Proteomic insights into the modulation of foetal neurogenesis by the anti-retroviral efavirenz
Master Thesis
2019
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Background: South African guidelines recommend that HIV-positive pregnant women immediately initiate antiretroviral therapy (efavirenz, emtricitabine, and tenofovir), regardless of trimester. Efavirenz causes central nervous system neuropathy and has been linked to birth defects such as encephalocoele. Cohort studies of HIV-uninfected children exposed to antiretroviral treatment in utero report minor learning delays but are inconclusive. Non-transformed human derived neuroepithelial stem (NES) represent a unique pre-clinical model in which to investigate the effects of efavirenz on the developing neural system. Efavirenz-induced global cellular molecular changes may be characterised using mass spectrometry (MS). Aims: To optimise an MS-based efavirenz extraction and detection assay, and to investigate efavirenzinduced NES proteomic responses. Methods: A TSQ Vantage triple quadrupole mass spectrometer was employed to optimise targeted detection of efavirenz extracted from cultured cells and supernatant. Cells were cultured for 72 hours, incorporating a 24-hourly efavirenz treatment. Efavirenz concentration dynamics were assessed over this period, and cells were harvested every 24 hours for discovery proteomic analysis using a Q-Exactive quadrupole-Orbitrap mass spectrometer. Results: Drug extraction with acetonitrile was selected as the optimal extraction and detection technique. In cell culture, efavirenz concentration increased after 24 hours and decreased after 48 hours. A total of 1663 protein groups were identified, with 26, 39, and 80 protein groups differentially expressed 24, 48, and 72 hours respectively post EFV treatment. The most significantly enriched deregulated pathways included cholesterol biosynthesis, mRNA splicing, and JAK/STAT and Wnt signalling. Conclusions: Efavirenz-altered protein expression reflects functional pathway perturbations, which may contribute to clinically-observed neurological effects. Orthoganal and in vivo confirmation is required.
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Albeldas, C. 2019. Proteomic insights into the modulation of foetal neurogenesis by the anti-retroviral efavirenz.