The association between single-nucleotide polymorphisms within type 1 interferon pathway genes and human immunodeficiency virus type 1 viral load in antiretroviral-naïve participants.

BACKGROUND: Human genetic contribution to HIV progression remains inadequately explained. The type 1 interferon (IFN) pathway is important for host control of HIV and variation in type 1 IFN genes may contribute to disease progression. This study assessed the impact of variations at the gene and pathway level of type 1 IFN on HIV-1 viral load (VL). METHODS: Two cohorts of antiretroviral (ART) naïve participants living with HIV (PLWH) with either early (START) or advanced infection (FIRST) were analysed separately. Type 1 IFN genes (n = 17) and receptor subunits (IFNAR1, IFNAR2) were examined for both cumulated type 1 IFN pathway analysis and individual gene analysis. SKAT-O was applied to detect associations between the genotype and HIV-1 study entry viral load (log10 transformed) as a proxy for set point VL; P-values were corrected using Bonferroni (P < 0.0025). RESULTS: The analyses among those with early infection included 2429 individuals from five continents. The median study entry HIV VL was 14,623 (IQR 3460-45100) copies/mL. Across 673 SNPs within 19 type 1 IFN genes, no significant association with study entry VL was detected. Conversely, examining individual genes in START showed a borderline significant association between IFNW1, and study entry VL (P = 0.0025). This significance remained after separate adjustments for age, CD4+ T-cell count, CD4+/CD8+ T-cell ratio and recent infection. When controlling for population structure using linear mixed effects models (LME), in addition to principal components used in the main model, this was no longer significant (p = 0.0244). In subgroup analyses stratified by geographical region, the association between IFNW1 and study entry VL was only observed among African participants, although, the association was not significant when controlling for population structure using LME. Of the 17 SNPs within the IFNW1 region, only rs79876898 (A > G) was associated with study entry VL (p = 0.0020, beta = 0.32; G associated with higher study entry VL than A) in single SNP association analyses. The findings were not reproduced in FIRST participants. CONCLUSION: Across 19 type 1 IFN genes, only IFNW1 was associated with HIV-1 study entry VL in a cohort of ART-naïve individuals in early stages of their infection, however, this was no longer significant in sensitivity analyses that controlled for population structures using LME.

Modeling and Analysis of HIV-1 Pol Polyprotein as a Case Study for Predicting Large Polyprotein Structures.

Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). HIV protease, reverse transcriptase, and integrase are targets of current drugs to treat the disease. However, anti-viral drug-resistant strains have emerged quickly due to the high mutation rate of the virus, leading to the demand for the development of new drugs. One attractive target is Gag-Pol polyprotein, which plays a key role in the life cycle of HIV. Recently, we found that a combination of M50I and V151I mutations in HIV-1 integrase can suppress virus release and inhibit the initiation of Gag-Pol autoprocessing and maturation without interfering with the dimerization of Gag-Pol. Additional mutations in integrase or RNase H domain in reverse transcriptase can compensate for the defect. However, the molecular mechanism is unknown. There is no tertiary structure of the full-length HIV-1 Pol protein available for further study. Therefore, we developed a workflow to predict the tertiary structure of HIV-1 NL4.3 Pol polyprotein. The modeled structure has comparable quality compared with the recently published partial HIV-1 Pol structure (PDB ID: 7SJX). Our HIV-1 NL4.3 Pol dimer model is the first full-length Pol tertiary structure. It can provide a structural platform for studying the autoprocessing mechanism of HIV-1 Pol and for developing new potent drugs. Moreover, the workflow can be used to predict other large protein structures that cannot be resolved via conventional experimental methods.

Transcriptionally-active "defective" HIV-1 proviruses and their association with immunological non-response to antiretroviral therapy.

A subset of antiretroviral therapy-treated persons with HIV, referred to as immunological non-responders (INRs), fails to normalize CD4+ T-cell numbers. In a case-control study involving 26 INRs (CD4<250 cells/µL) and 25 immunological responders (IRs, CD4≥250 cells/µL), we evaluated the potential contribution of transcriptionally-competent "defective" HIV-1 proviruses to poor CD4+ T-cell recovery. Compared to the responders, the INRs had higher levels of cell-associated HIV-RNA (p=0.034) and higher percentages of HLA-DR+CD4+ T-cells (p<0.001). While not encoding replication-competent viruses, the RNA transcripts frequently encoded HIV-1 Gag-p17 and Nef proteins. These transcripts and/or resulting proteins may activate pathway(s) leading to the immunological non-response phenotype.

Discovery of a Novel Intron in US10/US11/US12 of HSV-1 Strain 17.

Herpes Simplex Virus type 1 (HSV-1) infects humans and causes a variety of clinical manifestations. Many HSV-1 genomes have been sequenced with high-throughput sequencing technologies and the annotation of these genome sequences heavily relies on the known genes in reference strains. Consequently, the accuracy of reference strain annotation is critical for future research and treatment of HSV-1 infection. In this study, we analyzed RNA-Seq data of HSV-1 from NCBI databases and discovered a novel intron in the overlapping coding sequence (CDS) of US10 and US11, and the 3' UTR of US12 in strain 17, a commonly used HSV-1 reference strain. To comprehensively understand the shared US10/US11/US12 intron structure, we used US11 as a representative and surveyed all US11 gene sequences from the NCBI nt/nr database. A total of 193 high-quality US11 sequences were obtained, of which 186 sequences have a domain of uninterrupted tandemly repeated RXP (Arg-X-Pro) in the C-terminus half of the protein. In total, 97 of the 186 sequences encode US11 protein with the same length of the mature US11 in strain 17:26 of them have the same structure of US11 and can be spliced as in strain 17; 71 of them have transcripts that are the same as mature US11 mRNA in strain 17. In total, 76 US11 gene sequences have either canonical or known noncanonical intron border sequences and may be spliced like strain 17 and obtain mature US11 CDS with the same length. If not spliced, they will have extra RXP repeats. A tandemly repeated RXP domain was proposed to be essential for US11 to bind with RNA and other host factors. US10 protein sequences from the same strains have also been studied. The results of this study show that even a frequently used reference organism may have errors in widely used databases. This study provides accurate annotation of the US10, US11, and US12 gene structure, which will build a more solid foundation to study expression regulation of the function of these genes.

The C-Terminal Domain of RNase H and the C-Terminus Amino Acid Residue Regulate Virus Release and Autoprocessing of a Defective HIV-1 Possessing M50I and V151I Changes in Integrase.

Previously, we reported that an HIV-1 variant containing Met-to-Ile change at codon 50 and Val-to-Ile mutation at codon 151 of integrase (IN), HIV(IN:M50I/V151I), was an impaired virus. Despite the mutations being in IN, the virus release was significantly suppressed (p < 0.0001) and the initiation of autoprocessing was inhibited; the mechanism of the defect remains unknown. In the current study, we attempted to identify the critical domains or amino acid (aa) residue(s) that promote defects in HIV(IN:M50I/V151I), using a series of variants, including truncated or aa-substituted RNase H (RH) or IN. The results demonstrated that virus release and the initiation of autoprocessing were regulated by the C-terminal domains (CTDs) of RH and IN. Further studies illustrated that Asp at codon 109 of RH CTD and Asp at the C terminus of IN induces the defect. This result indicated that the CTDs of RH and IN in GagPol and particular aa positions in RH and IN regulated the virus release and the initiation of autoprocessing, and these sites could be potential targets for the development of new therapies.

The Association of Baseline Plasma SARS-CoV-2 Nucleocapsid Antigen Level and Outcomes in Patients Hospitalized With COVID-19.

BACKGROUND: Levels of plasma SARS-CoV-2 nucleocapsid (N) antigen may be an important biomarker in patients with COVID-19 and enhance our understanding of the pathogenesis of COVID-19. OBJECTIVE: To evaluate whether levels of plasma antigen can predict short-term clinical outcomes and identify clinical and viral factors associated with plasma antigen levels in hospitalized patients with SARS-CoV-2. DESIGN: Cross-sectional study of baseline plasma antigen level from 2540 participants enrolled in the TICO (Therapeutics for Inpatients With COVID-19) platform trial from August 2020 to November 2021, with additional data on day 5 outcome and time to discharge. SETTING: 114 centers in 10 countries. PARTICIPANTS: Adults hospitalized for acute SARS-CoV-2 infection with 12 days or less of symptoms. MEASUREMENTS: Baseline plasma viral N antigen level was measured at a central laboratory. Delta variant status was determined from baseline nasal swabs using reverse transcriptase polymerase chain reaction. Associations between baseline patient characteristics and viral factors and baseline plasma antigen levels were assessed using both unadjusted and multivariable modeling. Association between elevated baseline antigen level of 1000 ng/L or greater and outcomes, including worsening of ordinal pulmonary scale at day 5 and time to hospital discharge, were evaluated using logistic regression and Fine-Gray regression models, respectively. RESULTS: Plasma antigen was below the level of quantification in 5% of participants at enrollment, and 1000 ng/L or greater in 57%. Baseline pulmonary severity of illness was strongly associated with plasma antigen level, with mean plasma antigen level 3.10-fold higher among those requiring noninvasive ventilation or high-flow nasal cannula compared with room air (95% CI, 2.22 to 4.34). Plasma antigen level was higher in those who lacked antispike antibodies (6.42 fold; CI, 5.37 to 7.66) and in those with the Delta variant (1.73 fold; CI, 1.41 to 2.13). Additional factors associated with higher baseline antigen level included male sex, shorter time since hospital admission, decreased days of remdesivir, and renal impairment. In contrast, race, ethnicity, body mass index, and immunocompromising conditions were not associated with plasma antigen levels. Plasma antigen level of 1000 ng/L or greater was associated with a markedly higher odds of worsened pulmonary status at day 5 (odds ratio, 5.06 [CI, 3.41 to 7.50]) and longer time to hospital discharge (median, 7 vs. 4 days; subhazard ratio, 0.51 [CI, 0.45 to 0.57]), with subhazard ratios similar across all levels of baseline pulmonary severity. LIMITATIONS: Plasma samples were drawn at enrollment, not hospital presentation. No point-of-care test to measure plasma antigen is currently available. CONCLUSION: Elevated plasma antigen is highly associated with both severity of pulmonary illness and clinically important patient outcomes. Multiple clinical and viral factors are associated with plasma antigen level at presentation. These data support a potential role of ongoing viral replication in the pathogenesis of SARS-CoV-2 in hospitalized patients. PRIMARY FUNDING SOURCE: U.S. government Operation Warp Speed and National Institute of Allergy and Infectious Diseases.

QuasiSeq: profiling viral quasispecies via self-tuning spectral clustering with PacBio long sequencing reads.

MOTIVATION: The existence of quasispecies in the viral population causes difficulties for disease prevention and treatment. High-throughput sequencing provides opportunity to determine rare quasispecies and long sequencing reads covering full genomes reduce quasispecies determination to a clustering problem. The challenge is high similarity of quasispecies and high error rate of long sequencing reads. RESULTS: We developed QuasiSeq using a novel signature-based self-tuning clustering method, SigClust, to profile viral mixtures with high accuracy and sensitivity. QuasiSeq can correctly identify quasispecies even using low-quality sequencing reads (accuracy <80%) and produce quasispecies sequences with high accuracy (≥99.55%). Using high-quality circular consensus sequencing reads, QuasiSeq can produce quasispecies sequences with 100% accuracy. QuasiSeq has higher sensitivity and specificity than similar published software. Moreover, the requirement of the computational resource can be controlled by the size of the signature, which makes it possible to handle big sequencing data for rare quasispecies discovery. Furthermore, parallel computation is implemented to process the clusters and further reduce the runtime. Finally, we developed a web interface for the QuasiSeq workflow with simple parameter settings based on the quality of sequencing data, making it easy to use for users without advanced data science skills. AVAILABILITY AND IMPLEMENTATION: QuasiSeq is open source and freely available at https://github.com/LHRI-Bioinformatics/QuasiSeq. The current release (v1.0.0) is archived and available at https://zenodo.org/badge/latestdoi/340494542. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update).

DAVID is a popular bioinformatics resource system including a web server and web service for functional annotation and enrichment analyses of gene lists. It consists of a comprehensive knowledgebase and a set of functional analysis tools. Here, we report all updates made in 2021. The DAVID Gene system was rebuilt to gain coverage of more organisms, which increased the taxonomy coverage from 17 399 to 55 464. All existing annotation types have been updated, if available, based on the new DAVID Gene system. Compared with the last version, the number of gene-term records for most annotation types within the updated Knowledgebase have significantly increased. Moreover, we have incorporated new annotations in the Knowledgebase including small molecule-gene interactions from PubChem, drug-gene interactions from DrugBank, tissue expression information from the Human Protein Atlas, disease information from DisGeNET, and pathways from WikiPathways and PathBank. Eight of ten subgroups split from Uniprot Keyword annotation were assigned to specific types. Finally, we added a species parameter for uploading a list of gene symbols to minimize the ambiguity between species, which increases the efficiency of the list upload and eliminates confusion for users. These current updates have significantly expanded the Knowledgebase and enhanced the discovery power of DAVID.

Complete Genome Sequence of Herpes Simplex Virus 2 Strain G.

Herpes simplex virus type 2 (HSV-2) is a common causative agent of genital tract infections. Moreover, HSV-2 and HIV infection can mutually increase the risk of acquiring another virus infection. Due to the high GC content and highly repetitive regions in HSV-2 genomes, only the genomes of four strains have been completely sequenced (HG52, 333, SD90e, and MS). Strain G is commonly used for HSV-2 research, but only a partial genome sequence has been assembled with Illumina sequencing reads. In the current study, we de novo assembled and annotated the complete genome of strain G using PacBio long sequencing reads, which can span the repetitive regions, analyzed the 'α' sequence, which plays key roles in HSV-2 genome circulation, replication, cleavage, and packaging of progeny viral DNA, identified the packaging signals homologous to HSV-1 within the 'α' sequence, and determined both termini of the linear genome and cleavage site for the process of concatemeric HSV-2 DNA produced via rolling-circle replication. In addition, using Oxford Nanopore Technology sequencing reads, we visualized four HSV-2 genome isomers at the nucleotide level for the first time. Furthermore, the coding sequences of HSV-2 strain G have been compared with those of HG52, 333, and MS. Moreover, phylogenetic analysis of strain G and other diverse HSV-2 strains has been conducted to determine their evolutionary relationship. The results will aid clinical research and treatment development of HSV-2.

Responses to a Neutralizing Monoclonal Antibody for Hospitalized Patients With COVID-19 According to Baseline Antibody and Antigen Levels : A Randomized Controlled Trial.

BACKGROUND: In a randomized, placebo-controlled, clinical trial, bamlanivimab, a SARS-CoV-2-neutralizing monoclonal antibody, given in combination with remdesivir, did not improve outcomes among hospitalized patients with COVID-19 based on an early futility assessment. OBJECTIVE: To evaluate the a priori hypothesis that bamlanivimab has greater benefit in patients without detectable levels of endogenous neutralizing antibody (nAb) at study entry than in those with antibodies, especially if viral levels are high. DESIGN: Randomized, placebo-controlled trial. (ClinicalTrials.gov: NCT04501978). SETTING: Multicenter trial. PATIENTS: Hospitalized patients with COVID-19 without end-organ failure. INTERVENTION: Bamlanivimab (7000 mg) or placebo. MEASUREMENTS: Antibody, antigen, and viral RNA levels were centrally measured on stored specimens collected at baseline. Patients were followed for 90 days for sustained recovery (defined as discharge to home and remaining home for 14 consecutive days) and a composite safety outcome (death, serious adverse events, organ failure, or serious infections). RESULTS: Among 314 participants (163 receiving bamlanivimab and 151 placebo), the median time to sustained recovery was 19 days and did not differ between the bamlanivimab and placebo groups (subhazard ratio [sHR], 0.99 [95% CI, 0.79 to 1.22]; sHR > 1 favors bamlanivimab). At entry, 50% evidenced production of anti-spike nAbs; 50% had SARS-CoV-2 nucleocapsid plasma antigen levels of at least 1000 ng/L. Among those without and with nAbs at study entry, the sHRs were 1.24 (CI, 0.90 to 1.70) and 0.74 (CI, 0.54 to 1.00), respectively (nominal P for interaction = 0.018). The sHR (bamlanivimab vs. placebo) was also more than 1 for those with plasma antigen or nasal viral RNA levels above median level at entry and was greatest for those without antibodies and with elevated levels of antigen (sHR, 1.48 [CI, 0.99 to 2.23]) or viral RNA (sHR, 1.89 [CI, 1.23 to 2.91]). Hazard ratios for the composite safety outcome (<1 favors bamlanivimab) also differed by serostatus at entry: 0.67 (CI, 0.37 to 1.20) for those without and 1.79 (CI, 0.92 to 3.48) for those with nAbs. LIMITATION: Subgroup analysis of a trial prematurely stopped because of futility; small sample size; multiple subgroups analyzed. CONCLUSION: Efficacy and safety of bamlanivimab may differ depending on whether an endogenous nAb response has been mounted. The limited sample size of the study does not allow firm conclusions based on these findings, and further independent trials are required that assess other types of passive immune therapies in the same patient setting. PRIMARY FUNDING SOURCE: U.S. government Operation Warp Speed and National Institute of Allergy and Infectious Diseases.

A Combination of M50I and V151I Polymorphic Mutations in HIV-1 Subtype B Integrase Results in Defects in Autoprocessing.

We have recently reported that a recombinant HIV-1NL4.3 containing Met-to-Ile change at codon 50 of integrase (IN) (IN:M50I) exhibits suppression of the virus release below 0.5% of WT HIV, and the released viral particles are replication-incompetent due to defects in Gag/GagPol processing by inhibition of the initiation of autoprocessing of GagPol polyproteins in the virions and leads to replication-incompetent viruses. The coexisting Ser-to-Asn change at codon 17 of IN or Asn-to-Ser mutation at codon 79 of RNaseH (RH) compensated the defective IN:M50I phenotype, suggesting that both IN and RH regulate an HIV infectability. In the current study, to elucidate a distribution of the three mutations during anti-retroviral therapy among patients, we performed a population analysis using 529 plasma virus RNA sequences obtained through the MiSeq. The result demonstrated that 14 plasma HIVs contained IN:M50I without the compensatory mutations. Comparing the sequences of the 14 viruses with that of the defective virus illustrated that only Val-to-Ile change at codon 151 of IN (IN:V151I) existed in the recombinant virus. This IN:V151I is known as a polymorphic mutation and was derived from HIVNL4.3 backbone. A back-mutation at 151 from Ile-to-Val in the defective virus recovered HIV replication capability, and Western Blotting assay displayed that the back-mutation restored Gag/GagPol processing in viral particles. These results demonstrate that a combination of IN:M50I and IN:V151I mutations, but not IN:M50I alone, produces a defective virus.

The Role of Ku70 as a Cytosolic DNA Sensor in Innate Immunity and Beyond.

Human Ku70 is a well-known endogenous nuclear protein involved in the non-homologous end joining pathway to repair double-stranded breaks in DNA. However, Ku70 has been studied in multiple contexts and grown into a multifunctional protein. In addition to the extensive functional study of Ku70 in DNA repair process, many studies have emphasized the role of Ku70 in various other cellular processes, including apoptosis, aging, and HIV replication. In this review, we focus on discussing the role of Ku70 in inducing interferons and proinflammatory cytokines as a cytosolic DNA sensor. We explored the unique structure of Ku70 binding with DNA; illustrated, with evidence, how Ku70, as a nuclear protein, responds to extracellular DNA stimulation; and summarized the mechanisms of the Ku70-involved innate immune response pathway. Finally, we discussed several new strategies to modulate Ku70-mediated innate immune response and highlighted some potential physiological insights based on the role of Ku70 in innate immunity.

Natural Occurring Polymorphisms in HIV-1 Integrase and RNase H Regulate Viral Release and Autoprocessing.

Recently, a genome-wide association study using plasma HIV RNA from antiretroviral therapy-naive patients reported that 14 naturally occurring nonsynonymous single-nucleotide polymorphisms (SNPs) in HIV derived from antiretrovirus drug-naive patients were associated with virus load (VL). Those SNPs were detected in reverse transcriptase, RNase H, integrase, envelope, and Nef. However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP and examined their replicative abilities. An HIV variant containing a Met-to-Ile change at codon 50 in integrase [HIV(IN:M50I)] was found as an impaired virus. Despite the mutation being in integrase, the virus release was significantly suppressed (P < 0.001). Transmission electron microscopy analysis revealed that abnormal bud accumulation on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins' autoprocessing in the HIV(IN:M50I) particles, although Förster resonance energy transfer (FRET) assay displayed that GagPol containing IN:M50I forms a homodimer with a similar efficiency with GagPol (wild type). The impaired maturation and replication were rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 of integrase and Asn-to-Ser change at codon 79 of RNase H. These data demonstrate that Gag and GagPol assembly, virus release, and autoprocessing are regulated by not only integrase but also RNase H. IMPORTANCE Nascent HIV-1 is a noninfectious viral particle. Cleaving Gag and GagPol polyproteins in the particle by mature HIV protease (PR), the nascent virus becomes an infectious virus. PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by self-cleavage is called autoprocessing. Here, during the evaluation of the roles of naturally emerging nonsynonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase GagPol. Other coexisting SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, recovered this defect, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.

MicroRNA Profiles in Monocyte-Derived Macrophages Generated by Interleukin-27 and Human Serum: Identification of a Novel HIV-Inhibiting and Autophagy-Inducing MicroRNA.

Interleukin-27 (IL-27) is a pleiotropic cytokine that influences the innate and adaptive immune systems. It inhibits viral infection and regulates the expression of microRNAs (miRNAs). We recently reported that macrophages differentiated from human primary monocytes in the presence of IL-27 and human AB serum resisted human immunodeficiency virus (HIV) infection and showed significant autophagy induction. In the current study, the miRNA profiles in these cells were investigated, especially focusing on the identification of novel miRNAs regulated by IL-27-treatment. The miRNA sequencing analysis detected 38 novel miRNAs. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed that IL-27 differentially regulated the expression of 16 of the 38 miRNAs. Overexpression of the synthesized miRNA mimics by transfection revealed that miRAB40 had potent HIV-inhibiting and autophagy-inducing properties. B18R, an interferon (IFN)-neutralization protein, partially suppressed both activities, indicating that the two functions were induced via IFN-dependent and -independent pathways. Although the target mRNA(s) of miRAB40 involving in the induction of both functions was unable to identify in this study, the discovery of miRAB40, a potential HIV-inhibiting and autophagy inducing miRNA, may provide novel insights into the miRNA (small none-coding RNA)-mediated regulation of HIV inhibition and autophagy induction as an innate immune response.

Genome-wide association study of high-sensitivity C-reactive protein, D-dimer, and interleukin-6 levels in multiethnic HIV+ cohorts.

OBJECTIVES: Elevated levels of interleukin-6 (IL-6), D-dimer, and C-reactive protein (hsCRP) are associated with increased incidence of comorbid disease and mortality among people living with HIV (PLWH). Prior studies suggest a genetic basis for these biomarker elevations in the general population. The study objectives are to identify the genetic basis for these biomarkers among PLWH. METHODS: Baseline levels of hsCRP, D-dimer, and IL-6, and single nucleotide polymorphisms (SNPs) were determined for 7768 participants in three HIV treatment trials. Single variant analysis was performed for each biomarker on samples from each of three ethnic groups [African (AFR), Admixed American (AMR), European (EUR)] within each trial including covariates relevant to biomarker levels. For each ethnic group, the results were pooled across trials, then further pooled across ethnicities. RESULTS: The transethnic analysis identified three, two, and one known loci associated with hsCRP, D-dimer, and IL-6 levels, respectively, and two novel loci, FGB and GCNT1, associated with D-dimer levels. Lead SNPs exhibited similar effects across ethnicities. Additionally, three novel, ethnic-specific loci were identified: CATSPERG associated with D-dimer in AFR and PROX1-AS1 and TRAPPC9 associated with IL-6 in AFR and AMR, respectively. CONCLUSION: Eleven loci associated with three biomarker levels were identified in PLWH from the three studies including six loci known in the general population and five novel loci associated with D-dimer and IL-6 levels. These findings support the hypothesis that host genetics may partially contribute to chronic inflammation in PLWH and help to identify potential targets for intervention of serious non-AIDS complications.

Profiles of MicroRNAs in Interleukin-27-Induced HIV-Resistant T Cells: Identification of a Novel Antiviral MicroRNA.

OBJECTIVES: Interleukin-27 (IL-27) is known as an anti-HIV cytokine. We have recently demonstrated that IL-27-pretreatment promotes phytohemagglutinin-stimulated CD4(+) T cells into HIV-1-resistant cells by inhibiting an uncoating step. PURPOSE: To further characterize the function of the HIV resistant T cells, we investigated profiles of microRNA in the cells using microRNA sequencing (miRNA-seq) and assessed anti-HIV effect of the microRNAs. METHODS: Phytohemagglutinin-stimulated CD4(+) T cells were treated with or without IL-27 for 3 days. MicroRNA profiles were analyzed using miRNA-seq. To assess anti-HIV effect, T cells or macrophages were transfected with synthesized microRNA mimics and then infected with HIVNL4.3 or HIVAD8. Anti-HIV effect was monitored by a p24 antigen enzyme-linked immunosorbent assay kit. interferon (IFN)-α, IFN-ß, or IFN-λ production was quantified using each subtype-specific enzyme-linked immunosorbent assay kit. RESULTS: A comparative analysis of microRNA profiles indicated that expression of known miRNAs was not significantly changed in IL-27-treated cells compared with untreated T cells; however, a total of 15 novel microRNAs (miRTC1 ∼ miRTC15) were identified. Anti-HIV assay using overexpression of each novel microRNA revealed that 10 nM miRTC14 (GenBank accession number: MF281439) remarkably suppressed HIV infection by (99.3 ± 0.27%, n = 9) in macrophages but not in T cells. The inhibition was associated through induction of >1000 pg/mL of IFN-αs and IFN-λ1. CONCLUSION: We discovered a total of 15 novel microRNAs in T cells and characterized that miRTC14, one of the novel microRNAs, was a potent IFN-inducing anti-HIV miRNA, implicating that regulation of the expression of miRTC14 may be a potent therapeutic tool for not only HIV but also other virus infection.

Core genome MLST and resistome analysis of Klebsiella pneumoniae using a clinically amenable workflow.

Whole genome sequencing (WGS) is replacing traditional microbiological typing methods for investigation of outbreaks in clinical settings. Here, we used a clinical microbiology laboratory core genome multilocus sequence typing (cgMLST) workflow to analyze 40 isolates of K. pneumoniae which are part of the Antimicrobial Resistance Leadership Group (ARLG) isolate collection, alongside 10 Mayo Clinic K. pneumoniae isolates, comparing results to those of pulsed-field gel electrophoresis (PFGE). Additionally, we used the WGS data to predict phenotypic antimicrobial susceptibility (AST). Thirty-one of 40 ARLG K. pneumoniae isolates belonged to the same PFGE type, all of which, alongside 3 isolates of different PFGE types, formed a large cluster by cgMLST. PFGE and cgMLST were completely concordant for the 10 Mayo Clinic K. pneumoniae isolates. For AST prediction, the overall agreement between phenotypic AST and genotypic prediction was 95.6%.

Predicting Antibiotic Resistance in Gram-Negative Bacilli from Resistance Genes.

We developed a rapid high-throughput PCR test and evaluated highly antibiotic-resistant clinical isolates of Escherichia coli (n = 2,919), Klebsiella pneumoniae (n = 1,974), Proteus mirabilis (n = 1,150), and Pseudomonas aeruginosa (n = 1,484) for several antibiotic resistance genes for comparison with phenotypic resistance across penicillins, cephalosporins, carbapenems, aminoglycosides, trimethoprim-sulfamethoxazole, fluoroquinolones, and macrolides. The isolates originated from hospitals in North America (34%), Europe (23%), Asia (13%), South America (12%), Africa (7%), or Oceania (1%) or were of unknown origin (9%). We developed statistical methods to predict phenotypic resistance from resistance genes for 49 antibiotic-organism combinations, including gentamicin, tobramycin, ciprofloxacin, levofloxacin, trimethoprim-sulfamethoxazole, ertapenem, imipenem, cefazolin, cefepime, cefotaxime, ceftazidime, ceftriaxone, ampicillin, and aztreonam. Average positive predictive values for genotypic prediction of phenotypic resistance were 91% for E. coli, 93% for K. pneumoniae, 87% for P. mirabilis, and 92% for P. aeruginosa across the various antibiotics for this highly resistant cohort of bacterial isolates.

Expression and prognostic value of matriptase in ovarian serous adenocarcinoma.

Previous studies have demonstrated that matriptase is involved in degradation of the extracellular matrix and angiogenesis, and is overexpressed in certain forms of epithelial cancer. The present study aimed to examine matriptase expression in ovarian serous adenocarcinoma, and to investigate its association with clinicopathological characteristics and patient prognosis. Matriptase expression was analyzed in 80 ovarian serous adenocarcinoma and 12 normal ovarian tissue samples by immunohistochemistry. All data were analyzed to evaluate the association between matriptase expression and clinicopathological parameters and overall survival. Immunohistochemistry demonstrated that matriptase protein was significantly overexpressed in the ovarian serous adenocarcinoma tissues compared with the normal ovarian tissues (P=0.0003). Furthermore, matriptase expression was significantly associated with clinical stage (P=0.0077) and lymph node metastasis (P=0.0111). Kaplan-Meier survival curves demonstrated that patients with positive matriptase expression had significantly greater survival times (P=0.0008). Matriptase expression is associated with early stage and a greater survival time; therefore, this protein may function as a novel diagnostic and prognostic marker.

Non-Lethal Type VIII Osteogenesis Imperfecta Has Elevated Bone Matrix Mineralization.

CONTEXT: Type VIII osteogenesis imperfecta (OI; OMIM 601915) is a recessive form of lethal or severe OI caused by null mutations in P3H1, which encodes prolyl 3-hydroxylase 1. OBJECTIVES: Clinical and bone material description of non-lethal type VIII OI. DESIGN: Natural history study of type VIII OI. SETTING: Pediatric academic research centers. PATIENTS: Five patients with non-lethal type VIII OI, and one patient with lethal type VIII OI. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Clinical examinations included bone mineral density, radiographs, and serum and urinary metabolites. Bone biopsy samples were analyzed for histomorphometry and bone mineral density distribution by quantitative backscattered electron imaging microscopy. Collagen biochemistry was examined by mass spectrometry, and collagen fibrils were examined by transmission electron microscopy. RESULTS: Type VIII OI patients have extreme growth deficiency, an L1-L4 areal bone mineral density Z-score of -5 to -6, and normal bone formation markers. Collagen from bone and skin tissue and cultured osteoblasts and fibroblasts have nearly absent 3-hydroxylation (1-4%). Collagen fibrils showed abnormal diameters and irregular borders. Bone histomorphometry revealed decreased cortical width and very thin trabeculae with patches of increased osteoid, although the overall osteoid surface was normal. Quantitative backscattered electron imaging showed increased matrix mineralization of cortical and trabecular bone, typical of other OI types. However, the proportion of bone with low mineralization was increased in type VIII OI bone, compared to type VII OI. CONCLUSIONS: P3H1 is the unique enzyme responsible for collagen 3-hydroxylation in skin and bone. Bone from non-lethal type VIII OI children is similar to type VII, especially bone matrix hypermineralization, but it has distinctive features including extremely thin trabeculae, focal osteoid accumulation, and an increased proportion of low mineralized bone.