Validation of Pyrosequencing for the Analysis of KRAS Mutations in Colorectal Cancer

The use of antibodies against epidermal growth factor receptor( EGFR) in conjunction with conventionalchemotherapy for metastatic colorectal cancer (CRC) in patients with KRAS wild-type tumors has beenproven to be efficacious. Recently, KRAS testing prior to anti-EGFR therapy has become mandatory formetastatic CRC patients. Although newly developed pyrosequencing is expected to be one of the highthroughput procedures detecting such mutations, the accuracy of the procedure has not been well evaluated.In the present study, we aimed to validate the accuracy, especially the potential for a false-negative result,in detecting KRAS mutations by pyrosequencing using cultured tumor cells. DNA extracted from culturedìNOZî gallbladder cancer cells( known to contain KRAS mutation G12V) at concentrations of 1%, 5%, 10%, and 25%, as well as 2 DNA samples extracted from a resected CRC specimen( known to contain anotherKRAS mutation, G12C) at concentrations of 5% and 25%, were prepared. We analyzed KRAS mutationalstatus and nonexistent and/or nonfunctional mutations of these 6 samples using pyrosequencing. TheKRAS mutation detection rates in the 4 NOZ samples( 1%, 5%, 10%, and 25%) were 0.37%, 2.79%, 5.28%,and 13.85%, respectively. Some artifacts of KRAS mutations unlikely to be present were detected in 1%samples of NOZ at a rate similar to that of the G12V mutation( G12C, 0.29%;G13C, 0.42%). Although theKRAS mutation G12C was detected at rates of 1.26% and 6.49% in samples with 5% and 25% DNA extractedfrom resected CRC specimen, respectively, no other type of KRAS mutation was detected in suchsamples. Pyrosequencing could not detect KRAS mutations correctly in the sample containing 1% DNA.This might cause false negatives. A sample mutated DNA concentration of at least 5% was necessary forprecise analyses by this procedure

Impact of clinical phenotypes on management and outcomes in European atrial fibrillation patients: a report from the ESC-EHRA EURObservational Research Programme in AF (EORP-AF) General Long-Term Registry

Background: Epidemiological studies in atrial fibrillation (AF) illustrate that clinical complexity increase the risk of major adverse outcomes. We aimed to describe European AF patients\u2019 clinical phenotypes and analyse the differential clinical course. Methods: We performed a hierarchical cluster analysis based on Ward\u2019s Method and Squared Euclidean Distance using 22 clinical binary variables, identifying the optimal number of clusters. We investigated differences in clinical management, use of healthcare resources and outcomes in a cohort of European AF patients from a Europe-wide observational registry. Results: A total of 9363 were available for this analysis. We identified three clusters: Cluster 1 (n = 3634; 38.8%) characterized by older patients and prevalent non-cardiac comorbidities; Cluster 2 (n = 2774; 29.6%) characterized by younger patients with low prevalence of comorbidities; Cluster 3 (n = 2955;31.6%) characterized by patients\u2019 prevalent cardiovascular risk factors/comorbidities. Over a mean follow-up of 22.5 months, Cluster 3 had the highest rate of cardiovascular events, all-cause death, and the composite outcome (combining the previous two) compared to Cluster 1 and Cluster 2 (all P <.001). An adjusted Cox regression showed that compared to Cluster 2, Cluster 3 (hazard ratio (HR) 2.87, 95% confidence interval (CI) 2.27\u20133.62; HR 3.42, 95%CI 2.72\u20134.31; HR 2.79, 95%CI 2.32\u20133.35), and Cluster 1 (HR 1.88, 95%CI 1.48\u20132.38; HR 2.50, 95%CI 1.98\u20133.15; HR 2.09, 95%CI 1.74\u20132.51) reported a higher risk for the three outcomes respectively. Conclusions: In European AF patients, three main clusters were identified, differentiated by differential presence of comorbidities. Both non-cardiac and cardiac comorbidities clusters were found to be associated with an increased risk of major adverse outcomes

The structure of resilience in irritable bowel syndrome and its improvement through hypnotherapy: Cross-sectional and prospective longitudinal data

Background: Resilience refers to a class of variables that are highly relevant to wellbeing and coping with stress, trauma, and chronic adversity. Despite its significance for health, resilience suffers from poor conceptual integration. Irritable bowel syndrome (IBS) is a functional disorder with altered psychological stress reactivity and a brain-gut-microbiota axis, which causes high levels of chronic strain. Gut-directed Hypnotherapy (GHT) is a standardized treatment for IBS aimed at improving resilience. An improvement of resilience as a result of GHT has been hypothesized but requires further investigation. The aims of the study were to validate the construct and develop an integrational measure of various resilience domains by dimensional reduction, and to investigate changes in resilience in IBS patients after GHT. Method: A total of N = 74 gastroenterology outpatients with IBS (Rome III criteria) were examined in 7 resilience domains, quality of life, psychological distress and symptom severity. Of these, n = 53 participated in 7 to 10 GHT group sessions (Manchester protocol). Post-treatment examinations were performed on average 10 months after last GHT session. Results: Resilience factors proved to be unidimensional in the total sample. Greater resilience (composite score of resilience domains) and quality of life, and lower symptom severity and psychological distress were found after treatment (n = 16). Similar differences were present in cross-sectional comparisons of n = 37 treated vs. n = 37 untreated patients. Conclusion: Resilience factors share a common psychological dimension and are functionally connected. The absence of maladaptive behaviours contributes to resilience. Improvements in resilience after hypnotherapy with parallel increases in quality of life and reduced psychological distress and symptom severity were observed. Independent replications with larger sample sizes and randomized controlled trials are needed.© 2018 Peter et a

Ethanol sensitizes hepatocytes for TGF-β-triggered apoptosis

Alcohol abuse is a global health problem causing a substantial fraction of chronic liver diseases. Abundant TGF-β—a potent pro-fibrogenic cytokine—leads to disease progression. Our aim was to elucidate the crosstalk of TGF-β and alcohol on hepatocytes. Primary murine hepatocytes were challenged with ethanol and TGF-β and cell fate was determined. Fluidigm RNA analyses revealed transcriptional effects that regulate survival and apoptosis. Mechanistic insights were derived from enzyme/pathway inhibition experiments and modulation of oxidative stress levels. To substantiate findings, animal model specimens and human liver tissue cultures were investigated. Results: On its own, ethanol had no effect on hepatocyte apoptosis, whereas TGF-β increased cell death. Combined treatment led to massive hepatocyte apoptosis, which could also be recapitulated in human HCC liver tissue treated ex vivo. Alcohol boosted the TGF-β pro-apoptotic gene signature. The underlying mechanism of pathway crosstalk involves SMAD and non-SMAD/AKT signaling. Blunting CYP2E1 and ADH activities did not prevent this effect, implying that it was not a consequence of alcohol metabolism. In line with this, the ethanol metabolite acetaldehyde did not mimic the effect and glutathione supplementation did not prevent the super-induction of cell death. In contrast, blocking GSK-3β activity, a downstream mediator of AKT signaling, rescued the strong apoptotic response triggered by ethanol and TGF-β. This study provides novel information on the crosstalk between ethanol and TGF-β. We give evidence that ethanol directly leads to a boost of TGF-β’s pro-apoptotic function in hepatocytes, which may have implications for patients with chronic alcoholic liver disease.© The Author(s) 201

Non-culture-based studies of the appendiceal microbiota: a systematic review - Methods-Reporting-Checklist_FM

Aims: To review studies examining the appendiceal microbiota and microbial changes in acute appendicitis. Methods: After a systematic literature search, 11 studies examining the appendiceal microbiota (414 samples) using non-culture-based methods were included. Results: The appendiceal microbiota showed decreased α-diversity compared with fecal microbiota. Inflamed and uninflamed appendices showed differences in β-diversity, and there was an increased abundance of oral-associated bacteria in inflamed versus uninflamed appendices. Conclusion: The appendiceal microbiota exhibits lower α-diversity than the fecal microbiota, with an increased abundance of oral-associated bacteria. Compared with uninflamed appendices, the appendix microbiota in acute appendicitis also showed increased abundance of oral-associated bacteria, but no bacterial profile unique to either complicated or uncomplicated appendicitis was found

Non-culture-based studies of the appendiceal microbiota: a systematic review - Methods-Reporting-Checklist_FM

Aims: To review studies examining the appendiceal microbiota and microbial changes in acute appendicitis. Methods: After a systematic literature search, 11 studies examining the appendiceal microbiota (414 samples) using non-culture-based methods were included. Results: The appendiceal microbiota showed decreased α-diversity compared with fecal microbiota. Inflamed and uninflamed appendices showed differences in β-diversity, and there was an increased abundance of oral-associated bacteria in inflamed versus uninflamed appendices. Conclusion: The appendiceal microbiota exhibits lower α-diversity than the fecal microbiota, with an increased abundance of oral-associated bacteria. Compared with uninflamed appendices, the appendix microbiota in acute appendicitis also showed increased abundance of oral-associated bacteria, but no bacterial profile unique to either complicated or uncomplicated appendicitis was found

Non-culture-based studies of the appendiceal microbiota: a systematic review - Table S1

Aims: To review studies examining the appendiceal microbiota and microbial changes in acute appendicitis. Methods: After a systematic literature search, 11 studies examining the appendiceal microbiota (414 samples) using non-culture-based methods were included. Results: The appendiceal microbiota showed decreased α-diversity compared with fecal microbiota. Inflamed and uninflamed appendices showed differences in β-diversity, and there was an increased abundance of oral-associated bacteria in inflamed versus uninflamed appendices. Conclusion: The appendiceal microbiota exhibits lower α-diversity than the fecal microbiota, with an increased abundance of oral-associated bacteria. Compared with uninflamed appendices, the appendix microbiota in acute appendicitis also showed increased abundance of oral-associated bacteria, but no bacterial profile unique to either complicated or uncomplicated appendicitis was found

Non-culture-based studies of the appendiceal microbiota: a systematic review - Supplementary material FM

Aims: To review studies examining the appendiceal microbiota and microbial changes in acute appendicitis. Methods: After a systematic literature search, 11 studies examining the appendiceal microbiota (414 samples) using non-culture-based methods were included. Results: The appendiceal microbiota showed decreased α-diversity compared with fecal microbiota. Inflamed and uninflamed appendices showed differences in β-diversity, and there was an increased abundance of oral-associated bacteria in inflamed versus uninflamed appendices. Conclusion: The appendiceal microbiota exhibits lower α-diversity than the fecal microbiota, with an increased abundance of oral-associated bacteria. Compared with uninflamed appendices, the appendix microbiota in acute appendicitis also showed increased abundance of oral-associated bacteria, but no bacterial profile unique to either complicated or uncomplicated appendicitis was found

Non-culture-based studies of the appendiceal microbiota: a systematic review - Table S1

Aims: To review studies examining the appendiceal microbiota and microbial changes in acute appendicitis. Methods: After a systematic literature search, 11 studies examining the appendiceal microbiota (414 samples) using non-culture-based methods were included. Results: The appendiceal microbiota showed decreased α-diversity compared with fecal microbiota. Inflamed and uninflamed appendices showed differences in β-diversity, and there was an increased abundance of oral-associated bacteria in inflamed versus uninflamed appendices. Conclusion: The appendiceal microbiota exhibits lower α-diversity than the fecal microbiota, with an increased abundance of oral-associated bacteria. Compared with uninflamed appendices, the appendix microbiota in acute appendicitis also showed increased abundance of oral-associated bacteria, but no bacterial profile unique to either complicated or uncomplicated appendicitis was found