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In this follow-up study, we investigated the abundance and compartmentalization of blood plasma extracellular miRNA (exmiRNA) into lipid-based carriers—blood plasma extracellular vesicles (EVs) and non-lipid-based carriers—extracellular condensates (ECs) during SIV infection. We also assessed how combination antiretroviral therapy (cART), administered in conjunction with phytocannabinoid delta-9-tetrahydrocannabinol (THC), altered the abundance and compartmentalization of exmiRNAs in the EVs and ECs of SIV-infected rhesus macaques (RMs). Unlike cellular miRNAs, exmiRNAs in blood plasma may serve as minimally invasive disease indicators because they are readily detected in stable forms. The stability of exmiRNAs in cell culture fluids and body fluids (urine, saliva, tears, cerebrospinal fluid (CSF), semen, blood) is based on their association with different carriers (lipoproteins, EVs, and ECs) that protect them from the activities of endogenous RNases. Here, we showed that in the blood plasma of uninfected control RMs, significantly less exmiRNAs were associated with EVs compared to the level (30% higher) associated with ECs, and that SIV infection altered the profile of EVs and ECs miRNAome (Manuscript 1). In people living with HIV (PLWH), host-encoded miRNAs regulate both host and viral gene expression, which may serve as indicators of disease or treatment biomarkers. The profile of miRNAs in blood plasma of PLWH (elite controllers versus viremic patients) are different, indicating that HIV may alter host miRNAome. However, there are no studies assessing the effect of cART or other substances used by PLWH, such as THC, on the abundance of exmiRNA and their association with EVs and ECs. Moreover, longitudinal exmiRNA profiles following SIV infection, treatment with THC, cART, or THC+cART remains unclear. Here, we serially analyzed miRNAs associated with blood plasma derived EVs and ECs. Methods: Paired EVs and ECs were separated from EDTA blood plasma of male Indian rhesus macaques (RMs) in five treatment groups, including VEH/SIV, VEH/SIV/cART, THC/SIV, THC/SIV/cART, or THC alone. Separation of EVs and ECs was achieved with the unparalleled nano-particle purification tool ─PPLC, a state-of-the-art, innovative technology equipped with gradient agarose bead sizes and a fast fraction collector that allows high resolution separation and retrieval of preparative quantities of sub-populations of extracellular structures. Global miRNA profiles of the paired EVs and ECs were determined with RealSeq Biosciences (Santa Cruz, CA) custom sequencing platform by conducting small RNA (sRNA)-seq. The sRNA-seq data were analyzed using various bioinformatic tools. Validation of key exmiRNA was performed using specific TaqMan microRNA stem-loop RT-qPCR assays. Results: We investigated the effect of cART, THC, or both cART and THC together on the abundance and compartmentalization of blood plasma exmiRNA in EVs and ECs in SIV-infected RMs. As shown in Manuscript 1 of this series, were in uninfected RMs, ~30% of exmiRNAs were associated with ECs, we confirmed in this follow up manuscript that exmiRNAs were present in both lipid-based carriers—EVs and non-lipid-based carriers—ECs, with 29.5 to 35.6% and 64.2 to 70.5 % being associated with EVs and ECs, respectively. Remarkably, the different treatments (cART, THC) have distinct effects on the enrichment and compartmentalization pattern of exmiRNAs. In the VEH/SIV/cART group, 12 EV-associated and 15 EC-associated miRNAs were significantly downregulated. EV-associated miR-206, a muscle-specific miRNA that is present in blood, was higher in the VEH/SIV/ART compared to the VEH/SIV group. ExmiR-139-5p that was implicated in endocrine resistance, focal adhesion, lipid and atherosclerosis, apoptosis, and breast cancer by miRNA-target enrichment analysis was significantly lower in VEH/SIV/cART compared to VEH/SIV, irrespective of the compartment. With respect to THC treatment, 5 EV-associated and 21 EC-associated miRNAs were significantly lower in the VEH/THC/SIV. EV-associated miR-99a-5p was higher in VEH/THC/SIV compared to VEH/SIV, while miR-335-5p counts were significantly lower in both EVs and ECs of THC/SIV compared to VEH/SIV. EVs from SIV/cART/THC combined treatment group have significant increases in the count of eight (miR-186-5p, miR-382-5p, miR-139-5p and miR-652, miR-10a-5p, miR-657, miR-140-5p, miR-29c-3p) miRNAs, all of which were lower in VEH/SIV/cART group. Analysis of miRNA-target enrichment showed that this set of eight miRNAs were implicated in endocrine resistance, focal adhesions, lipid and atherosclerosis, apoptosis, and breast cancer as well as cocaine and amphetamine addiction. In ECs and EVs, combined THC and cART treatment significantly increased miR-139-5p counts compared to VEH/SIV group. Significant alterations in these host miRNAs in both EVs and ECs in the untreated and treated (cART, THC, or both) RMs indicate the persistence of host responses to infection or treatments, and this is despite cART suppression of viral load and THC suppression of inflammation. To gain further insight into the pattern of miRNA alterations in EVs and ECs and to assess potential cause-and-effect relationships, we performed longitudinal miRNA profile analysis, measured in terms of months (1 and 5) post-infection (MPI). We uncovered miRNA signatures associated with THC or cART treatment of SIV-infected macaques in both EVs and ECs. While the number of miRNAs was significantly higher in ECs relative to EVs for all groups (VEH/SIV, SIV/cART, THC/SIV, THC/SIV/cART, and THC) longitudinally from 1 MPI to 5 MPI, treatment with cART and THC have longitudinal effects on the abundance and compartmentalization pattern of exmiRNAs in the two carriers. As shown in Manuscript 1 where SIV infection led to longitudinal suppression of EV-associated miRNA-128-3p, administration of cART to SIV-infected RMs did not increase miR-128-3p but resulted in longitudinal increases in six EV-associated miRNAs (miR-484, miR-107, miR-206, miR-184, miR-1260b, miR-6132). Furthermore, administration of cART to THC treated SIV-infected RMs resulted in a longitudinal decrease in three EV-associated miRNAs (miR-342-3p, miR-100-5p, miR181b-5p) and a longitudinal increase in three EC-associated miRNAs (miR-676-3p, miR-574-3p, miR-505-5p). The longitudinally altered miRNAs in SIV-infected RMs may indicate disease progression, while in the cART Group and the THC Group, the longitudinally altered miRNAs may serve as biomarkers of response to treatment. Conclusions: This paired EVs and ECs miRNAome analyses provided a comprehensive cross-sectional and longitudinal summary of the host exmiRNA responses to SIV infection and the impact of THC, cART, or THC and cART together on the miRNAome during SIV infection. Overall, our data point to previously unrecognized alterations in the exmiRNA profile in blood plasma following SIV infection. Our data also indicate that cART and THC treatment independently and in combination may alter both the abundance and the compartmentalization of several exmiRNA related to various disease and biological processes. Full article

Background: This is Manuscript 1 of a two-part Manuscript of the same series. Here, we present findings from our first set of studies on the abundance and compartmentalization of blood plasma extracellular microRNAs (exmiRNAs) into extracellular particles, including blood plasma extracellular vesicles (EVs) and extracellular condensates (ECs) in the setting of untreated HIV/SIV infection. The goals of the study presented in this Manuscript 1 are to (i) assess the abundance and compartmentalization of exmiRNAs in EVs versus ECs in the healthy uninfected state, and (ii) evaluate how SIV infection may affect exmiRNA abundance and compartmentalization in these particles. Considerable effort has been devoted to studying the epigenetic control of viral infection, particularly in understanding the role of exmiRNAs as key regulators of viral pathogenesis. MicroRNA (miRNAs) are small (~20–22 nts) non-coding RNAs that regulate cellular processes through targeted mRNA degradation and/or repression of protein translation. Originally associated with the cellular microenvironment, circulating miRNAs are now known to be present in various extracellular environments, including blood serum and plasma. While in circulation, miRNAs are protected from degradation by ribonucleases through their association with lipid and protein carriers, such as lipoproteins and other extracellular particles—EVs and ECs. Functionally, miRNAs play important roles in diverse biological processes and diseases (cell proliferation, differentiation, apoptosis, stress responses, inflammation, cardiovascular diseases, cancer, aging, neurological diseases, and HIV/SIV pathogenesis). While lipoproteins and EV-associated exmiRNAs have been characterized and linked to various disease processes, the association of exmiRNAs with ECs is yet to be made. Likewise, the effect of SIV infection on the abundance and compartmentalization of exmiRNAs within extracellular particles is unclear. Literature in the EV field has suggested that most circulating miRNAs may not be associated with EVs. However, a systematic analysis of the carriers of exmiRNAs has not been conducted due to the inefficient separation of EVs from other extracellular particles, including ECs. Methods: Paired EVs and ECs were separated from EDTA blood plasma of SIV-uninfected male Indian rhesus macaques (RMs, n = 15). Additionally, paired EVs and ECs were isolated from EDTA blood plasma of combination anti-retroviral therapy (cART) naïve SIV-infected (SIV+, n = 3) RMs at two time points (1- and 5-months post infection, 1 MPI and 5 MPI). Separation of EVs and ECs was achieved with PPLC, a state-of-the-art, innovative technology equipped with gradient agarose bead sizes and a fast fraction collector that allows high-resolution separation and retrieval of preparative quantities of sub-populations of extracellular particles. Global miRNA profiles of the paired EVs and ECs were determined with RealSeq Biosciences (Santa Cruz, CA) custom sequencing platform by conducting small RNA (sRNA)-seq. The sRNA-seq data were analyzed using various bioinformatic tools. Validation of key exmiRNAs was performed using specific TaqMan microRNA stem-loop RT-qPCR assays. Results: We showed that exmiRNAs in blood plasma are not restricted to any type of extracellular particles but are associated with lipid-based carriers—EVs and non-lipid-based carriers—ECs, with a significant (~30%) proportion of the exmiRNAs being associated with ECs. In the blood plasma of uninfected RMs, a total of 315 miRNAs were associated with EVs, while 410 miRNAs were associated with ECs. A comparison of detectable miRNAs within paired EVs and ECs revealed 19 and 114 common miRNAs, respectively, detected in all 15 RMs. Let-7a-5p, Let-7c-5p, miR-26a-5p, miR-191-5p, and let-7f-5p were among the top 5 detectable miRNAs associated with EVs in that order. In ECs, miR-16-5p, miR-451, miR-191-5p, miR-27a-3p, and miR-27b-3p, in that order, were the top detectable miRNAs in ECs. miRNA-target enrichment analysis of the top 10 detected common EV and EC miRNAs identified MYC and TNPO1 as top target genes, respectively. Functional enrichment analysis of top EV- and EC-associated miRNAs identified common and distinct gene-network signatures associated with various biological and disease processes. Top EV-associated miRNAs were implicated in cytokine–cytokine receptor interactions, Th17 cell differentiation, IL-17 signaling, inflammatory bowel disease, and glioma. On the other hand, top EC-associated miRNAs were implicated in lipid and atherosclerosis, Th1 and Th2 cell differentiation, Th17 cell differentiation, and glioma. Interestingly, infection of RMs with SIV revealed that the brain-enriched miR-128-3p was longitudinally and significantly downregulated in EVs, but not ECs. This SIV-mediated decrease in miR-128-3p counts was validated by specific TaqMan microRNA stem-loop RT-qPCR assay. Remarkably, the observed SIV-mediated decrease in miR-128-3p levels in EVs from RMs agrees with publicly available EV miRNAome data by Kaddour et al., 2021, which showed that miR-128-3p levels were significantly lower in semen-derived EVs from HIV-infected men who used or did not use cocaine compared to HIV-uninfected individuals. These findings confirmed our previously reported finding and suggested that miR-128 may be a target of HIV/SIV. Conclusions: In the present study, we used sRNA sequencing to provide a holistic understanding of the repertoire of circulating exmiRNAs and their association with extracellular particles, such as EVs and ECs. Our data also showed that SIV infection altered the profile of the miRNAome of EVs and revealed that miR-128-3p may be a potential target of HIV/SIV. The significant decrease in miR-128-3p in HIV-infected humans and in SIV-infected RMs may indicate disease progression. Our study has important implications for the development of biomarker approaches for various types of cancer, cardiovascular diseases, organ injury, and HIV based on the capture and analysis of circulating exmiRNAs. Full article

The neurological damage of Alzheimer’s disease (AD) is thought to be irreversible upon onset of dementia-like symptoms, as it takes years to decades for occult pathologic changes to become symptomatic. It is thus necessary to identify individuals at risk for the development of the disease before symptoms manifest in order to provide early intervention. Surrogate markers are critical for early disease detection, stratification of patients in clinical trials, prediction of disease progression, evaluation of response to treatment, and also insight into pathomechanisms. Here, we review the evidence for a number of microRNAs that may serve as biomarkers with possible mechanistic insights into the AD pathophysiologic processes, years before the clinical manifestation of the disease. Full article

Recent evidence suggests that testosterone deficiency can dramatically decrease the quality of sperm. MicroRNAs (miRNAs) are conserved mediators of post-transcriptional gene regulation in eukaryotes. However, the systemic regulation and function of miRNAs in sperm quality decline induced by testosterone deficiency has not been investigated. Here, we found that the sperm apoptosis was significantly enhanced and the sperm motility was dramatically decreased in hemicastrated pigs. We then used small RNA sequencing to detect miRNA profiles of sperm from pigs with prepubertal hemicastration (HC) and compared them with control libraries. We identified 16 differentially expressed (DE) miRNAs between the sperm of prepubertal HC and control (CT) pigs. Functional enrichment analysis indicated that the target genes of these DE miRNAs were mainly enriched in apoptosis-related pathways including the p53, mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin (mTOR) pathways. Furthermore, gain- and loss-of-function analyses demonstrated potential anti-apoptotic effects of the DE miRNAs miR-26a-5p and let-7g-5p on sperm cells. The luciferase reporter assay confirmed that PTEN and PMAIP1 are targets of miR-26a-5p and let-7g-5p, respectively. Spearman’s correlation analysis revealed significantly positive correlations between the sperm and its corresponding seminal plasma exosomes regarding the miRNA expression levels. In conclusion, testosterone deficiency-induced changes in the miRNA components of seminal plasma exosomes secreted by the genital tract may partially elucidate sperm miRNAome alterations, which are further responsible for the decline of sperm motility. Full article