subtype better than others. If a given drug does not work to treat a particular subtype, then it is useless in terms of treating patients that are infected with that strain of the virus. According to Singh, most drugs produced in the U.S. treat HIV subtype B, which is most prevalent in the developed world. However, patients in many other parts of the world, such as India, South Africa and Brazil are infected with subtype C of the virus. Singh says that 95% of patients in India are infected with subtype C.

“Because these patients are infected with a different strain of the virus, they, in many cases, do not respond to the type of drugs that are traditionally given to patients in the developed world,” he said. “Our job is figuring out why they don’t respond to that particular drug, and then coming up with something that can more effectively treat them.”

This practical application of his work is what’s most rewarding to Singh. His lab is able to provide concrete results that physicians then apply when making decisions about how to best treat their patients. Based on Singh’s work, physicians can better determine which type of drug would most likely effectively treat patients infected with a specific subtype of the virus, also considering the specific set of genetic variations and mutations that the patient may have. It is in this way that Singh’s work makes a very real difference in the world, and that is ultimately what keeps him so passionate about his work.

“To be able to apply what I work on in the lab and see it at work in real patients is very rewarding for me,” he said. “I am able to see the entire process unfold, starting with the very basic biochemistry all the way to direct application to the patients. Seeing that I’m making a real difference means everything to me.”

#IAmScience Kaitlyn Waters

January 25, 2020 By Mariah Cox in #IAmScience Tags: animal science, virology

By Mariah Cox | Bond LSC

It’s common knowledge that all Ph.D. candidates must complete research in preparation for a dissertation, but what happens when one’s faculty mentor moves to a different school before completion?

Kaitlyn Waters found herself in that situation as she was preparing for the final year of her Ph.D. program.

Waters, a current veterinary medical sciences Ph.D. candidate at Mississippi State University (MSU), is finishing her research in the Bond Life Sciences Center with primary investigator Henry Wan. Wan came to Mizzou last summer from MSU as a joint professor in the School of Medicine, the departments of Veterinary Pathobiology, and Electrical Engineering and Computer Science.

While a handful of Wan’s Ph.D. candidates graduated before he left MSU, Kaitlyn Waters and Mandy Guan were the last of the bunch to finish their degrees.

For Waters, it was a simple decision moving 500 miles from Starkville, Mississippi, to Columbia.

“It was actually pretty easy. Like personally and especially just school-wise and finishing the research,” Waters said. “The way he presented it was that we had more opportunities here to collaborate with more departments around campus and core facilities. It was definitely an easy decision.”

After graduation, Waters has a four-year fellowship with the Foreign Animal Disease Diagnostic Laboratory (FADDL) that begins at Plum Island in Connecticut and transitions to the National Bio and Agro-Defense Facility set to open in Manhattan, Kansas, in 2022.

“They’re opening up this new facility and they’re bringing in new scientists. They’re training us to be the, quote, unquote, ‘next group of scientists’ for that facility,” Waters said. “It’s a lot of responsibility, but it’s exciting.”

Before her move to Missouri, Waters had never lived far away from home. A Byram native, Waters only moved two-and-a-half hours away to attend MSU for her undergraduate and doctoral degrees.

“If I had done this right after finishing undergrad, I think it would have been difficult but because I’m older now, I am more comfortable being independent and relying on myself.”

While it was an important steppingstone for Waters’ post-graduation plans, she misses the football culture and food back home.

“I’m a big sports fan in general and I’ve noticed that Columbia still functions during football games, which is completely weird to me because Starkville shuts down. You can’t go anywhere near MSU’s campus on gameday without traffic being insane and people being everywhere,” Waters said.

Before Waters began the research path, she wanted to become a veterinarian, but those plans got derailed when she found out she did not get into veterinary school.

“When I didn’t get in it was kind of like, I have no backup plan,” Waters said. “Before joining Dr. Wan’s lab I had never held a pipette. I had heard about research, but I had no idea that it’s really what I wanted to do. I definitely don’t think I would be the person I am today if I had gone to veterinary school.”

From not being able to hold a pipette, Waters has come a long way since the beginning of her doctoral program. Waters’ dissertation focuses on the RNP complex, which are three polymerase proteins in the influenza genome that are responsible for the replication of viral genome and RNA. Specifically, she is looking to understand how the RNP complex efficiently and inefficiently replicate in nature to cause new viral strains.

According to Waters, her research path has given her a chance to learn and grow not only as a scientist, but also as a person that she might not have gotten back home as a veterinarian.

“If I had gotten into vet school and become a veterinarian, I would’ve stayed in Mississippi to practice as a clinician and, possibly, own my own clinic which would’ve been great. But now knowing I will have a Ph.D. in infectious diseases, I also know this path can take me anywhere in the word and I can see a lot more whereas if I’d been a veterinarian I would not be experiencing the things that I get the chance to experience now.”

At Plum Island, Waters will serve as a liaison between the computer scientists and the biologists and virologists. Her fellowship will allow her to collaborate with influential scientists and travel the world.

After the four years is up, Waters hopes to stay with the National Bio and Agro-Defense Facility.

“I don’t think I can get a better research experience anywhere else in the United States. At this moment, I plan on sticking with it and staying with them, but anything could happen.”

#IAmScience Karen Segovia

January 10, 2020 By Mariah Cox in #IAmScience Tags: influenza, virology

By Mariah Cox | Bond LSC

Karen Segovia wanted to work with animals the moment her childhood dog fell sick. With few veterinarians near the rural town in Perú where she grew up, she felt powerless to help, and that inspired her to eventually go to veterinary school.

But it was her preparation for her dissertation to meet the degree requirements at San Marcos National University veterinary school that refined Segovia’s focus on something smaller. Already interested in virology, her research narrowed in viruses and avian flu. A connection with a colleague led her to wild bird reservoirs to study samples for various avian viruses.

“I was going to the coast to sample wild bird feces on the beach and then we isolated several viruses in the south from wild birds,” Segovia said.

Segovia’s career path has taken a turn since then toward research with the realization that it has a bigger impact on the health of the animal population. After becoming a veterinarian, she had the opportunity to continue her education at the University of Georgia and continue doing research on avian viruses.

From there, she began a two-year postdoctoral fellowship at the United States Department of Agriculture (USDA) in Athens, Georgia. Most avian influenza research is centralized in the United States in Athens, Georgia, because of the states’ large poultry industry. With influenza research only being conducted in a select number of places throughout the U.S., Segovia was on the lookout for her next step.

“I was finishing my postdoc at the USDA and I was looking to broaden my horizon to increase my knowledge and skills working with influenza viruses” Segovia said. “Upon researching, I discovered that Dr. Wan was leading projects that were analyzing several host factors that can help us understand the mechanisms shaping the ecology of influenza viruses in different populations by using new innovative techniques”

Now Segovia is a senior research associate and lab manager in Henry Wan’s lab at Bond LSC. Her research focuses on detecting and characterizing avian viruses directly from clinical samples by the use of a different approach.

For Segovia, she sees her research as not only a benefit to the economy and public health but to a bigger picture.

“As a vet student, I went to poultry and swine farms often, you can clearly see how many losses it has when there is a disease present,” Segovia said. “It’s not just the economy, but people rely on livestock farms for their jobs and income. Research on influenza has a very big impact.”

Segovia’s work studying influenza has also allowed her to give back to her country.

“In the past, my country didn’t do much research, but last year I got contacted by my university to participate in a grant application sponsored by the World Bank,” Segovia said. “My home university received a grant and they needed two new graduated Ph.D. researchers and I was one of them. My university received a significant amount of money for three years and employed more people to help with research.”

In the future, Segovia plans to continue to help her home country with research efforts through collaborations and suggestions.

As for now, Segovia is focused on establishing herself here in Bond LSC, learning new techniques and discovering more about avian influenza.

#IAmScience Gilberto Perez

August 31, 2018 By Erica Overfelt in #IAmScience Tags: virology

By Erica Overfelt | Bond LSC

Research is like a puzzle, and figuring out how the pieces fit together is Gilberto Perez’s favorite part of research.

“In research you have to think about the steps,” Perez said. “Most likely, I’m not going to complete the puzzle in my lifetime, but it will help someone else put a piece in it and hopefully, down the road, it will be finished.”

Before coming to Mizzou, Perez had mindless jobs in high school and that’s why he likes the idea of a puzzle. He came here unsure of what he wanted to be involved in until he was introduced to a major in biochemistry. He took classes with Stefan Sarafianos then soon got involved with his lab at Bond LSC. When Sarafianos left Perez was under the direction Kamlendra Singh in the lab.

“Once I started to figure out how important research was, it showed me an important aspect of finding cures and vaccinations,” Perez said. “I like mysteries, and it’s a big giant mystery on what works and what doesn’t.”

When Perez first joined the lab his sophomore year he made solutions and buffers, now he uses those same buffers to make reaction mixes to see the efficacy in drug resistance in HIV. The lab tests which drugs react best to HIV viruses.

“I really like the work I do with virology,” Perez said. “It interests me how viruses work.”

Two years later, Perez sees how research extends outside of the lab.

“I always figured research was about taking steps to figure out what to do next, such as in life,” Perez said. “You take little chances here and there and figure out the outcomes, but sometimes you have to take a different approach. You see failures and it is not your fault but it is just life. It is about the persistence of life.”

Along with a new way of seeing life, the lab has also introduced Perez to different perspectives on various cultures.

“Our lab consists of three people and our cultures are all very different,” Perez said. “There are diverse minds, so it really helps out with communicating. They are more patient to be able to hear what I am saying, there are times I don’t make sense. Because of their patience I am able to articulate slowly.”

However, a new perspective isn’t the only thing the lab has given him. Perez was president of Association of Latina America Students last year and credits the lab for the confidence to run.

“I have become more social and have taken a lot of leadership roles,” Perez said. “I am becoming more independent, before I was very dependent on others. I wouldn’t take the initiative. I was really scared of failure and that is something research teaches you. I just have to accept all the mess ups I make.”

#IAmScience Rohit Rao

February 2, 2018 By Allison Scott in #IAmScience Tags: biology, virology

By Allison Scott | Bond LSC

“#IAmScience because I get to apply knowledge from the classroom to my research.”

There are a number of ways to get involved in research, but tennis probably doesn’t come to mind. Rohit Rao was practicing his serve alongside Kamal Singh in 2015 when the two began talking about science.

The junior biology and psychology double major expressed his interest in working in research, and Singh offered for him to join Stefan Sarafianos’ lab in the Bond Life Sciences Center.

“I got my first taste of research in high school and found a passion I didn’t know I had,” Rao said. “I wanted to continue to grow as a researcher when I went to college, and meeting Kamal was a pretty clear path to doing that.”

Rao understands the idea that research builds upon itself, which is why learning the basics before coming to Mizzou proved helpful.

“In high school, I did civil engineering research testing water quality from the Missouri River,” Rao said. “It was clearly something I could see myself doing for many years.”

The Columbia native is following in his family’s footsteps by pursuing science.

“My family is full of doctors and scientists, so having that has given me a greater understanding of what goes on,” Rao said. “I was never pushed into it, though, because it’s something I really want to do.”

After graduating next year, Rao plans on attending medical school and applying the knowledge he’s gained from all of his experiences.

“There are things I learn from the lab and then it’s taught in class, and there are things I learn in class that are helpful in lab,” Rao said. “There’s a big class-lab application interaction.”

Those applications have proved helpful for Rao while working with Singh. He has grown as both a scientist and a researcher since that conversation on the tennis courts years ago.

Now, he works with Human Immunodeficiency Virus (HIV) and contributes to the drug development process.

“We check the biochemical characterization of HIV proteins,” Rao said. “We run various reactions with the HIV proteins to determine their biological characteristics and how the virus mutates to become resistant to approved drugs. Once we do that, we can help choose drugs to overcome that resistance.”

This process serves as the precursor to clinical trials, which ultimately leads to drugs going on the market.

While the work is classified as basic research, Rao is happy to do his part.

“You can’t do applied research without the basic research,” Rao said. “In science, creating the foundation for others to build upon is critical.”

#IAmScience Marc Johnson

March 22, 2017 By Mary Jane Rogers in #IAmScience Tags: virology

By Mary Jane Rogers | Bond LSC

“#IAmScience because the mysteries of the natural world aren’t going to solve themselves.”

Since the third grade, Marc Johnson never wanted to be anything else but a mad scientist. What began as experimenting with sprouting seeds and chemistry sets has blossomed into a career in virology. Specifically, he studies the “moves and countermoves” of viral components, a few hundred thousand at time! His advice for people wondering if science is for them: “If you’ve ever stayed up until 4 in the morning to finish a puzzle, you might be a scientist.”

Sci over Pie with Dave Pintel

March 20, 2015 By Hannah Baldwin in Research Tags: Dave Pintel, Sci over Pie, strawberry rhubarb pie, virology

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