HIV-associated gut microbial alterations are dependent on host and geographic context.

HIV-associated changes in intestinal microbiota are believed to be important drivers of disease progression. However, the majority of studies have focused on populations in high-income countries rather than in developing regions where HIV burden is greatest. To better understand the impact of HIV on fecal microbiota globally, we compare the fecal microbial community of individuals in the U.S., Uganda, and Botswana. We identify significant bacterial taxa alterations with both treated and untreated HIV infection with a high degree of uniqueness in each cohort. HIV-associated taxa alterations are also significantly different between populations that report men who have sex with men (MSM) behavior and non-MSM populations. Additionally, while we find that HIV infection is consistently associated with higher soluble markers of immune activation, most specific bacterial taxa associated with these markers in each region are not shared and none are shared across all three geographic locations in our study. Our findings demonstrate that HIV-associated changes in fecal microbiota are overall distinct among geographical locations and sexual behavior groups, although a small number of taxa shared between pairs of geographic locations warrant further investigation, highlighting the importance of considering host context to fully assess the impact of the gut microbiome on human health and disease.

Expanding the ligandable proteome by paralog hopping with covalent probes.

More than half of the ~20,000 protein-encoding human genes have at least one paralog. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to a subset of paralogous proteins. Here, we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs that lack the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we mutated the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling (ABPP) that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-N112C-CCNE1 interaction into a NanoBRET-ABPP assay capable of identifying compounds that reversibly inhibit both N112C- and WT-CCNE1:CDK2 complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings thus provide a roadmap for leveraging electrophile-cysteine interactions to extend the ligandability of the proteome beyond covalent chemistry.

Complex Organisms Must Deal with Complex Threats: How Does Amphibian Conservation Deal with Biphasic Life Cycles?

The unprecedented rate of global amphibian decline is attributed to The Anthropocene, with human actions triggering the Sixth Mass Extinction Event. Amphibians have suffered some of the most extreme declines, and their lack of response to conservation actions may reflect challenges faced by taxa that exhibit biphasic life histories. There is an urgent need to ensure that conservation measures are cost-effective and yield positive outcomes. Many conservation actions have failed to meet their intended goals of bolstering populations to ensure the persistence of species into the future. We suggest that past conservation efforts have not considered how different threats influence multiple life stages of amphibians, potentially leading to suboptimal outcomes for their conservation. Our review highlights the multitude of threats amphibians face at each life stage and the conservation actions used to mitigate these threats. We also draw attention to the paucity of studies that have employed multiple actions across more than one life stage. Conservation programs for biphasic amphibians, and the research that guides them, lack a multi-pronged approach to deal with multiple threats across the lifecycle. Conservation management programs must recognise the changing threat landscape for biphasic amphibians to reduce their notoriety as the most threatened vertebrate taxa globally.

Cysteine dependence of Lactobacillus iners is a potential therapeutic target for vaginal microbiota modulation.

Vaginal microbiota composition affects many facets of reproductive health. Lactobacillus iners-dominated microbial communities are associated with poorer outcomes, including higher risk of bacterial vaginosis (BV), compared with vaginal microbiota rich in L. crispatus. Unfortunately, standard-of-care metronidazole therapy for BV typically results in dominance of L. iners, probably contributing to post-treatment relapse. Here we generate an L. iners isolate collection comprising 34 previously unreported isolates from 14 South African women with and without BV and 4 previously unreported isolates from 3 US women. We also report an associated genome catalogue comprising 1,218 vaginal Lactobacillus isolate genomes and metagenome-assembled genomes from >300 women across 4 continents. We show that, unlike L. crispatus, L. iners growth is dependent on L-cysteine in vitro and we trace this phenotype to the absence of canonical cysteine biosynthesis pathways and a restricted repertoire of cysteine-related transport mechanisms. We further show that cysteine concentrations in cervicovaginal lavage samples correlate with Lactobacillus abundance in vivo and that cystine uptake inhibitors selectively inhibit L. iners growth in vitro. Combining an inhibitor with metronidazole promotes L. crispatus dominance of defined BV-like communities in vitro by suppressing L. iners growth. Our findings enable a better understanding of L. iners biology and suggest candidate treatments to modulate the vaginal microbiota to improve reproductive health for women globally.

Antigen Presenting Cells Link the Female Genital Tract Microbiome to Mucosal Inflammation, With Hormonal Contraception as an Additional Modulator of Inflammatory Signatures.

The microbiome of the female genital tract (FGT) is closely linked to reproductive health outcomes. Diverse, anaerobe-dominated communities with low Lactobacillus abundance are associated with a number of adverse reproductive outcomes, such as preterm birth, cervical dysplasia, and sexually transmitted infections (STIs), including HIV. Vaginal dysbiosis is associated with local mucosal inflammation, which likely serves as a biological mediator of poor reproductive outcomes. Yet the precise mechanisms of this FGT inflammation remain unclear. Studies in humans have been complicated by confounding demographic, behavioral, and clinical variables. Specifically, hormonal contraception is associated both with changes in the vaginal microbiome and with mucosal inflammation. In this study, we examined the transcriptional landscape of cervical cell populations in a cohort of South African women with differing vaginal microbial community types. We also investigate effects of reproductive hormones on the transcriptional profiles of cervical cells, focusing on the contraceptive depot medroxyprogesterone acetate (DMPA), the most common form of contraception in sub-Saharan Africa. We found that antigen presenting cells (APCs) are key mediators of microbiome associated FGT inflammation. We also found that DMPA is associated with significant transcriptional changes across multiple cell lineages, with some shared and some distinct pathways compared to the inflammatory signature seen with dysbiosis. These results highlight the importance of an integrated, systems-level approach to understanding host-microbe interactions, with an appreciation for important variables, such as reproductive hormones, in the complex system of the FGT mucosa.

A proteome-wide atlas of lysine-reactive chemistry.

Recent advances in chemical proteomics have begun to characterize the reactivity and ligandability of lysines on a global scale. Yet, only a limited diversity of aminophilic electrophiles have been evaluated for interactions with the lysine proteome. Here, we report an in-depth profiling of >30 uncharted aminophilic chemotypes that greatly expands the content of ligandable lysines in human proteins. Aminophilic electrophiles showed disparate proteomic reactivities that range from selective interactions with a handful of lysines to, for a set of dicarboxaldehyde fragments, remarkably broad engagement of the covalent small-molecule-lysine interactions captured by the entire library. We used these latter 'scout' electrophiles to efficiently map ligandable lysines in primary human immune cells under stimulatory conditions. Finally, we show that aminophilic compounds perturb diverse biochemical functions through site-selective modification of lysines in proteins, including protein-RNA interactions implicated in innate immune responses. These findings support the broad potential of covalent chemistry for targeting functional lysines in the human proteome.

Modeling the temporal dynamics of cervicovaginal microbiota identifies targets that may promote reproductive health.

BACKGROUND: Cervicovaginal bacterial communities composed of diverse anaerobes with low Lactobacillus abundance are associated with poor reproductive outcomes such as preterm birth, infertility, cervicitis, and risk of sexually transmitted infections (STIs), including human immunodeficiency virus (HIV). Women in sub-Saharan Africa have a higher prevalence of these high-risk bacterial communities when compared to Western populations. However, the transition of cervicovaginal communities between high- and low-risk community states over time is not well described in African populations. RESULTS: We profiled the bacterial composition of 316 cervicovaginal swabs collected at 3-month intervals from 88 healthy young Black South African women with a median follow-up of 9 months per participant and developed a Markov-based model of transition dynamics that accurately predicted bacterial composition within a broader cross-sectional cohort. We found that Lactobacillus iners-dominant, but not Lactobacillus crispatus-dominant, communities have a high probability of transitioning to high-risk states. Simulating clinical interventions by manipulating the underlying transition probabilities, our model predicts that the population prevalence of low-risk microbial communities could most effectively be increased by manipulating the movement between L. iners- and L. crispatus-dominant communities. CONCLUSIONS: The Markov model we present here indicates that L. iners-dominant communities have a high probability of transitioning to higher-risk states. We additionally identify transitions to target to increase the prevalence of L. crispatus-dominant communities. These findings may help guide future intervention strategies targeted at reducing bacteria-associated adverse reproductive outcomes among women living in sub-Saharan Africa. Video Abstract.

Comparison of the Vaginal Microbiota in Postmenopausal Black and White Women.

BACKGROUND: We compared vaginal microbial communities in postmenopausal black and white women. METHODS: Shotgun sequencing of vaginal swabs from postmenopausal women self-identified as black or white was compared using MiRKAT. RESULTS: Vaginal community dominance by Lactobacillus crispatus or Lactobacillusgasseri was more common in 44 postmenopausal black women (n = 12, 27%) than among 44 matched white women (n = 2, 5%; P = .01). No individual taxa were significantly more abundant in either group. CONCLUSIONS: We identified small overall differences in vaginal microbial communities of black and white postmenopausal women. L. crispatus dominance was more common in black women. CLINICAL TRIALS REGISTRATION: NCT02516202 (MsFLASH05) and NCT01418209 (MsFLASH03).

Snapshots and ensembles of BTK and cIAP1 protein degrader ternary complexes.

Heterobifunctional chimeric degraders are a class of ligands that recruit target proteins to E3 ubiquitin ligases to drive compound-dependent protein degradation. Advancing from initial chemical tools, protein degraders represent a mechanism of growing interest in drug discovery. Critical to the mechanism of action is the formation of a ternary complex between the target, degrader and E3 ligase to promote ubiquitination and subsequent degradation. However, limited insights into ternary complex structures exist, including a near absence of studies on one of the most widely co-opted E3s, cellular inhibitor of apoptosis 1 (cIAP1). In this work, we use a combination of biochemical, biophysical and structural studies to characterize degrader-mediated ternary complexes of Bruton's tyrosine kinase and cIAP1. Our results reveal new insights from unique ternary complex structures and show that increased ternary complex stability or rigidity need not always correlate with increased degradation efficiency.

A Single Human VH-gene Allows for a Broad-Spectrum Antibody Response Targeting Bacterial Lipopolysaccharides in the Blood.

B cell receptors (BCRs) display a combination of variable (V)-gene-encoded complementarity determining regions (CDRs) and adaptive/hypervariable CDR3 loops to engage antigens. It has long been proposed that the former tune for recognition of pathogens or groups of pathogens. To experimentally evaluate this within the human antibody repertoire, we perform immune challenges in transgenic mice that bear diverse human CDR3 and light chains but are constrained to different human VH-genes. We find that, of six commonly deployed VH sequences, only those CDRs encoded by IGHV1-2∗02 enable polyclonal antibody responses against bacterial lipopolysaccharide (LPS) when introduced to the bloodstream. The LPS is from diverse strains of gram-negative bacteria, and the VH-gene-dependent responses are directed against the non-variable and universal saccrolipid substructure of this antigen. This reveals a broad-spectrum anti-LPS response in which germline-encoded CDRs naturally hardwire the human antibody repertoire for recognition of a conserved microbial target.

Discovery of Tyrosine Kinase 2 (TYK2) Inhibitor (PF-06826647) for the Treatment of Autoimmune Diseases.

Tyrosine kinase 2 (TYK2) is a member of the JAK kinase family that regulates signal transduction downstream of receptors for the IL-23/IL-12 pathways and type I interferon family, where it pairs with JAK2 or JAK1, respectively. On the basis of human genetic and emerging clinical data, a selective TYK2 inhibitor provides an opportunity to treat autoimmune diseases delivering a potentially differentiated clinical profile compared to currently approved JAK inhibitors. The discovery of an ATP-competitive pyrazolopyrazinyl series of TYK2 inhibitors was accomplished through computational and structurally enabled design starting from a known kinase hinge binding motif. With understanding of PK/PD relationships, a target profile balancing TYK2 potency and selectivity over off-target JAK2 was established. Lead optimization involved modulating potency, selectivity, and ADME properties which led to the identification of the clinical candidate PF-06826647 (22).

Complete Genome Sequences of Six Lactobacillus iners Strains Isolated from the Human Vagina.

Lactobacillus iners is a common member of the human vaginal microbiota, with a genome size smaller than that of other lactobacilli. Here, we report the complete genome sequences of six L. iners strains isolated from different vaginal swab specimens. Three strains were found to harbor ∼100-kbp plasmids, which were not known previously.

Sports chiropractors in Australia: a cross-sectional survey.

BACKGROUND: Whilst half of all Australian chiropractors report often treating athletes, there is insufficient evidence to characterise the sports chiropractor in Australia. OBJECTIVE: To perform a workforce survey of Sports Chiropractic Australia (SCA) members. METHODS: A 74-item web-based questionnaire collected information about practitioner and practice characteristics. Descriptive statistics summarised practitioner and patient characteristics, caseload and management approaches. RESULTS: SCA members were predominantly male (74%) with 11.3 (±8.4) years of clinical experience. Amateur or semi-professional sportspeople comprised 67% of SCA members' caseload. Athletes were most likely to present with a lower limb musculoskeletal condition (44%), followed by low back pain (34%). Nearly half (43%) of musculoskeletal conditions were co-managed with another healthcare practitioner. CONCLUSIONS: SCA members provided care for people of all sporting abilities, ranging from recreational to elite athletes, but most typically at the non-elite level. SCA members almost exclusively treat musculoskeletal conditions and apply various modalities in the management of athletes and sportspeople.

CONTEXTE: Bien que la moitié des chiropraticiens australiens affirment soigner souvent des athlètes, on ne dispose pas de données suffisantes pour caractériser les chiropraticiens du sport australiens. OBJECTIF: Mener une enquête sur l'effectif auprès des membres de la Sports Chiropractic Australia (SCA). MÉTHODOLOGIE: Un questionnaire en ligne comprenant 74 questions a servi à recueillir des données sur les caractéristiques du praticien et de sa clientèle. Des données statistiques descriptives ont résumé les caractéristiques du praticien et celles du patient, le nombre de cas et les méthodes thérapeutiques. RÉSULTATS: Les membres de la SCA étaient majoritairement des hommes (74 %) possédant 11,3 (± 8,4) années d'expérience en clinique. Les sportifs amateurs et les sportifs semi-professionnels constituaient 67 % des membres de la SCA. Les athlètes présentaient le plus souvent un trouble musculosquelettique à un membre inférieur (44 %) et des lombalgies (34 %). Presque la moitié (43 %) des troubles musculosquelettiques étaient aussi traités par un autre professionnel de la santé. CONCLUSIONS: Les membres de la SCA traitent des gens ayant toutes sortes de capacités sportives, à partir de sportifs amateurs jusqu'à des athlètes d'élite, mais le plus souvent à des sportifs de niveau non compétitif. Les membres de la SCA traitent presque exclusivement des troubles musculosquelettiques et utilisent diverses modalités en traitant des athlètes et des sportifs.

Tourist photographs as a scalable framework for wildlife monitoring in protected areas.

Protected areas are critical to conservation efforts in the face of rapid biodiversity declines [1]. Yet the resources for conservation are often limited and shared amongst many competing priorities [2]. As a consequence, even basic monitoring surveys are absent within most protected areas [3]. Although a range of wildlife monitoring methods exist, considerable focused survey effort is often required to yield accurate and precise estimates [4]. This makes monitoring difficult to sustain or replicate, limiting access to the data required for evidence-based conservation decisions. Citizen-scientists have been proposed as an important complement to the finite resources available for basic monitoring within protected areas [5]; however, the full potential of this approach has yet to be realised. Wildlife tourists and guides are especially focussed on encountering and photographing fauna and flora, yet the data collected in these efforts is rarely harnessed for conservation monitoring within protected areas. A detailed understanding of photographic tourism's potential role in wildlife monitoring has been lacking, but is essential for the development of new tools to harness the data being collected through tourism. Here, we demonstrate that tourist-contributed data can aid wildlife monitoring in protected areas by providing population estimates of large carnivores comparable to those from traditional survey methods. Our approach could capitalize upon the immense number of wildlife photographs being taken daily as part of the global > 30-billion USD, wildlife-based tourism industry.

Extensive transmission of microbes along the gastrointestinal tract.

The gastrointestinal tract is abundantly colonized by microbes, yet the translocation of oral species to the intestine is considered a rare aberrant event, and a hallmark of disease. By studying salivary and fecal microbial strain populations of 310 species in 470 individuals from five countries, we found that transmission to, and subsequent colonization of, the large intestine by oral microbes is common and extensive among healthy individuals. We found evidence for a vast majority of oral species to be transferable, with increased levels of transmission in colorectal cancer and rheumatoid arthritis patients and, more generally, for species described as opportunistic pathogens. This establishes the oral cavity as an endogenous reservoir for gut microbial strains, and oral-fecal transmission as an important process that shapes the gastrointestinal microbiome in health and disease.

Feeding responses of the golden jackal after reduction of anthropogenic food subsidies.

Little is known of the resources that limit or promote the rapidly expanding golden jackal (Canis aureus) population in Europe. We hypothesised that in an area of intensive big game hunting, a reduction of the main food resource (human subsidised big game viscera) would result in dietary switching. We used multivariate analyses to test whether the dietary composition of 200 jackal stomachs varied between two 2-yearly survey occasions, the first without big game viscera removal (availability of 68 kg viscera/year/km2) followed by a period with viscera removal (minimum of 50 kg of viscera/year/km2 removed). The proportion of empty stomachs and the stomach wet content weight did not differ between the two periods. Even after the reduction of food subsidies, the primary food of jackals was viscera and carrion from wild ungulates (frequency of occurrence: 45% vs. 30%; wet weight: 55% vs. 29%, respectively), and scavenging was not affected by season or sex. Log-linear analysis of frequency data revealed no significant differences between survey occasions in consumption of either food type. MANCOVA of wet weight data revealed that in the first period with food subsidies jackals consumed a higher proportion of adult wild boar (11.6% vs. 1.3%; from predation or scavenging), while juvenile wild boar (0 vs. 11.8%; from predation or scavenging), domestic animals (0.8% vs. 6.2%; mostly from scavenging) and invertebrates (2.6% vs. 4.1%) increased in the second period. The stomachs in the second survey occasion contained more varied food items, but the trophic niche was not significantly wider. The feeding responses of this mesopredator to the reduction of food subsidies were less pronounced than expected. Because in high big game density areas, wild ungulate carrion from different mortality causes are available in high quantities throughout the year, predator populations can be maintained despite the high amount of viscera removal.

Dual Inhibition of TYK2 and JAK1 for the Treatment of Autoimmune Diseases: Discovery of (( S)-2,2-Difluorocyclopropyl)((1 R,5 S)-3-(2-((1-methyl-1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)methanone (PF-06700841).

Cytokine signaling is an important characteristic of autoimmune diseases. Many pro-inflammatory cytokines signal through the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway. JAK1 is important for the γ-common chain cytokines, interleukin (IL)-6, and type-I interferon (IFN) family, while TYK2 in addition to type-I IFN signaling also plays a role in IL-23 and IL-12 signaling. Intervention with monoclonal antibodies (mAbs) or JAK1 inhibitors has demonstrated efficacy in Phase III psoriasis, psoriatic arthritis, inflammatory bowel disease, and rheumatoid arthritis studies, leading to multiple drug approvals. We hypothesized that a dual JAK1/TYK2 inhibitor will provide additional efficacy, while managing risk by optimizing selectivity against JAK2 driven hematopoietic changes. Our program began with a conformationally constrained piperazinyl-pyrimidine Type 1 ATP site inhibitor, subsequent work led to the discovery of PF-06700841 (compound 23), which is in Phase II clinical development (NCT02969018, NCT02958865, NCT03395184, and NCT02974868).

Delineating the role of cooperativity in the design of potent PROTACs for BTK.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.