SignificanceNucleic acids mediate storage and expression of genetic information. Extracellular DNA (exDNA) and exRNA are traces of nucleic acids released from cells into the extracellular environment. Their use as disease biomarkers has been limited by technical challenges in their isolation caused by abundant RNA- and DNA-degrading enzymes in biofluids. Using isolation protocols developed especially for biofluids, we generated plasma and serum exRNA reference profiles from 13 healthy volunteers over time and determined the effect of critical clinical parameters such as gender and fasting. Surprisingly, we encountered one participant with dramatically increased endocrine-origin exRNA contributions stable over 1 year and detectable in all of his samples, thereby demonstrating the robustness of this approach and the clinical potential of circulating RNAs as biomarkers.

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27Extracellular mRNAs (ex-mRNAs) potentially supersede extracellular miRNAs (ex-miRNAs) and other 28 RNA classes as biomarkers. Here, we present a comprehensive extracellular RNA (exRNA) study in 29 human blood circulation based on conventional small RNA-sequencing (sRNA-seq) and sRNA-seq after 30 T4 polynucleotide kinase (PNK) end-treatment of total exRNA isolated from serum and platelet-poor 31 EDTA, ACD, and heparin plasma. Applying strict criteria for read mapping and annotation, we found that 32 compared to conventional sRNA-seq PNK-treatment increased the detection of informative ex-mRNAs 33 reads up to 50-fold. Based on captured ex-mRNAs from healthy individuals, we concluded that the 34 exRNA pool is dominated by hematopoietic cells and platelets, with additional contribution from the 35 liver.

Efavirenz (EFV), the most popular nonnucleoside reverse transcriptase inhibitor, has been associated with mitochondrial dysfunction in most studies. However, in real life the prevalence of EFV-induced mitochondrial toxicity is relatively low. We hypothesized that the agents given in combination with EFV moderate the effect of EFV on mitochondrial function. To test this hypothesis, we cultured a human T lymphoblastoid cell line (CEM cells) with EFV alone and in combination with emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) to investigate the effects on mitochondrial respiration and function and cholesterol biosynthesis. There was a statistically significant concentration- and time-dependent apoptosis, reduction in mitochondrial membrane potential, and increase in production of reactive oxygen species in cells treated with either EVF alone or in combination with TDF plus FTC. Compared to dimethyl sulfoxide-treated cells, EFV-treated cells had significant reduction in oxygen consumption rate contributed by basal mitochondrial respiration and decreased protein expression of electron transport chain complexes (CI, CII, and CIV). Treatment with EFV resulted in a decrease in mitochondrial DNA content and perturbation of more coding genes ( = 13); among these were 11 genes associated with lipid or cholesterol biosynthesis. Our findings support the growing body of knowledge on the effects of EFV on mitochondrial respiration and function and cholesterol biosynthesis. Interestingly, combining TDF and FTC with EFV did not alter the effects of EFV on mitochondrial respiration and function and cholesterol biosynthesis. The gap between the prevalence of EFV-induced mitochondrial toxicity in and studies could be due to individual differences in the pharmacokinetics of EFV.

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Respiratory viruses can be transmitted through contact, droplet and airborne routes. Viruses that are not naturally airborne may be aerosolised during medical procedures and transmitted to healthcare workers. Most resource-limited healthcare settings lack complex air handling systems to filter air and create pressure gradients that are necessary for minimising viral transmission. This review explores the association between ventilation and the transmission of respiratory viruses like SAR-CoV-2. When used appropriately, both natural and mechanical ventilation can decrease the concentration of viral aerosols, thereby reducing transmission. Although mechanical ventilation systems are more efficient, installation and maintenance costs limit their use in resource-limited settings, whereas the prevailing climate conditions make natural ventilation less desirable. Cost-effective hybrid systems of natural and mechanical ventilation may overcome these limitations.

20Efavirenz (EFV), the most popular non-nucleoside reverse transcriptase inhibitor, has been 21 associated with mitochondrial dysfunction in most in vitro studies. However, in real life the 22 prevalence of EFV-induced mitochondrial toxicity is relatively low. We hypothesized that the 23 agents given in combination with EFV might moderate the effect of EFV on mitochondrial 24 function. To test this hypothesis, we cultured human T lymphoblastoid cell line (CEM cells) with 25 EFV alone and in combination with emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) 26 to investigate the effects on mitochondrial respiration and function and cholesterol biosynthesis. 27 There was a statistically significant concentration-and time-dependent apoptosis, reduction in 28 mitochondrial membrane potential (ΔΨ), and increase production of reactive oxygen species 29 (ROS) in cells treated with either EVF alone or in combination with TDF/FTC. EFV treated 30 cells compared to DMSO treated cells had significant reduction in oxygen consumption rate 31 (OCR) contributed by mitochondrial respiration, ATP production-linked respiration, and spare 32 respiratory capacity (SRC). Treatment with EFV resulted in a decrease in mitochondrial DNA 33 content, and perturbation of more coding genes (n=13); among these were 11 genes associated 34 with lipid or cholesterol biosynthesis. Our findings support the growing body of knowledge on 35 the effects of EFV on mitochondrial respiration and function and cholesterol biosynthesis. 36Interestingly, combining TDF/FTC with EFV did not alter the effects of EFV on mitochondrial 37 respiration and function and cholesterol biosynthesis. The gap between the prevalence of EFV-38 induced mitochondrial toxicity in vitro and in vivo studies may be explained by individual 39 differences in the pharmacokinetic of EFV.40 41 42 3 INTRODUCTION: 43Combination antiretroviral therapy (ART) has resulted in a decrease in AIDS-related 44 morbidity and mortality (1), though the therapeutic benefit of ART is often limited by delayed 45 toxicity (2). Although contemporary ART regimens compared to early ART regimens are less 46 toxic(3, 4), toxicity is still pervasive and affects a significant number of people living with HIV 47 (4, 5). In vitro studies demonstrated that inhibition of polymerase gamma (Pol-γ), enzyme 48 responsible for mitochondrial DNA replication, by nucleoside reverse transcriptase inhibitors 49 (NRTIs) leads to depletion of mitochondrial DNA (mtDNA) and subsequent mitochondrial 50 dysfunction(6, 7); the "Pol-γ inhibition" hypothesis. However, there is a growing body of 51 knowledge to suggest that ART-associated mitochondrial dysfunction cannot be explained solely 52 by Pol-γ inhibition (8, 9). For instance, other classes of ART such as protease inhibitors (PIs) and 53 non-nucleoside reverse transcriptase inhibitors (NNRTIs) do not inhibit Pol-γ and yet they cause 54 side effects akin to mitochondrial dysfunction(8, 10). Taken together, there must be alternative or 55 additional mechanisms...

Although antiretroviral therapy (ART) has resulted in a marked decrease in AIDS-related morbidity and mortality, the therapeutic benefit is often limited by side effects such as metabolic derangement such as lipodystrophy and hyperlipidemia and cardiovascular diseases. These side effects are pervasive in people living with HIV (PLWH). However, the underlying mechanisms are not completely understood. We investigated the effects of ART on cholesterol biosynthesis genes. This is a retrospective analysis of data and specimens collected during a cross-sectional, case-control study of ART-induced toxicity. Cases were HIV treatment-experienced individuals with HIV viral suppression and no diagnosis of ART-associated toxicity (n = 18), and controls were HIV-uninfected individuals (n = 18). The mRNA expressions of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) and ATP binding cassette transporter A1 (ABCA1) were significantly upregulated in cases (HIV+) compared to controls (HIV-), as well as the corresponding protein expression level of HMGCR. We observed dysregulation between sterol regulatory element-binding protein 2 (SREBP-2, sensory control) and HMGCR and low-density lipoprotein receptor (LDLR) pathways. Dysregulation of cholesterol biosynthesis genes may predate clinical manifestation of ART-induced lipid abnormalities.