Alterations in Abundance and Compartmentalization of miRNAs in Blood Plasma Extracellular Vesicles and Extracellular Condensates during HIV/SIV Infection and Its Modulation by Antiretroviral Therapy (ART) and Delta-9-Tetrahydrocannabinol (Δ9-THC).

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.

SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128.

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.

A Malaria Parasite Cross Reveals Genetic Determinants of Plasmodium falciparum Growth in Different Culture Media.

What genes determine in vitro growth and nutrient utilization in asexual blood-stage malaria parasites? Competition experiments between NF54, clone 3D7, a lab-adapted African parasite, and a recently isolated Asian parasite (NHP4026) reveal contrasting outcomes in different media: 3D7 outcompetes NHP4026 in media containing human serum, while NHP4026 outcompetes 3D7 in media containing AlbuMAX, a commercial lipid-rich bovine serum formulation. To determine the basis for this polymorphism, we conducted parasite genetic crosses using humanized mice and compared genome-wide allele frequency changes in three independent progeny populations cultured in media containing human serum or AlbuMAX. This bulk segregant analysis detected three quantitative trait loci (QTL) regions [on chromosome (chr) 2 containing aspartate transaminase AST; chr 13 containing EBA-140; and chr 14 containing cysteine protease ATG4] linked with differential growth in serum or AlbuMAX in each of the three independent progeny pools. Selection driving differential growth was strong (s = 0.10 - 0.23 per 48-hour lifecycle). We conducted validation experiments for the strongest QTL on chr 13: competition experiments between ΔEBA-140 and 3D7 wildtype parasites showed fitness reversals in the two medium types as seen in the parental parasites, validating this locus as the causative gene. These results (i) demonstrate the effectiveness of bulk segregant analysis for dissecting fitness traits in P. falciparum genetic crosses, and (ii) reveal intimate links between red blood cell invasion and nutrient composition of growth media. Use of parasite crosses combined with bulk segregant analysis will allow systematic dissection of key nutrient acquisition/metabolism and red blood cell invasion pathways in P. falciparum.

Genetic analysis of praziquantel response in schistosome parasites implicates a transient receptor potential channel.

Mass drug administration with praziquantel (PZQ) monotherapy is considered the mainstay for control and elimination of the parasites causing schistosomiasis in humans. This drug shows imperfect cure rates in the field, and parasites showing reduced PZQ response can be selected in the laboratory, but the extent of resistance in Schistosoma mansoni populations is unknown. We examined the genetic basis of the variation in response in a PZQ-selected S. mansoni population (SmLE-PZQ-R) in which 35% of the parasitic worms survive high-dose PZQ (73 micrograms per milliliter) treatment. We used genome-wide association to map loci underlying PZQ response and identified a transient receptor potential (Sm.TRPMPZQ) channel (Smp_246790) within the major chromosome 3 peak that is activated by nanomolar concentrations of PZQ. The PZQ response showed recessive inheritance and marker-assisted selection of parasites at a single Sm.TRPMPZQ SNP that produced populations of PZQ-enriched resistant (PZQ-ER) and PZQ-enriched sensitive (PZQ-ES) parasites, exhibiting >377-fold difference in PZQ response. The PZQ-ER parasites survived treatment in rodents at higher frequencies compared with PZQ-ES, and resistant parasites exhibited 2.25-fold lower expression of Sm.TRPMPZQ relative to sensitive parasites. Specific chemical blockers of Sm.TRPMPZQ enhanced PZQ resistance, whereas Sm.TRPMPZQ activators increased sensitivity. We surveyed Sm.TRPMPZQ sequence variations in 259 parasites from different global sites and identified one nonsense mutation that resulted in a truncated protein with no PZQ binding site. Our results demonstrate that Sm.TRPMPZQ underlies variation in PZQ responses in S. mansoni and provides an approach for monitoring emerging PZQ-resistant alleles in schistosome elimination programs.

The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput.

BACKGROUND: Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations. METHODS: Plasmodium falciparum cultures were synchronized with single layer Percoll during the schizont stage of the intraerythrocytic development cycle. Cultures were left to reinvade to early ring-stage and parasitaemia was quantified using flow cytometry. Cultures were diluted to 2% haematocrit and 0.5% parasitaemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700 nM of dihydroartemisinin or 0.02% dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples. RESULTS: 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of - 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date. CONCLUSIONS: eRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.