Wednesday, January 16, 2019

URSP Student James Erickson Examines How to Study Ebola VP40 Exosomes Part II

This past summer, I work on an OSCAR program ion Ebola VP40 and exosome research in Dr. Fatah Kashanchi’s molecular virology lab.  During this internship, I was mentored a bright Ph.D. candidate, Michelle Pleet, who taught me over the past 2 and half years various lab protocols and skills that I use to do my project today. In the fall of 2017, we had received plasmids from a collaborator who is well-established in the field. These plasmids are designed to produce a mutated form of the Ebola matrix protein (VP40), and we planned to test them in a series of experiments targeted towards determining the confirmations of VP40 necessary to enter into exosomes and have physiological effects in recipient cells. These experiments, however, were put on the back-burner until after our most recent Ebola VP40 and exosome review paper, on which I am a co-author, that is currently in progess. After learning about the URSP/OSCAR program, it became clear that the next step to advance me further in my scientific career was to head the start of the research into the Ebola VP40 mutant plasmids, and how the mutations in the VP40’s structure would affect the protein’s ability to become packaged into exosomes and affect recipient cells. As our recent Ebola paper was submitted earlier this year, we started the new research project by transfecting 293T cells with the 5 mutant VP40-producing plasmids. This was followed by Western blotting for intracellular VP40 protein levels to confirm that the cells took up the plasmids. The exosomes from these cells were next characterized by ZetaView analysis, which measures the size and concentration of extracellular vesicles by using the principles of Brownian motion. We also checked for the presence and levels of VP40 protein in the exosomes produced from the transfected cells, as well as other confirmatory exosomal markers by doing special Western blots using nanoparticles in order to concentrate the extracellular material. Ultimately, this led to 1- 2 Western blots being performed weekly to probe for and confirm the presence and levels of various host cell and mutant VP40 proteins. As at this point, I am fairly used to rigorous wet lab bench work, the experiments themselves were not difficult or particularly complicated to perform. Instead, the most eye-opening thing I learned this term was how much work is actually done to publish a paper, as all the time that wasn’t going to my OSCAR was spent on responding to the comments of reviewers from our recently submitted paper. This involved intensely researching the pre-existing literature as well as designing and running intricate control experiments to answer the reviewers’ pressing questions and concerns. All in all, I have learned a great deal this term, not only in terms of the biology we have explored in my project, but also in terms of gaining an overall better understanding of what it takes to become a successful independent researcher and scientific writer.