I was interested in BSURF because I had never worked in a research lab before, and I was curious about what it was like. I learned some new benchwork techniques and I learned how to use several different machines. Although some parts were tedious, I was never completely bored. A lot of lab work requires vigilance, whether it’s making sure you pipette a certain amount of buffer into each well or making sure the printer does what you want it to do after you run the print program. And when things went wrong, it felt like a puzzle. A frustrating puzzle, yes, but an interesting one as you mentally replay what you did and try to pinpoint what could have gone wrong.
I learned that 90% of the time, experiments don’t run the way you wanted them to, the reagents you need won’t be delivered when you want them to be, and the lab will run out of dichloromethane just when you need it for your experiment. It is easy to get discouraged, and I think in those moments it’s important to remember why you do research. The breast cancer and MRSA D4 assay projects I’m working on can help to democratize access to healthcare in low-resource settings. These projects have the potential to impact many lives, and I’m excited to be a part of that. I can see why people choose a lifelong career in research.
I appreciated my experiences outside of the lab, too. Despite having been in Durham for all of freshman year, I rarely went beyond 9th Street. This summer, I got to simultaneously explore the Durham area and try not to melt in the heat. I tried new restaurants that I can’t wait to return to this fall, I went to the local farmers’ market, and I finally saw a game at the Durham Bulls baseball stadium. I made some new friends in the BSURF program that I don’t think I would’ve met outside this program, and I pretended to be an adult by making dinner for myself and going to bed before midnight.
I want to thank those who made this summer as fun as it was – particularly Dr. Grunwald, Anna, the faculty members and graduate students who spoke to us, and last but certainly not least, Dan and Jake, my mentors. I also want to thank everyone at the Chilkoti Lab who supported and helped me this summer. This post shares the same title as my first blog post because I truly do see the end of the program as a beginning of my adventures in research. I really liked being in a lab this summer, and while my ultimate career goal of increasing worldwide healthcare access hasn’t changed, how I get there might. There are career options that I haven’t considered before, like pursuing a Ph.D. or working in a lab in industry. I look forward to continuing on my adventures in research this fall and beyond.
A component of the BSURF program that sets it apart from just a summer research experience is the opportunity to hear interesting and informative talks given by Duke faculty members. All of them are PIs and were kind enough to take time out of their busy schedules to talk to us about their research and their career paths.
One talk that stood out to me was Dr. Silva’s. The Silva Lab studies the ubiquitin-proteasome system and its role in cellular oxidative stress response. I just learned about ubiquitin-proteasome system this past semester in Molecular Biology. We mostly focused on its role in keeping the cell healthy by degrading proteins. It was interesting to learn that this system also has a role in oxidative stress response and that there was a lot of nuance in the mechanisms of this system.
During his talk, Dr. Silva also gave us a lot of advice about what he learned in his journey to where he is now. He earned his undergraduate and Ph.D. degrees from the University of Sao Paolo. He then completed his postdoctoral training at New York University before coming to Duke. He had a lot of advice for us, especially if we were interested in a path that led to a career in academia. He told us what he wished he had known and considered when he was in our position. Some specific advice was about criteria to consider when applying to and choosing a graduate program. He emphasized the importance of connecting with the people you work with, whether that be as a mentor, a friend, or as part of the larger network of people you know in science.
All of the different faculty members’ journeys in science were very different from each other. They studied at different institutions, followed different timelines, and some even earned an M.D. in addition to a Ph.D. Despite none of their paths being the same, there was one unifying trait: they all absolutely love their research. When they talk about it, you can tell that it immediately excites them. I think when you find something that brings so much personal joy and fulfillment, you know that it is what you’re meant to be doing. I am grateful to these faculty members for sharing their joy and passion with us.
Breast cancer is the leading cause of cancer mortality among women. The majority of cases and resulting deaths occur in low-resource settings. Effective breast cancer treatment requires a detailed assessment of the tumor. Unfortunately, the lack of clinical infrastructure in low-resource settings prohibits the use of traditional breast cancer pathological methods. The D4 assay is a miniaturized, self-contained assay that can measure protein biomarkers from complex biological milieu with high sensitivity and specificity without the need for equipment (other than a smartphone) and can be performed with minimal user training. Previously, a D4 assay for HER2 detection from fine needle aspirate (FNA) samples was developed. However, due to variation in FNA sampling, it is important to normalize HER2 concentration in clinical settings. GADPH, a housekeeping protein, provides a normalization standard. Thus, I constructed a multiplexed assay that simultaneously detects HER2 and GADPH. The assay was tested for cross-reactivity and optimal antibody concentrations. We found that there is minimal cross reactivity between HER2 and GADPH. Furthermore, multiplexing does not compromise the analytical performance of the test. The ability to multiplex HER2 and GADPH will make the D4 assay a more accurate diagnostic tool to enable effective breast cancer treatment.
What I do in the lab depends on the day, but all of my activities revolve around glass slides. Some days, I have to do my least favorite task: polymerizing the glass slides. The procedure isn’t particularly difficult or unpleasant, it is just time-consuming. The upside is that once I polymerize two batches of slides, I don’t have to do it again for awhile.
Other days are all about printing the antibodies on the slides. In the morning, I will take some polymerized slides to the Shared Materials Instrumentation Facility (SMIF) cleanroom to use the microarray printer. I have to wear a full body suit over my clothes, complete with a head cover, shoe covers, and a surgical mask. The SMIF printer is really precise and can print tiny spots of capture antibody. I print 24 assays onto each glass slide. Then I take my printed slides back to our lab to use the Biodot printer. The Biodot is also a non-contact inkjet printer, but we can use it to print trehalose pads and detection antibody as larger dots surrounding the capture antibody spots. Then I leave the slides in a vacuum dessication chamber overnight.
The next day, I will test the assays that I printed. This means spiking a liquid, usually a buffer or fetal bovine serum, with different concentrations of the protein the assay is meant to detect. These serially diluted solutions are pipetted onto the slides and incubated for about one hour. Then I put the slides in a wash buffer, centrifuge them dry, and analyze them with our lab scanner. The computer program we use scans and quantifies the fluorescence on the assays. All of this data is collected, sorted in Excel, and able to be made into a dose response curve. The dose response curve is the big make or break moment. What you hope to see is that as the analyte concentration increases, so does the fluorescence intensity on the assay. If the dose response curve looks weird or doesn’t have the expected limit of detection, I have to think about what went wrong and try again.
My days usually follow one of those three basic patterns. Of course, each day is still a little different. Sometimes there are lab meetings, free food leftover from other events, or lunches with friends. Most days the grad students bring their dogs to our office space, so I get to play with them whenever I have a free moment. The people I work with are friendly and patiently answer my questions about where things are and what I’m supposed to do. My only complaint is that because we work with delicate antibodies printed onto glass slides, I live in constant fear of dropping slides (or God forbid, a whole box of 30 slides!) or smudging the antibody spots. It keeps me on my toes.
This past week, everyone gave short chalk talks on the research they’re doing this summer. After a week of listening to seventeen different chalk talks, I gained new knowledge about a myriad of different research topics. One of the projects that caught my attention was Jaan’s research on dystonia, a movement disorder that causes muscles to uncontrollably contract. She explained a complicated topic clearly and articulately, and in those eight minutes I learned a lot of new information.
When cells experience high stress, the protein pathway eIF2α typically responds by triggering phosphorylation. A hypothesis is that in dystonia patients, stress does not cause as much eIF2α phosphorylation as there should be. However, whether targeting eIF2α signaling can mitigate the symptoms of dystonia remains uncertain. Furthermore, it is important to identify the exact parts of the brain that are vulnerable to altered eIF2α signaling.
Jaan is researching whether mice with dystonia have a dysregulated eIF2α pathway using western blot. By examining the brain tissue of the mice for eIF2α expression at various times throughout development, she can study whether there is a period of susceptibility in which pathway dysregulation happens. She can also investigate which specific brain regions have altered eIF2α signaling.
While listening to all of my peers present, I was surprised by how many of them are researching the brain. All of the neurology projects were studying different proteins, pathways, and brain regions. The impression I formed was that the details of how and why our brains function mostly remain a mystery to us. But that mystery has an exciting allure about it. After all, the heart of research is that despite all the science and knowledge we’ve discovered, there is still so much more that we don’t know.
Dr. Ashutosh Chilkoti is a professor, the chair of Duke’s Biomedical Engineering department, a PI of his own lab, an entrepreneur, a mentor, and much more. Surprisingly, he says he’s never had goals or a five-year plan. He just takes advantage of the opportunities that come his way.
As an undergraduate, Dr. Chilkoti studied Chemical Engineering at the Indian Institute of Technology in Delhi. He didn’t initially intend to go into science, and he might have studied history or literature. But after passing the IIT entrance exam, the people around him encouraged him to take advantage of this opportunity, so he did. Then, he earned his Ph.D. in chemical engineering at the University of Washington and stayed there as a postdoc to study bioengineering. When I asked why he pivoted away from chemical engineering to bioengineering, he said it wasn’t much of a pivot. His Ph.D. mentor ran a lab that focused on bioengineering, so he was used to applying his knowledge to bioengineering problems. From Seattle, Dr. Chilkoti came to Duke, where he still is today.
Research brings the act of invention and discovery, and Dr. Chilkoti finds it really gratifying to do something nobody has ever done before. But his job as a PI goes beyond scientific discovery. To him, the best part of his job is mentoring graduate students and postdocs as they grow and develop as professionals and scientists. His only qualm with science is the difficulty in obtaining funding. He fears that the scarcity of funding discourages young scientists from becoming professors and researchers because they don’t want to spend their careers constantly writing grant proposals and looking for money.
His advice for a student who is interested in research is to find the “flavor” of science that you are both interested in and good at. The only way to do that is to try new things. Some people are good at the theory; they discover with their paper and pencil without ever stepping foot in a lab. Some people prefer a more tactile path and work in a wet lab, pipetting and culturing cells, or in a dry lab, building machines and devices. As long as you find the kind of science you like to do, the specific research projects will follow.
Modern clinical medicine relies heavily on the blood-based diagnostic tests that measure the amount of protein biomarkers present in circulation to make clinical decisions. In hospital settings, this is most commonly done by enzyme-linked immunosorbent assay (ELISA). While accurate and sensitive, ELISA requires considerable resources, infrastructure, and expertise to perform. The D4 assay is a miniaturized, self-contained assay that can measure protein biomarkers in blood with ELISA-like performance without the need for equipment other than a smartphone and can be performed with minimal user training. Assay reagents are inkjet printed onto a glass chip coated with a special “zero-background” polymer coating, which acts to minimize biomolecular noise (making the assay very sensitive) and stabilize the reagents even without refrigeration. The assay is user-friendly because adding a liquid sample (blood, serum, cell lysate) to D4 chips automatically drives the assay to completion. Furthermore, the assay is very portable since a cellphone-based detector utilizes the phone’s camera lens to readout the fluorescence on the D4 assay. For a more in-depth explanation of how the D4 system works, read Joh, D.Y., et al. Because of the EpiView-D4 system’s portability, it has huge implications for healthcare in low-resource settings like Liberia.
Over the summer, I’m helping with two D4 assay projects. The first project is developing a point of care test for breast cancer. I’m optimizing a D4 assay that detects HER2, a protein found on breast cancer cells; this protein is important because if breast cancers are found to be HER2-positive, then they are likely to respond to so-called “anti-HER2” cancer drugs (e.g. trastuzumab), which is potentially life-saving. In the long-term, my goal is to make a multiplexed assay that targets the four major clinically-relevant biomarkers for breast cancer: these are ER, PR, HER2, and Ki67. This capability is standard care in the United States, but unavailable in many developing countries (where the majority of breast cancer deaths now occur). By having a single, low-cost, and user-friendly assay that profiles all four markers simultaneously, clinicians in the developing world will be able to match different breast tumors to the medications which they are most likely to respond to. The second project is developing a point-of-care test that rapidly identifies Methicillin-resistant Staphylococcus aureus (MRSA). MRSA can resist antibiotics due to expression of an altered penicillin-binding protein, PBP2a. Current methods for identifying MRSA versus methicillin-sensitive strains are based on culturing the bacteria, and this typically requires at least a day or longer. If a point of care D4 assay can be developed to detect PBP2a, MRSA diagnosis and treatment could become more efficient.
My ultimate career goal is to contribute to democratizing healthcare access, especially in low-resource settings around the world. I’m interested in how engineering and science can help me to reach this goal. There are many ways to do that, including working in industry and volunteering with an organization like Engineers Without Borders, becoming a device technician or field worker in a remote setting, or working in a lab to design cost-effective medical devices that don’t require lots of resources and infrastructure to operate. I didn’t know what specific path I want to take, so I applied to BSURF to see what it was like to work in a lab. I thought I could learn more about what being in a research lab looks like and if I was interested in pursuing research and lab work.
I know it’s only been one week, but I think working in the Chilkoti lab is fun. There are just so many things about the lab that are new to me. I have learned how to work with new tools and machines, polymerize a surface, and conjugate antibodies. After someone in the lab introduced me to all of the procedures and patiently answered all of my questions, I’ve had to practice doing these tasks on my own, which is equal parts daunting and exciting. This past week I successfully polymerized some slides, but the whole time I was so nervous that I was going to mess it up.
By the end of these 8 weeks, I’m hoping to be able to make a usable “D4” assay (more on what that means next week) on my own and to feel confident in my ability to contribute to the ongoing projects of the lab. I am sure there will be many mistakes along the way and asking questions with embarrassingly simple answers. But I know that with these less-than-ideal moments comes growth and knowledge. I look forward to what the rest of the summer has in store.
Me with some freshly conjugated antibodies.