EvoPipes.net: Bioinformatic Tools for Ecological and Evolutionary Genomics

Recent increases in the production of genomic data are yielding new opportunities and challenges for biologists. Among the chief problems posed by next-generation sequencing are assembly and analyses of these large data sets. Here we present an online server, http://EvoPipes.net, that provides access to a wide range of tools for bioinformatic analyses of genomic data oriented for ecological and evolutionary biologists. The EvoPipes.net server includes a basic tool kit for analyses of genomic data including a next-generation sequence cleaning pipeline (SnoWhite), scaffolded assembly software (SCARF), a reciprocal best-blast hit ortholog pipeline (RBH Orthologs), a pipeline for reference protein-based translation and identification of reading frame in transcriptome and genomic DNA (TransPipe), a pipeline to identify gene families and summarize the history of gene duplications (DupPipe), and a tool for developing SSRs or microsatellites from a transcriptome or genomic coding sequence collection (findSSR). EvoPipes.net also provides links to other software developed for evolutionary and ecological genomics, including chromEvol and NU-IN, as well as a forum for discussions of issues relating to genomic analyses and interpretation of results. Overall, these applications provide a basic bioinformatic tool kit that will enable ecologists and evolutionary biologists with relatively little experience and computational resources to take advantage of the opportunities provided by next-generation sequencing in their systems

V3 Loop Truncations in HIV-1 Envelope Impart Resistance to Coreceptor Inhibitors and Enhanced Sensitivity to Neutralizing Antibodies

The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (ΔV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds

Three-Year Follow-up of 2-Dose Versus 3-Dose HPV Vaccine

BACKGROUND AND OBJECTIVES: Human papillomavirus (HPV) antibody responses to the 9-valent abstract human papillomavirus (9vHPV) vaccine among girls and boys (aged 9–14 years) receiving 2- dose regimens (months 0, 6 or 0, 12) were noninferior to a 3-dose regimen (months 0, 2, 6) in young women (aged 16–26 years) 4 weeks after last vaccination in an international, randomized, open-label trial (NCT01984697). We assessed response durability through month 36. METHODS: Girls received 2 (months 0 and 6 [0, 6]: n = 301; months 0 and 12 [0, 12]: n = 151) or 3 doses (months 0,2, and 6 [0, 2, 6]: n = 301); boys received 2 doses ([0, 6]: n = 301; [0, 12]: n = 150); and young women received 3 doses ([0, 2, 6]: n = 314) of 9vHPV vaccine. Anti-HPV geometric mean titers (GMTs) were assessed by competitive Luminex immunoassay (cLIA) and immunoglobulin G-Luminex immunoassay (IgG-LIA) through month 36. RESULTS: Anti-HPV GMTs were highest 1 month after the last 9vHPV vaccine regimen dose, decreased sharply during the subsequent 12 months, and then decreased more slowly. GMTs 2 to 2.5 years after the last regimen dose in girls and boys given 2 doses were generally similar to or greater than GMTs in young women given 3 doses. Across HPV types, most boys and girls who received 2 doses (cLIA: 81%–100%; IgG-LIA: 91%–100%) and young women who received 3 doses (cLIA: 78%–98%; IgG-LIA: 91%–100%) remained seropositive 2 to 2.5 years after the last regimen dose. CONCLUSIONS: Antibody responses persisted through 2 to 2.5 years after the last dose of a 2-dose 9vHPV vaccine regimen in girls and boys. In girls and boys, antibody responses generated by 2 doses administered 6 to 12 months apart may be sufficient to induce high-level protective efficacy through at least 2 years after the second dose.Medicin

Indicators of Mental Disorders in UK Biobank – A comparison of approaches

Objectives: For many research cohorts, it is not practical to provide a “gold‐standard” mental health diagnosis. It is therefore important for mental health research that potential alternative measures for ascertaining mental disorder status are understood. Methods: Data from UK Biobank in those participants who had completed the online Mental Health Questionnaire (n = 157,363) were used to compare the classification of mental disorder by four methods: symptom‐based outcome (self‐complete based on diagnostic interviews), self‐reported diagnosis, hospital data linkage, and self‐report medication. Results: Participants self‐reporting any psychiatric diagnosis had elevated risk of any symptom‐based outcome. Cohen's κ between self‐reported diagnosis and symptom‐based outcome was 0.46 for depression, 0.28 for bipolar affective disorder, and 0.24 for anxiety. There were small numbers of participants uniquely identified by hospital data linkage and medication. Conclusion: Our results confirm that ascertainment of mental disorder diagnosis in large cohorts such as UK Biobank is complex. There may not be one method of classification that is right for all circumstances, but an informed and transparent use of outcome measure(s) to suit each research question will maximise the potential of UK Biobank and other resources for mental health research

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

The role of the HIV-1 V3 loop in determining coreceptor tropism and modulating sensitivity to coreceptor inhibitors

In order for an HIV-1 virion to infect a host cell, the gp120 subunit of envelope, the only viral protein exposed on the surface of the virion, first attaches to the cellular receptor CD4 and subsequently to a chemokine coreceptor, either CCR5 or CXCR4. Tropism for these coreceptors is principally determined by the V3 loop, which is a 35-residue region bound by a disulphide bond and exposed on the surface of gp120. While V3 is known to be critical for coreceptor binding, V3-coreceptor interactions and the structural basis for CCR5 or CXCR4 specificity remain elusive. We hypothesized that subdomains within V3 determine coreceptor tropism. Using the dual-tropic virus HIV-1R3A, which uses both CCR5 and CXCR4, we made an extensive panel of small V3 deletions and evaluated these mutants for their effects on tropism and sensitivity to coreceptor antagonists. Small deletions on either side of the V3 base abrogated R5 tropism. The resulting X4-tropic mutants exhibited enhanced sensitivity to the CXCR4 antagonist AMD3100. Only a deletion of residues 9 to 12 in the V3 stem ablated X4 tropism. Remarkably, this R5-tropic mutant showed complete resistance to a panel of CCR5 antagonists, including vicriviroc and aplaviroc. Our findings suggest that subdomains in V3 differentially affect R5 and X4 tropism and modulate sensitivity to coreceptor antagonists. Small molecule CCR5 inhibitors are being used clinically to treat HIV-1-infected patients, so in order to study mechanisms of resistance to this new drug class, we further characterized a variant of the drug-resistant Δ9-12 mutant, termed Δ9-12a. Since CCR5 antagonists disrupt the gp120-CCR5 extracellular loop interaction, we hypothesized that Δ9-12a is more dependent on the CCR5 amino terminus (NT). Our results indicate that Δ9-12a does have an increased reliance on the CCR5 NT, exhibits increased sensitivity to several broadly neutralizing monoclonal antibodies, including b12 and 4E10, and is capable of using aplaviroc-bound CCR5. This detailed characterization suggests that increased dependence on the CCR5 NT may represent a mechanism by which HIV-1 Envs acquire resistance to CCR5 antagonists. In addition, envelopes such as Δ9-12a may be useful for identifying new drugs that target the gpl20-CCR5 NT interaction

The role of the HIV-1 V3 loop in determining coreceptor tropism and modulating sensitivity to coreceptor inhibitors

In order for an HIV-1 virion to infect a host cell, the gp120 subunit of envelope, the only viral protein exposed on the surface of the virion, first attaches to the cellular receptor CD4 and subsequently to a chemokine coreceptor, either CCR5 or CXCR4. Tropism for these coreceptors is principally determined by the V3 loop, which is a 35-residue region bound by a disulphide bond and exposed on the surface of gp120. While V3 is known to be critical for coreceptor binding, V3-coreceptor interactions and the structural basis for CCR5 or CXCR4 specificity remain elusive. We hypothesized that subdomains within V3 determine coreceptor tropism. Using the dual-tropic virus HIV-1R3A, which uses both CCR5 and CXCR4, we made an extensive panel of small V3 deletions and evaluated these mutants for their effects on tropism and sensitivity to coreceptor antagonists. Small deletions on either side of the V3 base abrogated R5 tropism. The resulting X4-tropic mutants exhibited enhanced sensitivity to the CXCR4 antagonist AMD3100. Only a deletion of residues 9 to 12 in the V3 stem ablated X4 tropism. Remarkably, this R5-tropic mutant showed complete resistance to a panel of CCR5 antagonists, including vicriviroc and aplaviroc. Our findings suggest that subdomains in V3 differentially affect R5 and X4 tropism and modulate sensitivity to coreceptor antagonists. Small molecule CCR5 inhibitors are being used clinically to treat HIV-1-infected patients, so in order to study mechanisms of resistance to this new drug class, we further characterized a variant of the drug-resistant Δ9-12 mutant, termed Δ9-12a. Since CCR5 antagonists disrupt the gp120-CCR5 extracellular loop interaction, we hypothesized that Δ9-12a is more dependent on the CCR5 amino terminus (NT). Our results indicate that Δ9-12a does have an increased reliance on the CCR5 NT, exhibits increased sensitivity to several broadly neutralizing monoclonal antibodies, including b12 and 4E10, and is capable of using aplaviroc-bound CCR5. This detailed characterization suggests that increased dependence on the CCR5 NT may represent a mechanism by which HIV-1 Envs acquire resistance to CCR5 antagonists. In addition, envelopes such as Δ9-12a may be useful for identifying new drugs that target the gpl20-CCR5 NT interaction

The role of the HIV-1 V3 loop in determining coreceptor tropism and modulating sensitivity to coreceptor inhibitors

In order for an HIV-1 virion to infect a host cell, the gp120 subunit of envelope, the only viral protein exposed on the surface of the virion, first attaches to the cellular receptor CD4 and subsequently to a chemokine coreceptor, either CCR5 or CXCR4. Tropism for these coreceptors is principally determined by the V3 loop, which is a 35-residue region bound by a disulphide bond and exposed on the surface of gp120. While V3 is known to be critical for coreceptor binding, V3-coreceptor interactions and the structural basis for CCR5 or CXCR4 specificity remain elusive. We hypothesized that subdomains within V3 determine coreceptor tropism. Using the dual-tropic virus HIV-1R3A, which uses both CCR5 and CXCR4, we made an extensive panel of small V3 deletions and evaluated these mutants for their effects on tropism and sensitivity to coreceptor antagonists. Small deletions on either side of the V3 base abrogated R5 tropism. The resulting X4-tropic mutants exhibited enhanced sensitivity to the CXCR4 antagonist AMD3100. Only a deletion of residues 9 to 12 in the V3 stem ablated X4 tropism. Remarkably, this R5-tropic mutant showed complete resistance to a panel of CCR5 antagonists, including vicriviroc and aplaviroc. Our findings suggest that subdomains in V3 differentially affect R5 and X4 tropism and modulate sensitivity to coreceptor antagonists. Small molecule CCR5 inhibitors are being used clinically to treat HIV-1-infected patients, so in order to study mechanisms of resistance to this new drug class, we further characterized a variant of the drug-resistant Δ9-12 mutant, termed Δ9-12a. Since CCR5 antagonists disrupt the gp120-CCR5 extracellular loop interaction, we hypothesized that Δ9-12a is more dependent on the CCR5 amino terminus (NT). Our results indicate that Δ9-12a does have an increased reliance on the CCR5 NT, exhibits increased sensitivity to several broadly neutralizing monoclonal antibodies, including b12 and 4E10, and is capable of using aplaviroc-bound CCR5. This detailed characterization suggests that increased dependence on the CCR5 NT may represent a mechanism by which HIV-1 Envs acquire resistance to CCR5 antagonists. In addition, envelopes such as Δ9-12a may be useful for identifying new drugs that target the gpl20-CCR5 NT interaction

Effects of Partial Deletions within the Human Immunodeficiency Virus Type 1 V3 Loop on Coreceptor Tropism and Sensitivity to Entry Inhibitors▿

The human immunodeficiency virus type 1 (HIV-1) V3 loop is critical for coreceptor binding and principally determines tropism for the CCR5 and CXCR4 coreceptors. The recent crystallographic resolution of V3 shows that its base is closely associated with the conserved coreceptor binding site on the gp120 core, whereas more distal regions protrude toward the cell surface, likely mediating interactions with coreceptor extracellular loops. However, these V3-coreceptor interactions and the structural basis for CCR5 or CXCR4 specificity are poorly understood. Using the dual-tropic virus HIV-1R3A, which uses both CCR5 and CXCR4, we sought to identify subdomains within V3 that selectively mediate R5 or X4 tropism. An extensive panel of V3 mutants was evaluated for effects on tropism and sensitivity to coreceptor antagonists. Mutations on either side of the V3 base (residues 3 to 8 and 26 to 33) ablated R5 tropism and made the resulting X4-tropic Envs more sensitive to the CXCR4 inhibitor AMD3100. When mutations were introduced within the V3 stem, only a deletion of residues 9 to 12 on the N-terminal side ablated X4 tropism. Remarkably, this R5-tropic Δ9-12 mutant was completely resistant to several small-molecule inhibitors of CCR5. Envs with mutations in the V3 crown (residues 13 to 20) remained dual tropic. Similar observations were made for a second dual-tropic isolate, HIV-189.6. These findings suggest that V3 subdomains can be identified that differentially affect R5 and X4 tropism and modulate sensitivity to CCR5 and CXCR4 inhibitors. These studies provide a novel approach for probing V3-coreceptor interactions and mechanisms by which these interactions can be inhibited