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.