This research experience was at the Ponce Health Sciences University (previously known as Ponce School of Medicine) in ponce, Puerto Rico.
The Ponce Health Sciences University is a nationally-accredited university inaugurated in 1977, it issues graduate degrees in Medicine (MD), Clinical Psychology (PsyD and PhD), Biomedical Sciences (PhD), Medical Sciences (MSMS), and Public Health (MPH and DrPH).
In here I performed work with Dr. Martin Hill from the Pharmaceutical and Toxicology Department on the effect of Ascorbic Acid in HIV-1 infected CD4+ T-Cells concentrating in DNA damage.
José M. Santiago Echevarria; Lenin Godoy, BS; Elinette Albino, PhD; Martin Hill, PhD
Abstract
Human immunodeficiency virus-1 (HIV-1) attacks the immune system by infecting and depleting CD4+ T-cells. HIV infection induces cellular oxidative stress which potentially leads to DNA damage. Although no cure has been found for HIV-1, antiretroviral treatment (ART) can control its viral load. Since impoverished countries have limited access to ART, complementary, inexpensive treatments need to be further studied. Studies have shown that micronutrients have a positive effect in limiting HIV-1 pathogenesis. Ascorbic acid (vitamin C) is a known antioxidant micronutrient that can be found in fruits, vegetables and as a dietary supplement. This study aims to assess the effect of ascorbic acid in HIV-1 infected CD4+ T-cells in terms of viral load reduction and protection from oxidative stress-induced DNA damage. We hypothesize that, upon exposure to ascorbic acid, there will be a reduction in viral load and oxidative stress-induced DNA damage in infected CD4+ T-cells. The devised method consists of exposing HIV-1 infected and non-infected CD4+ T-cells to ascorbic acid at concentrations ranging from 2 to 140 µg/ml. At 24, 48 and 72 hours of incubation viral load was measured through qRT-PCR; cell number and viability were assessed with trypan blue dying using an AutoT4 Cellometer and DNA oxidative stress damage was measured at 72 hours through the Comet assay. Comparing the DNA oxidative stress damage and viral counts of HIV-1 infected CD4+ T-cells upon exposure to ascorbic acid can provide a significant insight into the use of this antioxidant in HIV-1 infected CD4+ T-cells in limiting its viral replication.
In this Researched Experience I gained deep knowledge of laboratory techniques including and not limited to:
- Cell culture care
- qRT-PCR for Viral RNA
- ELISA
- Alkaline gel electrophoresis
- Polyacrylamide gel electrophoresis
- Agarose gel electrophoresis
- Infection methods with HIV-1 for Jurkat T-cell line
Also encountering troubleshooting in the following areas:
- Low cell replication due to low addition of phyto-haemagglutining
- Automatic cell counter low precision due to cell clumping
- Comet Assay contamination due to accidental use of old reagents
- ELISA readings above calibration curve due to unexpected high concentrations of sample
Without a doubt I learned many new protocols and applications for classroom theory during this internship. Results were presented in the STEP-UP Summer Research Symposium at the National Institute of Health (Aug 2015).
This research experience received funding by the NIH STEP-UP program and Ponce Health Sciences University. The support of the Molecular and Genomics Core (NIMHD Grant MD007579) is acknowledge.
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