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.