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Small extracellular vesicles (sEV) are nano-sized (40-150 nm), membrane-encapsulated vesicles that are released by essentially all cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules, including microRNA (miRNA) and other small non-coding RNA (ncRNA), that can alter the phenotype of recipient cells. sEV are present in essentially all extracellular biofluids, including serum, urine and saliva, and offer a new avenue for discovery and development of novel biomarkers of various disease states and exposures. The objective of this study was to systematically interrogate similarities and differences between sEV ncRNA derived from saliva, serum and urine, as well as cell-free small ncRNA (cf-ncRNA) from serum. Saliva, urine and serum were concomitantly collected from 4 healthy donors to mitigate potential bias that can stem from interpersonal and temporal variability. sEV were isolated from each respective biofluid, along with cf-RNA from serum. sEV were isolated from the respective biofluids via differential ultracentrifugation with a 30% sucrose cushion to minimize protein contamination. Small RNA-sequencing was performed on each sample, and cluster analysis was performed based on ncRNA profiles. While some similarities existed in terms of sEV ncRNA cargo across biofluids, there are also notable differences in ncRNA class and ncRNA secretion, with sEV in each biofluid bearing a unique ncRNA profile, including major differences in composition by ncRNA class. We conclude that sEV ncRNA cargo varies according to biofluid, so thus should be carefully selected and interpreted when designing or contrasting translational or epidemiological studies., Competing Interests: The authors have declared that no competing interests exist.

Aim: The goal of this study was to comprehensively interrogate and map DNA methylation across 16 CpG-dense regions previously associated with oral and pharyngeal squamous cell carcinoma (OPSCC). Materials & methods: Targeted multiplex bisulfite amplicon sequencing was performed on four OPSCC cell lines and primary non-neoplastic oral epithelial cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for a subset of associated genes.  Results: There was clear differential methylation between one or more OPSCC cell lines and control cells for the majority of CpG-dense regions. Conclusion: Targeted multiplex bisulfite amplicon sequencing allowed us to efficiently map methylation across the entire region of interest with a high degree of sensitivity and helps shed light on novel differentially methylated regions that may have value as biomarkers of OPSCC.