A hotspot for posttranslational modifications on the androgen receptor dimer interface drives pathology and anti-androgen resistance.

Mutations of the androgen receptor (AR) associated with prostate cancer and androgen insensitivity syndrome may profoundly influence its structure, protein interaction network, and binding to chromatin, resulting in altered transcription signatures and drug responses. Current structural information fails to explain the effect of pathological mutations on AR structure-function relationship. Here, we have thoroughly studied the effects of selected mutations that span the complete dimer interface of AR ligand-binding domain (AR-LBD) using x-ray crystallography in combination with in vitro, in silico, and cell-based assays. We show that these variants alter AR-dependent transcription and responses to anti-androgens by inducing a previously undescribed allosteric switch in the AR-LBD that increases exposure of a major methylation target, Arg761. We also corroborate the relevance of residues Arg761 and Tyr764 for AR dimerization and function. Together, our results reveal allosteric coupling of AR dimerization and posttranslational modifications as a disease mechanism with implications for precision medicine.

The mineralocorticoid receptor modulates timing and location of genomic binding by glucocorticoid receptor in response to synthetic glucocorticoids in keratinocytes.

Glucocorticoids (GCs) exert potent antiproliferative and anti-inflammatory properties, explaining their therapeutic efficacy for skin diseases. GCs act by binding to the GC receptor (GR) and the mineralocorticoid receptor (MR), co-expressed in classical and non-classical targets including keratinocytes. Using knockout mice, we previously demonstrated that GR and MR exert essential nonoverlapping functions in skin homeostasis. These closely related receptors may homo- or heterodimerize to regulate transcription, and theoretically bind identical GC-response elements (GRE). We assessed the contribution of MR to GR genomic binding and the transcriptional response to the synthetic GC dexamethasone (Dex) using control (CO) and MR knockout (MREKO ) keratinocytes. GR chromatin immunoprecipitation (ChIP)-seq identified peaks common and unique to both genotypes upon Dex treatment (1 h). GREs, AP-1, TEAD, and p53 motifs were enriched in CO and MREKO peaks. However, GR genomic binding was 35% reduced in MREKO , with significantly decreased GRE enrichment, and reduced nuclear GR. Surface plasmon resonance determined steady state affinity constants, suggesting preferred dimer formation as MR-MR > GR-MR ~ GR-GR; however, kinetic studies demonstrated that GR-containing dimers had the longest lifetimes. Despite GR-binding differences, RNA-seq identified largely similar subsets of differentially expressed genes in both genotypes upon Dex treatment (3 h). However, time-course experiments showed gene-dependent differences in the magnitude of expression, which correlated with earlier and more pronounced GR binding to GRE sites unique to CO including near Nr3c1. Our data show that endogenous MR has an impact on the kinetics and differential genomic binding of GR, affecting the time-course, specificity, and magnitude of GC transcriptional responses in keratinocytes.

The multivalency of the glucocorticoid receptor ligand-binding domain explains its manifold physiological activities.

The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor, which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. We identify four distinct homodimerization interfaces on the GR-LBD surface, which can associate into 20 topologically different homodimers. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution, quantitative fluorescence microscopy in living cells, and transcriptomic analyses. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work illustrates the unique flexibility of GR's LBD and suggests different dimeric conformations within cells. In addition, we unveil pathophysiologically relevant quaternary assemblies of the receptor with important implications for glucocorticoid action and drug design.

Structure of the homodimeric androgen receptor ligand-binding domain.

The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor.

Efficacy of a videoconferencing intervention compared with standard postnatal care at primary care health centres in Catalonia.

OBJECTIVE: to evaluate the efficacy of an intervention combining videoconferencing and telephone contact compared to standard post partum care of recent mothers attending health centres in Catalonia were recorded. DESIGN: multicentre, randomised parallel controlled clinical trial. PARTICIPANTS AND SETTING: 1598 post partum women with Internet access attending eight 'Attention to Sexual and Reproductive Health' (Catalan acronym ASSIR) units at Primary Health Care centres, in Catalonia (Spain). INTERVENTION: at each of the eight ASSIR units, 100 women were randomly assigned to the intervention group (IG) and 100 to the control group (CG). Women in the IG could consult midwives by videoconference or telephone and could also receive standard care. Women in the control group received standard care from midwives at their health centres or at home. MEASURES: number and type of visits, reasons for consultation, type of feeding at six weeks and women's satisfaction with the intervention on a scale of 1 to 5. FINDINGS: 1401 women were studied (80.9% of the initial sample), 683 in the IG and 718 in the CG. Two hundred and seventy-six women (40.4%) used videoconferencing or telephone in the IG. The mean total visits, virtual and face-to-face, was higher in IG women than in controls (2.74 versus 1.22). IG women made fewer visits to the health centre (mean=1) than CG women (mean=1.17). Both differences were statistically significant, with p<0.001 and p=0.002 respectively. The prevalence of breast feeding was similar in the two groups (IG 64.5%, and CG 65.4%). The mean overall satisfaction of women with midwife care was very high in both groups (IG 4.77, CG 4.76). CONCLUSIONS AND IMPLICATIONS FOR THE PRACTICE: virtual care via videoconferencing is effective for post partum women. It reduces the number of health centre visits and allows mothers to consult health staff immediately and from their own home.

Diagnosis delay and follow-up strategies in colorectal cancer. Prognosis implications: a study protocol.

BACKGROUND: Controversy exists with regard to the impact that the different components of diagnosis delay may have on the degree of invasion and prognosis in patients with colorectal cancer. The follow-up strategies after treatment also vary considerably. The aims of this study are: a) to determine if the symptoms-to-diagnosis interval and the treatment delay modify the survival of patients with colorectal cancer, and b) to determine if different follow-up strategies are associated with a higher survival rate. METHODS/DESIGN: Multi-centre study with prospective follow-up in five regions in Spain (Galicia, Balearic Islands, Catalonia, Aragón and Valencia) during the period 2010-2012. Incident cases are included with anatomopathological confirmation of colorectal cancer (International Classification of Diseases 9th revision codes 153-154) that formed a part of a previous study (n = 953).At the time of diagnosis, each patient was given a structured interview. Their clinical records will be reviewed during the follow-up period in order to obtain information on the explorations and tests carried out after treatment, and the progress of these patients.Symptoms-to-diagnosis interval is defined as the time calculated from the diagnosis of cancer and the first symptoms attributed to cancer. Treatment delay is defined as the time elapsed between diagnosis and treatment. In non-metastatic patients treated with curative intention, information will be obtained during the follow-up period on consultations performed in the digestive, surgery and oncology departments, as well as the endoscopies, tumour markers and imaging procedures carried out.Local recurrence, development of metastases in the follow-up, appearance of a new tumour and mortality will be included as outcome variables.Actuarial survival analysis with Kaplan-Meier curves, Cox regression and competitive risk survival analysis will be performed. DISCUSSION: This study will make it possible to verify if the different components of delay have an impact on survival rate in colon cancer and rectal cancer. In consequence, this multi-centre study will be able to detect the variability present in the follow-up of patients with colorectal cancer, and if this variability modifies the prognosis. Ideally, this study could determine which follow-up strategies are associated with a better prognosis in colorectal cancer.

LCA as a decision support tool for the environmental improvement of the operation of a municipal wastewater treatment plant.

Life cycle assessment (LCA) methodology is used to evaluate the environmental profile of a product or process from its origin to its final destination. In this paper we used LCA to evaluate the current situation of a wastewater treatment plant and identify improvement alternatives. Currently, the highest environmental impacts are caused by the stages of the plant with the highest energy consumption, the use of biogas from anaerobic digestion (95% burned in torch) and the final destination of the sludge (98.6% for agricultural use and 1.4% for compost). We propose four alternatives for biogas applications and five alternatives for sludge applications and compare them to the current situation. The alternatives were incorporated in a decision support system to identify and prioritize the most positive environmental option. Using biogas to produce electricity or a combination of electricity and heat provided the best environmental options since the energy produced would be enough to supply all the stages of the plant, thus reducing their environmental impact. The best environmental option for the final destination of the sludge is to combine the current situation (fertilizer replacement) with use of the sludge in a cement plant (as a replacement for fuel and raw material).