Week 8

During this last week, I was able to complete my primary research project for the summer, which was to elucidate the genetic basis for resistance to a novel proteasome inhibitor compound in plasmodium falciparum parasites.  Through sequencing of isolated proteasome beta subunit genes, I was able to identify several point mutations in resistant parasite strains that may be responsible for altering the conformational state of the proteasome and thus prevent the proteasome inhibitor from binding.  The next step will be to use CRISPR to induce the identified point mutations in wild type parasite strains and subsequently assessing for the induction of resistance.  Ultimately, knowledge accrued from these studies will facilitate the development of next generation anti-malarial compounds, for which there is a pressing need as current drugs such as artemisinin are becoming less and less effective. 

I also started a mini-project my last week which involved mimicking the mechanical filtration of the spleen in vitro using a matrix of metallic microspheres.  While normal red blood cells are highly deformable, red blood cells infected with plasmodium falciparum decrease in deformability as parasites mature.  This change is believed to be caused by interactions between parasite secreted proteins and the cellular cytoskeleton.  In vivo, the inter-endothelial slits of the spleen trap red blood cells with low deformability, which is the mechanism by which senescent red blood cells are removed from circulation.  This mechanism can be recapitulated in vitro using a matrix of microspheres of sizes 5-25 microns.  Red blood cells flowing through the matrix are forced to undergo dumbbell shaped deformations in the inter-sphere spaces, which is the same type of deformation that occurs during splenic filtration.  In this manner, normal red blood cells can pass through the matrix while red blood cells filled with late stage parasites have a high probability of being retained.  This filtration technique can be used as an alternative to sorbitol-based parasite stage synchronization and can also be used to study other hemolytic diseases such as babesia.  Preliminary experiments conducted using the set-up shown below showed that the microsphere matrix does retain a significant amount of late stage parasite containing red blood cells.  This was a good starting point and Dr. Kirkman’s group will continue to improve and optimize the process.

Filtration set-up

Metallic microsphere layer on pipette filter

Filtration in action

Overall, my immersion term has been an amazing experience and I had a blast spending the summer in New York City.  It was great having the opportunity to interact with clinicians, residents, and fellows and also just being able to enjoy activities the city has to offer during my downtime.  I was able to pick up some molecular biology knowledge through my research and the clinical shadowing portion has granted me extensive insight into how infectious disease care is implemented when an advanced medical infrastructure is available.  Hopefully, I’ll be able to use this knowledge to brainstorm clever ways to bring about equivalent levels of care in low-resource settings for my PhD work.