Homeobox regulator Wilms Tumour 1 is displaced by androgen receptor at cis-regulatory elements in the endometrium of PCOS patients.

Decidualisation, the process whereby endometrial stromal cells undergo morphological and functional transformation in preparation for trophoblast invasion, is often disrupted in women with polycystic ovary syndrome (PCOS) resulting in complications with pregnancy and/or infertility. The transcription factor Wilms tumour suppressor 1 (WT1) is a key regulator of the decidualization process, which is reduced in patients with PCOS, a complex condition characterized by increased expression of androgen receptor in endometrial cells and high presence of circulating androgens. Using genome-wide chromatin immunoprecipitation approaches on primary human endometrial stromal cells, we identify key genes regulated by WT1 during decidualization, including homeobox transcription factors which are important for regulating cell differentiation. Furthermore, we found that AR in PCOS patients binds to the same DNA regions as WT1 in samples from healthy endometrium, suggesting dysregulation of genes important to decidualisation pathways in PCOS endometrium due to competitive binding between WT1 and AR. Integrating RNA-seq and H3K4me3 and H3K27ac ChIP-seq metadata with our WT1/AR data, we identified a number of key genes involved in immune response and angiogenesis pathways that are dysregulated in PCOS patients. This is likely due to epigenetic alterations at distal enhancer regions allowing AR to recruit cofactors such as MAGEA11, and demonstrates the consequences of AR disruption of WT1 in PCOS endometrium.

Transcriptomic analysis reveals the anti-cancer effect of gestational mesenchymal stem cell secretome.

The environment created during embryogenesis contributes to reducing aberrations that drive structural malformations and tumorigenesis. In this study, we investigate the anti-cancer effect of mesenchymal stem cells (MSCs) derived from 2 different gestational tissues, the amniotic fluid (AF) and the chorionic villi (CV), with emphasis on their secretome. Transcriptomic analysis was performed on patient-derived AF- and CV-MSCs collected during prenatal diagnosis and identified both mRNAs and lncRNAs, involved in tissue homeostasis and inhibiting biological processes associated with the etiology of aggressive cancers while regulating immune pathways shown to be important in chronic disorders. Secretome enrichment analysis also identified soluble moieties involved in target cell regulation, tissue homeostasis, and cancer cell inhibition through the highlighted Wnt, TNF, and TGF-ß signaling pathways. Transcriptomic data were experimentally confirmed through in vitro assays, by evaluating the anti-cancer effect of the media conditioned by AF- and CV-MSCs and the exosomes derived from them on ovarian cancer cells, revealing inhibitory effects in 2D (by reducing cell viability and inducing apoptosis) and in 3D conditions (by negatively interfering with spheroid formation). These data provide molecular insights into the potential role of gestational tissues-derived MSCs as source of anti-cancer factors, paving the way for the development of therapeutics to create a pro-regenerative environment for tissue restoration following injury, disease, or against degenerative disorders.

Transcriptomic analysis of stem cells from chorionic villi uncovers the impact of chromosomes 2, 6 and 22 in the clinical manifestations of Down syndrome.

BACKGROUND: Down syndrome (DS) clinical multisystem condition is generally considered the result of a genetic imbalance generated by the extra copy of chromosome 21. Recent discoveries, however, demonstrate that the molecular mechanisms activated in DS compared to euploid individuals are more complex than previously thought. Here, we utilize mesenchymal stem cells from chorionic villi (CV) to uncover the role of comprehensive functional genomics-based understanding of DS complexity. METHODS: Next-generation sequencing coupled with bioinformatic analysis was performed on CV obtained from women carrying fetuses with DS (DS-CV) to reveal specific genome-wide transcriptional changes compared to their euploid counterparts. Functional assays were carried out to confirm the biological processes identified as enriched in DS-CV compared to CV (i.e., cell cycle, proliferation features, immunosuppression and ROS production). RESULTS: Genes located on chromosomes other than the canonical 21 (Ch. 2, 6 and 22) are responsible for the impairment of life-essential pathways, including cell cycle regulation, innate immune response and reaction to external stimuli were found to be differentially expressed in DS-CV. Experimental validation confirmed the key role of the biological pathways regulated by those genes in the etiology of such a multisystem condition. CONCLUSIONS: NGS dataset generated in this study highlights the compromised functionality in the proliferative rate and in the innate response of DS-associated clinical conditions and identifies DS-CV as suitable tools for the development of specifically tailored, personalized intervention modalities.

Bromodomain inhibitor i-BET858 triggers a unique transcriptional response coupled to enhanced DNA damage, cell cycle arrest and apoptosis in high-grade ovarian carcinoma cells.

BACKGROUND: Ovarian cancer has a specific unmet clinical need, with a persistently poor 5-year survival rate observed in women with advanced stage disease warranting continued efforts to develop new treatment options. The amplification of BRD4 in a significant subset of high-grade serous ovarian carcinomas (HGSC) has led to the development of BET inhibitors (BETi) as promising antitumour agents that have subsequently been evaluated in phase I/II clinical trials. Here, we describe the molecular effects and ex vivo preclinical activities of i-BET858, a bivalent pan-BET inhibitor with proven in vivo BRD inhibitory activity. RESULTS: i-BET858 demonstrates enhanced cytotoxic activity compared with earlier generation BETis both in cell lines and primary cells derived from clinical samples of HGSC. At molecular level, i-BET858 triggered a bipartite transcriptional response, comprised of a 'core' network of genes commonly associated with BET inhibition in solid tumours, together with a unique i-BET858 gene signature. Mechanistically, i-BET858 elicited enhanced DNA damage, cell cycle arrest and apoptotic cell death compared to its predecessor i-BET151. CONCLUSIONS: Overall, our ex vivo and in vitro studies indicate that i-BET858 represents an optimal candidate to pursue further clinical validation for the treatment of HGSC.

In silico enhancer mining reveals SNS-032 and EHMT2 inhibitors as therapeutic candidates in high-grade serous ovarian cancer.

BACKGROUND: Epigenomic dysregulation has been linked to solid tumour malignancies, including ovarian cancers. Profiling of re-programmed enhancer locations associated with disease has the potential to improve stratification and thus therapeutic choices. Ovarian cancers are subdivided into histological subtypes that have significant molecular and clinical differences, with high-grade serous carcinoma representing the most common and aggressive subtype. METHODS: We interrogated the enhancer landscape(s) of normal ovary and subtype-specific ovarian cancer states using publicly available data. With an initial focus on H3K27ac histone mark, we developed a computational pipeline to predict drug compound activity based on epigenomic stratification. Lastly, we substantiated our predictions in vitro using patient-derived clinical samples and cell lines. RESULTS: Using our in silico approach, we highlighted recurrent and privative enhancer landscapes and identified the differential enrichment of a total of 164 transcription factors involved in 201 protein complexes across the subtypes. We pinpointed SNS-032 and EHMT2 inhibitors BIX-01294 and UNC0646 as therapeutic candidates in high-grade serous carcinoma, as well as probed the efficacy of specific inhibitors in vitro. CONCLUSION: Here, we report the first attempt to exploit ovarian cancer epigenomic landscapes for drug discovery. This computational pipeline holds enormous potential for translating epigenomic profiling into therapeutic leads.

Selenium nanoparticles modulate histone methylation via lysine methyltransferase activity and S-adenosylhomocysteine depletion.

At physiological levels, the trace element selenium plays a key role in redox reactions through the incorporation of selenocysteine in antioxidant enzymes. Selenium has also been evaluated as a potential anti-cancer agent, where selenium nanoparticles have proven effective, and are well tolerated in vivo at doses that are toxic as soluble Se. The use of such nanoparticles, coated with either serum albumin or the naturally occurring alkaline polysaccharide chitosan, also serves to enhance biocompatibility and bioavailability. Here we demonstrate a novel role for selenium in regulating histone methylation in ovarian cancer cell models treated with inorganic selenium nanoparticles coated with serum albumin or chitosan. As well as inducing thioredoxin reductase expression, ROS activity and cancer cell cytotoxicity, coated nanoparticles caused significant increases in histone methylation. Specifically, selenium nanoparticles triggered an increase in the methylation of histone 3 at lysines K9 and K27, histone marks involved in both the activation and repression of gene expression, thus suggesting a fundamental role for selenium in these epigenetic processes. This direct function was confirmed using chemical inhibitors of the histone lysine methyltransferases EZH2 (H3K27) and G9a/EHMT2 (H3K9), both of which blocked the effect of selenium on histone methylation. This novel role for selenium supports a distinct function in histone methylation that occurs due to a decrease in S-adenosylhomocysteine, an endogenous inhibitor of lysine methyltransferases, the metabolic product of methyl-group transfer from S-adenosylmethionine in the one-carbon metabolism pathway. These observations provide important new insights into the action of selenium nanoparticles. It is now important to consider both the classic antioxidant and novel histone methylation effects of this key redox element in its development in cancer therapy and other applications.

Subdiffusion model for granular discharge in a submerged silo.

Silo discharge has been extensively studied for decades although questions remain regarding the nature of the velocity field, particularly for submerged systems. In this work, fluid-driven granular drainage was performed in a quasi-two-dimensional silo with grains submerged in fluid. While the observed Gaussian velocity profiles were generally consistent with current diffusion models, the diffusion length was found to significantly decrease with height in contrast to the increases previously seen in dry silos. We propose a phenomenological anomalous diffusion model for the spreading of the flow upwards in the cell, with the fluid-driven flows we study here falling in the category of subdiffusive behavior. As the viscous characteristics of the system were amplified, the diffusion length increased and the shape of the flowing zone in the silo changed, deviating further from the parabolic form predicted by traditional normal diffusion models, in effect becoming more subdiffusive as quantified by a decreasing diffusion exponent.

High content, quantitative AFM analysis of the scalable biomechanical properties of extracellular vesicles.

Extracellular vesicles (EVs) are studied extensively as natural biomolecular shuttles and for their diagnostic and therapeutic potential. This exponential rise in interest has highlighted the need for highly robust and reproducible approaches for EV characterisation. Here we optimise quantitative nanomechanical tools and demonstrate the advantages of EV population screening by atomic force microscopy (AFM). Our high-content informatics analytical tools are made available for use by the EV community for widespread, standardised determination of structural stability. Ultracentrifugation (UC) and sonication, the common mechanical techniques used for EV isolation and loading respectively, are used to demonstrate the utility of optimised PeakForce-Quantitative Nano Mechanics (PF-QNM) analysis. EVs produced at an industrial scale exhibited biochemical and biomechanical alterations after exposure to these common techniques. UC resulted in slight increases in physical dimensions, and decreased EV adhesion concurrent with a decrease in CD63 content. Sonicated EVs exhibited significantly reduced levels of CD81, a decrease in size, increased Young's modulus and decreased adhesive force. These biomechanical and biochemical changes highlight the effect of EV sample preparation techniques on critical properties linked to EV cellular uptake and biological function. PF-QNM offers significant additional information about the structural information of EVs following their purification and downstream processing, and the analytical tools will ensure consistency of analysis of AFM data by the EV community, as this technique continues to become more widely implemented.

Data Driven Cell Cycle Model to Quantify the Efficacy of Cancer Therapeutics Targeting Specific Cell-Cycle Phases From Flow Cytometry Results.

Many chemotherapeutic drugs target cell processes in specific cell cycle phases. Determining the specific phases targeted is key to understanding drug mechanism of action and efficacy against specific cancer types. Flow cytometry experiments, combined with cell cycle phase and division round specific staining, can be used to quantify the current cell cycle phase and number of mitotic events of each cell within a population. However, quantification of cell interphase times and the efficacy of cytotoxic drugs targeting specific cell cycle phases cannot be determined directly. We present a data driven computational cell population model for interpreting experimental results, where in-silico populations are initialized to match observable results from experimental populations. A two-stage approach is used to determine the efficacy of cytotoxic drugs in blocking cell-cycle phase transitions. In the first stage, our model is fitted to experimental multi-parameter flow cytometry results from untreated cell populations to identify parameters defining probability density functions for phase transitions. In the second stage, we introduce a blocking routine to the model which blocks a percentage of attempted transitions between cell-cycle phases due to therapeutic treatment. The resulting model closely matches the percentage of cells from experiment in each cell-cycle phase and division round. From untreated cell populations, interphase and intermitotic times can be inferred. We then identify the specific cell-cycle phases that cytotoxic compounds target and quantify the percentages of cell transitions that are blocked compared with the untreated population, which will lead to improved understanding of drug efficacy and mechanism of action.

Hand and foot surgery rates in rheumatoid arthritis have declined from 1986 to 2011, but large-joint replacement rates remain unchanged: results from two UK inception cohorts.

OBJECTIVE: To assess whether there have been any secular changes in orthopedic interventions in patients with rheumatoid arthritis (RA) since 1986, as examined in 2 early rheumatoid arthritis (RA) inception cohorts with up to 25 years of followup. METHODS: The study examined orthopedic data from the UK Early RA Study (1986-1999, 9 centers; n = 1,465) and the UK Early RA Network (2002-2012, 23 centers; n = 1,236) with linkage to national data sets (Hospital Episode Statistics, National Joint Registry, and Office of National Statistics). Clinical and laboratory measures and hand and foot radiographs were standardized and obtained yearly in both cohorts. The use of disease-modifying antirheumatic drugs (DMARDs), corticosteroids, and biologic therapies reflected the contemporary conventional practices and guidelines of the time frames examined. Recruitment years were grouped into 6 periods, and interventions were classified into major, intermediate, and minor categories. RESULTS: A total of 1,602 orthopedic surgical procedures were performed in 770 patients (29%) over a maximum of 25 years of followup. The 25-year cumulative incidence rate of major interventions was 21.7% (range 19.4-24.0%), and that of intermediate interventions was 21.5% (range 17.8-25.5%). There was a decline in the 10-year cumulative incidence of intermediate surgeries over time (P < 0.001), but not of major/minor surgery. This decline coincided with a gradual shift from sequential monotherapy to combination DMARD therapies and biologic agents in recent recruitment periods. CONCLUSION: Orthopedic surgery is an important and common outcome in RA. Only the rates of hand/foot surgery showed a consistent decline from 1986 to 2011. Possible explanations include differences in the pathophysiologic processes affecting the joints, variations in the responses to therapy between large-joint and small-joint destructive processes, and changes in service provision and thresholds for surgery over time.