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The Structural Genomics Consortium is an international open science research organization with a focus on accelerating early-stage drug discovery, namely hit discovery and optimization. We, as many others, believe that artificial intelligence (AI) is poised to be a main accelerator in the field. The question is then how to best benefit from recent advances in AI and how to generate, format and disseminate data to enable future breakthroughs in AI-guided drug discovery. We present here the recommendations of a working group composed of experts from both the public and private sectors. Robust data management requires precise ontologies and standardized vocabulary while a centralized database architecture across laboratories facilitates data integration into high-value datasets. Lab automation and opening electronic lab notebooks to data mining push the boundaries of data sharing and data modeling. Important considerations for building robust machine-learning models include transparent and reproducible data processing, choosing the most relevant data representation, defining the right training and test sets, and estimating prediction uncertainty. Beyond data-sharing, cloud-based computing can be harnessed to build and disseminate machine-learning models. Important vectors of acceleration for hit and chemical probe discovery will be (1) the real-time integration of experimental data generation and modeling workflows within design-make-test-analyze (DMTA) cycles openly, and at scale and (2) the adoption of a mindset where data scientists and experimentalists work as a unified team, and where data science is incorporated into the experimental design. Artificial intelligence is greatly accelerating research in drug discovery, but its development is still hindered by the lack of available data. Here the authors present data management and data science recommendations to help reach AI’s potential in the field.

We investigated the mechanisms by which T-cell cytokines are able to influence the Toll-like receptor (TLR)-induced, vitamin D-dependent antimicrobial pathway in human monocytes. T-cell cytokines differentially influenced TLR2/1-induced expression of the antimicrobial peptides cathelicidin and DEFB4, being up-regulated by IFN-γ, down-regulated by IL-4, and unaffected by IL-17. The Th1 cytokine IFN-γ up-regulated TLR2/1 induction of 25-hydroxyvitamin D-1α-hydroxylase (i.e., CYP27B1), leading to enhanced bioconversion of 25-hydroxyvitamin D₃ (25D₃) to its active metabolite 1,25D₃. In contrast, the Th2 cytokine IL-4, by itself and in combination with the TLR2/1 ligand, induced catabolism of 25D₃ to the inactive metabolite 24,25D₃, and was dependent on expression of vitamin D-24-hydroxylase (i.e., CYP24A1). Therefore, the ability of T-cell cytokines to differentially control monocyte vitamin D metabolism represents a mechanism by which cell-mediated immune responses can regulate innate immune mechanisms to defend against microbial pathogens.

One aspirational goal of computational chemistry is to predict potent and drug-like binders for any protein, such that only those that bind are synthesized. In this Roadmap, we describe the launch of Critical Assessment of Computational Hit-finding Experiments (CACHE), a public benchmarking project to compare and improve small-molecule hit-finding algorithms through cycles of prediction and experimental testing. Participants will predict small-molecule binders for new and biologically relevant protein targets representing different prediction scenarios. Predicted compounds will be tested rigorously in an experimental hub, and all predicted binders as well as all experimental screening data, including the chemical structures of experimentally tested compounds, will be made publicly available and not subject to any intellectual property restrictions. The ability of a range of computational approaches to find novel binders will be evaluated, compared and openly published. CACHE will launch three new benchmarking exercises every year. The outcomes will be better prediction methods, new small-molecule binders for target proteins of importance for fundamental biology or drug discovery and a major technological step towards achieving the goal of Target 2035, a global initiative to identify pharmacological probes for all human proteins. Critical Assessment of Computational Hit-finding Experiments (CACHE) is a public benchmarking project to compare and improve computational small-molecule hit-finding approaches through cycles of prediction, compound synthesis and experimental testing. By that, CACHE will enable a more efficient and effective approach to hit identification and drug discovery.

The ability of Mycobacterium leprae to upregulate miRNA-21 provides an effective mechanism for the pathogen to escape from the vitamin D–dependent antimicrobial pathway. Leprosy provides a model to investigate mechanisms of immune regulation in humans, given that the disease forms a spectrum of clinical presentations that correlate with host immune responses. Here we identified 13 miRNAs that were differentially expressed in the lesions of subjects with progressive lepromatous (L-lep) versus the self-limited tuberculoid (T-lep) disease. Bioinformatic analysis revealed a significant enrichment of L-lep–specific miRNAs that preferentially target key immune genes downregulated in L-lep versus T-lep lesions. The most differentially expressed miRNA in L-lep lesions, hsa-mir-21, was upregulated in Mycobacterium leprae –infected monocytes. By directly downregulating Toll-like receptor 2/1 heterodimer (TLR2/1)-induced CYP27B1 and IL1B expression as well as indirectly upregulating interleukin-10 (IL-10), hsa-mir-21 inhibited expression of the genes encoding two vitamin D–dependent antimicrobial peptides, CAMP and DEFB4A . Conversely, knockdown of hsa-mir-21 in M. leprae –infected monocytes enhanced expression of CAMP and DEFB4A and restored TLR2/1-mediated antimicrobial activity against M. leprae . Therefore, the ability of M. leprae to upregulate hsa-mir-21 targets multiple genes associated with the immunologically localized disease form, providing an effective mechanism to escape from the vitamin D–dependent antimicrobial pathway.

Recurrent vulvovaginal candidiasis (RVVC) is a common condition characterized by frequent relapses, often without a clearly identifiable cause. Fluconazole (FLZ) is the standard treatment but concerns about emerging resistance and drug interaction highlight the need for alternative therapies. Chlorhexidine gluconate (CHG), known for its antifungal and biofilm-disrupting properties, has been proposed as a potential alternative. To evaluate the efficacy, tolerability, and microbiome impact of a vaginal CHG formulation compared to oral FLZ in the treatment of RVVC caused by Candida albicans. An open label randomized non-inferiority trial was conducted to compare vaginal CHG and FLZ treatments. Primary outcome was negative cultures for C. albicans. Resistance profiles and changes in the vaginal microbiome composition were also assessed. The study was terminated early due to local irritation associated with CHG and the study design was transitioned into a pilot study. CHG treatment showed comparable efficacy to FLZ in clearing C. albicans infections and preventing recurrences, although the sample size was limited. All 11 participants in the FLZ group cleared the infection after one week treatment, compared to 9 out 11 in the CHG group. No harmful changes to the vaginal microbiome were observed in the CGH or FLZ group. FLZ promoted a shift toward Lactobacillus crispatus dominance, unlike CHG. Notably, 16% of C. albicans isolates exhibited reduced susceptibility or resistance to FLZ. Due to the limited number of participants, we cannot conclusively determine whether CHG is non-inferior to FLZ in terms of efficacy for clearing acute C. albicans infections or preventing recurrences. While the current CHG formulation caused local irritation and is not suitable for clinical use, its antifungal and biofilm-inhibiting properties remain promising. Further development of less irritative CHG formulations may offer a valuable alternative for RVVC treatment, particularly in the context of rising FLZ resistance.

Depot medroxyprogesterone acetate (DMPA) is an injectable hormonal contraceptive used by millions of women worldwide. However, experimental studies have associated DMPA use with genital epithelial barrier disruption and mucosal influx of human immunodeficiency virus (HIV) target cells. We explored the underlying molecular mechanisms of these findings. Ectocervical biopsies and cervicovaginal lavage (CVL) specimens were collected from HIV-seronegative Kenyan sex workers using DMPA ( n = 32) or regularly cycling controls ( n = 64). Tissue samples were assessed by RNA-sequencing and quantitative imaging analysis, whereas protein levels were measured in CVL samples. The results suggested a DMPA-associated upregulation of genes involved in immune regulation, including genes associated with cytokine-mediated signaling and neutrophil-mediated immunity. A transcription factor analysis further revealed DMPA-associated upregulation of RELA and NFKB1 which are involved in several immune activation pathways. Several genes significantly downregulated in the DMPA versus the control group were involved in epithelial structure and function, including genes encoding keratins, small proline-rich proteins, and cell-cell adhesion proteins. Pathway analyses indicated DMPA use was associated with immune activation and suppression of epithelium development, including keratinization and cornification processes. The cervicovaginal microbiome composition ( Lactobacillus dominant and non- Lactobacillus dominant) had no overall interactional impact on the DMPA associated tissue gene expression. Imaging analysis verified that DMPA use was associated with an impaired epithelial layer as illustrated by staining for the selected epithelial junction proteins E-cadherin, desmoglein-1 and claudin-1. Additional staining for CD4 + cells revealed a more superficial location of these cells in the ectocervical epithelium of DMPA users versus controls. Altered protein levels of SERPINB1 and ITIH2 were further observed in the DMPA group. Identification of specific impaired epithelial barrier structures at the gene expression level, which were verified at the functional level by tissue imaging analysis, illustrates mechanisms by which DMPA adversely may affect the integrity of the genital mucosa.

The cervicovaginal microbiome is a key biological determinant of human immunodeficiency virus (HIV) susceptibility, but its underlying impact on the ectocervical transcriptional landscape is unclear. Ectocervical tissue samples from Kenyan female sex workers were categorized into pre-defined cervicovaginal microbiome groups based on dominant compositions: Lactobacillus crispatus/acidophilus , Lactobacillus iners , Gardnerella , and ‘highly diverse’. The tissue samples (n = 21) were assessed using spatial transcriptomics, revealing three epithelial, one mixed border, and nine submucosal gene clusters. Differential gene expression analysis across the microbiome groups and gene clusters identified 3,771 unique genes. The highly diverse microbiome group associated with the largest differences, mostly located near the epithelial basal membrane, encompassing genes involved in epithelial maintenance, submucosal extracellular matrix structures, and immune function. The L. crispatus/acidophilus -dominated group was identified by genes involved in active immune engagement, supporting mucosal barrier integrity. Weighted gene co-expression analysis confirmed tissue-wide altered gene expression associated with all microbiome groups and with individual bacterial taxa. Despite the assumption that microbiome colonization is restricted to the luminal surface, the transcriptional landscape was affected throughout the mucosa, with the most pronounced effect near both sides of the basal membrane. This broad association with the mucosal barrier integrity could affect susceptibility to HIV acquisition.

Chronic systemic immune activation significantly influences human immunodeficiency virus (HIV) disease progression. Despite evidence of a pro-inflammatory environment in the genital tract of HIV-infected women, comprehensive investigations into cervical tissue from this region remain limited. Similarly, the consequences of chronic HIV infection on the integrity of the female genital epithelium are poorly understood, despite its importance in HIV transmission and replication. Ectocervical biopsies were obtained from HIV-seropositive (n = 14) and HIV-seronegative (n = 47) female Kenyan sex workers. RNA sequencing and bioimage analysis of epithelial junction proteins (E-cadherin, desmoglein-1, claudin-1, and zonula occludens-1) were conducted, along with CD4 staining. RNA sequencing revealed upregulation of immunoregulatory genes in HIV-seropositive women, primarily associated with heightened T cell activity and interferon signaling, which further correlated with plasma viral load. Transcription factor analysis confirmed the upregulation of pro-inflammatory transcription factors, such as RELA , NFKB1 , and IKZF3 , which facilitates HIV persistence in T cells. Conversely, genes and pathways associated with epithelial barrier function and structure were downregulated in the context of HIV. Digital bioimage analysis corroborated these findings, revealing significant disruption of various epithelial junction proteins in ectocervical tissues of the HIV-seropositive women. Thus, chronic HIV infection associated with ectocervical inflammation, characterized by induced T cell responses and interferon signaling, coupled with epithelial disruption. These alterations may influence HIV transmission and heighten susceptibility to other sexually transmitted infections. These findings prompt exploration of therapeutic interventions to address HIV-related complications and mitigate the risk of sexually transmitted infection transmission.

Natural-product derived lectins can function as potent viral inhibitors with minimal toxicity as shown in vitro and in small animal models. We here assessed the effect of rectal application of an anti-HIV lectin-based microbicide Q-Griffithsin (Q-GRFT) in rectal tissue samples from rhesus macaques. E-cadherin + cells, CD4 + cells and total mucosal cells were assessed using in situ staining combined with a novel customized digital image analysis platform. Variations in cell numbers between baseline, placebo and Q-GRFT treated samples were analyzed using random intercept linear mixed effect models. The frequencies of rectal E-cadherin + cells remained stable despite multiple tissue samplings and Q-GRFT gel (0.1%, 0.3% and 1%, respectively) treatment. Whereas single dose application of Q-GRFT did not affect the frequencies of rectal CD4 + cells, multi-dose Q-GRFT caused a small, but significant increase of the frequencies of intra-epithelial CD4 + cells (placebo: median 4%; 1% Q-GRFT: median 7%) and of the CD4 + lamina propria cells (placebo: median 30%; 0.1–1% Q-GRFT: median 36–39%). The resting time between sampling points were further associated with minor changes in the total and CD4 + rectal mucosal cell levels. The results add to general knowledge of in vivo evaluation of anti-HIV microbicide application concerning cellular effects in rectal mucosa.

Background The majority of studies characterizing female genital tract microbiota have focused on luminal organisms, while the presence and impact of tissue-adherent ectocervical microbiota remain incompletely understood. Studies of luminal and tissue-associated bacteria in the gastrointestinal tract suggest that these communities may have distinct roles in health and disease. Here, we performed a multi-omics characterization of paired luminal and tissue samples collected from a cohort of Kenyan female sex workers. Results We identified a tissue-adherent bacterial microbiome, with a higher alpha diversity than the luminal microbiome, in which dominant genera overall included Gardnerella and Lactobacillus , followed by Prevotella , Atopobium , and Sneathia . About half of the L. iners- dominated luminal samples had a corresponding Gardnerella- dominated tissue microbiome. Broadly, the tissue-adherent microbiome was associated with fewer differentially expressed host genes than the luminal microbiome. Gene set enrichment analysis revealed that L. crispatus- dominated tissue-adherent communities were associated with protein translation and antimicrobial activity, whereas a highly diverse microbial community was associated with epithelial remodeling and pro-inflammatory pathways. Tissue-adherent communities dominated by L. iners and Gardnerella were associated with lower host transcriptional activity. Tissue-adherent microbiomes dominated by Lactobacillus and Gardnerella correlated with host protein profiles associated with epithelial barrier stability, although with a more pro-inflammatory profile for the Gardnerella -dominated microbiome group. Tissue samples with a highly diverse composition had a protein profile representing cell proliferation and pro-inflammatory activity. Conclusion We identified ectocervical tissue-adherent bacterial communities in all study participants of a female sex worker cohort. These communities were distinct from cervicovaginal luminal microbiota in a significant proportion of individuals. We further revealed that bacterial communities at both sites correlated with distinct host gene expression and protein levels. The tissue-adherent bacterial community could possibly act as a reservoir that seed the lumen with less optimal, non- Lactobacillus , bacteria. 1vNfGdLTcr4_vB6d-MCta- Video Abstract