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NMDA receptor-dependent glutamate excitotoxicity in human embryonic stem cell-derived neurons

AbstractThanks to the development of efficient differentiation strategies, human pluripotent stem cells (HPSC) offer the opportunity for modelling neuronal injury and dysfunction in human neurons in vitro. Critically, the effective use of HPSC-derived neural cells in disease-modelling and potentially cell replacement therapies hinges on an understanding of the biology of these cells, specifically their development, subtype specification and responses to neurotoxic signalling mediators. Here, we generated neurons from human embryonic stem cells and characterised the development of vulnerability to glutamate excitotoxicity, a key contributor to neuronal injury in several acute and chronic neurodegenerative disorders. Over two months of differentiation we observed a gradual increase in responsiveness of neurons to glutamate-induced Ca2+ influx, attributable to NMDA receptor activity. This increase was concomitant with an increase in expression of mRNA encoding NMDA and AMPA receptor subunits. Differentiated neurons were vulnerable to glutamate excitotoxicity in a dose-dependent manner, which was reduced by NMDA receptor antagonists

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

BCL2 in breast cancer: a favourable prognostic marker across molecular subtypes and independent of adjuvant therapy received

Background: Breast cancer is heterogeneous and the existing prognostic classifiers are limited in accuracy, leading to unnecessary treatment of numerous women. B-cell lymphoma 2 (BCL2), an antiapoptotic protein, has been proposed as a prognostic marker, but this effect is considered to relate to oestrogen receptor (ER) status. This study aimed to test the clinical validity of BCL2 as an independent prognostic marker. Methods: Five studies of 11 212 women with early-stage breast cancer were analysed. Individual patient data included tumour size, grade, lymph node status, endocrine therapy, chemotherapy and mortality. BCL2, ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) levels were determined in all tumours. A Cox model incorporating the time-dependent effects of each variable was used to explore the prognostic significance of BCL2. Results: In univariate analysis, ER, PR and BCL2 positivity was associated with improved survival and HER2 positivity with inferior survival. For ER and PR this effect was time dependent, whereas for BCL2 and HER2 the effect persisted over time. In multivariate analysis, BCL2 positivity retained independent prognostic significance (hazard ratio (HR) 0.76, 95% confidence interval (CI) 0.66-0.88, P<0.001). BCL2 was a powerful prognostic marker in ER (HR 0.63, 95% CI 0.54-0.74, P<0.001) and ER disease (HR 0.56, 95% CI 0.48-0.65, P<0.001), and in HER2 (HR 0.55, 95% CI 0.49-0.61, P<0.001) and HER2 disease (HR 0.70, 95% CI 0.57-0.85, P<0.001), irrespective of the type of adjuvant therapy received. Addition of BCL2 to the Adjuvant! Online prognostic model, for a subset of cases with a 10-year follow-up, improved the survival prediction (P<0.0039). Conclusions: BCL2 is an independent indicator of favourable prognosis for all types of early-stage breast cancer. This study establishes the rationale for introduction of BCL2 immunohistochemistry to improve prognostic stratification. Further work is now needed to ascertain the exact way to apply BCL2 testing for risk stratification and to standardise BCL2 immunohistochemistry for this application. © 2010 Cancer Research UK All rights reserved

An Accurate Mass Determination for Kepler-1655b, a Moderately Irradiated World with a Significant Volatile Envelope

Funding: A.C.C. acknowledges support from STFC consolidated grant number ST/M001296/1. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant Agreement No. 313014 (ETAEARTH).We present the confirmation of a small, moderately-irradiated (F= 155±7 F⊕) Neptune with a substantial gas envelope in a P=11.8728787±0.0000085-day orbit about a quiet, Sun-like G0V star Kepler-1655. Based on our analysis of the Kepler light curve, we determined Kepler-1655b’s radius to be 2.213±0.082 R⊕. We acquired 95 high-resolution spectra with TNG/HARPS-N, enabling us to characterize the host star and determine an accurate mass for Kepler-1655b of 5.0±^3.1_2.8 M⊕ via Gaussian-process regression. Our mass determination excludes an Earth-like composition with 98% confidence. Kepler-1655b falls on the upper edge of the evaporation valley, in the relatively sparsely occupied transition region between rocky and gas-rich planets. It is therefore part of a population of planets that we should actively seek to characterize further.PostprintPeer reviewe

Influenza Virus A Infection of Human Monocyte and Macrophage Subpopulations Reveals Increased Susceptibility Associated with Cell Differentiation

Influenza virus infection accounts for significant morbidity and mortality world-wide. Interactions of the virus with host cells, particularly those of the macrophage lineage, are thought to contribute to various pathological changes associated with poor patient outcome. Development of new strategies to treat disease therefore requires a detailed understanding of the impact of virus infection upon cellular responses. Here we report that human blood-derived monocytes could be readily infected with the H3N2 influenza virus A/Udorn/72 (Udorn), irrespective of their phenotype (CD14++/CD16−, CD14++/CD16+ or CD14dimCD16++), as determined by multi-colour flow cytometry for viral haemagglutinin (HA) expression and cell surface markers 8–16 hours post infection. Monocytes are relatively resistant to influenza-induced cell death early in infection, as approximately 20% of cells showed influenza-induced caspase-dependent apoptosis. Infection of monocytes with Udorn also induced the release of IL-6, IL-8, TNFα and IP-10, suggesting that NS1 protein of Udorn does not (effectively) inhibit this host defence response in human monocytes. Comparative analysis of human monocyte-derived macrophages (Mph) demonstrated greater susceptibility to human influenza virus than monocytes, with the majority of both pro-inflammatory Mph1 and anti-inflammatory/regulatory Mph2 cells expressing viral HA after infection with Udorn. Influenza infection of macrophages also induced cytokine and chemokine production. However, both Mph1 and Mph2 phenotypes released comparable amounts of TNFα, IL-12p40 and IP-10 after infection with H3N2, in marked contrast to differential responses to LPS-stimulation. In addition, we found that influenza virus infection augmented the capacity of poorly phagocytic Mph1 cells to phagocytose apoptotic cells by a mechanism that was independent of either IL-10 or the Mer receptor tyrosine kinase/Protein S pathway. In summary, our data reveal that influenza virus infection of human macrophages causes functional alterations that may impact on the process of resolution of inflammation, with implications for viral clearance and lung pathology

Symptom-based stratification of patients with primary Sjögren's syndrome: multi-dimensional characterisation of international observational cohorts and reanalyses of randomised clinical trials

Background: Heterogeneity is a major obstacle to developing effective treatments for patients with primary Sjögren's syndrome. We aimed to develop a robust method for stratification, exploiting heterogeneity in patient-reported symptoms, and to relate these differences to pathobiology and therapeutic response. / Methods: We did hierarchical cluster analysis using five common symptoms associated with primary Sjögren's syndrome (pain, fatigue, dryness, anxiety, and depression), followed by multinomial logistic regression to identify subgroups in the UK Primary Sjögren's Syndrome Registry (UKPSSR). We assessed clinical and biological differences between these subgroups, including transcriptional differences in peripheral blood. Patients from two independent validation cohorts in Norway and France were used to confirm patient stratification. Data from two phase 3 clinical trials were similarly stratified to assess the differences between subgroups in treatment response to hydroxychloroquine and rituximab. / Findings: In the UKPSSR cohort (n=608), we identified four subgroups: Low symptom burden (LSB), high symptom burden (HSB), dryness dominant with fatigue (DDF), and pain dominant with fatigue (PDF). Significant differences in peripheral blood lymphocyte counts, anti-SSA and anti-SSB antibody positivity, as well as serum IgG, κ-free light chain, β2-microglobulin, and CXCL13 concentrations were observed between these subgroups, along with differentially expressed transcriptomic modules in peripheral blood. Similar findings were observed in the independent validation cohorts (n=396). Reanalysis of trial data stratifying patients into these subgroups suggested a treatment effect with hydroxychloroquine in the HSB subgroup and with rituximab in the DDF subgroup compared with placebo. / Interpretation: Stratification on the basis of patient-reported symptoms of patients with primary Sjögren's syndrome revealed distinct pathobiological endotypes with distinct responses to immunomodulatory treatments. Our data have important implications for clinical management, trial design, and therapeutic development. Similar stratification approaches might be useful for patients with other chronic immune-mediated diseases. / Funding: UK Medical Research Council, British Sjogren's Syndrome Association, French Ministry of Health, Arthritis Research UK, Foundation for Research in Rheumatology

Homologous recombination DNA repair defects in PALB2-associated breast cancers

© 2019, The Author(s). Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display bi-allelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2-associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD