The bHLH transcription factor Mist1 is required to maintain exocrine pancreas cell organization and acinar cell identity

The pancreas is a complex organ that consists of separate endocrine and exocrine cell compartments. Although great strides have been made in identifying regulatory factors responsible for endocrine pancreas formation, the molecular regulatory circuits that control exocrine pancreas properties are just beginning to be elucidated. In an effort to identify genes involved in exocrine pancreas function, we have examined Mist1, a basic helix-loop-helix transcription factor expressed in pancreatic acinar cells. Mist1-null (Mist1KO) mice exhibit extensive disorganization of exocrine tissue and intracellular enzyme activation. The exocrine disorganization is accompanied by increases in p8, RegI/PSP, and PAP1/RegIII gene expression, mimicking the molecular changes observed in pancreatic injury. By 12 m, Mist1KO mice develop lesions that contain cells coexpressing acinar and duct cell markers. Analysis of the factors involved in cholecystokinin (CCK) signaling reveal inappropriate levels of the CCK receptor A and the inositol-1,4,5-trisphosphate receptor 3, suggesting that a functional defect exists in the regulated exocytosis pathway of Mist1KO mice. Based on these observations, we propose that Mist1KO mice represent a new genetic model for chronic pancreas injury and that the Mist1 protein serves as a key regulator of acinar cell function, stability, and identity

Activation of protein kinase Cδ leads to increased pancreatic acinar cell dedifferentiation in the absence of MIST1

Pancreatic ductal adenocarcinoma (PDAC) has a 5 year survival rate post-diagnosis of \u3c 5%. Individuals with chronic pancreatitis (CP) are 20-fold more likely to develop PDAC, making it a significant risk factor for PDAC. While the relationship for the increased susceptibility to PDAC is unknown, loss of the acinar cell phenotype is common to both pathologies. Pancreatic acinar cells can dedifferentiate or trans-differentiate into a number of cell types including duct cells, β cells, hepatocytes and adipocytes. Knowledge of the molecular pathways that regulate this plasticity should provide insight into PDAC and CP. MIST1 (encoded by Bhlha15 in mice) is a transcription factor required for complete acinar cell maturation. The goal of this study was to examine the plasticity of acinar cells that do not express MIST1 (Mist1 -/-). The fate of acinar cells from C57Bl6 or congenic Mist1 -/- mice expressing an acinar specific, tamoxifen-inducible Cre recombinase mated to Rosa26 reporter LacZ mice (Mist1CreERT/- R26r) was determined following culture in a three-dimensional collagen matrix. Mist1CreERT/- R26r acini showed increased acinar dedifferentiation, formation of ductal cysts and transient increases in PDX1 expression compared to wild-type acinar cells. Other progenitor cell markers, including Foxa1, Sox9, Sca1 and Hes1, were elevated only in Mist1-/- cultures. Analysis of protein kinase C (PKC) isoforms by western blot and immunofluorescence identified increased PKCε accumulation and nuclear localization of PKCδ that correlated with increased duct formation. Treatment with rottlerin, a PKCδ-specific inhibitor, but not the PKCε-specific antagonist εV1-2, reduced acinar dedifferentiation, progenitor gene expression and ductal cyst formation. Immunocytochemistry on CP or PDAC tissue samples showed reduced MIST1 expression combined with increased nuclear PKCδ accumulation. These results suggest that the loss of MIST1 is a common event during PDAC and CP and events that affect MIST1 function and expression may increase susceptibility to these pathologies. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Activating Transcription Factor 3 Promotes Loss of the Acinar Cell Phenotype in Response to Cerulein-Induced Pancreatitis in Mice

Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of \u3e30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result

Non-Markovian Dynamics and Entanglement of Two-level Atoms in a Common Field

We derive the stochastic equations and consider the non-Markovian dynamics of a system of multiple two-level atoms in a common quantum field. We make only the dipole approximation for the atoms and assume weak atom-field interactions. From these assumptions we use a combination of non-secular open- and closed-system perturbation theory, and we abstain from any additional approximation schemes. These more accurate solutions are necessary to explore several regimes: in particular, near-resonance dynamics and low-temperature behavior. In detuned atomic systems, small variations in the system energy levels engender timescales which, in general, cannot be safely ignored, as would be the case in the rotating-wave approximation (RWA). More problematic are the second-order solutions, which, as has been recently pointed out, cannot be accurately calculated using any second-order perturbative master equation, whether RWA, Born-Markov, Redfield, etc.. This latter problem, which applies to all perturbative open-system master equations, has a profound effect upon calculation of entanglement at low temperatures. We find that even at zero temperature all initial states will undergo finite-time disentanglement (sometimes termed "sudden death"), in contrast to previous work. We also use our solution, without invoking RWA, to characterize the necessary conditions for Dickie subradiance at finite temperature. We find that the subradiant states fall into two categories at finite temperature: one that is temperature independent and one that acquires temperature dependence. With the RWA there is no temperature dependence in any case.Comment: 17 pages, 13 figures, v2 updated references, v3 clarified results and corrected renormalization, v4 further clarified results and new Fig. 8-1

The Absence of MIST1 Leads to Increased Ethanol Sensitivity and Decreased Activity of the Unfolded Protein Response in Mouse Pancreatic Acinar Cells

Background: Alcohol abuse is a leading cause of pancreatitis in humans. However, rodent models suggest that alcohol only sensitizes the pancreas to subsequent insult, indicating that additional factors play a role in alcohol-induced pancreatic injury. The goal of this study was to determine if an absence of MIST1, a transcription factor required for complete differentiation of pancreatic acinar cells in mice, increased the sensitivity to alcohol. Methods: Two to four month-old mice lacking MIST1 (Mist1 2/2) or congenic C57 Bl6 mice were placed on a Lieber-DeCarli diet (36 % of total kcal from ethanol and fat), a control liquid diet (36 % kcal from fat) or a regular breeding chow diet (22% kcal from fat). After six weeks, pancreatic morphology was assessed. Biochemical and immunofluorescent analysis was used to assess mediators of the unfolded protein response (UPR). Results: Ethanol-fed Mist1 2/2 mice developed periductal accumulations of inflammatory cells that did not appear in wild type or control-fed Mist1 2/2 mice. Wild type mice fed diets high in ethanol or fat showed enhancement of the UPR based on increased accumulation of peIF2a and spliced XBP1. These increases were not observed in Mist1 2/2 pancreatic tissue, which had elevated levels of UPR activity prior to diet exposure. Indeed, exposure to ethanol resulted in a reduction of UPR activity in Mist1 2/2 mice. Conclusions: Our findings suggest that an absence of MIST1 increases the sensitivity to ethanol that correlated wit

Identifying care-home residents in routine healthcare datasets:a diagnostic test accuracy study of five methods

Background: there is no established method to identify care-home residents in routine healthcare datasets. Methods matching patient’s addresses to known care-home addresses have been proposed in the UK, but few have been formally evaluated. Study design: prospective diagnostic test accuracy study. Methods: four independent samples of 5,000 addresses from Community Health Index (CHI) population registers were sampled for two NHS Scotland Health Boards on 1 April 2017, with one sample of adults aged ≥65 years and one of all residents. To derive the reference standard, all 20,000 addresses were manually adjudicated as ‘care-home address’ or not. The performance of five methods (NHS Scotland assigned CHI Institution Flag, exact address matching, postcode matching, Phonics and Markov) was evaluated compared to the reference standard. Results: the CHI Institution Flag had a high PPV 97–99% in all four test sets, but poorer sensitivity 55–89%. Exact address matching failed in every case. Postcode matching had higher sensitivity than the CHI flag 78–90%, but worse PPV 77–85%. Area under the receiver operating curve values for Phonics and Markov scores were 0.86–0.95 and 0.93–0.98, respectively. Phonics score with cut-off ≥13 had PPV 92–97% with sensitivity 72–87%. Markov PPVs were 90–95% with sensitivity 69–90% with cut-off ≥29.6. Conclusions: more complex address matching methods greatly improve identification compared to the existing NHS Scotland flag or postcode matching, although no method achieved both sensitivity and positive predictive value > 95%. Choice of method and cut-offs will be determined by the specific needs of researchers and practitioners

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Comprehensive Molecular Characterization of Pheochromocytoma and Paraganglioma

SummaryWe report a comprehensive molecular characterization of pheochromocytomas and paragangliomas (PCCs/PGLs), a rare tumor type. Multi-platform integration revealed that PCCs/PGLs are driven by diverse alterations affecting multiple genes and pathways. Pathogenic germline mutations occurred in eight PCC/PGL susceptibility genes. We identified CSDE1 as a somatically mutated driver gene, complementing four known drivers (HRAS, RET, EPAS1, and NF1). We also discovered fusion genes in PCCs/PGLs, involving MAML3, BRAF, NGFR, and NF1. Integrated analysis classified PCCs/PGLs into four molecularly defined groups: a kinase signaling subtype, a pseudohypoxia subtype, a Wnt-altered subtype, driven by MAML3 and CSDE1, and a cortical admixture subtype. Correlates of metastatic PCCs/PGLs included the MAML3 fusion gene. This integrated molecular characterization provides a comprehensive foundation for developing PCC/PGL precision medicine

MUSiC : a model-unspecific search for new physics in proton-proton collisions at root s=13TeV

Results of the Model Unspecific Search in CMS (MUSiC), using proton-proton collision data recorded at the LHC at a centre-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 35.9 fb(-1), are presented. The MUSiC analysis searches for anomalies that could be signatures of physics beyond the standard model. The analysis is based on the comparison of observed data with the standard model prediction, as determined from simulation, in several hundred final states and multiple kinematic distributions. Events containing at least one electron or muon are classified based on their final state topology, and an automated search algorithm surveys the observed data for deviations from the prediction. The sensitivity of the search is validated using multiple methods. No significant deviations from the predictions have been observed. For a wide range of final state topologies, agreement is found between the data and the standard model simulation. This analysis complements dedicated search analyses by significantly expanding the range of final states covered using a model independent approach with the largest data set to date to probe phase space regions beyond the reach of previous general searches.Peer reviewe

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe