The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells.

An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells.We thank Ludovic Vallier for constitutive Nanog-EpiSC, Gabrielle Brons for 129S2 EpiSC, Prim Singh for H3K9me3 antibody, Maria Elena Torres Padilla for TALE-mClover and luciferase plasmids, Wellcome Trust Sanger Institute for pCyL43 plasmid and Andras Nagy for PB-TET and rtTA plasmids. We are grateful to David Oxley and Judith Webster Novo et al. for mass spectrometry support, Simon Walker for imaging support and Anne Segonds- Pichon for statistical advice. We thank Wolf Reik and Jon Houseley for comments on the manuscript and members of Wolf Reik’s group for helpful discussions. P.J.R.-G. is supported by the Wellcome Trust [WT093736], BBSRC [M022285] and the European Commission Network of Excellence EpiGeneSys [HEALTH-F4-2010-257082]. The work was also supported with funds from the Canadian Institutes of Health Research to J.E. [Team Grant EPS-129129] and D.P.B.-J. D.P.B-J. holds the Canada Research Chair in Molecular and Cellular Imaging. I.C. is supported by the MRC

Identification and functional characterisation of CRK12:CYC9, a novel cyclin-dependent kinase (CDK)-cyclin complex in Trypanosoma brucei

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively

Satellite repeat transcripts modulate heterochromatin condensates and safeguard chromosome stability in mouse embryonic stem cells

Heterochromatin maintains genome integrity and function, and is organised into distinct nuclear domains. Some of these domains are proposed to form by phase separation through the accumulation of HP1ɑ. Mouse heterochromatin contains noncoding major satellite repeats (MSR), which are highly transcribed in mouse embryonic stem cells (ESCs). Here, we report that MSR transcripts can drive the formation of HP1ɑ droplets in vitro, and modulate heterochromatin into dynamic condensates in ESCs, contributing to the formation of large nuclear domains that are characteristic of pluripotent cells. Depleting MSR transcripts causes heterochromatin to transition into a more compact and static state. Unexpectedly, changing heterochromatin’s biophysical properties has severe consequences for ESCs, including chromosome instability and mitotic defects. These findings uncover an essential role for MSR transcripts in modulating the organisation and properties of heterochromatin to preserve genome stability. They also provide insights into the processes that could regulate phase separation and the functional consequences of disrupting the properties of heterochromatin condensates

An intranasal selective antisense oligonucleotide impairs lung cyclooxygenase-2 production and improves inflammation, but worsens airway function, in house dust mite sensitive mice

<p>Abstract</p> <p>Background</p> <p>Despite its reported pro-inflammatory activity, cyclooxygenase (COX)-2 has been proposed to play a protective role in asthma. Accordingly, COX-2 might be down-regulated in the airway cells of asthmatics. This, together with results of experiments to assess the impact of COX-2 blockade in ovalbumin (OVA)-sensitized mice in vivo, led us to propose a novel experimental approach using house dust mite (HDM)-sensitized mice in which we mimicked altered regulation of COX-2.</p> <p>Methods</p> <p>Allergic inflammation was induced in BALBc mice by intranasal exposure to HDM for 10 consecutive days. This model reproduces spontaneous exposure to aeroallergens by asthmatic patients. In order to impair, but not fully block, COX-2 production in the airways, some of the animals received an intranasal antisense oligonucleotide. Lung COX-2 expression and activity were measured along with bronchovascular inflammation, airway reactivity, and prostaglandin production.</p> <p>Results</p> <p>We observed impaired COX-2 mRNA and protein expression in the lung tissue of selective oligonucleotide-treated sensitized mice. This was accompanied by diminished production of mPGE synthase and PGE₂in the airways. In sensitized mice, the oligonucleotide induced increased airway hyperreactivity (AHR) to methacholine, but a substantially reduced bronchovascular inflammation. Finally, mRNA levels of hPGD synthase remained unchanged.</p> <p>Conclusion</p> <p>Intranasal antisense therapy against COX-2 in vivo mimicked the reported impairment of COX-2 regulation in the airway cells of asthmatic patients. This strategy revealed an unexpected novel dual effect: inflammation was improved but AHR worsened. This approach will provide insights into the differential regulation of inflammation and lung function in asthma, and will help identify pharmacological targets within the COX-2/PG system.</p

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4

Background: Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E2 (PGE2) are probably also released and could explain the refractory period observed in patients with EIB. Objective: This study aimed to evaluate the protective effect of PGE2 on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction. Methods: We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE2 and prostanoid receptor (EP) antagonists for EP1-4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed. Results: Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE2 significantly reduced mannitol-induced degranulation through EP2 and EP4 receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone. Conclusions:Our data show a protective role for the PGE2 receptors EP2 and EP4 following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition

Identification of Pathogenicity-Related Genes in the Vascular Wilt Fungus Verticillium dahliae by Agrobacterium tumefaciens-Mediated T-DNA Insertional Mutagenesis

Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacteriumtumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae

A systematic review of the effect of retention methods in population-based cohort studies

Background: Longitudinal studies are of aetiological and public health relevance but can be undermined by attrition. The aim of this paper was to identify effective retention strategies to increase participation in population-based cohort studies. Methods: Systematic review of the literature to identify prospective population-based cohort studies with health outcomes in which retention strategies had been evaluated. Results: Twenty-eight studies published up to January 2011 were included. Eleven of which were randomized controlled trials of retention strategies (RCT). Fifty-seven percent of the studies were postal, 21% in-person, 14% telephone and 7% had mixed data collection methods. A total of 45 different retention strategies were used, categorised as 1) incentives, 2) reminder methods, repeat visits or repeat questionnaires, alternative modes of data collection or 3) other methods. Incentives were associated with an increase in retention rates, which improved with greater incentive value. Whether cash was the most effective incentive was not clear from studies that compared cash and gifts of similar value. The average increase in retention rate was 12% for reminder letters, 5% for reminder calls and 12% for repeat questionnaires. Ten studies used alternative data collection methods, mainly as a last resort. All postal studies offered telephone interviews to non-responders, which increased retention rates by 3%. Studies that used face-to-face interviews increased their retention rates by 24% by offering alternative locations and modes of data collection. Conclusions: Incentives boosted retention rates in prospective cohort studies. Other methods appeared to have a beneficial effect but there was a general lack of a systematic approach to their evaluation

Effect of sitagliptin on cardiovascular outcomes in type 2 diabetes

BACKGROUND: Data are lacking on the long-term effect on cardiovascular events of adding sitagliptin, a dipeptidyl peptidase 4 inhibitor, to usual care in patients with type 2 diabetes and cardiovascular disease. METHODS: In this randomized, double-blind study, we assigned 14,671 patients to add either sitagliptin or placebo to their existing therapy. Open-label use of antihyperglycemic therapy was encouraged as required, aimed at reaching individually appropriate glycemic targets in all patients. To determine whether sitagliptin was noninferior to placebo, we used a relative risk of 1.3 as the marginal upper boundary. The primary cardiovascular outcome was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for unstable angina. RESULTS: During a median follow-up of 3.0 years, there was a small difference in glycated hemoglobin levels (least-squares mean difference for sitagliptin vs. placebo, -0.29 percentage points; 95% confidence interval [CI], -0.32 to -0.27). Overall, the primary outcome occurred in 839 patients in the sitagliptin group (11.4%; 4.06 per 100 person-years) and 851 patients in the placebo group (11.6%; 4.17 per 100 person-years). Sitagliptin was noninferior to placebo for the primary composite cardiovascular outcome (hazard ratio, 0.98; 95% CI, 0.88 to 1.09; P<0.001). Rates of hospitalization for heart failure did not differ between the two groups (hazard ratio, 1.00; 95% CI, 0.83 to 1.20; P = 0.98). There were no significant between-group differences in rates of acute pancreatitis (P = 0.07) or pancreatic cancer (P = 0.32). CONCLUSIONS: Among patients with type 2 diabetes and established cardiovascular disease, adding sitagliptin to usual care did not appear to increase the risk of major adverse cardiovascular events, hospitalization for heart failure, or other adverse events