Implication of DNA methylation, CTCF and BORIS factors in the transcriptional regulation of the human telomerase catalytic subunit hTERT

RESUME La télomérase confère une durée de vie illimitée et est réactivée dans la plupart des cellules tumorales. Sa sous-unité catalytique hTERT est définie comme le facteur limitant pour son activation. De l'identification de facteurs liant la région régulatrice d'hTERT, au rôle de la méthylation de l'ADN et de la modification des histones, de nombreux modèles de régulation ont été suggérés. Cependant, aucun de ces modèles n'a pu expliquer l'inactivation de la télomérase dans la plupart des cellules somatiques et sa réactivation dans la majorité des cellules tumorales. De plus, les observations contradictoires entre le faible niveau d'expression d'ARN messager d'hTERT dans les cellules télomérase-positives et la très forte activité transcriptionnelle du promoteur d'hTERT en transfection restent incomprises. Dans cette étude, nous avons montré que la région proximale du gène hTERT (exon 1 et 2) était impliquée dans la répression de l'activité de son promoteur. Nous avons identifié le facteur CTCF comme étant un inhibiteur du promoteur d'hTERT, en se liant au niveau de son premier exon. La méthylation de l'exon 1 du gène hTERT, couramment observée dans les tumeurs mais pas dans les cellules normales, empêcherait la liaison de CTCF. L'étude du profil de méthylation du promoteur d'hTERT indique qu'une partie du promoteur reste déméthylée et qu'elle semble suffisante pour permettre une faible activité transcriptionnelle du gène hTERT. Ainsi, la méthylation particulière des régions régulatrices d'hTERT inhibe la liaison de CTCF tout en permettant une faible transcription du gène. Cependant, dans certaines cellules tumorales, le promoteur et la région proximale du gène hTERT ne sont pas méthylés. Dans les lignées cellulaires tumorales de tesitcules et d'ovaires, l'inhibition de CTCF est contrée par son paralogue BORIS, qui se lie aussi au niveau de l'exon 1 d'hTERT, mais permet ainsi l'activation du promoteur. L'étude de l'expression du gène BORIS montre qu'il est exclusivement exprimé dans les tissus normaux de testicules et d'ovaires jeunes, ainsi qu'à différents niveaux dans la plupart des tumeurs. Sa transcription est sous le contrôle de deux promoteurs. Le promoteur proximal est régulé par méthylation et un transcrit alternatif majoritaire, délété de l'exon 6, est trouvé lorsque ce promoteur est actif. Tous ces résultats conduisent à un modèle de régulation du gène hTERT qui tient compte du profil épigénétique du gène et qui permet d'expliquer le faible taux de transcription observé in vivo. De plus, l'expression de BORIS dans les cancers et son implication dans l'activation du gène hTERT pourrait permettre de comprendre les phénomènes de dérégulation épigénétique et d'immortalisation qui ont lieu durant la tumorigenèse. SUMMARY Telomerase confers an unlimited lifespan, and is reactivated in most tumor cells. The catalytic subunit of telomerase, hTERT, is defined as the limiting factor for telomerase activity. Between activators and repressors that bind to the hTERT 5' regulatory region, and the role of CpG methylation and histone acetylation, an abundance of regulatory models have been suggested. None of these models can explain the silence of telomerase in most somatic cells and its reactivation in tumor cells. Moreover, the contradictory observations of the low level of hTERT mRNA in telomerase-positive cells and the high transcriptional activity of the hTERT promoter in transfection experiments remain unresolved. In this study, we demonstrated that the proximal exonic region of the hTERT gene (exon 1 and 2) is involved in the inhibition of its promoter. We identified the protein CTCF as the inhibitor of the hTERT promoter, through its binding to the first exon. The methylation of the first exon region, which is often observed in cancer cells but not in noimal cells, represses CTCF binding. Study of hTERT promoter methylation shows a partial demethylation sufficient to activate the transcription of the hTERT gene. Therefore, we demonstrated that the particular methylation profile of the hTERT regulatory sequences inhibits the binding of CTCF, while it allows a low transcription of the gene. Nevertheless, in some tumor cells, the promoter and the proximal exonic region of hTERT are unmethylated. In testicular and ovarian cancer cell lines, CTCF inhibition is counteracted by its BORIS paralogue that also binds the hTERT first exon but allows the promoter activation. The study of BORIS gene regulation showed that this factor is exclusively expressed in normal tissue of testis and ovary of young woman, as well as in almost all tumors with different levels. Two promoters were found to induce its transcription. The proximal promoter was regulated by methylation. Moreover, a major alternative transcript, deleted of the exon 6, is detected when this promoter is active. All these results lead to a model for hTERT regulation that takes into account the epigenetic profile of the gene and provides an explanation for the low transcriptional level observed in vivo. BORIS expression in cancers and its implication in hTERT activation might also permit the understanding of epigenetic deregulation and immortalization phenomena that occur during tumorigenesis.

Novel micropatterns mechanically control fibrotic reactions at the surface of silicone implants.

Over the past decade, various implantable devices have been developed to treat diseases that were previously difficult to manage such diabetes, chronic pain, and neurodegenerative disorders. However, translation of these novel technologies into clinical practice is often difficult because fibrotic encapsulation and/or rejection impairs device function after body implantation. Ideally, cells of the host tissue should perceive the surface of the implant being similar to the normal extracellular matrix. Here, we developed an innovative approach to provide implant surfaces with adhesive protein micropatterns. The patterns were designed to promote adhesion of fibroblasts and macrophages by simultaneously suppressing fibrogenic activation of both cell types. In a rat model, subcutaneously implanted silicone pads provided with the novel micropatterns caused 6-fold lower formation of inflammatory giant cells compared with clinical grade, uncoated, or collagen-coated silicone implants. We further show that micropatterning of implants resulted in 2-3-fold reduced numbers of pro-fibrotic myofibroblast by inhibiting their mechanical activation. Our novel approach allows controlled cell attachment to implant surfaces, representing a critical advance for enhanced biointegration of implantable medical devices.

Observer reactions to workplace mistreatment

This study explored observer reactions to workplace interpersonal mistreatment using an inductive analysis approach. I conducted 32 interviews with a wide sample of working professionals from various backgrounds and industries to examine how observers react to the unfolding process of workplace interpersonal mistreatment incidents. Specifically, the goal of this study was to gain a deeper and closer understanding of observer reaction processes by examining first-hand accounts of employees who have witnessed co-workers being mistreated by others. I generated typologies of reported observer affective, cognitive, and behavioral reactions that emerged from their stories, and I identified what employees believe are important factors that inhibit or enable intervention. Results reveal that a majority of employees are not inclined to intervene during an ongoing mistreatment incident, and that observers who intervened during the incident reported different appraisal processes than observers who only intervened afterwards, or who did not intervene at all. From these personal accounts of observing workplace mistreatment, I interpreted that observers generally react to interpersonal mistreatment incidents in two phases, and that how targets reacted after an incident was an important trigger that propelled observers to become involved afterwards, even if they did not have the desire or the intention to do so. These findings have implications for current theories on observer intervention to mistreatment in the workplace.

Age and adverse drug reactions from psychopharmacological treatment: data from the AMSP drug surveillance programme in Switzerland.

QUESTION UNDER STUDY: The frequency of severe adverse drug reactions (ADRs) from psychotropic drugs was investigated in hospitalised psychiatric patients in relation to their age. Specifically, the incidence of ADRs in patients up to 60 years was compared to that of patients older than 60 years. METHODS: Prescription rates of psychotropic drugs and reports of severe ADRs were collected in psychiatric hospitals in Switzerland between 2001 and 2010. The data stem from the drug surveillance programme AMSP. RESULTS: A total of 699 patients exhibited severe ADRs: 517 out of 28,282 patients up to 60 years (1.8%); 182 out of 11,446 elderly patients (1.6%, ns). Logistic regression analyses showed a significantly negative relationship between the incidence of ADRs and patients' age in general and in particular for weight gain, extrapyramidal motor system (EPMS) symptoms, increased liver enzymes and galactorrhoea. A significantly negative relationship was observed for age and the dosages of olanzapine, quetiapine, risperidone, valproic acid and lamotrigine. When comparing age groups, frequency of ADRs was lower in general for antipsychotic drugs and anticonvulsants, in particular for valproic acid in the elderly. Weight gain was found to be lower in the elderly for antipsychotic drugs, in particular for olanzapine. For the group of mood-stabilising anticonvulsants (carbamazepine, lamotrigine and valproic acid) the elderly exhibited a lower incidence of reported allergic skin reactions. CONCLUSION: The results suggest that for psychiatric inpatients the incidence of common severe ADRs (e.g., weight gain or EPMS symptoms) arising from psychotropic medication decreases with the age of patients.

2015 Update on Acute Adverse Reactions to Gadolinium based Contrast Agents in Cardiovascular MR. Large Multi-National and Multi-Ethnical Population Experience With 37788 Patients From the EuroCMR Registry.

OBJECTIVES: Specifically we aim to demonstrate that the results of our earlier safety data hold true in this much larger multi-national and multi-ethnical population. BACKGROUND: We sought to re-evaluate the frequency, manifestations, and severity of acute adverse reactions associated with administration of several gadolinium- based contrast agents during routine CMR on a European level. METHODS: Multi-centre, multi-national, and multi-ethnical registry with consecutive enrolment of patients in 57 European centres. RESULTS: During the current observation 37,788 doses of Gadolinium based contrast agent were administered to 37,788 patients. The mean dose was 24.7 ml (range 5-80 ml), which is equivalent to 0.123 mmol/kg (range 0.01 - 0.3 mmol/kg). Forty-five acute adverse reactions due to contrast administration occurred (0.12%). Most reactions were classified as mild (43 of 45) according to the American College of Radiology definition. The most frequent complaints following contrast administration were rashes and hives (15 of 45), followed by nausea (10 of 45) and flushes (10 of 45). The event rate ranged from 0.05% (linear non-ionic agent gadodiamide) to 0.42% (linear ionic agent gadobenate dimeglumine). Interestingly, we also found different event rates between the three main indications for CMR ranging from 0.05% (risk stratification in suspected CAD) to 0.22% (viability in known CAD). CONCLUSIONS: The current data indicate that the results of the earlier safety data hold true in this much larger multi-national and multi-ethnical population. Thus, the "off-label" use of Gadolinium based contrast in cardiovascular MR should be regarded as safe concerning the frequency, manifestation and severity of acute events.

MetaNetX/MNXref - reconciliation of metabolites and biochemical reactions to bring together genome-scale metabolic networks.

MetaNetX is a repository of genome-scale metabolic networks (GSMNs) and biochemical pathways from a number of major resources imported into a common namespace of chemical compounds, reactions, cellular compartments-namely MNXref-and proteins. The MetaNetX.org website (http://www.metanetx.org/) provides access to these integrated data as well as a variety of tools that allow users to import their own GSMNs, map them to the MNXref reconciliation, and manipulate, compare, analyze, simulate (using flux balance analysis) and export the resulting GSMNs. MNXref and MetaNetX are regularly updated and freely available.