A data science roadmap for open science organizations engaged in early-stage drug discovery

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

Target 2035-update on the quest for a probe for every protein

Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome

Innate immunity atherosclerosis : Signaling pattern recognition receptors and an antimicrobial peptide

Atherosclerosis, a disease characterized by fibroinflammatory lipid lesions in the arterial wall, is the leading cause of mortality in the Western world. For decades research has been aimed at dissecting the pathogenesis of this multifactorial disease. Recently vascular inflammation induced by both innate and adaptive immune responses has been shown to be important for the disease development. Innate immunity is the primary host defense to infections, and elicited by the recognition of microbial ligands by pattern recognition receptors (PRRs). Infections have been implicated in the pathogenesis of atherosclerosis and its complications such as myocardial infarction (MI). The aim of this thesis was to elucidate the function of innate immunity in human atherosclerosis. Specific objectives were to investigate expression and functional relevance of two types of PRRs: Toll-like receptors (TLRs) and nucleotide oligomerization domain protein 1 (NOD1) and NOD2, and to characterize the antimicrobial peptide, LL-37, an innate host defense peptide with effector and regulatory functions in innate immunity, in the disease. We investigated the expression of 9 TLRs in human atherosclerotic lesions and normal vessels. Our data show that endothelial cells of normal artery express low levels of TLRs. However, TLR1, TLR2 and TLR4, were upregulated both at the mRNA and protein level in atherosclerotic lesions compared to normal arteries. TLR2 and TLR4 were expressed by macrophages and some endothelial cells, and co-localized with activated NF-κB signaling in the lesions. These findings suggest that TLRs on endothelial cells constitute the first line of vascular defense against infection, and may be linked to the inflammatory response in atherosclerosis. To assess the impact of TLR4-mediated innate immunity on the risk of MI, the Stockholm Heart Epidemiology Program (SHEEP) was screened for two functional polymorphisms in the TLR4 gene. Hyporesponsive TLR4 variants were associated with a higher risk for MI in men. Although the underlying mechanisms remain to be elucidated, this genetic study indicates that alteration in TLR4- mediated innate immunity may affect progression of coronary artery disease. In addition to TLRs, vascular endothelial cells also express basal levels of the intracellular PRRs, NOD1 and NOD2. Yet, NOD2 was markedly increased in atherosclerotic lesions and expressed predominantly by macrophages and some endothelial cells. NOD2 expression in human endothelial cells could be induced by proinflammatory cytokines and activation of NOD2 induced the expression of intercellular adhesion molecule 1 (ICAM-1) and monocyte chemoattractant protein 1 (MCP-1), two proteins with important roles in the recruitment and activation of leukocytes during vascular inflammation. These findings imply that NOD2 might be a key mechanism for sensing intracellular infection in atherogenesis. In an effort to identify innate effectors, we analyzed antimicrobial peptides and found that LL-37 was produced in atherosclerotic lesions, predominantly by macrophages. Expression of the murine homologue of LL-37 could be induced in macrophages by Chlamydia pneumoniae, a bacterium associated with cardiovascular disease. Stimulation of endothelial cells with LL-37 led to dose-dependent induction of a limited set of proinflammatory genes, signifying a novel proinflammatory pathway involved in disease development. Although LL-37 exhibits broad microbicidal activity, this peptide had no microbicidal effect on Chlamydia pneumoniae. In summary, our results reveal that the endothelium is the first line vascular defense of the artery. It is characterized by expression of a spectrum of PRRs, capable of sensing extracellular as well as intracellular pathogens. Whether infections contribute to atherosclerosis remains controversial but the results of this thesis suggest that the machinery for microbial recognition and effector functions is further enhanced in atherosclerosis. By identifying a large array of PRRs in atherosclerosis together with the characterization of the antimicrobial peptide, LL-37, the work of this thesis hopefully adds novel understanding of innate immunity in the disease

MicroRNA-21 targets the vitamin D-dependent antimicrobial pathway in leprosy.

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

Regular Use of Depot Medroxyprogesterone Acetate Causes Thinning of the Superficial Lining and Apical Distribution of Human Immunodeficiency Virus Target Cells in the Human Ectocervix

Background The hormonal contraceptive depot medroxyprogesterone acetate (DMPA) may be associated with an increased risk of acquiring human immunodeficiency virus (HIV). We hypothesize that DMPA use influences the ectocervical tissue architecture and HIV target cell localization. Methods Quantitative image analysis workflows were developed to assess ectocervical tissue samples collected from DMPA users and control subjects not using hormonal contraception. Results Compared to controls, the DMPA group exhibited a significantly thinner apical ectocervical epithelial layer and a higher proportion of CD4(+)CCR5(+) cells with a more superficial location. This localization corresponded to an area with a nonintact E-cadherin net structure. CD4(+)Langerin(+) cells were also more superficially located in the DMPA group, although fewer in number compared to the controls. Natural plasma progesterone levels did not correlate with any of these parameters, whereas estradiol levels were positively correlated with E-cadherin expression and a more basal location for HIV target cells of the control group. Conclusions DMPA users have a less robust epithelial layer and a more apical distribution of HIV target cells in the human ectocervix, which could confer a higher risk of HIV infection. Our results highlight the importance of assessing intact genital tissue samples to gain insights into HIV susceptibility factors. By developing quantitative image analysis workflows, we revealed that women taking the hormonal contraceptive depot medroxyprogesterone acetate had a less robust ectocervical epithelium and a more superficial distribution of HIV target cells. This could confer a higher risk of HIV infection

Regular use of depot medroxyprogesterone acetate causes thinning of the superficial lining and apical distribution of HIV target cells in the human ectocervix.

BACKGROUND: The hormonal contraceptive depot medroxyprogesterone acetate (DMPA) may be associated with an increased risk of acquiring human immunodeficiency virus (HIV). We hypothesize that DMPA use influences the ectocervical tissue architecture and HIV target cell localization. METHODS: Quantitative image analysis workflows were developed to assess ectocervical tissue samples collected from DMPA users and control subjects not using hormonal contraception. RESULTS: Compared to controls, the DMPA group exhibited a significantly thinner apical ectocervical epithelial layer and a higher proportion of CD4+CCR5+ cells with a more superficial location. This localization corresponded to an area with a non-intact E-cadherin net structure. CD4+Langerin+ cells were also more superficially located in the DMPA group, while fewer in number compared to the controls. Natural plasma progesterone levels did not correlate with any of these parameters, whereas estradiol levels were positively correlated with E-cadherin expression and a more basal location for HIV target cells of the control group. CONCLUSIONS: DMPA users have a less robust epithelial layer and a more apical distribution of HIV target cells in the human ectocervix, which could confer a higher risk of HIV infection. Our results highlight the importance of assessing intact genital tissue samples to gain insights into HIV susceptibility factors

Distinct cervical tissue-adherent and luminal microbiome communities correlate with mucosal host gene expression and protein levels in Kenyan sex workers

Abstract 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. Video Abstrac

Impact of Q-Griffithsin anti-HIV microbicide gel in non-human primates : In situ analyses of epithelial and immune cell markers in rectal mucosa

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

NOD2-Mediated Innate Immune Signaling Regulates the Eicosanoids in Atherosclerosis

<p>Objective-The activity of eicosanoid pathways is critical to the inflammatory and immune responses that are associated with the progression of atherosclerosis. Yet, the signals that regulate these pathways are poorly understood. Here, we address whether the innate immune signals of nucleotide-binding oligomerization domain-containing protein (NOD) 2 affect eicosanoids metabolism in atherosclerosis.</p><p>Approach and Results-Analysis of human carotid plaques revealed that NOD2 was abundantly expressed at both mRNA and protein levels by endothelial cells and macrophages. Stimulation of NOD2 in ex vivo-cultured carotid plaques by muramyl dipeptide, an extrinsic ligand of NOD2, led to release of prostaglandin E-2, upregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, and to downregulation of cyclooxygenase-1. NOD2 was coexpressed with cyclooxygenase-2 in lesional macrophages. NOD2-induced cyclooxygenase-2 expression in macrophages was dependent on p38 mitogen-activated protein kinase activation and was mediated by interleukin-1 beta and tumor necrosis factor-alpha. Selective lipidomic analysis of the eicosanoids released by the carotid plaques characterized the metabolites of 12-, 5-, and 15-lipoxygenase as the predominant eicosanoids that were produced by the atherosclerotic lesion in the absence of additional stimuli. Unlike the prostaglandin E-2 pathway, metabolic activity of the lipoxygenase pathways was not altered on the short-term activation of NOD2 in carotid plaques.</p><p>Conclusions-These results suggest that atherosclerosis may involve enhanced NOD2-mediated innate immunity. Activation of NOD2 preferentially upregulates the prostaglandin E-2 pathway. Nevertheless, lipoxygenase pathways, such as 12-lipoxygenase, predominate the basal synthesis and metabolism of eicosanoids in atherosclerotic plaques. These findings provide new insights into the regulation of eicosanoids in atherosclerosis.</p>