Effects of creatine supplementation on housekeeping genes in human skeletal muscle using real-time RT-PCR.

The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (C(T)) values were established for beta-actin, beta2-microglobulin (beta2M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between C(T) values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw C(T) values and the linear value of 2(-C(T)), respectively. Interassay variability was 2.3% for raw C(T) values and 34% for the linear value of 2(-C(T)). We also examined the expression of various housekeeping genes in human skeletal muscle at days 0, 1, and 5 following oral supplementation with either creatine or a placebo employing a double-blind crossover study design. Treatments were separated by a 5-wk washout period. Immediately following each muscle sampling, subjects performed two 30-s all-out bouts on a cycle ergometer. Creatine supplementation increased (P < 0.05) muscle total creatine content above placebo levels; however, there were no changes (P > 0.05) in C(T) values across the supplementation periods for any of the genes. Nevertheless, 95% confidence intervals showed that GAPDH was variable, whereas beta-actin, beta2M, and CYC were the least varying genes. Normalization of the data to these housekeeping genes revealed variable behavior for beta2M with more stable expressions for both beta-actin and CYC. We conclude that, using real-time RT-PCR, beta-actin or CYC may be used as housekeeping genes to study gene expression in human muscle in experiments employing short-term creatine supplementation combined with high-intensity exercise

Learning from errors:Assessing final year medical students' reflection on safety improvement, five year cohort study

Abstract Background Investigation of real incidents has been consistently identified by expert reviews and student surveys as a potentially valuable teaching resource for medical students. The aim of this study was to adapt a published method to measure resident doctors’ reflection on quality improvement and evaluate this as an assessment tool for medical students. Methods The design is a cohort study. Medical students were prepared with a tutorial in team based learning format and an online Managing Incident Review course. The reliability of the modified Mayo Evaluation of Reflection on Improvement tool (mMERIT) was analysed with Generalizability G-theory. Long term sustainability of assessment of incident review with mMERIT was tested over five consecutive years. Results A total of 824 students have completed an incident review using 167 incidents from NHS Tayside’s online reporting system. In order to address the academic practice gap students were supervised by Senior Charge Nurses or Consultants on the wards where the incidents had been reported. Inter-rater reliability was considered sufficiently high to have one assessor for each student report. There was no evidence of a gradient in student marks across the academic year. Marks were significantly higher for students who used Section Questions to structure their reports compared with those who did not. In Year 1 of the study 21 (14%) of 153 mMERIT reports were graded as concern. All 21 of these students achieved the required standard on resubmission. Rates of resubmission were lower (3% to 7%) in subsequent years. Conclusions We have shown that mMERIT has high reliability with one rater. mMERIT can be used by students as part of a suite of feedback to help supplement their self-assessment on their learning needs and develop insightful practice to drive their development of quality, safety and person centred professional practice. Incident review addresses the need for workplace based learning and use of real life examples of mistakes, which has been identified by previous studies of education about patient safety in medical schools

Single-step ambient-air synthesis of graphene from renewable precursors as electrochemical genosensor

©The Author(s) 2017. Thermal chemical vapour deposition techniques for graphene fabrication, while promising, are thus far limited by resource-consuming and energy-intensive principles. In particular, purified gases and extensive vacuum processing are necessary for creating a highly controlled environment, isolated from ambient air, to enable the growth of graphene films. Here we exploit the ambient-air environment to enable the growth of graphene films, without the need for compressed gases. A renewable natural precursor, soybean oil, is transformed into continuous graphene films, composed of single-to-few layers, in a single step. The enabling parameters for controlled synthesis and tailored properties of the graphene film are discussed, and a mechanism for the ambient-air growth is proposed. Furthermore, the functionality of the graphene is demonstrated through direct utilization as an electrode to realize an effective electrochemical genosensor. Our method is applicable to other types of renewable precursors and may open a new avenue for low-cost synthesis of graphene films

The identification of informative genes from multiple datasets with increasing complexity

Background In microarray data analysis, factors such as data quality, biological variation, and the increasingly multi-layered nature of more complex biological systems complicates the modelling of regulatory networks that can represent and capture the interactions among genes. We believe that the use of multiple datasets derived from related biological systems leads to more robust models. Therefore, we developed a novel framework for modelling regulatory networks that involves training and evaluation on independent datasets. Our approach includes the following steps: (1) ordering the datasets based on their level of noise and informativeness; (2) selection of a Bayesian classifier with an appropriate level of complexity by evaluation of predictive performance on independent data sets; (3) comparing the different gene selections and the influence of increasing the model complexity; (4) functional analysis of the informative genes. Results In this paper, we identify the most appropriate model complexity using cross-validation and independent test set validation for predicting gene expression in three published datasets related to myogenesis and muscle differentiation. Furthermore, we demonstrate that models trained on simpler datasets can be used to identify interactions among genes and select the most informative. We also show that these models can explain the myogenesis-related genes (genes of interest) significantly better than others (P < 0.004) since the improvement in their rankings is much more pronounced. Finally, after further evaluating our results on synthetic datasets, we show that our approach outperforms a concordance method by Lai et al. in identifying informative genes from multiple datasets with increasing complexity whilst additionally modelling the interaction between genes. Conclusions We show that Bayesian networks derived from simpler controlled systems have better performance than those trained on datasets from more complex biological systems. Further, we present that highly predictive and consistent genes, from the pool of differentially expressed genes, across independent datasets are more likely to be fundamentally involved in the biological process under study. We conclude that networks trained on simpler controlled systems, such as in vitro experiments, can be used to model and capture interactions among genes in more complex datasets, such as in vivo experiments, where these interactions would otherwise be concealed by a multitude of other ongoing events

Platelet kinetics after slow versus standard transfusions: A pilot study

Background. Platelet transfusion is required in the acute phase of some thrombocytopenic disorders in order to prevent potentially dangerous hemorrhages. The purpose of this study was to assess the increase in platelet count following a slow platelet transfusion. Methods. Patients suffering from thrombocytopenia due to various underlying diseases were enrolled in the prospective pilot feasibility trial and were randomly divided into two groups. Standard platelet transfusion was administered in one group, while slow transfusion was used in the other. The platelet count was examined at 1 hour, 24 hours, and 1 week following the transfusions. Results. Although the platelet count was higher following 1 hour after transfusion via the standard method, the count tended to be higher 1 week after the transfusion in the slow transfusion group. This difference, however, only turned out to be statistically significant amongst females. Conclusion. A therapy of slow platelet transfusion might be more effective for the prevention of platelet loss. Further studies will be required to strengthen this hypothesis

Exercise physiological responses to drug treatments in chronic thromboembolic pulmonary hypertension

We tested the hypothesis that patients with chronic thromboembolic pulmonary hypertension (CTEPH) deemed inoperable were more likely to respond to PAH drugs than those CTEPH deemed operable, using cardiopulmonary exercise testing (CPX). We analyzed CPX data of all CTEPH patients who were treated with PAH drugs and had undergone CPX testing pre- and post-treatment at a single Pulmonary Hypertension center between February 2009 and March 2013. Suitability for pulmonary endarterectomy (PEA) was decided at a PEA expert center. The inoperable group included 16 patients and the operable group 26 patients. There was no difference in demographics and baseline hemodynamics between the groups. Unlike the operable group, after drug treatment inoperable patients had a significantly higher peak VO2 (p<0.001), workload (p=0.002) and oxygen pulse (p<0.001). In terms of gas exchange, there was an overall net trend towards improved VE/VCO2 in the inoperable group, with an increased PaCO2 (p=0.01), suggesting reduced hyperventilation. No changes were seen in the operable patients. In conclusion, treatment with PAH drug therapies reveals important pathophysiological differences between inoperable and operable CTEPH, with significant pulmonary vascular and cardiac responses in inoperable disease. Drug effects on exercise function seen in inoperable CTEPH cannot be translated to all forms of CTEPH

Transmembrane helix dynamics of bacterial chemoreceptors supports a piston model of signalling.

Transmembrane α-helices play a key role in many receptors, transmitting a signal from one side to the other of the lipid bilayer membrane. Bacterial chemoreceptors are one of the best studied such systems, with a wealth of biophysical and mutational data indicating a key role for the TM2 helix in signalling. In particular, aromatic (Trp and Tyr) and basic (Arg) residues help to lock α-helices into a membrane. Mutants in TM2 of E. coli Tar and related chemoreceptors involving these residues implicate changes in helix location and/or orientation in signalling. We have investigated the detailed structural basis of this via high throughput coarse-grained molecular dynamics (CG-MD) of Tar TM2 and its mutants in lipid bilayers. We focus on the position (shift) and orientation (tilt, rotation) of TM2 relative to the bilayer and how these are perturbed in mutants relative to the wildtype. The simulations reveal a clear correlation between small (ca. 1.5 Å) shift in position of TM2 along the bilayer normal and downstream changes in signalling activity. Weaker correlations are seen with helix tilt, and little/none between signalling and helix twist. This analysis of relatively subtle changes was only possible because the high throughput simulation method allowed us to run large (n = 100) ensembles for substantial numbers of different helix sequences, amounting to ca. 2000 simulations in total. Overall, this analysis supports a swinging-piston model of transmembrane signalling by Tar and related chemoreceptors

The effectiveness of public health interventions to reduce the health impact of climate change:a systematic review of systematic reviews

Climate change is likely to be one of the most important threats to public health in the coming years. Yet despite the large number of papers considering the health impact of climate change, few have considered what public health interventions may be of most value in reducing the disease burden. We aimed to evaluate the effectiveness of public health interventions to reduce the disease burden of high priority climate sensitive diseases

Interleukin-6 gene (IL-6): a possible role in brain morphology in the healthy adult brain

Background: Cytokines such as interleukin 6 (IL-6) have been implicated in dual functions in neuropsychiatric disorders. Little is known about the genetic predisposition to neurodegenerative and neuroproliferative properties of cytokine genes. In this study the potential dual role of several IL-6 polymorphisms in brain morphology is investigated. Methodology: In a large sample of healthy individuals (N = 303), associations between genetic variants of IL-6 (rs1800795; rs1800796, rs2069833, rs2069840) and brain volume (gray matter volume) were analyzed using voxel-based morphometry (VBM). Selection of single nucleotide polymorphisms (SNPs) followed a tagging SNP approach (e.g., Stampa algorigthm), yielding a capture 97.08% of the variation in the IL-6 gene using four tagging SNPs. Principal findings/results: In a whole-brain analysis, the polymorphism rs1800795 (−174 C/G) showed a strong main effect of genotype (43 CC vs. 150 CG vs. 100 GG; x = 24, y = −10, z = −15; F(2,286) = 8.54, puncorrected = 0.0002; pAlphaSim-corrected = 0.002; cluster size k = 577) within the right hippocampus head. Homozygous carriers of the G-allele had significantly larger hippocampus gray matter volumes compared to heterozygous subjects. None of the other investigated SNPs showed a significant association with grey matter volume in whole-brain analyses. Conclusions/significance: These findings suggest a possible neuroprotective role of the G-allele of the SNP rs1800795 on hippocampal volumes. Studies on the role of this SNP in psychiatric populations and especially in those with an affected hippocampus (e.g., by maltreatment, stress) are warranted.Bernhard T Baune, Carsten Konrad, Dominik Grotegerd, Thomas Suslow, Eva Birosova, Patricia Ohrmann, Jochen Bauer, Volker Arolt, Walter Heindel, Katharina Domschke, Sonja Schöning, Astrid V Rauch, Christina Uhlmann, Harald Kugel and Udo Dannlowsk