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The HIV-1 envelope (Env) undergoes conformational changes during infection. Broadly neutralizing antibodies (bNAbs) are typically isolated by using soluble Env trimers, which do not capture all Env states. To address these limitations, we devised a vesicular stomatitis virus (VSV)-based probe to display membrane-embedded Env trimers and isolated five bNAbs from two chronically infected donors, M4008 and M1214. Donor B cell receptor (BCR) repertoires identified two bNAb lineages, M4008_N1 and M1214_N1, that class-switched to immunoglobulin G (IgG) and IgA. Variants of these bNAbs reconstituted as IgA demonstrated broadly neutralizing activity, and the IgA fraction of M1214 plasma conferred neutralization. M4008_N1 epitope mapping revealed a glycan-independent V3 epitope conferring tier 2 virus neutralization. A 4.86-angstrom-resolution cryogenic electron microscopy (cryo-EM) structure of M1214_N1 complexed with CH505 SOSIP revealed another elongated epitope, the V2V5 corridor, extending from V2 to V5. Overall, the VSVENV probe identified bNAb lineages with neutralizing IgG and IgA members targeting distinct sites of HIV-1 Env vulnerability.

Penile acquisition of HIV accounts for most infections among men globally. Nevertheless, candidate HIV interventions for men advance to clinical trials without preclinical efficacy data, due primarily to a paucity of relevant animal models of penile HIV infection. Using our recently developed macaque model, we show that a single subcutaneous administration of broadly neutralizing antibody (bNAb) 10-1074 conferred durable protection against repeated penile exposures to simian-human immunodeficiency virus (SHIVSF162P3). Macaques co-administered bNAbs 10-1074 and 3BNC117, or 3BNC117 alone, also exhibited significant protection against repeated vaginal SHIVAD8-EO exposures. Regression modeling estimated that individual plasma bNAb concentrations of 5 mu gml(-1) correlated with >= 99.9% relative reduction in SHIV infection probability via penile (10-1074) or vaginal (10-1074 or 3BNC117) challenge routes. These results demonstrate that comparably large reductions in penile and vaginal SHIV infection risk among macaques were achieved at clinically relevant plasma bNAb concentrations and inform dose selection for the development of bNAbs as long-acting pre-exposure prophylaxis candidates for use by men and women.

This tutorial and the accompanying poster (10.1038/s41596-020-0307-7) provide a guide for performing quantitative fluorescence imaging using confocal microscopy. It includes advice and troubleshooting information from sample preparation and microscope setup to data analysis and statistics. When used appropriately, a confocal fluorescence microscope is an excellent tool for making quantitative measurements in cells and tissues. The confocal microscope's ability to block out-of-focus light and thereby perform optical sectioning through a specimen allows the researcher to quantify fluorescence with very high spatial precision. However, generating meaningful data using confocal microscopy requires careful planning and a thorough understanding of the technique. In this tutorial, the researcher is guided through all aspects of acquiring quantitative confocal microscopy images, including optimizing sample preparation for fixed and live cells, choosing the most suitable microscope for a given application and configuring the microscope parameters. Suggestions are offered for planning unbiased and rigorous confocal microscope experiments. Common pitfalls such as photobleaching and cross-talk are addressed, as well as several troubling instrumentation problems that may prevent the acquisition of quantitative data. Finally, guidelines for analyzing and presenting confocal images in a way that maintains the quantitative nature of the data are presented, and statistical analysis is discussed. A visual summary of this tutorial is available as a poster (10.1038/s41596-020-0307-7).

The pathogenesis of hidradenitis suppurativa (HS) centers around Th17/Treg dysfunction illustrated by lesional elevation of IL-17A, IL-6, and other inflammatory mediators resulting in a chronic feed-forward inflammatory cascade. Similar inflammatory mechanisms have been identified in psoriasis and rheumatoid arthritis (RA) in which traditional immunosuppressants (including methotrexate) are routinely used with reasonable levels of disease control. Methotrexate's mechanism of action in these instances includes downregulation of the Th17 axis via alterations in dendritic cell and T-cell activity and maturation. Published data suggest methotrexate in an ineffective therapy in HS, which does not pair with our current understanding of the mechanisms of disease. The reasons behind this, including are discussed. Some HS patients may benefit from drugs such as methotrexate, and acknowledgment of the potential of disease heterogeneity will allow exploration of which factors may enable identification of such individuals.

Background. MK-8591 (4'-ethynyl-2-fluoro-2'-deoxyadenosine [EFdA]) is a novel reverse transcriptase-translocation inhibitor. Methods. We assessed MK-8591 as preexposure prophylaxis in the rhesus macaque model of intrarectal challenge with simian/human immunodeficiency virus (SHIV). In study 1, 8 rhesus macaques received 3.9 mg/kg of MK-8591 orally on day 0 and once weekly for the next 14 weeks. Eight controls were treated with vehicle. All rhesus macaques were challenged with SHIV109CP3 on day 6 and weekly for up to 12 challenges or until infection was confirmed. The dose of MK-8591 was reduced to 1.3 and 0.43 mg/kg/week in study 2 and further to 0.1 and 0.025 mg/kg/week in study 3. In studies 2 and 3, each dose was given up to 6 times once weekly, and animals were challenged 4 times once weekly with SHIV109CP3. Results. Control macaques were infected after a median of 1 challenge (range, 1-4 challenges). All treated animals in studies 1 and 2 were protected, consistent with a 41.5-fold lower risk of infection (P < .0001, by the log-rank test). In study 3, at a 0.1-mg/kg dose, 2 rhesus macaques became infected, consistent with a 7.2-fold lower risk of infection (P = .0003, by the log-rank test). The 0.025-mg/kg dose offered no protection. Conclusions. These data support MK-8591's potential as a preexposure prophylaxis agent.

Mu-opioid receptors (MORs) mediate the rewarding properties of oxycodone and other prescription opioid medications, which have played a central role in the current opioid epidemic in the United States. The human mu-opioid receptor gene (OPRM1) contains a functional single nucleotide polymorphism (SNP), A118G, which has been associated with altered opioid addiction risk, however the mechanisms responsible for this are not well understood. To explore this, we examined oxycodone conditioned place preference (CPP) and self-administration behavior (SA) in A112G mice, which possess a functionally analogous SNP in the mouse mu-opioid receptor gene (Oprm1). For CPP, male and female A112G mice homozygous for the A112 (wild-type; AA) or G112 (GG) allele were conditioned with doses of 1 and 3 mg/kg across an 8-day period. For SA, mice were allowed to self administer oxycodone (unit dose 0.25 mg/kg/infusion, FR1) for 4h/day for 10 consecutive days. We observed no effects of genotype or sex on conditioned place preference behavior. Oxycodone 3 mg/kg increased locomotor activity in AA mice but not GG mice, and both male and female GG mice self-administered significantly more oxycodone compared to their wild-type AA littermates. These studies suggest that the G allele promotes greater opioid intake, which may underlie greater opioid addiction morbidity in G-allele carriers.

T-cell receptor (TCR)-based therapeutic cells and agents have emerged as a new class of effective cancer therapies. These therapies work on cells that express intracellular cancer-associated proteins by targeting peptides displayed on MHC receptors. However, cross-reactivities of these agents to off-target cells and tissues have resulted in serious, sometimes fatal, adverse events. We have developed a high-throughput genetic platform (termed "PresentER") that encodes MHC-I peptide minigenes for functional immunologic assays and determines the reactivities of TCR-like therapeutic agents against large libraries of MHC-I ligands. In this article, we demonstrated that PresentER could be used to identify the on-and-off targets of T cells and TCR-mimic (TCRm) antibodies using in vitro coculture assays or binding assays. We found dozens of MHC-I ligands that were cross-reactive with two TCRm antibodies and two native TCRs and that were not easily predictable by other methods.

Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFF) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase, resulting in defects in protein prenylation. Yet, it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID. Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and A TRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Last, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare nonsynonymous coding variants in patients with ONJ or an AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people.

An accurate, comprehensive reference sequence library maximizes information gained from environmental DNA (eDNA) metabarcoding of marine fishes. Here, we used a regional checklist and early results from an ongoing eDNA time series to target mid-Atlantic U.S. coastal fishes lacking reference sequences. We obtained 60 specimens representing 31 species from NOM trawl surveys and institutional collections, and analyzed 12S and COI barcode regions, the latter to confirm specimen identification. Combined with existing GenBank accessions, the enhanced 12S dataset covered most (74%) of 341 fishes on New Jersey State checklist including 95% of those categorized abundant or common. For eDNA time series, we collected water samples approximately twice monthly for 24 months at an ocean and a bay site in New Jersey. Metabarcoding was performed using separate 12S primer sets targeting bony and cartilaginous fishes. Bioinformatic analysis of Illumina MiSeq fastq files with the augmented library yielded exact matches for 90% of the 104 fish amplicon sequence variants generated from field samples. Newly obtained reference sequences revealed two southern U.S. species as relatively common warm season migrants: Gulf kingfish (Menticirrhus littoralis) and Brazilian cownose ray (Rhinoptera brasiliensis). A beach wrack specimen corroborated the local presence of Brazilian cownose ray. Our results highlight the value of strengthening reference libraries and demonstrate that eDNA can help detect range shifts including those of species overlooked by traditional surveys.

Athukoralage et al. (2020) identify a new anti-CRISPR (Acr) that degrades cA(4), a cyclic oligo-adenylate second messenger produced during the type III CRISPR immune response. This provides an effective way by which invaders can bypass downstream CRISPR effectors that rely on this signaling molecule.