Mass spectrometry-based proteomic platforms for better understanding of SARS-CoV-2 induced pathogenesis and potential diagnostic approaches.

While protein-protein interaction is the first step of the SARS-CoV-2 infection, recent comparative proteomic profiling enabled the identification of over 11,000 protein dynamics, thus providing a comprehensive reflection of the molecular mechanisms underlying the cellular system in response to viral infection. Here we summarize and rationalize the results obtained by various mass spectrometry (MS)-based proteomic approaches applied to the functional characterization of proteins and pathways associated with SARS-CoV-2-mediated infections in humans. Comparative analysis of cell-lines versus tissue samples indicates that our knowledge in proteome profile alternation in response to SARS-CoV-2 infection is still incomplete and the tissue-specific response to SARS-CoV-2 infection can probably not be recapitulated efficiently by in vitro experiments. However, regardless of the viral infection period, sample types, and experimental strategies, a thorough cross-comparison of the recently published proteome, phosphoproteome, and interactome datasets led to the identification of a common set of proteins and kinases associated with PI3K-Akt, EGFR, MAPK, Rap1, and AMPK signaling pathways. Ephrin receptor A2 (EPHA2) was identified by 11 studies including all proteomic platforms, suggesting it as a potential future target for SARS-CoV-2 infection mechanisms and the development of new therapeutic strategies. We further discuss the potentials of future proteomics strategies for identifying prognostic SARS-CoV-2 responsive age-, gender-dependent, tissue-specific protein targets.

Potential biomarkers to detect traumatic brain injury by the profiling of salivary extracellular vesicles.

Traumatic brain injury (TBI) is a common cause of death and acquired disability in adults and children. Identifying biomarkers for mild TBI (mTBI) that can predict functional impairments on neuropsychiatric and neurocognitive testing after head trauma is yet to be firmly established. Extracellular vesicles (EVs) are known to traffic from the brain to the oral cavity and can be detected in saliva. We hypothesize the genetic profile of salivary EVs in patients who have suffered head trauma will differ from normal healthy controls, thus constituting a unique expression signature for mTBI. We enrolled a total of 54 subjects including for saliva sampling, 23 controls with no history of head traumas, 16 patients enrolled from an outpatient concussion clinic, and 15 patients from the emergency department who had sustained a head trauma within 24 hr. We performed real-time PCR of the salivary EVs of the 54 subjects profiling 96 genes from the TaqMan Human Alzheimer's disease array. Real-time PCR analysis revealed 57 (15 genes, p < 0.05) upregulated genes in emergency department patients and 56 (14 genes, p < 0.05) upregulated genes in concussion clinic patients when compared with controls. Three genes were upregulated in both the emergency department patients and concussion clinic patients: CDC2, CSNK1A1, and CTSD ( p < 0.05). Our results demonstrate that salivary EVs gene expression can serve as a viable source of biomarkers for mTBI. This study shows multiple Alzheimer's disease genes present after an mTBI.

HIV Infection, HCV Coinfection, and Alcohol Use: Associations with Microbial Translocation and Immune Activation.

BACKGROUND: Human immunodeficiency virus (HIV) infection and heavy drinking independently promote microbial translocation and inflammation. However, it is not known how alcohol use may affect these processes in people living with HIV (PLWH). This study tested the hypothesis that alcohol exacerbates innate immune dysfunction in PLWH. METHODS: Participants were 75 PLWH and 34 uninfected controls. Groups were recruited to have similar proportions of nondrinkers, moderate drinkers, and heavy drinkers. Substance use data and plasma samples were collected at up to 3 visits over a 5-year study period. Recent alcohol use was assessed with the Timeline Followback Interview. Biomarkers of microbial translocation (lipopolysaccharide, LPS) and immune activation (lipopolysaccharide binding protein, LBP; soluble CD14, sCD14; soluble CD163, sCD163) were quantified using enzyme-linked immunosorbent assays. Analyses tested 2 hypotheses: (i) that biomarker levels would be significantly higher in PLWH than controls with comparable alcohol use and (ii) that current alcohol use would exacerbate biomarker elevations in PLWH. The second analysis included the interaction of alcohol use with hepatitis C virus (HCV) coinfection. RESULTS: Groups were matched on alcohol use, smoking, and other drug use. All biomarkers were significantly higher in PLWH relative to controls (LBP: p = 0.005; LPS: p = 0.014; sCD14: p < 0.001; sCD163: p < 0.001). In PLWH, alcohol use showed a significant, positive association with sCD163, but not with other biomarkers. However, the interaction of alcohol use with HCV coinfection was significant for all biomarkers (LBP: p = 0.002; LPS: p = 0.026; sCD14: p = 0.0004; sCD163: p = 0.001). In pairwise tests with sequential Bonferroni correction, HIV/HCV coinfected individuals who drank heavily had significantly higher sCD163 compared to coinfected nondrinkers and to HIV monoinfected nondrinkers, moderate drinkers, and heavy drinkers (ps < 0.005). Coinfected moderate drinkers had significantly higher sCD163 than each monoinfected group (ps < 0.003). In addition, sCD14 was significantly higher in coinfected moderate drinkers than coinfected nondrinkers (p = 0.027). CONCLUSIONS: As predicted, PLWH had higher levels of LBP, LPS, sCD14, and sCD163 than uninfected individuals with similar alcohol use. In PLWH, alcohol by itself was significantly associated only with higher sCD163. However, heavy or moderate alcohol use was associated with elevations in macrophage activation (sCD163) and monocyte activation (sCD14) in HIV/HCV coinfected individuals.

Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- and tissue-specific microRNAs.

Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤ 0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stem-loop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage- and/or tissue-specific miRNAs that are uncharacterized.

Lactobacillus-derived extracellular vesicles enhance host immune responses against vancomycin-resistant enterococci.

BACKGROUND: Probiotic bacteria are known to modulate host immune responses against various pathogens. Recently, extracellular vesicles (EVs) have emerged as potentially important mediators of host-pathogen interactions. In this study, we explored the role of L. plantarum derived EVs in modulating host responses to vancomycin-resistant Enterococcus faecium (VRE) using both Caenorhabditis elegans and human cells. RESULTS: Our previous work has shown that probiotic conditioning C. elegans with L. acidophilus NCFM prolongs the survival of nematodes exposed to VRE. Similarly, L. plantarum WCFS1 derived extracellular vesicles (LDEVs) also significantly protected the worms against VRE infection. To dissect the molecular mechanisms of this EV-induced protection, we found that treatment of C. elegans with LDEVs significantly increased the transcription of host defense genes, cpr-1 and clec-60. Both cpr-1 and clec-60 have been previously reported to have protective roles against bacterial infections. Incubating human colon-derived Caco-2 cells with fluorescent dye-labeled LDEVs confirmed that LDEVs could be transported into the mammalian cells. Furthermore, LDEV uptake was associated with significant upregulation of CTSB, a human homologous gene of cpr-1, and REG3G, a human gene that has similar functions to clec-60. CONCLUSIONS: We have found that EVs produced from L. plantarum WCFS1 up-regulate the expression of host defense genes and provide protective effects on hosts. Using probiotic-derived EVs instead of probiotic bacteria themselves, this study provides a new direction to treat antimicrobial resistant pathogens, such as VRE.

The impact of pregnancy on anti-HIV activity of cervicovaginal secretions.

BACKGROUND: Mucosal immunity of the female genital tract plays a critical role in defense against sexually transmitted infections like HIV. Pregnancy is associated with both structural and immunologic alterations in the genital mucosa, but the impact of these changes on its ability to suppress HIV infection is unknown. Current epidemiologic data are conflicting as to whether pregnancy increases the risk of HIV acquisition. OBJECTIVE: The purpose of this study was to define the association between antimicrobial peptides and chemokines in cervicovaginal secretions and in vitro HIV infectivity among pregnant and nonpregnant women. STUDY DESIGN: Forty pregnant and 37 nonpregnant women were enrolled in a prospective longitudinal cohort study at a single tertiary care women's hospital in Providence, RI. Cervicovaginal lavage was performed at each study visit. For pregnant women, study visits occurred once per trimester, and there was an optional postpartum visit. For nonpregnant women, study visits occurred across a single cycle that was timed to occur in the proliferative, ovulatory, and secretory phases based on the presumption of a regular menstrual cycle. The impact of cervicovaginal lavage on HIV infectivity was evaluated using a TZM-bl assay and compared between pregnant and nonpregnant women for each visit. The previously validated TZM-bl assay, which uses a luciferase reporting gene to indicate HIV infection of TZM-bl cells, was measured with a luminometer with higher relative light units that indicate greater levels of in vitro HIV infection. Immune mediators were measured with a multiplex bead assay. HIV infectivity and median concentration of each mediator were compared between pregnant and nonpregnant groups with the Wilcoxon rank sum test. RESULTS: Cervicovaginal fluid from pregnant and nonpregnant women significantly decreased HIV infectivity in both groups compared with positive control (virus only; P<.01), but infectivity was not different between groups (P≥.44). During the second and third trimesters, pregnant women experienced suppression of several cervicovaginal immune mediators that included human beta defensin-2; lactoferrin; macrophage inflammatory protein-3α; regulated on activation, normally T-cell expressed and secreted; and stromal cell-derived factor-1 (all P≤.05). The antimicrobial peptide elafin was significantly correlated with HIV infectivity in both groups across all visits, except at the postpartum visit in the pregnant group (n=16). Secretory leukocyte protease inhibitor also was correlated significantly with infectivity across all visits, but in nonpregnant women only (P≤.03). CONCLUSION: Cervicovaginal secretions from both pregnant and nonpregnant women contain immune mediators that are associated with HIV infectivity in an in vitro assay; however, infectivity was not different between pregnant and nonpregnant groups. If pregnant women are at increased risk for HIV infection, it is unlikely to be mediated by alterations in the effectiveness of these protective secretions.

Alcohol use predicts elevation in inflammatory marker soluble CD14 in men living with HIV.

Independently, HIV infection and heavy alcohol use increase microbial translocation (MT) of gut products into systemic circulation. MT and consequent immune response have been linked to chronic inflammation and a host of negative health outcomes in individuals living with HIV. However, previous research has not systematically investigated the immune correlates of heavy drinking specifically within the HIV-positive population. This pilot study investigated MT and immune activation as a function of alcohol use in 21 HIV-positive men who met NIAAA criteria for heavy drinking. Participants averaged 46.7 ± 8.5 (mean ± standard deviation) years of age, 12.2 ± 9.2 years since HIV diagnosis, 337 ± 158 CD4 nadir, and 643 ± 245 current CD4 count. All participants were virologically suppressed on antiretroviral therapy. Data on alcohol use and immune function were collected at baseline and three-month follow-up. Plasma concentrations of markers of MT and immune activation (lipopolysaccharide (LPS), soluble CD14 (sCD14), endotoxin core antibody immunoglobulin M (EndoCAb)) were measured using enzyme-linked immunosorbent assays. Generalized estimating equation models tested alcohol use variables as predictors of LPS, sCD14, and EndoCAb levels. Greater quantity and frequency of drinking significantly predicted higher sCD14 levels (p's < .01). Conversely, longer duration of abstinence from alcohol significantly predicted lower sCD14 levels (p < .001). These results remained significant after controlling for age, HIV duration, smoking status, current CD4 count, CD4 nadir, and antiretroviral drug type. In addition, participants with ≥50% relative reduction in drinks per week showed a significant decrease (p < .05) in sCD14 from baseline to three-month follow-up. This pilot study provides preliminary evidence that heavy drinking may increase a key inflammatory marker in HIV-infected individuals with suppressed infection.

Glycogen synthase kinase 3 beta inhibits microRNA-183-96-182 cluster via the ß-Catenin/TCF/LEF-1 pathway in gastric cancer cells.

Glycogen synthase kinase 3 beta (GSK3ß) is a critical protein kinase that phosphorylates numerous proteins in cells and thereby impacts multiple pathways including the ß-Catenin/TCF/LEF-1 pathway. MicroRNAs (miRs) are a class of noncoding small RNAs of ∼22 nucleotides in length. Both GSK3ß and miR play myriad roles in cell functions including stem cell development, apoptosis, embryogenesis and tumorigenesis. Here we show that GSK3ß inhibits the expression of miR-96, miR-182 and miR-183 through the ß-Catenin/TCF/LEF-1 pathway. Knockout of GSK3ß in mouse embryonic fibroblast cells increases expression of miR-96, miR-182 and miR-183, coinciding with increases in the protein level and nuclear translocation of ß-Catenin. In addition, overexpression of ß-Catenin enhances the expression of miR-96, miR-182 and miR-183 in human gastric cancer AGS cells. GSK3ß protein levels are decreased in human gastric cancer tissue compared with surrounding normal gastric tissue, coinciding with increases of ß-Catenin protein, miR-96, miR-182, miR-183 and primary miR-183-96-182 cluster (pri-miR-183). Furthermore, suppression of miR-183-96-182 cluster with miRCURY LNA miR inhibitors decreases the proliferation and migration of AGS cells. Knockdown of GSK3ß with siRNA increases the proliferation of AGS cells. Mechanistically, we show that ß-Catenin/TCF/LEF-1 binds to the promoter of miR-183-96-182 cluster gene and thereby activates the transcription of the cluster. In summary, our findings identify a novel role for GSK3ß in the regulation of miR-183-96-182 biogenesis through ß-Catenin/TCF/LEF-1 pathway in gastric cancer cells.

Quantitative proteomic analysis of exosomes from HIV-1-infected lymphocytic cells.

HIV-1 infection causes profound effects both inside and outside of cells through multiple mechanisms, including those mediated by exosomes. Using the technique of stable isotope labeling by amino acids in cell culture, we compared protein expression patterns in the exosomal compartment of HIV-1-infected and -uninfected lymphocytic H9 cells. Of 770 proteins identified in two independent sets of exosomal samples, 14 proteins were found to be differentially expressed in the exosomal fraction of HIV-1-infected cells versus -uninfected controls. Gene Ontology survey and DAVID analysis revealed that identified proteins were enriched for functional categories such as binding. Of these 14 proteins, three immunomodulatory molecules were reproducibly identified in both replicates and included ADP-ribosyl cyclase 1 (CD38), L-lactate dehydrogenase B chain (LDHB), and Annexin A5 (ANXA5). In addition to previously reported HIV-1 associations with CD38 and LDHB, new interactions were identified and validated for ANXA5, CD38, and LDHB, which were found to bind to HIV-1 p24 and Tat. In summary, our studies reveal that exosomes released from HIV-1-infected cells are composed of a unique and quantitatively different protein signature and harbor regulatory molecules that impact the processes of cellular apoptosis (ANXA5 and LDHB) and proliferation (CD38).

A new stem cell biology: the continuum and microvesicles.

The hierarchical models of stem cell biology have been based on work first demonstrating pluripotental spleen-colony-forming units, then showing progenitors with many differentiation fates assayed in in vitro culture; there followed the definition and separation of "stem cells" using monoclonal antibodies to surface epitopes and fluorescent-activated cell characterization and sorting (FACS). These studies led to an elegant model of stem cell biology in which primitive dormant G0 stem cells with tremendous proliferative and differentiative potential gave rise to progressively more restricted and differentiated classes of stem/progenitor cells, and finally differentiated marrow hematopoietic cells. The holy grail of hematopoietic stem cell biology became the purification of the stem cell and the clonal definition of this cell. Most recently, the long-term repopulating hematopoietic stem cell (LT-HSC) has been believed to be a lineage negative sca-1+C-kit+ Flk3- and CD150+ cell. However, a series of studies over the past 10 years has indicated that murine marrow stem cells continuously change phenotype with cell cycle passage. We present here studies using tritiated thymidine suicide and pyronin-Hoechst FACS separations indicating that the murine hematopoietic stem cell is a cycling cell. This would indicate that the hematopoietic stem cell must be continuously changing in phenotype and, thus, could not be purified. The extant data indicate that murine marrow stem cells are continually transiting cell cycle and that the purification has discarded these cycling cells. Further in vivo BrdU studies indicate that the "quiescent" LT-HSC in G0 rapidly transits cycle. Further complexity of the marrow stem cell system is indicated by studies on cell-derived microvesicles showing that they enter marrow cells and transcriptionally alter their cell fate and phenotype. Thus, the stem cell model is a model of continuing changing potential tied to cell cycle and microvesicle exposure. The challenge of the future is to define the stem cell population, not purify the stem cell. We are at the beginning of elucidation of quantum stemomics.

Phosphorylation of the RNase III enzyme Drosha at Serine300 or Serine302 is required for its nuclear localization.

The RNaseIII enzyme Drosha plays a pivotal role in microRNA (miRNA) biogenesis by cleaving primary miRNA transcripts to generate precursor miRNA in the nucleus. The RNA binding and enzymatic domains of Drosha have been characterized and are on its C-terminus. Its N-terminus harbors a nuclear localization signal. Using a series of truncated Drosha constructs, we narrowed down the segment responsible for nuclear translocation to a domain between aa 270 and aa 390. We further identified two phosphorylation sites at Serine300 (S300) and Serine302 (S302) by mass spectrometric analysis. Double mutations of S→A at S300 and S302 completely disrupted nuclear localization. Single mutation of S→A at S300 or S302, however, had no effect on nuclear localization indicating that phosphorylation at either site is sufficient to locate Drosha to the nucleus. Furthermore, mimicking phosphorylation status by mutating S→E at S300 and/or S→D at S302 restored nuclear localization. Our findings add a further layer of complexity to the molecular anatomy of Drosha as it relates to miRNA biogenesis.

Atypical transcription of microRNA gene fragments.

MicroRNAs (miRNAs) are short ( approximately 22 nt) RNAs that impact gene expression by sequence-specific interactions with messenger RNA or promoter sequences of genomic DNA. Ectopic expression of miRNAs can be accomplished by placing fragments of the corresponding miRNA precursor under the control of RNA polymerase II or III (RNAP II/III). Here, we report that, in the absence of exogenous promoters, DNA fragments incorporating miRNA precursors can be delivered directly into a variety of human cells and give rise to the corresponding mature miRNA. Notably, the transcription of these miRNA DNA fragments appears resistant to conventional inhibitors of RNAP I/II/III activity. Taken together, our findings suggest the existence of a previously unrecognized atypical transcription program for miRNA precursor sequences.

Intercellular transfer of proteins as identified by stable isotope labeling of amino acids in cell culture.

We tracked the extracellular fate of proteins of pulmonary origin using the technique of stable isotope labeling of amino acids in cell culture (SILAC) in cell-impermeable Transwell culture systems. We find that irradiation to murine lung and lung-derived cells induces their release of proteins that are capable of entering neighboring cells, including primary murine bone marrow cells as well as prostate cancer and hematopoietic cell lines. The functional classification of transferred proteins was broad and included transcription factors, mediators of basic cellular processes and components of the nucleosome remodeling and deacetylase complex, including metastasis associated protein 3 and retinoblastoma-binding protein 7. In further analysis we find that retinoblastoma-binding protein 7 is a transcriptional activator of E-cadherin and that its intercellular transfer leads to decreased gene expression of downstream targets such as N-cadherin and vimentin. SILAC-generated data sets offer a valuable tool to identify and validate potential paracrine networks that may impact relevant biologic processes associated with phenotypic and genotypic signatures of health and disease.

Neurotoxicity of HIV-1 Tat is attributed to its penetrating property.

We have recently engineered an exosomal Tat (Exo-Tat) which can activate latent HIV-1 in resting CD4+ T lymphocytes from antiretroviral treated HIV-1 infected patients. HIV-1 Tat protein can penetrate cell membrane freely and secrete into extracellular medium. Exo-Tat loses this penetrating property. HIV-1 Tat protein can damage the synaptic membranes contributing to the development of dementia in HIV-1 infected patients. To investigate whether the penetrating property attributes to synaptic damage in vivo, we have generated adeno-associated viruses AAV-Tat and AAV-Exo-Tat viruses. Vehicle control or AAV viruses (1 × 1012 GC/mouse in 200 µl PBS) were injected into Balb/cj mice via tail veins. The mRNA and protein expression levels in blood, brain, heart, intestine, kidney, liver, lung, muscle and spleen were determined on day 21. Intravenously injected AAV-Tat or AAV-Exo-Tat mainly infects liver and heart. Short-term expression of Tat or Exo-Tat doesn't change the expression levels of neuronal cytoskeletal marker ß3-tubulin and synaptic marker postsynaptic density 95 protein (PSD-95). Wild-type Tat, but not Exo-Tat, reduces the expression level of synaptic marker synaptophysin significantly in mice, indicating that penetrating property of HIV-1 Tat protein attributes to synaptic damage.

Depot medroxyprogesterone acetate administration increases cervical CCR5+CD4+ T cells and induces immunosuppressive milieu at the cervicovaginal mucosa.

OBJECTIVES: Depot medroxyprogesterone acetate (Depo-Provera) is the most commonly used injectable hormone contraceptive in Sub-Saharan Africa where HIV incidence is high. We determined the impact of Depo-Provera on cervical immune cells and mediators in healthy women. METHODS: In this longitudinal study, vaginal, endocervical, and rectal swabs were collected at baseline (visit 1), 1 month (visit 2), and 3 months (visit 3) after Depo-Provera injection. Cervical cells were collected by cytobrush and immune markers on cervical CD4 T cells were analyzed by multicolor flow cytometry at three different visits. The levels of immune mediators in cytobrush supernatants as well as vaginal, cervical, and rectal secretions from swabs were analyzed by multiplex assays and ELISA. RESULTS: Compared with baseline levels, we found a significant increase in the frequency of cervical CCR5CD4 T cells and a significant decrease in the frequency of cervical central memory CD4 T cells. Depo-Provera treatment had little effect on expression of immune mediators in rectal mucosa but significantly suppressed numerous immune mediators at cervicovaginal mucosa. Levels of MCP-1, G-CSF, IL-6, IL-10, GM-CSF, and IP-10 were significantly decreased in both vaginal and cervical secretions after Depo-Provera injection. In cervical samples collected by cytobrush, we found reduced levels of 22 of 25 immune mediators after Depo-Provera injection. Changes in immune mediators differed between vaginal and cervical mucosa, demonstrating compartment-specific responses. CONCLUSION: Depo-Provera altered immune profiles of cervical CD4 T cells and suppressed host immune response at cervicovaginal mucosa, suggesting its likely effect on transmission of sexually transmitted infections including HIV.

Inflammation-related gene expression profiles of salivary extracellular vesicles in patients with head trauma.

At present, there is no reliable biomarker for the diagnosis of traumatic brain injury (TBI). Studies have shown that extracellular vesicles released by damaged cells into biological fluids can be used as potential biomarkers for diagnosis of TBI and evaluation of TBI severity. We hypothesize that the genetic profile of salivary extracellular vesicles in patients with head trauma differs from that in uninjured subjects. Findings from this hypothesis would help investigate the severity of TBI. This study included 19 subjects, consisting of seven healthy controls who denied history of head trauma, six patients diagnosed with concussion injury from an outpatient concussion clinic, and six patients with TBI who received treatment in the emergency department within 24 hours after injury. Real-time PCR analysis of salivary extracellular vesicles in participants was performed using TaqMan Human Inflammation array. Gene expression analysis revealed nine upregulated genes in emergency department patients (LOX5, ANXA3, CASP1, IL2RG, ITGAM, ITGB2, LTA4H, MAPK14, and TNFRSF1A) and 13 upregulated genes in concussion clinic patients compared with healthy participants (ADRB1, ADRB2, BDKRB1, HRH1, HRH2, LTB4R2, LTB4R, PTAFR, CYSLTR1, CES1, KLK1, MC2R, and PTGER3). Each patient group had a unique profile. Comparison between groups showed that 15 inflammation-related genes had significant expression change. Our results indicate that inflammation biomarkers can be used for diagnosis of TBI and evaluation of disease severity. This study was approved by the Institutional Review Board on December 18, 2015 (approval No. 0078-12) and on June 9, 2016 (approval No. 4093-16).

SARS-CoV-2 and HIV coinfection: clinical experience from Rhode Island, United States.

INTRODUCTION: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has infected >6 million people worldwide since December 2019. Global reports of HIV/SARS-CoV-2 coinfection are limited. To better understand the impact of the coronavirus disease 2019 (COVID-19) pandemic on persons with HIV and improve their care, we present an outpatient and inpatient clinical experience of HIV/SARS-CoV-2 coinfection from Rhode Island, US. METHODS: We describe outpatient and inpatient preparedness for the COVID-19 pandemic, and present a case series of all known patients with HIV/SARS-CoV-2 coinfection at The Miriam Hospital and Rhode Island Hospital, and The Miriam Hospital Infectious Diseases and Immunology Center, in Providence, Rhode Island, US. RESULTS AND DISCUSSION: The Infectious Diseases and Immunology Center rapidly prepared for outpatient and inpatient care of persons with HIV and SARS-CoV-2. Between 30 March and 20 May 2020, 27 patients with HIV were diagnosed with SARS-CoV-2. Twenty were male, six female and one transgender female; average age was 49 years; 13/27 were Hispanic and 6/27 were African American. All had HIV viral load <200 copies/mL and were on antiretroviral therapy with CD4 count range 87 to 1441 cells/µL. Twenty-six of the 27 had common COVID-19 symptoms for one to twenty-eight days and most had other co-morbidities and/or risk factors. Nine of the 27 were hospitalized for one to thirteen days; of those, three lived in a nursing home, six received remdesivir through a clinical trial or emergency use authorization and tolerated it well; eight recovered and one died. Overall, 17% of known Center people had HIV/SARS-CoV-2 coinfection, whereas the comparable state-wide prevalence was 9%. CONCLUSIONS: We highlight challenges of outpatient and inpatient HIV care in the setting of the COVID-19 pandemic and present the largest detailed case series to date from the United States on HIV/SARS-CoV-2 coinfection, adding to limited global reports. The aggregated clinical findings suggest that the clinical presentation and outcomes of COVID-19 appear consistent with those without HIV. Whether SARS-CoV-2 infection is more frequent among persons with HIV remains to be determined. More data are needed as we develop our understanding of how HIV and antiretroviral therapy are affected by or have an impact on this pandemic.

Genotypic HIV type-1 drug resistance among patients with immunological failure to first-line antiretroviral therapy in south India.

BACKGROUND: HIV type-1 (HIV-1) monitoring in resource-limited settings relies on clinical and immunological assessment. The objective of this study was to study the frequency and pattern of reverse transcriptase (RT) drug resistance among patients with immunological failure (IF) to first-line therapy. METHODS: A cross-sectional study of 228 patients with IF was done, of which 126 were drug-naive (group A) when starting highly active antiretroviral therapy (HAART) and 102 were exposed to mono/dual therapy prior to HAART initiation (group B). A validated in-house genotyping method and Stanford interpretation was used. Means, sd, median and frequencies (as percentages) were used to indicate the patient characteristics in each group. The chi(2) test and Fisher's exact test were used to compare categorical variables as appropriate. All analyses were performed using SPSS software, version 13.0. P-values <0.05 were considered to be statistically significant. RESULTS: RT drug resistance mutations were found in 92% and 96% of patients in groups A and B, respectively. Median (interquartile range) CD4(+) T-cell count at failure was 181 cells/microl (18-999) and time to failure was 40 months (2-100). M184V (80% versus 75%), thymidine analogue mutations (63% versus 74%), Y181C (39% versus 39%) and K103N (29% versus 39%) were predominant RT mutations in both groups. Extensive nucleoside reverse transcriptase inhibitor cross-resistance mutations were observed in 51% and 26% of patients in group B and A, respectively. CONCLUSIONS: Alternative strategies for initial therapy and affordable viral load monitoring could reduce resistance accumulations and preserve available drugs for future options in resource-limited settings.

Impact of exosomal HIV-1 Tat expression on the human cellular proteome.

HIV-1 exists in a latent form in all infected patients. When antiretroviral therapy is stopped, viral replication resumes. The HIV-1 Tat protein is a potent activator of viral transcription. Our previous work has demonstrated that exosomal formulations of Tat can reverse HIV-1 latency in primary CD4+ T lymphocytes isolated from long term antiretroviral treated individuals suggesting a potential role for Tat as a therapeutic HIV-1 Latency Reversal Agent (LRA). Here, we employed the label-free proteomic approach for profiling the proteomic changes associated with exosomal Tat production in human cell lines. Comparative proteomic analysis revealed that >30% peptides were differentially expressed in abundance in the Tat-expressing cell line compared with relevant controls. As expected, many of the known Tat-interactor proteins were upregulated. Tat expression also led to the upregulation of antioxidant proteins suggesting Tat-mediates an oxidative burst. Gene ontology and pathway analyses of these differentially expressed proteins showed enrichment of extracellular vesicular exosome and spliceosome localized proteins and proteins involved with transcriptional and translational mechanisms. Our work suggests that HIV-1 Tat expression leads to perturbations in cellular protein expression. In vivo administration of Tat using HIV/SIV animal models needs to be performed to assess the physiologic significance of Tat-induced proteomic changes prior to developing HIV-1 Tat as an LRA.

Proteomic Analysis of Exosomes and Its Application in HIV-1 Infection.

Exosomes are 30-100 nm extracellular vesicles secreted from late endosomes by various types of cells. Numerous studies have suggested that exosomes play significant roles in human immunodeficiency virus 1 (HIV-1) biogenesis. Proteomics coupled with exosome fractionation has been successfully used to identify various exosomal proteins and helped to uncover the interactions between exosomes and HIV-1. To inform the current progress in the intersection of exosome, proteomics, and HIV-1, this review is focused on: i) analyzing different exosome isolation, purification methods, and their implications in HIV-1 studies; ii) evaluating the roles of various proteomic techniques in defining exosomal contents; iii) discussing the research and clinical applications of proteomics and exosome in HIV-1 biology.