Redesign of a WW Domain Peptide for Selective Recognition of Single-Stranded DNA

A β-sheet mini-protein based on the FBP11 WW1 domain sequence has been redesigned for the molecular recognition of ssDNA. A previous report showed that a β-hairpin peptide dimer, (WKWK)2, binds ssDNA with low micromolar affinity but with little selectivity over duplex DNA. This report extends those studies to a three-stranded β-sheet mini-protein designed to mimic the OB-fold. The new peptide binds ssDNA with low micromolar affinity and shows about 10-fold selectivity for ssDNA over duplex DNA. The redesigned peptide no longer binds its native ligand, the polyproline helix, confirming that the peptide has been redesigned for the function of binding ssDNA. Structural studies provide evidence that this peptide consists of a well structured β-hairpin made of Strands 2&3 with a less structured first strand that provides affinity for ssDNA but does not improve the stability of the full peptide. These studies provide insight into protein-DNA interactions as well as a novel example of protein-redesign

Structural insights into the calcium-dependent interaction between calbindin-D28K and caspase-3

AbstractThe regulation of apoptosis involves a complicated cascade requiring numerous protein interactions including the pro-apoptotic executioner protein caspase-3 and the anti-apoptotic calcium-binding protein calbindin-D28K. Using isothermal titration calorimetry, we show that calbindin-D28K binds caspase-3 in a Ca2+-dependent fashion. Molecular docking and conformational sampling studies of the Ca2+-loaded capase-3/calbindin-D28K interaction were performed in order to isolate potentially crucial intermolecular contacts. Residues in the active site loops of caspase-3 and EF-hands 1 and 2 of calbindin-D28K were shown to be critical to the interaction. Based on these studies, a model is proposed to help understand how calbindin-D28K may deactivate caspase-3 upon binding.Structured summary of protein interactionsCalbindin-D28K and Caspase-3 bind by isothermal titration calorimetry (View interaction

Methods to Describe Referral Patterns in a Canadian Primary Care Electronic Medical Record Database: Modelling Multilevel Count Data

Background:  A referral from a family physician (FP) to a specialist is an inflection point in the patient journey, with potential implications for clinical outcomes and health policy. Primary care electronic medical record (EMR) databases offer opportunities to examine referral patterns. Until recently, software techniques were not available to model these kinds of multi-level count data. Objective:  To establish methodology for determining referral rates from FPs to medical specialists using the Canadian Primary Care Sentinel Surveillance Network (CPCSSN) EMR database. Method: Retrospective cohort study, mixed effects and multi-level negative binomial regression modelling with 87,258 eligible patients between 2007 and 2012. Mean referrals compared by patient sex, age, chronic conditions, FP visits, and urban/rural practice location.  Proportion of variance in referral rates attributable to the patient and practice levels. Results:  On average, males had 0.26, and females 0.31 referrals in a 12-month period.  Referrals were significantly higher for females, increased with age, FP visits, and number of chronic conditions (p<.0001). Overall, 14% of the variance in referrals could be attributed to the practice level, and 86% to patient level characteristics. Conclusions:  Both patient and practice characteristics influenced referral patterns. The methodologic insights gained from this study have relevance to future studies on many research questions that utilize count data, both within primary care and broader health services research. The utility of the CPCSSN database will continue to increase in tandem with data quality improvements, providing a valuable resource to study Canadian referral patterns over time

Maximizing the Impact of Digital Media Campaigns to Promote Smoking Cessation: A Case Study of the California Tobacco Control Program and the California Smokers’ Helpline

Background and Purpose: Digital media (e.g., banner ads, video ads) is often used to encourage smoking cessation by increasing quitline call volume through direct promotion to smokers or indirect promotion through smoker proxies. This process evaluation study highlights the use of digital media in a proxy-targeted campaign to promote the California Smokers’ Helpline to health care professionals. Methods: Data were collected from October 2009 to September 2012. We describe the iterative development of the campaign’s digital media activities and report campaign summaries of web metrics (website visits, webinar registrations, downloads of online materials, online orders for promotional materials) and media buy (gross impressions) tracking data. Results: The campaign generated more than 2.7 million gross impressions from digital media sources over three campaign waves. Online orders for promotional materials increased almost 40% over the course of the campaign. Conclusion: A clearly defined campaign strategy ensured that there was a systematic approach in developing and implementing campaign activities. It also ensured that lessons learned from previous waves were incorporated; one lesson included the frequent rotation of new ad content to keep the target audience engaged

Australian general practice trainees’ exposure to ophthalmic problems and implications for training: A cross-sectional analysis

INTRODUCTION: Eye conditions are common presentations in Australian general practice, with the potential for serious sequelae. Pre-vocational ophthalmology training for General Practitioner (GP) trainees is limited. AIM: To describe the rate, nature and associations of ophthalmic problems managed by Australian GP trainees, and derive implications for education and training. METHODS: Cross-sectional analysis from an ongoing cohort study of GP trainees’ clinical consultations. Trainees recorded demographic, clinical and educational details of consecutive patient consultations. Descriptive analyses report trainee, patient and practice demographics. Proportions of all problems managed in these consultations that were ophthalmology-related were calculated with 95% confidence intervals (CI). Associations were tested using simple logistic regression within the generalised estimating equations (GEE) framework. RESULTS: In total, 884 trainees returned data on 184,476 individual problems or diagnoses from 118,541 encounters. There were 2649 ophthalmology-related problems, equating to 1.4% (95% CI: 1.38-1.49) of all problems managed. The most common eye presentations were conjunctivitis (32.5% of total problems), eyelid problems (14.9%), foreign body (5.3%) and dry eye (4.7%). Statistically significant associations were male trainee; male patient and patient aged 14 years or under; the problem being new and the patient being new to both trainee and practice; urban and of higher socioeconomic status practice location; the practice nurse not being involved; planned follow up not arranged; referral made; in-consultation information sought; and learning goals generated. DISCUSSION: Trainees have comparable ophthalmology exposure to established GPs. However, associations with referral and information-seeking suggest GP trainees find ophthalmic problems challenging, reinforcing the critical importance of appropriate training

A trans-homologue interaction between reciprocally imprinted miR-127 and Rtl1 regulates placenta development.

The paternally expressed imprinted retrotransposon-like 1 (Rtl1) is a retrotransposon-derived gene that has evolved a function in eutherian placentation. Seven miRNAs, including miR-127, are processed from a maternally expressed antisense Rtl1 transcript (Rtl1as) and regulate Rtl1 levels through RNAi-mediated post-transcriptional degradation. To determine the relative functional role of Rtl1as miRNAs in Rtl1 dosage, we generated a mouse specifically deleted for miR-127. The miR-127 knockout mice exhibit placentomegaly with specific defects within the labyrinthine zone involved in maternal-fetal nutrient transfer. Although fetal weight is unaltered, specific Rtl1 transcripts and protein levels are increased in both the fetus and placenta. Phenotypic analysis of single (ΔmiR-127/Rtl1 or miR-127/ΔRtl1) and double (ΔmiR-127/ΔRtl1) heterozygous miR-127- and Rtl1-deficient mice indicate that Rtl1 is the main target gene of miR-127 in placental development. Our results demonstrate that miR-127 is an essential regulator of Rtl1, mediated by a trans-homologue interaction between reciprocally imprinted genes on the maternally and paternally inherited chromosomes.This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) and Medical Research Council (MRC) and EU FP7 Marie Curie Action 290123 (INGENIUM). This work was partly funded by a National Health and Medical Research Council (NHMRC) CJ Martin Biomedical Fellowship to A.N.S.P.This is the accepted manuscript. The final version is available at http://dev.biologists.org/content/early/2015/07/01/dev.121996.abstract

V2: Integrated management of rainwater for crop-livestock agroecosystems

With mixed crop-livestock systems projected to remain the main providers of food in the coming decades, opportunities exist for smallholders to participate and benefit from emerging crop and livestock markets in the Volta Basin. This project intends to identify, evaluate, adapt, and disseminate best-fit integrated rainwater management strategies (RMS), targeted to different biophysical and socio-economic domains. The integrated RMS are comprised of technological solutions, directed at different components of the agroecosystems, underpinned by enabling institutional and policy environments and linked to market incentives that can drive adoptio

Loss of ELK1 has differential effects on age-dependent organ fibrosis and integrin expression

ETS domain-containing protein-1 (ELK1) is a transcription factor important in regulating αvβ6 integrin expression. αvβ6 integrins activate the profibrotic cytokine Transforming Growth Factor β1 (TGFβ1) and are increased in the alveolar epithelium in idiopathic pulmonary fibrosis (IPF). IPF is a disease associated with aging and therefore we hypothesised that aged animals lacking Elk1 globally would develop spontaneous fibrosis in organs where αvβ6 mediated TGFβ activation has been implicated. Here we identify that Elk1-knockout (Elk1−/0) mice aged to one year developed spontaneous fibrosis in the absence of injury in both the lung and the liver but not in the heart or kidneys. The lungs of Elk1−/0 aged mice demonstrated increased collagen deposition, in particular collagen 3α1, located in small fibrotic foci and thickened alveolar walls. Despite the liver having relatively low global levels of ELK1 expression, Elk1−/0 animals developed hepatosteatosis and fibrosis. The loss of Elk1 also had differential effects on Itgb1, Itgb5 and Itgb6 expression in the four organs potentially explaining the phenotypic differences in these organs. To understand the potential causes of reduced ELK1 in human disease we exposed human lung epithelial cells and murine lung slices to cigarette smoke extract, which lead to reduced ELK1 expression andmay explain the loss of ELK1 in human disease. These data support a fundamental role for ELK1 in protecting against the development of progressive fibrosis via transcriptional regulation of beta integrin subunit genes, and demonstrate that loss of ELK1 can be caused by cigarette smoke

Retrospective and prospective assessments of gambling-related behaviors across the female menstrual cycle

Background and aims: Despite increases in female gambling, little research investigates female-specific factors affecting gambling behavior (GB). Although research suggests that some addictive behaviors may fluctuate across menstrual cycle phase (MCP), gambling requires further investigation. In two studies, we examined associations between MCP and three risky GBs: time spent gambling, money spent gambling, and the probability of consuming alcohol while gambling. Associations between MCP and negative affect were also examined in Study 2. We predicted that, consistent with self-medication theory, increases in negative affect (Study 2) and risky GBs (Studies 1 and 2) would occur premenstrually/menstrually relative to other phases. Methods: Data were obtained from 33 female gamblers using a retrospective timeline followback procedure (Study 1) and from 20 female gamblers using a prospective 32-day, daily diary method (Study 2). In Study 2, salivary progesterone levels verified self-reported MCP validity. Results: Findings revealed significant, but somewhat inconsistent, MCP effects on GBs across studies. The self-medication hypothesis was partially supported. Increases relative to another MCP(s) were found for alcohol consumption while gambling premenstrually, time spent gambling menstrually/premenstrually, money spent gambling menstrually, and negative affect premenstrually. Unexpectedly, findings more consistently indicated that GBs increased during ovulation, suggestive of enhanced reward sensitivity. Progesterone assays validated self-reported MCP (Study 2). Discussion and conclusions: The results suggest a role of ovarian hormones on negative affect and GBs in females. This research could lead to the identification of female-specific factors affecting gambling and the development of more effective interventions for females with, or at risk for, problematic gambling

Mycobacterium tuberculosis ClpP Proteases Are Co-transcribed but Exhibit Different Substrate Specificities

PMCID: PMC3613350This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited