Stephanie Melchor and Dr. John Bell, Department of Physiology and Developmental Biology
Well, to be perfectly honest, this project has been fraught with disaster since the beginning. We obtained 400 microliters of the immunosuppressive strain of Minute Virus of Mice (MVMi) at 1 x 109 pfu/mL from Dr. David J. Pintel at the University of Missouri-Columbia. MVMi was the only virus we could find that would infect our cell line (S49 mouse T-lymphoma cells. Because S49 cells are suspension cells and not adherent, they do not form plaques when infected with a virus, which makes it more difficult to quantify how many cells have been successfully infected with the virus. Instead, we attempted to perform a TCID50 assay, where one scores samples as either + or – for evidence of an infection at different concentrations of the virus and titrates the virus that way. Unfortunately, cytopathic effects of the virus were confounded when our cells became contaminated with some sort of fungus. We tried a few more times to observe the effects of the viral infection on a cell culture, but every time, our cells (even the control cells) exhibited the effects of fungal contamination instead of a viral infection.
We planned on setting that test aside for a while and wanted to move straight on to our experiments with our propidium iodide timecourses. Initially, the results looked promising. Cells that were exposed to the virus and then sPLA2 (secretory phospholipase A II) seemed to die much more quickly through hydrolysis by the phospholipase than non-infected cells that were also exposed to the phospholipase. This seemed to support our hypothesis that sPLA2 is able to recognize and target the membranes of virally infected cells, killing them before the virus can propagate within the cell, similar to how it is sensitive to other events that cause trauma to the cell membrane (such as oxidation and phosphatidylserine exposure). However, our astute lab manager realized that all of my experiments were run on Fridays, which is when our cells were the most crowded and unhealthy. They were more likely to die by hydrolysis of sPLA2 (and probably more likely to be infected by MVMi) by the sheer virtue (or vice) of just being very unhealthy. When I started doing identical experiments earlier in the week when the cells were at their healthiest, we saw no observable effect of increased sPLA2 hydrolysis on infected cells. In fact, in several trials, the cells exposed to the virus seemed to have prolonged lives, perhaps because the MVMi was blocking an sPLA2 binding site on the cell’s plasma membrane. I would like to do more experiments to see if this effect is really reproducible and if so, why it happened.
At this point in our experimentation, we decided to suspend the project. It was supposed to be for my Honors Thesis and I was running out of time to get interesting and publishable data to write a thesis around by my December 2012 graduation. Since this project wasn’t producing any really conclusive data, I was planning on writing my thesis on research I had done the previous summer, which did lead to a poster at the 2012 Biophysics Convention in San Diego and later publication.1 I eventually decided that graduating with Honors was not worth rewriting an Honors Thesis proposal and spending hours upon hours writing and preparing to defend a thesis that my heart wasn’t in (and that I didn’t care about at all), especially because I was still so devastated about my project not yielding positive results.
I spent the rest of the summer helping other students in my lab with their projects and then had to quit when the fall semester started because my school schedule was not conducive to lab work. Had I had more time before leaving the lab, I definitely would have pursued the project further. I would have done more research (and perhaps collaborated more with Dr. Pintel in Missouri) on the cytopathic effects of MVMi on S49 cells and figured out a way to avoid contamination in my cultures so that I could see those cytopathic effects and conducted a successful TCID50 assay. I would have pursued the other legs of my proposed experiments, which included using fluorescence spectroscopy and flow cytometry to identify candidates for membrane changes induced by the infecting virus, such as using Alexafluor-Annexin to measure phosphatidylserine (PS) because the probe only fluoresces when bound to exposed PS.2 I would have also done some fluorescence microscopy as well.
Even though the results of my experiment were disappointing and time constraints forced us to drop the project, I learned so much about actually doing science. I learned how to do a real literature review. I learned how to be independent and to contact other researchers on my own, not just through my adviser. I learned how to troubleshoot and how to be flexible with my experimentation. And I learned that sometimes, research can’t support the hypothesis, and that’s okay. Most importantly (I know that this is corny and not scientific at all, but this is what I learned), I learned that I have a passion for research that I didn’t know I had before. Even though I ended up quitting, the fact that I was so emotionally involved in my research and so devastated when I had to stop reminded me that I do, in fact, have drive and commitment, and that was really reassuring to remember at the end of my undergraduate career when I was burned out and jaded.