DNA Binding in High Salt: Analysing the Salt Dependence of Replication Protein A3 from the Halophile Haloferax volcanii

Halophilic archaea maintain intracellular salt concentrations close to saturation to survive in high-salt environments and their cellular processes have adapted to function under these conditions. Little is known regarding halophilic adaptation of the DNA processing machinery, particularly intriguing since protein-DNA interactions are classically salt sensitive. To investigate such adaptation, we characterised the DNA-binding capabilities of recombinant RPA3 from Haloferax volcanii (HvRPA3). Under physiological salt conditions (3M KCl), HvRPA3 is monomeric, binding 18 nucleotide ssDNA with nanomolar affinity, demonstrating that RPAs containing the single OB-fold/zinc finger architecture bind with broadly comparable affinity to two OB-fold/zinc finger RPAs. Reducing the salt concentration to 1M KCl induces dimerisation of the protein, which retains its ability to bind DNA. On circular ssDNA, two concentration-dependent binding modes are observed. Conventionally, increased salt concentration adversely affects DNA binding but HvRPA3 does not bind DNA in 0.2M KCl, although multimerisation may occlude the binding site. The single N-terminal OB-fold is competent to bind DNA in the absence of the C-terminal zinc finger, albeit with reduced affinity. This study represents the first quantitative characterisation of DNA binding in a halophilic protein in extreme salt concentrations

Neuroendocrine Reactivity During the Naturalistic Stress of an Outward Bound Course

Recent studies have found that subjects with high hostility propensity have greater catecholamine (CA) and cortisol (Cf) levels during laboratory stressors. The present investigation sought to determine if similar patterns would be found in the naturalistic setting of an Outward Bound experience, and if high versus low aerobic fitness would influence CA and CT levels in this setting. Sixty-six individuals volunteered for the study from participants who had registered for a nine-day \u27adult intensive\u27 Outward Bound course. Thirty-one subjects were selected from those who volunteered. The Cook-Medley Hostility Inventory was completed by each subject prior to arriving for their course and aerobic fitness estimates were completed within the first six hours of the course using the University of Houston Non-Exercise Test Stress levels were quantified by urinary CA and CT excretion. Three to five hour urine samples were collected for the following activities: rockclimbing/rappelling, whitewater canoeing, ropes course, off-trail backpacking, commuting from airport to base camp and back, and one night-time sleep sample. At the beginning of each activity period, all subjects voided and at the end of the three to five hour period urine was collected, measured for total volume, and a sample was taken and ftw.en within two hours of collection. The urine was assayed by high-pressure liquid chromatography with electrochemical detection to determine levels of excreted epinephrine (E), norepinephrine (NE), and cortisol (CT). The CA and CT were then quantified by mg of creatinine. Of the 31 subjects (14 females and 17 males), nine were classfied as high hostile (HiHo) and 22 as average to low hostile (LoHo). The classifications for aerobic fitness were: 26 average to high fit (HF) with an estimated max V02 greater than 39 ml/kg for women and 46 ml/kg for men, and five low fit (LF) subjects with lower estimated V02 max. The data were analyzed by the two levels of hostility and two levels of aerobic fitness using univariate ANOVA with repeated measures across activity periods. The analysis of hostility revealed significant CT excretion differences between hostility levels for four of the nine activity periods with the LoHo subjects excreting higher CT levels. The four activities where this was found were: off-trail backpacking, (F=4.74,

Family-Owned Newspapers: Filling Niches in Local U.S. Communities

Citation: Powers, A., Sohn, A. B., Briggs-Bunting, J. (2016) Family-Owned Newspapers: Filling Niches in Local U.S. Communities. Journal of Media Business Studies. 2(11)Since small town newspapers are facing increased competition and technological changes that are threatening their survival, the purpose of this paper was to analyze the strategy-making activities of these organizations from an ecological perspective. Findings indicate that family-owned newspapers were finding stability in retaining their core print businesses while migrating content to the web. Too many variations from the norm appeared to weaken financial footholds and were often eventually abandoned. Such behaviors indicate a rationale for upholding a “tried-and-true” approach in newspapers. While profits may fluctuate, the uncertainty that occurred with variation seemed to play a role in increasing the threat of failure for these small, family-owned newspapers

Reliability of Observational Assessment Methods for Outcome-based Assessment of Surgical Skill: Systematic Review and Meta-analyses

BACKGROUND: Reliable performance assessment is a necessary prerequisite for outcome-based assessment of surgical technical skill. Numerous observational instruments for technical skill assessment have been developed in recent years. However, methodological shortcomings of reported studies might negatively impinge on the interpretation of inter-rater reliability. OBJECTIVE: To synthesize the evidence about the inter-rater reliability of observational instruments for technical skill assessment for high-stakes decisions. DESIGN: A systematic review and meta-analysis were performed. We searched Scopus (including MEDLINE) and Pubmed, and key publications through December, 2016. This included original studies that evaluated reliability of instruments for the observational assessment of technical skills. Two reviewers independently extracted information on the primary outcome (the reliability statistic), secondary outcomes, and general information. We calculated pooled estimates using multilevel random effects meta-analyses where appropriate. RESULTS: A total of 247 documents met our inclusion criteria and provided 491 inter-rater reliability estimates. Inappropriate inter-rater reliability indices were reported for 40% of the checklists estimates, 50% of the rating scales estimates and 41% of the other types of assessment instruments estimates. Only 14 documents provided sufficient information to be included in the meta-analyses. The pooled Cohen's kappa was.78 (95% CI 0.69-0.89, p < 0.001) and pooled proportion agreement was 0.84 (95% CI 0.71-0.96, p < 0.001). A moderator analysis was performed to explore the influence of type of assessment instrument as a possible source of heterogeneity. CONCLUSIONS AND RELEVANCE: For high-stakes decisions, there was often insufficient information available on which to base conclusions. The use of suboptimal statistical methods and incomplete reporting of reliability estimates does not support the use of observational assessment instruments for technical skill for high-stakes decisions. Interpretations of inter-rater reliability should consider the reliability index and assessment instrument used. Reporting of inter-rater reliability needs to be improved by detailed descriptions of the assessment process

Editor\u27s Introduction and Acknowledgments

An introduction to the journal Research in Outdoor Education, volume 7 is presented by the authors on behalf of the Coalition for Education in the Outdoors (CEO) Research Committee