A novel dual-fluorescence strategy for functionally validating microRNA targets in 3-prime untranslated regions: regulation of the inward rectifier potassium channel Kir2.1 by miR-212.

Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K+ channel Kir2.1 (KCNJ2) which is dysregulated in cardiac and vascular disorders. The 3'UTR was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known downregulator of Kir2.1 expression, and was used to investigate targeting of the Kir2.1 3'UTR by miR-212. Red/green ratio was lower in miR-212-expressing cells compared to non-targeting controls, an effect that was attenuated by mutating the predicted target site. MiR-212 also reduced inward rectifier current and Kir2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

Assessment Method for a Power Analysis to Identify Differentially Expressed Pathways

Gene expression data can provide a very rich source of information for elucidating the biological function on the pathway level if the experimental design considers the needs of the statistical analysis methods. The purpose of this paper is to provide a comparative analysis of statistical methods for detecting the differentially expression of pathways (DEP). In contrast to many other studies conducted so far, we use three novel simulation types, producing a more realistic correlation structure than previous simulation methods. This includes also the generation of surrogate data from two large-scale microarray experiments from prostate cancer and ALL. As a result from our comprehensive analysis of 41,004 parameter configurations, we find that each method should only be applied if certain conditions of the data from a pathway are met. Further, we provide method-specific estimates for the optimal sample size for microarray experiments aiming to identify DEP in order to avoid an underpowered design. Our study highlights the sensitivity of the studied methods on the parameters of the system. © 2012 Tripahti and Emmert-Streib.

Genetic variants influencing human aging from late-onset Alzheimer's disease (LOAD) genome-wide association studies (GWAS)

Genetics plays a crucial role in human aging with up to 30% of those living to the mid-80s being determined by genetic variation. Survival to older ages likely entails an even greater genetic contribution. There is increasing evidence that genes implicated in age-related diseases, such as cancer and neuronal disease, play a role in affecting human life span. We have selected the 10 most promising late-onset Alzheimer's disease (LOAD) susceptibility genes identified through several recent large genome-wide association studies (GWAS). These 10 LOAD genes (APOE, CLU, PICALM, CR1, BIN1, ABCA7, MS4A6A, CD33, CD2AP, and EPHA1) have been tested for association with human aging in our dataset (1385 samples with documented age at death [AAD], age range: 58-108 years; mean age at death: 80.2) using the most significant single nucleotide polymorphisms (SNPs) found in the previous studies. Apart from the APOE locus (rs2075650) which showed compelling evidence of association with risk on human life span (p = 5.27 × 10(-4)), none of the other LOAD gene loci demonstrated significant evidence of association. In addition to examining the known LOAD genes, we carried out analyses using age at death as a quantitative trait. No genome-wide significant SNPs were discovered. Increasing sample size and statistical power will be imperative to detect genuine aging-associated variants in the future. In this report, we also discuss issues relating to the analysis of genome-wide association studies data from different centers and the bioinformatic approach required to distinguish spurious genome-wide significant signals from real SNP associations.

A test for psychometric function shift

A nonparametric, small-sample-size test for the homogeneity of two psychometric functions against the left- and right-shift alternatives has been developed. The test is designed to determine whether it is safe to amalgamate psychometric functions obtained in different experimental sessions. The sum of the lower and upper p-values of the exact (conditional) Fisher test for several 2 × 2 contingency tables (one for each point of the psychometric function) is employed as the test statistic. The probability distribution of the statistic under the null (homogeneity) hypothesis is evaluated to obtain corresponding p-values. Power functions of the test have been computed by randomly generating samples from Weibull psychometric functions. The test is free of any assumptions about the shape of the psychometric function; it requires only that all observations are statistically independent. © 2011 Psychonomic Society, Inc.

Residues of nitrofuran antibiotic parent compounds and metabolites in eyes of broiler chickens

Nitrofuran antibiotic residues in food continue to be of international concern. The finding of sources of semicarbazide (SEM), other than through the misuse of nitrofurazone, present a challenge to the use of SEM as a definitive marker residue for this drug. Detection of intact (parent) nitrofurazone would avoid confusion over the source of SEM residues. Broiler chickens were fed sub-therapeutic nitrofuran-containing diets and their tissues were analysed for parent compounds and metabolites by liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS). Depletion half-lives in muscle were longer for tissue-bound metabolite residues, 3.4 days - 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) - to 4.5 days (SEM), than total metabolite residues, 2.0 days (AOZ) to 3.2 days (SEM). Metabolite concentrations were higher in eyes than in muscle. Metabolite half-lives in eyes ranged from 8.5 days (1-aminohydantoin (AHD)) to 20.3 days (SEM). Nitrofuran parent compounds were also detected in eyes. Furaltadone was detected in single eyes after 21 days' withdrawal of a 6 mg kg -1 furaltadone diet. When 50 eyes from broilers containing metabolites in muscle close to the 1 µg kg -1 minimum required performance level (MRPL) were pooled into single samples, 1.2 ng of furazolidone and 31.1 ng of furaltadone were detected, but nitrofurazone was not detected due to the long depletion half-life of SEM in muscle. Further studies are required to improve LC-MS/MS nitrofurazone sensitivity and refine the sample size necessary to use nitrofurazone detection in pooled eyes as a complement to SEM detection in muscle.

Occupational stress in the chef profession

Purpose – The purpose of this paper is to consider the occupational stress experienced by chefs and the moderating influence of coping behaviour and locus of control on stress outcomes. Design/methodology/approach – A total of 40 working chefs were surveyed. They were asked to complete an occupational stress questionnaire, the General Health Questionnaire. Differences were
sought between gender and locus of control and stress measures, and correlations were carried out between key variables. Findings – The reported stress was markedly higher than in previous research. Excessive workload,
feeling undervalued and communication issues were common and bullying and threats of violence were present for some. Unexpectedly, locus of control was unable to predict stress experiences. Explanations were offered, such as the possibility that those who perceive they have a strong sense of control may believe that this, paradoxically, affords them the opportunity to engage in unhealthy behaviours. Research limitations/implications – Limitations of the research include the influence of the wider environment, specifically the history of political violence in the province, and its possible effect on stress outcomes. However, this may be negated by the many positive effects peace has brought over the last decade. In drawing conclusions it is important to note the limitations of the sample size and the self-reporting nature of survey responses. Further research could usefully incorporate well-being as well as stress measures, including physiological ones. It would be worth exploring further how one’s sense of control affects perceptions of stress and, in turn, the coping behaviours engaged in. Practical implications – Practical implications include the need for managers and head chefs to provide more feedback to employees, to validate their good work and to foster a supportive working environment. Norms in the working environment endorsing aggressive behaviour must be challenged.
Staff appraisals should consider the need to have work that involves variety and challenge, especially where changes involve increases in workload. Originality/value – This paper identifies some important ingredients to reduce distress and it will be of value to chefs and other kitchen staff and, more broadly, to those involved in people management.

Diet of Black-legged Kittiwakes (Rissa tridactyla) feeding chicks at two Irish colonies highlights the importance of clupeids

We examined regurgitates from Black-legged Kittiwakes during the early chick-rearing period over two breeding seasons at two colonies in Ireland where diet has not been studied previously. Clupeids were the dominant food source at both colonies, which contrasts with other studies throughout the British Isles that suggest Kittiwakes feed mainly on sandeels during this period. Our study is limited
by sample size and restricted to the early breeding season, but suggests a link between reproductive success and dietary composition which should be investigated further.

High-contrast imaging search for planets and brown dwarfs around the most massive stars in the solar neighborhood

There has been a long-standing discussion in the literature as to whether core accretion or disk instability is the dominant mode of planet formation. Over the last decade, several lines of evidence have been presented showing that core accretion is most likely the dominant mechanism for the close-in population of planets probed by radial velocity and transits. However, this does not by itself prove that core accretion is the dominant mode for the total planet population, since disk instability might conceivably produce and retain large numbers of planets in the far-out regions of the disk. If this is a relevant scenario, then the outer massive disks of B-stars should be among the best places for massive planets and brown dwarfs to form and reside. In this study, we present high-contrast imaging of 18 nearby massive stars of which 15 are in the B2-A0 spectral-type range and provide excellent sensitivity to wide companions. By comparing our sensitivities to model predictions of disk instability based on physical criteria for fragmentation and cooling, and using Monte Carlo simulations for orbital distributions, we find that ~85% of such companions should have been detected in our images on average. Given this high degree of completeness, stringent statistical limits can be set from the null-detection result, even with the limited sample size. We find that

Genome scan meta-analysis of schizophrenia and bipolar disorder, part II:Schizophrenia

Schizophrenia is a common disorder with high heritability and a 10-fold increase in risk to siblings of probands. Replication has been inconsistent for reports of significant genetic linkage. To assess evidence for linkage across studies, rank-based genome scan meta-analysis (GSMA) was applied to data from 20 schizophrenia genome scans. Each marker for each scan was assigned to 1 of 120 30-cM bins, with the bins ranked by linkage scores (1 = most significant) and the ranks averaged across studies (R(avg)) and then weighted for sample size (N(sqrt)[affected casess]). A permutation test was used to compute the probability of observing, by chance, each bin's average rank (P(AvgRnk)) or of observing it for a bin with the same place (first, second, etc.) in the order of average ranks in each permutation (P(ord)). The GSMA produced significant genomewide evidence for linkage on chromosome 2q (PAvgRnk

Harnessing the complexity of gene expression data from cancer: from single gene to structural pathway methods

High-dimensional gene expression data provide a rich source of information because they capture the expression level of genes in dynamic states that reflect the biological functioning of a cell. For this reason, such data are suitable to reveal systems related properties inside a cell, e.g., in order to elucidate molecular mechanisms of complex diseases like breast or prostate cancer. However, this is not only strongly dependent on the sample size and the correlation structure of a data set, but also on the statistical hypotheses tested. Many different approaches have been developed over the years to analyze gene expression data to (I) identify changes in single genes, (II) identify changes in gene sets or pathways, and (III) identify changes in the correlation structure in pathways. In this paper, we review statistical methods for all three types of approaches, including subtypes, in the context of cancer data and provide links to software implementations and tools and address also the general problem of multiple hypotheses testing. Further, we provide recommendations for the selection of such analysis methods.

Variation of clinical outcomes used in glaucoma randomised controlled trials:a systematic review

Purpose: In randomised clinical trials (RCTs) the selection of appropriate outcomes is crucial to the assessment of whether one intervention is better than another. The purpose of this review is to identify different clinical outcomes reported in glaucoma trials.

Methods We conducted a systematic review of glaucoma RCTs. A sample or selection of glaucoma trials were included bounded by a time frame (between 2006 and March 2012). Only studies in English language were considered. All clinical measured and reported outcomes were included. The possible variations of clinical outcomes were defined prior to data analysis. Information on reported clinical outcomes was tabulated and analysed using descriptive statistics. Other data recorded included type of intervention and glaucoma, duration of the study, defined primary outcomes, and outcomes used for sample size calculation, if nominated.

Results The search strategy identified 4323 potentially relevant abstracts. There were 315 publications retrieved, of which 233 RCTs were included. A total of 967 clinical measures were reported. There were large variations in the definitions used to describe different outcomes and their measures. Intraocular pressure was the most commonly reported outcome (used in 201 RCTs, 86%) with a total of 422 measures (44%). Safety outcomes were commonly reported in 145 RCTs (62%) whereas visual field outcomes were used in 38 RCTs (16%).

Conclusions There is a large variation in the reporting of clinical outcomes in glaucoma RCTs. This lack of standardisation may impair the ability to evaluate the evidence of glaucoma interventions.

Model selection for prognostic time-to-event gene signature discovery with applications in early breast cancer data

Model selection between competing models is a key consideration in the discovery of prognostic multigene signatures. The use of appropriate statistical performance measures as well as verification of biological significance of the signatures is imperative to maximise the chance of external validation of the generated signatures. Current approaches in time-to-event studies often use only a single measure of performance in model selection, such as logrank test p-values, or dichotomise the follow-up times at some phase of the study to facilitate signature discovery. In this study we improve the prognostic signature discovery process through the application of the multivariate partial Cox model combined with the concordance index, hazard ratio of predictions, independence from available clinical covariates and biological enrichment as measures of signature performance. The proposed framework was applied to discover prognostic multigene signatures from early breast cancer data. The partial Cox model combined with the multiple performance measures were used in both guiding the selection of the optimal panel of prognostic genes and prediction of risk within cross validation without dichotomising the follow-up times at any stage. The signatures were successfully externally cross validated in independent breast cancer datasets, yielding a hazard ratio of 2.55 [1.44, 4.51] for the top ranking signature.

The relationship between breastfeeding and postpartum weight change-a systematic review and critical evaluation

Pregnancy and the postpartum period is a time of increased vulnerability for retention of excess body fat in women. Breastfeeding (BF) has been shown to have many health benefits for both mother and baby; however, its role in postpartum weight management is unclear. Our aim was to systematically review and critically appraise the literature published to date in relation to the impact of BF on postpartum weight change, weight retention and maternal body composition. Electronic literature searches were carried out using MEDLINE, EMBASE, PubMed, Web of Science, BIOSIS, CINAHL and British Nursing Index. The search covered publications up to 12 June 2012 and included observational studies (prospective and retrospective) carried out in BF mothers (either exclusively or as a subgroup), who were 2 years postpartum and with a body mass index (BMI) >18.5 kg m(-2), with an outcome measure of change in weight (including weight retention) and/or body composition. Thirty-seven prospective studies and eight retrospective studies were identified that met the selection criteria; studies were stratified according to study design and outcome measure. Overall, studies were heterogeneous, particularly in relation to sample size, measurement time points and in the classification of BF and postpartum weight change. The majority of studies reported little or no association between BF and weight change (n=27, 63%) or change in body composition (n=16, 89%), although this seemed to depend on the measurement time points and BF intensity. However, of the five studies that were considered to be of high methodological quality, four studies demonstrated a positive association between BF and weight change. This systematic review highlights the difficulties of examining the association between BF and weight management in observational research. Although the available evidence challenges the widely held belief that BF promotes weight loss, more robust studies are needed to reliably assess the impact of BF on postpartum weight management.International Journal of Obesity advance online publication, 20 August 2013; doi:10.1038/ijo.2013.132.

A randomized trial of paired tutoring in elementary schools:effects on self-esteem

This paper reports data on self-esteem collected during a large-scale randomized trial of paired reading and Duolog Maths with primary school children (The Fife Peer-Learning Project). Self-report measures were analysed to assess the effect of a range of organisational variables on measured self-esteem. The presentation reports findings from a sample of reading-only schools in the first year of the project. Statistically significant gains in self-esteem were found across a range of conditions. Patterns of scores pointed to differences between same-age and cross-age conditions, and highlighted the influence of role played (tutor v tutee). In addition to the findings summarized here, it is hoped that preliminary findings from year two of the project - with an increased sample size - will also be shared. © 2009.

Seasonality of cardiovascular risk factors: an analysis including over 230 000 participants in 15 countries

Objective: To assess the seasonality of cardiovascular risk factors (CVRF) in a large set of population-based studies.

Methods: Cross-sectional data from 24 population-based studies from 15 countries, with a total sample size of 237 979 subjects. CVRFs included Body Mass Index (BMI) and waist circumference; systolic (SBP) and diastolic (DBP) blood pressure; total, high (HDL) and low (LDL) density lipoprotein cholesterol; triglycerides and glucose levels. Within each study, all data were adjusted for age, gender and current smoking. For blood pressure, lipids and glucose levels, further adjustments on BMI and drug treatment were performed.

Results: In the Northern and Southern Hemispheres, CVRFs levels tended to be higher in winter and lower in summer months. These patterns were observed for most studies. In the Northern Hemisphere, the estimated seasonal variations were 0.26 kg/m2 for BMI, 0.6 cm for waist circumference, 2.9 mm Hg for SBP, 1.4 mm Hg for DBP, 0.02 mmol/L for triglycerides, 0.10 mmol/L for total cholesterol, 0.01 mmol/L for HDL cholesterol, 0.11 mmol/L for LDL cholesterol, and 0.07 mmol/L for glycaemia. Similar results were obtained when the analysis was restricted to studies collecting fasting blood samples. Similar seasonal variations were found for most CVRFs in the Southern Hemisphere, with the exception of waist circumference, HDL, and LDL cholesterol.

Conclusions: CVRFs show a seasonal pattern characterised by higher levels in winter, and lower levels in summer. This pattern could contribute to the seasonality of CV mortality.