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Endometriosis is a common, inflammatory pain disorder comprised of disease in the pelvis and abnormal uterine lining and ovarian function that affects ∼200 million women of reproductive age worldwide and up to 50% of those with pelvic pain and/or infertility. Existing medical treatments for endometriosis-related pain are often ineffective, with individuals experiencing minimal or transient pain relief or intolerable side effects limiting long-term use - thus underscoring the pressing need for new drug treatment strategies. In this study, we applied a computational drug repurposing pipeline to endometrial gene expression data in the setting of endometriosis and controls in an unstratified manner as well as stratified by disease stage and menstrual cycle phase in order to identify potential therapeutics from existing drugs, based on expression reversal. Out of the 3,131 unique genes differentially expressed by at least one of six endometriosis signatures, only 308, or 9.8%, were in common. Similarities were more pronounced when looking at therapeutic predictions: 221 out of 299 drugs identified across the six signatures, or 73.9%, were shared, and the majority of predicted compounds were concordant across disease stage-stratified and cycle phase-stratified signatures. Our pipeline returned many known treatments as well as novel candidates. We selected the NSAID fenoprofen, the top therapeutic candidate for the unstratified signature and among the top-ranked drugs for the stratified signatures, for further investigation. Our drug target network analysis shows that fenoprofen targets PPARG and PPARA which affect the growth of endometrial tissue, as well as PTGS2 (i.e., COX2), an enzyme induced by inflammation with significantly increased gene expression demonstrated in patients with endometriosis who experience severe dysmenorrhea. NSAIDs are widely prescribed for endometriosis-related dysmenorrhea and nonmenstrual pelvic pain. Our analysis of clinical records across University of California healthcare systems revealed that while NSAIDs have been commonly prescribed to the 61,306 patients identified with diagnoses of endometriosis, dysmenorrhea, or chronic pelvic pain (36,543, 59.61%), fenoprofen was infrequently prescribed to those with these conditions (5, 0.008%). We tested the effect of fenoprofen in an established rat model of endometriosis and determined that it successfully alleviated endometriosis-associated vaginal hyperalgesia, a surrogate marker for endometriosis-related pain. These findings validate fenoprofen as a potential endometriosis therapeutic and suggest the utility of future investigation into additional drug targets identified.

Endometriosis is a leading cause of pain and infertility affecting millions of women globally. Identifying biologic and genetic effects on DNA methylation (DNAm) in endometrium increases understanding of mechanisms that influence gene regulation predisposing to endometriosis and offers an opportunity for novel therapeutic target discovery. Herein, we characterize variation in endometrial DNAm and its association with menstrual cycle phase, endometriosis, and genetic variants through analysis of genome-wide genotype data and methylation at 759,345 DNAm sites in endometrial samples from 984 deeply-phenotyped participants. We identify significant differences in DNAm profiles between menstrual cycle phases and at four DNAm sites between stage III/IV endometriosis and controls. We estimate that 15.4% of the variation in endometriosis is captured by DNAm, and identify DNAm networks associated with endometriosis. DNAm quantitative trait locus (mQTL) analysis identified 118,185 independentcis-mQTL including some tissue-specific effects. We find significant differences in DNAm profiles between endometriosis sub- phenotypes and a significant association between genetic regulation of methylation in endometrium and disease risk, providing functional evidence for genomic targets contributing to endometriosis risk and pathogenesis.

Research questionAdenomyosis is a common uterine disorder of uncertain causes. Can transcriptomic analyses of the endometrium and myometrium reveal potential mechanisms underlying adenomyosis pathogenesis?DesignTranscriptomic profiles of eutopic endometrium and myometrium from women with and without diffuse adenomyosis and with symptomatic FIGO type 2-5 fibroids in the proliferative phase of the menstrual cycle were assessed using RNA sequencing and bioinformatic analysis. Differentially expressed genes (DEG) and potential pathways were validated by quantitative reverse transcription polymerase chain reaction, immunoblotting and Masson staining, using additional clinical samples.ResultsTop biological processes in the endometrium of women with versus without adenomyosis, enriched from DEG, comprised inflammation, extracellular matrix (ECM) organization, collagen degradation and hyaluronan synthesis, which are key in cell migration and cell movement. Top biological processes enriched from DEG in the myometrium of women with versus without adenomyosis revealed ECM organization dysfunction, abnormal sensory pain perception and gamma aminobutyric acid (GABA) synaptic transmission. Dysregulation of prolactin signalling was also enriched in eutopic endometrium and in the myometrium of women with adenomyosis.ConclusionsOverall, our results support the invasive endometrium theory in the pathogenesis of adenomyosis, in which inflammation induces ECM remodelling resulting in a track for subsequent endometrial collective cell migration and onset of adenomyosis. Moreover, abnormal myometrial GABA synaptic transmission may contribute to dysmenorrhoea in women with adenomyosis and is a possible target for novel therapeutic development. Prolactin signalling abnormalities may serve as another opportunity for therapeutic intervention.

INTRODUCTION: Although the genome is often called the blueprint of an organism, it is perhaps more accurate to describe it as a parts list composed of the various genes that may or may not be used in the different cell types of a multicellular organism. Although nearly every cell in the body has essentially the same genome, each cell type makes different use of that genome and expresses a subset of all possible genes. This has motivated efforts to characterize the molecular composition of various cell types within humans and multiple model organisms, both by transcriptional and proteomic approaches. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. RATIONALE: One caveat to current approaches to make cell atlases is that individual organs are often collected at different locations, collected from different donors, and processed using different protocols. Controlled comparisons of cell types between different tissues and organs are especially difficult when donors differ in genetic background, age, environmental exposure, and epigenetic effects. To address this, we developed an approach to analyzing large numbers of organs from the same individual. RESULTS: We collected multiple tissues from individual human donors and performed coordinated single-cell transcriptome analyses on live cells. The donors come from a range of ethnicities, are balanced by gender, have a mean age of 51 years, and have a variety of medical backgrounds. Tissue experts used a defined cell ontology terminology to annotate cell types consistently across the different tissues, leading to a total of 475 distinct cell types with reference transcriptome profiles. The full dataset can be explored online with the cellxgene tool. Data were collected for the bladder, blood, bone marrow, eye, fat, heart, kidney, large intestine, liver, lung, lymph node, mammary, muscle, pancreas, prostate, salivary gland, skin, small intestine, spleen, thymus, tongue, trachea, uterus, and vasculature. Fifty-nine separate specimens in total were collected, processed, and analyzed, and 483,152 cells passed quality control filtering. On a per-compartment basis, the dataset includes 264,824 immune cells, 104,148 epithelial cells, 31,691 endothelial cells, and 82,478 stromal cells. Working with live cells, as opposed to isolated nuclei, ensured that the dataset includes all mRNA transcripts within the cell, including transcripts that have been processed by the cell’s splicing machinery, thereby enabling insight into variation in alternative splicing. The Tabula Sapiens also provided an opportunity to densely and directly sample the human microbiome throughout the gastrointestinal tract. The intestines from two donors were sectioned into five regions: the duodenum, jejunum, ileum, and ascending and sigmoid colon. Each section was transected, and three to nine samples were collected from each location, followed by amplification and sequencing of the 16S ribosomal RNA gene. CONCLUSION: The Tabula Sapiens has revealed discoveries relating to shared behavior and subtle, organ-specific differences across cell types. We found T cell clones shared between organs and characterized organ-dependent hypermutation rates among B cells. Endothelial cells and macrophages are shared across tissues, often showing subtle but clear differences in gene expression. We found an unexpectedly large and diverse amount of cell type–specific RNA splice variant usage and discovered and validated many previously undefined splices. The intestinal microbiome was revealed to have nonuniform species distributions down to the 3-inch (7.62-cm) length scale. These are but a few examples of how the Tabula Sapiens represents a broadly useful reference to deeply understand and explore human biology at cellular resolution.

Frequent exposure to mechanistic damages, pathological ingression, and chronic inflammation leads to recurrent cell death in the gut epithelium. Intestinal stem cells (ISCs) that reside in crypt-specific niches have an unprecedented role in gut epithelium renewal. ISC also facilitates the formation of mature intestinal epithelial cells (IECs) through regular differentiation and renewal in short turnover cycles. Interestingly, oxidative stress (OS) prevalent in the gut has a dominant role in the regulation of ISC proliferation and development. However, it is unclear, which axis OS controls the cellular signaling and underlying molecular mechanism to drive ISC turnover and regeneration cycle. Therefore, this review provides a comprehensive overview of the present understanding of OS generation in the gut, relatively directing the ISC development and regeneration under a conditional cellular environment. Additionally, the focus has been drawn on intestinal nutritional state and its related alteration on OS and its effect on ISCs. Moreover, recent findings and new approaches are emphasized herewith to enhance the present understanding of the mechanisms that direct universal ISC characteristics. Intestinal stem cells (ISC) form the basis of all repair mechanisms that help in the proliferation of the gut through their constant renewal and replacement. This activity is closely regulated in the ISC niche and is modulated by several extrinsic as well as intrinsic factors. Reactive Oxygen Species (ROS) form one of the major factors that influence ISC formation. The levels of ROS in the gut influence stem cell renewal ROS itself however is further influenced by several other factors such as the microbiota concerning the gut and immune cells which in turn also influence one another by various cross-talk mechanisms. Diet also forms an important part of this crosstalk. It also regulates the levels of ROS in the gut and helps in the proliferation of the ISC cells and their overall turnover rate.

The uterine lining (endometrium) exhibits a pro-inflammatory phenotype in women with endometriosis, resulting in pain, infertility, and poor pregnancy outcomes. The full complement of cell types contributing to this phenotype has yet to be identified, as most studies have focused on bulk tissue or select cell populations. Herein, through integrating whole-tissue deconvolution and single cell RNAseq, we comprehensively characterized immune and nonimmune cell types in endometrium of women with or without disease and their dynamic changes across the menstrual cycle. We designed metrics to evaluate specificity of deconvolution signatures that resulted in single cell identification of 13 novel signatures for immune cell subtypes in healthy endometrium. Guided by statistical metrics, we identified contributions of endometrial epithelial, endothelial, plasmacytoid dendritic cells, classical dendritic cells, monocytes, macrophages, and granulocytes to the endometrial pro-inflammatory phenotype, underscoring roles for nonimmune as well as immune cells to the dysfunctionality of this tissue.Teaser SentenceCell type deconvolution and single cell RNAseq analysis identify altered endometrial cellular compositions in women with endometriosis

Adenomyosis is a prevalent, estrogen-dependent uterine disorder wherein endometrial cells are abnormally present in the myometrium and are surrounded by hyperplastic/hypertrophic smooth muscle. Its etiology is unclear, although endometrial cell invasion into the myometrium has been postulated. RNA methylation, particularly N6-methyladenosine (m6A), plays an important role in regulating various physiological processes and invasive disorders. The goal of this in silico and lab-based experimental study was to explore a possible role for m6A in adenomyosis. Gene expression profiles of both the endometrium and myometrium of women with adenomyosis (cases) and without disease (controls) were obtained from the publicly available Gene Expression Omnibus (GEO) database. In the endometrium, STRING database analysis revealed that METTL3 functions as a “hub” gene of m6A RNA methylation regulators, and the genes involved in m6A regulation, including METTL3, FTO, ZC3H13, and YTHDC1 expression, were significantly decreased in cases versus controls. Functional, co-expression, and correlational analyses of endometrium from cases versus controls revealed decreased total m6A levels, induced by METTL3, and the downstream elevated insulin−like growth factor−1(IGF1) and D-Dopachrome Tautomerase (DDT), with the latter two having known functions in epithelial proliferation and cell migration, which are important processes in the pathogenesis of adenomyosis in endometrium. m6A RNA methylation regulators, including RBM15/15B, ALKBH5, FTO, YTHDF1/2, KIAA1429, HNRNPC, METTL3, ZC3H13, and YTHDC2, were also differentially expressed in the myometrium from cases versus controls. We validated decreased total m6A levels and differential expression of m6A RNA methylation regulators in the myometrium of patients with adenomyosis using qRT-PCR, immunohistochemistry and tissues available from our biorepository. Possible target genes, including cadherin 3(CDH3), sodium channelβ-subunit 4 (SCN4B), and placenta-specific protein 8 (PLAC8), which are involved in cell adhesion, muscle contraction and immune response in the myometrium of adenomyosis patients were also validated. Thus, through extensive public database mining and validation of select genes, this study, for the first time, implicates m6A and its methylation regulators in the pathogenesis of adenomyosis. Follow on functional studies are anticipated to elucidate mechanisms involving m6A and its regulators and down-stream effectors in the pathogenesis of this enigmatic reproductive disorder and potentially identify druggable targets to control its associated symptoms.

Benzo[a]pyrene (B[a]P), a major human carcinogen in combustion products such as cigarette smoke and diesel exhaust, is metabolically activated into DNA-reactive metabolites via three different enzymatic pathways. The pathways are the anti-(+)-benzo[a]pyrene 7,8-diol 9,10-epoxide pathway (P450/epoxide hydrolase catalyzed) (B[a]PDE), the benzo[a]pyrene o-quinone pathway (aldo ketose reductase (AKR) catalyzed) and the B[a]P radical cation pathway (P450 peroxidase catalyzed). We used a yeast p53 mutagenesis system to assess mutagenesis by B[a]P radical cations. Because radical cations are short-lived, they were generated in situ by reacting B[a]P with cumene hydroperoxide (CuOOH) and horse radish peroxidase (HRP) and then monitoring the generation of the more stable downstream products, B[a]P-1,6-dione and B[a]P-3,6-dione. On the basis of B[a]P-1,6 and 3,6-dione formation, approximately 4 μM of radical cation was generated. In the mutagenesis assays, the radical cations produced in situ showed a dose-dependent increase in mutagenicity from 0.25 μM to 10 μM B[a]P with no significant increase seen with further escalation to 50 μM B[a]P. However, mutagenesis was 200-fold less than with the AKR pathway derived B[a]P, 7-8-dione. Mutant p53 plasmids, which yield red colonies, were recovered from the yeast to study the pattern and spectrum of mutations. The mutation pattern observed was G to T (31%) > G to C (29%) > G to A (14%). The frequency of codons mutated by the B[a]P radical cations was essentially random and not enriched at known cancer hotspots. The quinone products of radical cations, B[a]P-1,6-dione and B[a]P-3,6-dione were more mutagenic than the radical cation reactions, but still less mutagenic than AKR derived B[a]P-7,8-dione. We conclude that B[a]P radical cations and their quinone products are weakly mutagenic in this yeast-based system compared to redox cycling PAH o-quinones.

Tobacco smoke exposure stimulates the expression of genes that are likely to be involved in the metabolism of its combustion products such as polycyclic aromatic hydrocarbons (PAH). Four of the smoke induced genes are aldo-keto reductases (AKR), enzymes that metabolically activate PAH to PAH o-quinones. Alternatively, PAHs are metabolized to (±)-anti-diol epoxides, such as (±)-anti-benzo[a]pyrene diol epoxide ((±)-anti-BPDE)), by the combined action of P4501A1/1B1 and epoxide hydrolase. (±)-anti-BPDE forms DNA adducts directly, while PAH o-quinones cause DNA damage by oxidative stress through a futile redox cycle. To address the role of AKRs in PAH cytotoxicity, we compared the cytotoxicity of PAH metabolites and the effects of overexpressing AKR1A1 in lung cells. (±)-anti-BPDE and B[a]P-7,8-trans-dihydrodiol, an intermediate in (±)-anti-BPDE metabolism, are toxic to A549 cells at concentrations with an IC(50) of ∼2 μM. In contrast, the PAH o-quinone B[a]P-7,8-dione was about 10-fold less toxic to A549 cells with an IC(50)20 μM. Similar differences in cytoxicity were observed with two other PAH o-quinones (benz[a]anthracene-3,4-dione and 7,12-dimethylbenz[a]anthracene-3,4-dione) compared with their respective diol-epoxide counterparts (BA-3,4-diol-1,2-epoxide and DMBA-3,4-diol-1,2-epoxide). In addition, both anti-BPDE and B[a]P-7,8-trans-dihydrodiol induced p53 expression ∼6 h post-treatment at concentrations as low as 1 μM consistent with extensive DNA damage. B[a]P-7,8-dione treatment did not induce p53 but generated reactive oxygen species (ROS) in A549 cells and induced the expression of oxidative response genes in H358 cells. We also observed that overexpression of AKR1A1 in H358 cells, which otherwise have low levels of AKR expression, protected cells 2-10-fold from the toxic effects of B[a]P-7,8-trans-dihydrodiol. These data suggest that overexpression of AKRs may protect lung cancer cells from the acute toxic effects of PAH.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants, so widespread that it is impossible for anyone to avoid exposure to them. They are the products of partial combustion, and exposure comes from the fossil fuels that we use to drive our cars, cook our food, warm our home, and fuel our industry. Other exposure comes from tobacco smoke and oil spills. PAHs are responsible for more cancers, primarily lung cancers, than any other carcinogen. The most studied PAHs are unsubstituted multi-ring structures. These compounds are not DNA reactive, and must be modified to become carcinogenic. Metabolic pathways modify PAH during the detoxification process. Three pathways have been extensively documented: the diol-epoxide pathway, the radical cation pathway, and the o -quinone pathway. Some PAHs are generated during combustion in oxygenated forms, usually as quinones. Quinones also form as PAHs are exposed to sunlight, for example, in an oil spill. These metabolic products damage DNA through distinct mechanisms: diol epoxides form bulky adducts with DNA, radical cations form depurinating adducts and quinones undergo futile redox cycling to generate reactive oxygen. Here, we discuss the evidence for these pathways in PAH carcinogenesis.

Cancer is initiated and maintained by the accumulation of numerous mutations in specific genes. Rarely, the mutations are inherited but more commonly they occur via exposure to chemical carcinogens. Mutant genes fall into two categories: tumor suppressors and oncogenes. The most commonly mutated tumor suppressor is p53, while one of the most commonly mutated oncogenes is Ras. p53 induces cell cycle arrest and apoptosis in cells that have undergone DNA damage. Mutations in p53 usually result in inactivation of its normal transcriptional ability, leading to unregulated growth of damaged cells. In contrast, mutations in Ras are activating mutations that lead to increased cell proliferation. Among the more commonly studied carcinogens are polycyclic aromatic hydrocarbons (PAH), which are combustion products found in cigarette smoke, and aflatoxins, which are produced by molds. These are not carcinogenic until metabolically activated to compounds known as ultimate carcinogens, which can damage DNA, leading to mutations. Molecular epidemiology and experimental carcinogenesis with Ras and p53 have led to an understanding of the roles of metabolic activation and genetic selection in cancer.

A two-step procedure for preparing 2-alkyl-1,3-butadienes is described. Cuprate addition to commercially available 1,4-dibromo-2-butene yields 3-alkyl-4-bromo-1-butene, a product of SN2′ substitution. Dehydrohalogenation gives 2-alkyl-1,3-butadienes.

A two-step procedure for preparing 2-alkyl-1,3-butadienes is described. Cuprate addition to commercially available 1,4-dibromo-2-butene yields 3-alkyl-4-bromo-1-butene, a product of S(N)2' substitution. Dehydrohalogenation gives 2-alkyl-1,3-butadienes.

Dihydrosiloles are easily prepared from 1,3-dienes and dichlorosilanes, even on kilogram scale. Asymmetric hydroboration of a 3-alkyl-1,5-dihydrosilole, prepared from a 2-alkyl-1,3-diene, followed by treatment with aqueous HF results in Peterson fragmentation, forming optically active 3-alkyl-4-fluorosilyl-1-butenes. The fluorosilanes are stable to moisture but very reactive toward nucleophiles. In addition, they can be converted to nucleophilic silyllithium reagents.

The extraordinary and ordinary refractive indices, ne and no and the densities of four liquid crystals 4-pentylphenyl trans-4′-pentylcyclohexanecarboxylate, 4-ethoxyphenyl trans-4′-propylcyclohexanecarboxylate, 4-ethoxyphenyl trans-4′-butylcyclohexanecarboxylate, and 4-methoxyphenyl trans-4′-pentylcyclohexanecarboxylate have been reported at different temperatures. The effective polarizabilities αe and αo in the nematic phase have been calculated using 1) Neugebauer and 2) Vuks methods. The order parameter S calculated from the two methods were comparable. The dielectric constants e|| and e⊥, parallel and perpendicular to the long axis of the molecules have been measured at 1 kHz at different temperatures. The order parameters for the liquid crystals were then evaluated from dielectric anisotropy Δe(=e||−e⊥), polarizability anisotropy Δα(=αe−αo) and dipole moment μ, using Maier and Meier equation and were found only slightly less than those obtained from refractive indices method. It was concluded that Ma...

The dielectric permittivities e|| and e⊥ and the refractive indices ne, no of five 4-cyano-4′-alkyl-1,1′-bicyclohexyls (CCH 2, CCH 3, CCH 4, CCH 5, CCH 7 where 2, 3, 4, 5, 7 means ethyl, propyl, butyl, pentyl, heptyl respectively) have been measured at different temperatures in the nematic and isotropic phases. The orientational order parameter from the refractive indices data, calculated from (1) Vuks’ method and (2) Neugebauer’s method were in good agreement. At any reduced temperature, the order parameter for CCH 2 is largest and that of CCH 7 is smallest and those for the others are in between them. Unlike for cyanobiphenyls or cyclohexylbenzenes, the dielectric permittivity e|| is obtained for \overset→E⊥\overset→H and e⊥ for E||H, where \overset→E is the electric and \overset→H is the magnetic field, and has been explained as due to these molecules having negative diamagnetic anisotropy. The dielectric anisotropy, Δe and optical anisotropy Δn for all the CCH’s is much less than that of the correspon...

The refractive indices and densities of six liquid crystals—two alkylcyanobiphenyls, three alkoxycyanobiphenyls and one mixture of two alkylcyanobiphenyls have been reported as a function of temperature. The effective polarizabilities αeand αo in the nematic phase calculated using the methods due to Neugebauer, Vuks and Saupe and Maier, are found to be appreciably different, though the order parameter S evaluated with those values of (αe - αo) are in good agreement. The preference of one method over the other two is discussed.