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From eukaryotes to prokaryotes, all cells secrete extracellular vesicles (EVs) as part of their regular homeostasis, intercellular communication, and cargo disposal. Accumulating evidence suggests that small EVs carry functional small RNAs, potentially serving as extracellular messengers and liquid‐biopsy markers. Yet, the complete transcriptomic landscape of EV‐associated small RNAs during disease progression is poorly delineated due to critical limitations including the protocols used for sequencing, suboptimal alignment of short reads (20–50 nt), and uncharacterized genome annotations—often denoted as the 'dark matter' of the genome. In this study, we investigate the EV‐associated small unannotated RNAs that arise from endogenous genes and are part of the genomic 'dark matter', which may play a key emerging role in regulating gene expression and translational mechanisms. To address this, we created a distinct small RNAseq dataset from human prostate cancer & benign tissues, and EVs derived from blood (pre‐ & post‐prostatectomy), urine, and human prostate carcinoma epithelial cell line. We then developed an unsupervised data‐based bioinformatic pipeline that recognizes biologically relevant transcriptional signals irrespective of their genomic annotation. Using this approach, we discovered distinct EV‐RNA expression patterns emerging from the un‐annotated genomic regions (UGRs) of the transcriptomes associated with tissue‐specific phenotypes. We have named these novel EV‐associated small RNAs as 'EV‐UGRsʼ or "EV‐dark matter". Here, we demonstrate that EV‐UGR gene expressions are downregulated by ∼100 fold (FDR < 0.05) in the circulating serum EVs from aggressive prostate cancer subjects. Remarkably, these EV‐UGRs expression signatures were regained (upregulated) after radical prostatectomy in the same follow‐up patients. Finally, we developed a stem‐loop RT‐qPCR assay that validated prostate cancer‐specific EV‐UGRs for selective fluid‐based diagnostics. Overall, using an unsupervised data driven approach, we investigate the 'dark matter' of EV‐transcriptome and demonstrate that EV‐UGRs carry tissue‐specific Information that significantly alters pre‐ and post‐prostatectomy in the prostate cancer patients. Although further validation in randomized clinical trials is required, this new class of EV‐RNAs hold promise in liquid‐biopsy by avoiding highly invasive biopsy procedures in prostate cancer. [ABSTRACT FROM AUTHOR]

Importance: SARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or organ dysfunction after the acute phase of infection, termed Post-Acute Sequelae of SARS-CoV-2 (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are poorly understood. The objectives of the Researching COVID to Enhance Recovery (RECOVER) tissue pathology study (RECOVER-Pathology) are to: (1) characterize prevalence and types of organ injury/disease and pathology occurring with PASC; (2) characterize the association of pathologic findings with clinical and other characteristics; (3) define the pathophysiology and mechanisms of PASC, and possible mediation via viral persistence; and (4) establish a post-mortem tissue biobank and post-mortem brain imaging biorepository. Methods: RECOVER-Pathology is a cross-sectional study of decedents dying at least 15 days following initial SARS-CoV-2 infection. Eligible decedents must meet WHO criteria for suspected, probable, or confirmed infection and must be aged 18 years or more at the time of death. Enrollment occurs at 7 sites in four U.S. states and Washington, DC. Comprehensive autopsies are conducted according to a standardized protocol within 24 hours of death; tissue samples are sent to the PASC Biorepository for later analyses. Data on clinical history are collected from the medical records and/or next of kin. The primary study outcomes include an array of pathologic features organized by organ system. Causal inference methods will be employed to investigate associations between risk factors and pathologic outcomes. Discussion: RECOVER-Pathology is the largest autopsy study addressing PASC among US adults. Results of this study are intended to elucidate mechanisms of organ injury and disease and enhance our understanding of the pathophysiology of PASC. [ABSTRACT FROM AUTHOR]