Whole-genome sequencing for national surveillance of Shiga toxin–producing Escherichia coli O157

Background. National surveillance of gastrointestinal pathogens, such as Shiga toxin–producing Escherichia coli O157 (STEC O157), is key to rapidly identifying linked cases in the distributed food network to facilitate public health interventions. In this study, we used whole-genome sequencing (WGS) as a tool to inform national surveillance of STEC O157 in terms of identifying linked cases and clusters and guiding epidemiological investigation. Methods. We retrospectively analyzed 334 isolates randomly sampled from 1002 strains of STEC O157 received by the Gastrointestinal Bacteria Reference Unit at Public Health England, Colindale, in 2012. The genetic distance between each isolate, as estimated by WGS, was calculated and phylogenetic methods were used to place strains in an evolutionary context. Results. Estimates of linked clusters representing STEC O157 outbreaks in England and Wales increased by 2-fold when WGS was used instead of traditional typing techniques. The previously unidentified clusters were often widely geographically distributed and small in size. Phylogenetic analysis facilitated identification of temporally distinct cases sharing common exposures and delineating those that shared epidemiological and temporal links. Comparison with multi locus variable number tandem repeat analysis (MLVA) showed that although MLVA is as sensitive as WGS, WGS provides a more timely resolution to outbreak clustering. Conclusions. WGS has come of age as a molecular typing tool to inform national surveillance of STEC O157; it can be used in real time to provide the highest strain-level resolution for outbreak investigation. WGS allows linked cases to be identified with unprecedented specificity and sensitivity that will facilitate targeted and appropriate public health investigations

The development, application and analysis of an enhanced recovery programme for major oesophagogastric resection

Background: Enhanced recovery programmes improve outcomes in surgery, but their implementation after upper gastrointestinal resection has been limited. The aim of this study was to compare short-term outcomes for patients undergoing oesophagogastric surgery in an enhanced recovery programme (EROS). Methods: EROS was developed after a multidisciplinary meeting by multiple rounds of revision. EROS was applied to all patients undergoing major upper GI resection at a university teaching hospital in the UK from 20/9/13, with data reviewed at 18/09/15. EROS was assessed to identify predictors for compliance. Results: One hundred six patients underwent major upper GI resection including 81 oesophagectomies, 24 gastrectomies and 1 colonic interposition graft. Major complications (ClavienDindo≥3) occurred in 12 patients with 1 in-hospital death. Thirty-five patients (44%) were discharged on target day 8 of the EROS programme. Age and complications were independently associated with missing this discharge target. Conclusion: Enhanced recovery is feasible and safe after major upper gastrointestinal surgery.<br/

Fibroblasts derived from oesophageal adenocarcinoma differ in DNA methylation profile from normal oesophageal fibroblasts

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Oesophageal adenocarcinoma (OAC) is increasing in incidence and has a poor prognosis. Tumour derived fibroblasts (TDFs) differ functionally from normal fibroblasts (NDFs), and play a pivotal role in cancer. Many of the differences persist through subculture. We measured the DNA methylation profiles of 10 TDFs from OAC with 12 NDF from normal oesophageal mucosa using Infinium HumanMethylation450 Beadchips and found they differed in multidimensional scaling analysis. We identified 4,856 differentially methylated CpGs (DMCs, adjusted p  0.15), of which 3,243 (66.8%) were hypomethylated in TDFs compared to NDFs. Hypermethylated DMCs were enriched at transcription start sites (TSSs) and in CpG islands, and depleted in transcriptional enhancers. Gene ontology analysis of genes with DMCs at TSSs revealed an enrichment of genes involved in development, morphogenesis, migration, adhesion, regulation of processes and response to stimuli. Alpha-smooth muscle actin (α-SMA) is a marker of activated fibroblasts and a poor prognostic indicator in OAC. Hypomethylated DMCs were observed at the TSS of transcript variant 2 of α-SMA, which correlated with an increase in α-SMA protein expression. These data suggest that DNA methylation may contribute to the maintenance of the TDF phenotype

Loss of murine Paneth cell function alters the immature intestinal microbiome and mimics changes seen in neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) remains the leading cause of gastrointestinal morbidity and mortality in premature infants. Human and animal studies suggest a role for Paneth cells in NEC pathogenesis. Paneth cells play critical roles in host-microbial interactions and epithelial homeostasis. The ramifications of eliminating Paneth cell function on the immature host-microbial axis remains incomplete. Paneth cell function was depleted in the immature murine intestine using chemical and genetic models, which resulted in intestinal injury consistent with NEC. Paneth cell depletion was confirmed using histology, electron microscopy, flow cytometry, and real time RT-PCR. Cecal samples were analyzed at various time points to determine the effects of Paneth cell depletion with and without Klebsiella gavage on the microbiome. Deficient Paneth cell function induced significant compositional changes in the cecal microbiome with a significant increase in Enterobacteriacae species. Further, the bloom of Enterobacteriaceae species that occurs is phenotypically similar to what is seen in human NEC. This further strengthens our understanding of the importance of Paneth cells to intestinal homeostasis in the immature intestine

ExoMol molecular line lists - XIV. The rotation-vibration spectrum of hot SO₂

Sulphur dioxide is well-known in the atmospheres of planets and satellites, where its presence is often associated with volcanism, and in circumstellar envelopes of young and evolved stars as well as the interstellar medium. This work presents a line list of 1.3 billion $^{32}$S$^{16}$O$_2$ vibration-rotation transitions computed using an empirically-adjusted potential energy surface and an ab initio dipole moment surface. The list gives complete coverage up to 8000 cm$^{-1}$ (wavelengths longer than 1.25 $\mu$m) for temperatures below 2000 K. Infrared absorption cross sections are recorded at 300 and 500 C are used to validated the resulting ExoAmes line list. The line list is made available in electronic form as supplementary data to this article and at www.exomol.com.Comment: Submitted to MNRA

Cancer associated fibroblasts predict for poor outcome and promote periostin-dependent invasion in oesophageal adenocarcinoma

Interactions between cancer cells and cancer-associated fibroblasts (CAF) play an important role in tumour development and progression. In this study we investigated the functional role of CAF in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of EAC patients (183 patients) for CAF markers related to disease mortality. We characterized CAF and normal oesophageal fibroblasts (NOF) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3-D organotypic culture and xenograft models were used to examine effects on EAC cell function, and dissect molecular mechanisms regulating invasion. Most EAC (93%) contained CAF with a myofibroblastic (?-SMA-positive) phenotype, which correlated significantly with poor survival (p?=?0.016; HR 7. 1 (1.7-29.4). Primary CAF, isolated from EAC, have a contractile, myofibroblastic phenotype, and promote EAC cell invasion in vitro (Transwell assays, p?=?&lt;0.05; organotypic culture, p?&lt;?0.001) and in vivo (p?=?&lt;0.05). In vitro, this pro-invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAF, and acts as a ligand for EAC cell integrins ?v?3 and ?v?5, promoting activation of the PI3kinase/Akt pathway. In patient samples, periostin expression at the tumour cell/stromal interface correlates with poor overall and disease-free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF-secreted periostin and EAC-expressed integrins results in PI3 kinase/Akt activation and increased tumour cell invasion. Most EAC contain a myofibroblastic CAF-rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patient

The AUGIS Survival Predictor: Prediction of Long-Term and Conditional Survival After Esophagectomy Using Random Survival Forests.

OBJECTIVE: The aim of this study was to develop a predictive model for overall survival after esophagectomy using pre/postoperative clinical data and machine learning. SUMMARY BACKGROUND DATA: For patients with esophageal cancer, accurately predicting long-term survival after esophagectomy is challenging. This study investigated survival prediction after esophagectomy using a RandomSurvival Forest (RSF) model derived from routine data from a large, well-curated, national dataset. METHODS: Patients diagnosed with esophageal adenocarcinoma or squamous cell carcinoma between 2012 and 2018 in England and Wales who underwent an esophagectomy were included. Prediction models for overall survival were developed using the RSF method and Cox regression from 41 patient and disease characteristics. Calibration and discrimination (time-dependent area under the curve) were validated internally using bootstrap resampling. RESULTS: The study analyzed 6399 patients, with 2625 deaths during follow-up. Median follow-up was 41 months. Overall survival was 47.1% at 5 years. The final RSF model included 14 variables and had excellent discrimination with a 5-year time-dependent area under the receiver operator curve of 83.9% [95% confidence interval (CI) 82.6%-84.9%], compared to 82.3% (95% CI 81.1%-83.3%) for the Cox model. The most important variables were lymph node involvement, pT stage, circumferential resection margin involvement (tumor at < 1 mm from cut edge) and age. There was a wide range of survival estimates even within TNM staging groups, with quintiles of prediction within Stage 3b ranging from 12.2% to 44.7% survival at 5 years. CONCLUSIONS: An RSF model for long-term survival after esophagectomy exhibited excellent discrimination and well-calibrated predictions. At a patient level, it provides more accuracy than TNM staging alone and could help in the delivery of tailored treatment and follow-up

Modulation of the tumour promoting functions of cancer associated fibroblasts by phosphodiesterase type 5 inhibition increases the efficacy of chemotherapy in human preclinical models of esophageal adenocarcinoma

Background and aims: Esophageal adenocarcinoma (EAC) is chemoresistant in the majority of cases. The tumor-promoting biology of cancer associated fibroblasts (CAF) make them a target for novel therapies. Phosphodiesterase type 5 inhibitors (PDE5i) have been shown to regulate the activated fibroblast phenotype in benign disease. We investigated the potential for CAF modulation in EAC using PDE5i to enhance the efficacy of chemotherapy. Methods: EAC fibroblasts were treated with PDE5i and phenotypic effects examined using immunoblotting, immunohistochemistry, gel contraction, transwell invasion, organotypics, single cell RNAseq and shotgun proteomics. The combination of PDE5i with standard-of-care chemotherapy (Epirubicin, 5-Fluorouracil and Cisplatin) was tested for safety and efficacy in validated near-patient model systems (3D tumor growth assays (3D-TGAs) and patient derived xenograft (PDX) mouse models). Results: PDE5i treatment reduced alpha-SMA expression in CAFs by 50% (p<0.05), associated with a significant reduction in the ability of CAFs to contract collagen-1 gels and induce cancer cell invasion, (p<0.05). RNAseq and proteomic analysis of CAF and EAC cell lines revealed PDE5i specific regulation of pathways related to fibroblast activation and tumor promotion. 3D-TGA assays confirmed the importance of stromal cells to chemoresistance in EAC, which could be attenuated by PDE5i. Chemotherapy+PDE5i in PDX-bearing mice was safe and significantly reduced PDX tumor volume (p<0.05). Conclusion: PDE5 is a candidate for clinical trials to alter the fibroblast phenotype in esophageal cancer. We demonstrate the specificity of PDE5i for fibroblasts to prevent transdifferentiation and revert the CAF phenotype. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient in vitro and in vivo PDX-based model systems