Functional expression of two glucose transporter isoforms from the parasitic protozoan Leishmania enriettii

The parasitic protozoan Leishmania enriettii contains a family of tandemly repeated genes, designated Pro-1, that encode proteins with significant sequence similarity to mammalian facilitative glucose transporters. Pro-1 mRNAs are expressed almost exclusively in the promastigote or insect stage of the parasite life cycle. The Pro-1 tandem repeat encodes two isoforms of the putative transporter, iso-1 and iso-2, which have identical predicted amino acid sequences except for their NH2-terminal hydrophilic domains. We have now expressed both iso-1 and iso-2 by microinjecting their RNAs into Xenopus oocytes and assaying these oocytes for transport of various radiolabeled ligands. Both iso-1 and iso-2 transport [3H]2-deoxy-D-glucose, confirming that each protein is a bona fide glucose transporter. Each isoform also transports fructose and, to a much lesser degree, mannose. Compounds which inhibit 2-deoxy-D-glucose transport in L. enriettii promastigotes also inhibit transport in the microinjected oocytes expressing each isoform, indicating that the substrate specificities and pharmacological properties of each isoform are similar to those measured for 2-deoxy-D-glucose transport in intact parasites. The Km for transport of 2-deoxyglucose in oocytes expressing iso-1 is similar to that for oocytes expressing iso-2. These results reveal that both transporter isoforms have closely related functional properties and that the difference in their structures may serve some other purpose such as differential subcellular localization

Submarine Pier Side Weapons Handling Risk Assessment

Dr. Warren Vaneman, Prof. Joseph Sweeney, Dr. Gary Langford, Mr. Gary Parker, Mr. Chris Wolfgeher, LCDR Willima HarleyThis research examines new methods to assess and improve Physical Protection Systems (PPS), paying specific attention to a Navy Level 3 Restricted Areas, a special type of industrial and refit zone that normally handles high value units such as aircraft carriers and ballistic missile submarines, utilizing Model Based Systems Engineering (MBSE) and System of Systems (SoS) theory to create a framework which couples architectural level PPS design with detailed discrete event security assessment and prediction techniques in order to provide decision makers and acquisition authorities a more quantitative and effective method to holistically understand a PPS and the PPS�s internal and external interactions, allowing for improved capability and vulnerability analysis and the formulation of sound acquisition decisions.Naval Research ProgramPrepared for: Navy Strategic Systems Program (SSP) Sponsor: Mr. Brent Flaskerud, SP3015, Nuclear Weapon Security (NWS) Modeling and Simulation (M&S) and Demonstration

Expression capable library for studies of Neisseria gonorrhoeae, version 1.0

Background The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system. Results The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci. Conclusion This archived set of Gateway® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome

Pilot trial and process evaluation of a multilevel smoking prevention intervention in further education settings

Background: Preventing smoking uptake among young people is a public health priority. Further education (FE) settings provide access to the majority of 16- to 18-year-olds, but few evaluations of smoking prevention interventions have been reported in this context to date. Objectives: To evaluate the feasibility and acceptability of implementing and trialling a new multilevel smoking prevention intervention in FE settings. Design: Pilot cluster randomised controlled trial and process evaluation. Setting: Six UK FE institutions. Participants: FE students aged 16–18 years. Intervention: ‘The Filter FE’ intervention. Staff working on Action on Smoking and Health Wales’ ‘The Filter’ youth project applied existing staff training, social media and youth work resources in three intervention settings, compared with three control sites with usual practice. The intervention aimed to prevent smoking uptake by restricting the sale of tobacco to under-18s in local shops, implementing tobacco-free campus policies, training FE staff to deliver smoke-free messages, publicising The Filter youth project’s online advice and support services, and providing educational youth work activities. Main outcome measures: (1) The primary outcome assessed was the feasibility and acceptability of delivering and trialling the intervention. (2) Qualitative process data were analysed to explore student, staff and intervention team experiences of implementing and trialling the intervention. (3) Primary, secondary and intermediate (process) outcomes and economic evaluation methods were piloted. Data sources: New students at participating FE settings were surveyed in September 2014 and followed up in September 2015. Qualitative process data were collected via interviews with FE college managers (n = 5) and the intervention team (n = 6); focus groups with students (n = 11) and staff (n = 5); and observations of intervention settings. Other data sources were semistructured observations of intervention delivery, intervention team records, ‘mystery shopper’ audits of local shops and college policy documents. Results: The intervention was not delivered as planned at any of the three intervention settings, with no implementation of some community- and college-level components, and low fidelity of the social media component across sites. Staff training reached 28 staff and youth work activities were attended by 190 students across the three sites (< 10% of all eligible staff and students), with low levels of acceptability reported. Implementation was limited by various factors, such as uncertainty about the value of smoking prevention activities in FE colleges, intervention management weaknesses and high turnover of intervention staff. It was feasible to recruit, randomise and retain FE settings. Prevalence of weekly smoking at baseline was 20.6% and was 17.2% at follow-up, with low levels of missing data for all pilot outcomes. Limitations: Only 17% of eligible students participated in baseline and follow-up surveys; the representativeness of student and staff focus groups is uncertain. Conclusions: In this study, FE settings were not a supportive environment for smoking prevention activities because of their non-interventionist institutional cultures promoting personal responsibility. Weaknesses in intervention management and staff turnover also limited implementation. Managers accept randomisation but methodological work is required to improve student recruitment and retention rates if trials are to be conducted in FE settings. Trial registration: Current Controlled Trials ISRCTN19563136. Funding: This project was funded by the National Institute for Health Research (NIHR) Public Health Research programme and will be published in full in Public Health Research; Vol. 5, No. 8. See the NIHR Journals Library website for further project information. It was also funded by the Big Lottery Fund

Whole-genome microarrays of fission yeast: characteristics, accuracy, reproducibility, and processing of array data

Background: The genome of the fission yeast Schizosaccharomyces pombe has recently been sequenced, setting the stage for the post-genomic era of this increasingly popular model organism. We have built fission yeast microarrays, optimised protocols to improve array performance, and carried out experiments to assess various characteristics of microarrays.|Results: We designed PCR primers to amplify specific probes (180-500 bp) for all known and predicted fission yeast genes, which are printed in duplicate onto separate regions of glass slides together with control elements (similar to13,000 spots/slide). Fluorescence signal intensities depended on the size and intragenic position of the array elements, whereas the signal ratios were largely independent of element properties. Only the coding strand is covalently linked to the slides, and our array elements can discriminate transcriptional direction. The microarrays can distinguish sequences with up to 70% identity, above which cross-hybridisation contributes to the signal intensity. We tested the accuracy of signal ratios and measured the reproducibility of array data caused by biological and technical factors. Because the technical variability is lower, it is best to use samples prepared from independent biological experiments to obtain repeated measurements with swapping of fluorochromes to prevent dye bias. We also developed a script that discards unreliable data and performs a normalization to correct spatial artefacts.|Conclusions: This paper provides data for several microarray properties that are rarely measured. The results define critical parameters for microarray design and experiments and provide a framework to optimise and interpret array data. Our arrays give reproducible and accurate expression ratios with high sensitivity. The scripts for primer design and initial data processing as well as primer sequences and detailed protocols are available from our website.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

The correlation between reading and mathematics ability at age twelve has a substantial genetic component

Dissecting how genetic and environmental influences impact on learning is helpful for maximizing numeracy and literacy. Here we show, using twin and genome-wide analysis, that there is a substantial genetic component to children’s ability in reading and mathematics, and estimate that around one half of the observed correlation in these traits is due to shared genetic effects (so-called Generalist Genes). Thus, our results highlight the potential role of the learning environment in contributing to differences in a child’s cognitive abilities at age twelve

A physical activity, nutrition and oral health intervention in nursery settings:Process evaluation of the NAP SACC UK feasibility cluster RCT

Background The nutrition and physical activity self-assessment for childcare (NAP SACC) intervention has demonstrated effectiveness in the USA. A feasibility randomised controlled trial was conducted in England to adapt the intervention to the UK context. An embedded process evaluation focused on three key questions. 1. Was it feasible and acceptable to implement the intervention as planned? 2. How did the intervention affect staff and parent mediators? 3. Were the trial design and methods acceptable? Methods Twelve nurseries in south-west England were recruited and randomised to intervention or control. The intervention comprised: NAP SACC UK Partner (Health Visitor) support to nurseries to review practice and policies against best practice, and then set goals to improve physical activity, nutrition and oral health; two staff training workshops; and a web-based parent support element. The process evaluation comprised: observations of Partner training (n = 1), Partner/manager meetings (n = 5) and staff workshops (n = 10); semi-structured interviews with Partners (n = 4), managers (n = 12), staff (n = 4) and parents (n = 20); analysis of self-assessment forms, goal setting forms and Partner logbooks; and assessment of staff and parent knowledge, motivation and self-efficacy mediators. Results Overall, NAP SACC UK was feasible to implement and acceptable to nursery staff, managers, Partners and parents. The intervention was implemented as planned in five of the six intervention nurseries. Partners and managers appreciated the opportunity to review and improve nursery practices and valued the relationship forged between them. Staff rated the training workshops highly, despite attending outside of working hours. Most goals set by nurseries were achieved. However, Partners raised concerns about Health Visitors’ capacity to deliver the intervention in any subsequent roll out. Mediator scores improved in all but two areas in intervention staff and parents, with decreases or minimal changes in the control group. The web-based parent element was not well used and should be removed from any subsequent trial. The trial methods were acceptable to managers, staff, Partners and parents. Conclusions Implementing and evaluating a physical activity and nutrition intervention in nursery settings is feasible and acceptable. A full RCT of NAP SACC UK (with appropriate modifications) is warranted

Genome-wide association study identifies a variant in HDAC9 associated with large vessel ischemic stroke

Genetic factors have been implicated in stroke risk but few replicated associations have been reported. We conducted a genome-wide association study (GWAS) in ischemic stroke and its subtypes in 3,548 cases and 5,972 controls, all of European ancestry. Replication of potential signals was performed in 5,859 cases and 6,281 controls. We replicated reported associations between variants close to PITX2 and ZFHX3 with cardioembolic stroke, and a 9p21 locus with large vessel stroke. We identified a novel association for a SNP within the histone deacetylase 9(HDAC9) gene on chromosome 7p21.1 which was associated with large vessel stroke including additional replication in a further 735 cases and 28583 controls (rs11984041, combined P = 1.87×10−11, OR=1.42 (95% CI) 1.28-1.57). All four loci exhibit evidence for heterogeneity of effect across the stroke subtypes, with some, and possibly all, affecting risk for only one subtype. This suggests differing genetic architectures for different stroke subtypes