Expression Screening of Fusion Partners from an E. coli Genome for Soluble Expression of Recombinant Proteins in a Cell-Free Protein Synthesis System

While access to soluble recombinant proteins is essential for a number of proteome studies, preparation of purified functional proteins is often limited by the protein solubility. In this study, potent solubility-enhancing fusion partners were screened from the repertoire of endogenous E. coli proteins. Based on the presumed correlation between the intracellular abundance and folding efficiency of proteins, PCR-amplified ORFs of a series of highly abundant E. coli proteins were fused with aggregation-prone heterologous proteins and then directly expressed for quantitative estimation of the expression efficiency of soluble translation products. Through two-step screening procedures involving the expression of 552 fusion constructs targeted against a series of cytokine proteins, we were able to discover a number of endogenous E. coli proteins that dramatically enhanced the soluble expression of the target proteins. This strategy of cell-free expression screening can be extended to quantitative, global analysis of genomic resources for various purposes.National Research Foundation of KoreaKorea (South). Ministry of Education, Science and Technology (MEST) (grant 2011K000841)Korea (South). Ministry of Education, Science and Technology (MEST) (grant 2011-0027901

Physician perceptions of primary prevention: qualitative base for the conceptual shaping of a practice intervention tool

BACKGROUND: A practice intervention must have its basis in an understanding of the physician and practice to secure its benefit and relevancy. We used a formative process to characterize primary care physician attitudes, needs, and practice obstacles regarding primary prevention. The characterization will provide the conceptual framework for the development of a practice tool to facilitate routine delivery of primary preventive care. METHODS: A focus group of primary care physician Opinion Leaders was audio-taped, transcribed, and qualitatively analyzed to identify emergent themes that described physicians' perceptions of prevention in daily practice. RESULTS: The conceptual worth of primary prevention, including behavioral counseling, was high, but its practice was significantly countered by the predominant clinical emphasis on and rewards for secondary care. In addition, lack of health behavior training, perceived low self-efficacy, and patient resistance to change were key deterrents to primary prevention delivery. Also, the preventive focus in primary care is not on cancer, but on predominant chronic nonmalignant conditions. CONCLUSIONS: The success of the future practice tool will be largely dependent on its ability to "fit" primary prevention into the clinical culture of diagnoses and treatment sustained by physicians, patients, and payers. The tool's message output must be formatted to facilitate physician delivery of patient-tailored behavioral counseling in an accurate, confident, and efficacious manner. Also, the tool's health behavior messages should be behavior-specific, not disease-specific, to draw on shared risk behaviors of numerous diseases and increase the likelihood of perceived salience and utility of the tool in primary care

Dynamic Changes in Protein Functional Linkage Networks Revealed by Integration with Gene Expression Data

Response of cells to changing environmental conditions is governed by the dynamics of intricate biomolecular interactions. It may be reasonable to assume, proteins being the dominant macromolecules that carry out routine cellular functions, that understanding the dynamics of protein∶protein interactions might yield useful insights into the cellular responses. The large-scale protein interaction data sets are, however, unable to capture the changes in the profile of protein∶protein interactions. In order to understand how these interactions change dynamically, we have constructed conditional protein linkages for Escherichia coli by integrating functional linkages and gene expression information. As a case study, we have chosen to analyze UV exposure in wild-type and SOS deficient E. coli at 20 minutes post irradiation. The conditional networks exhibit similar topological properties. Although the global topological properties of the networks are similar, many subtle local changes are observed, which are suggestive of the cellular response to the perturbations. Some such changes correspond to differences in the path lengths among the nodes of carbohydrate metabolism correlating with its loss in efficiency in the UV treated cells. Similarly, expression of hubs under unique conditions reflects the importance of these genes. Various centrality measures applied to the networks indicate increased importance for replication, repair, and other stress proteins for the cells under UV treatment, as anticipated. We thus propose a novel approach for studying an organism at the systems level by integrating genome-wide functional linkages and the gene expression data

Exploring Web-Based University Policy Statements on Plagiarism by Research-Intensive Higher Education Institutions

Plagiarism may distress universities in the US, but there is little agreement as to exactly what constitutes plagiarism. While there is ample research on plagiarism, there is scant literature on the content of university policies regarding it. Using a systematic sample, we qualitatively analyzed 20 Carnegie-classified universities that are “Very High in Research.” This included 15 public state universities and five high-profile private universities. We uncovered highly varied and even contradictory policies at these institutions. Notable policy variations existed for verbatim plagiarism, intentional plagiarism and unauthorized student collaboration at the studied institutions. We conclude by advising that the American Association of University Professors (AAUP), the American Association of Colleges and Universities (AACU) and others confer and come to accord on the disposition of these issues

Definitions, Foundations and Associations of Physical Literacy: A Systematic Review

Background: The concept of physical literacy has stimulated increased research attention in recent years—being deployed in physical education, sport participation, and the promotion of physical activity. Independent research groups currently operationalize the construct differently. Objective The purpose of this systematic review was to conduct a systematic review of the physical literacy construct,as reflected in contemporary research literature. Methods: Five databases were searched using the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines for systematic reviews. Inclusion criteria were English language, peer reviewed, published by March 2016, and seeking to conceptualize physical literacy. Articles that met these criteria were analysed in relation to three core areas: properties/attributes, philosophicalfoundations and theoretical associations with other constructs. A total of 50 published articles met the inclusion criteria and were analysed qualitatively using inductive thematic analysis.Results: The thematic analysis addressed the three core areas. Under definitions, core attributes that define physical literacy were identified, as well as areas of conflict between different approaches currently being adopted. One relatively clear philosophical approach was prominent in approximately half of the papers, based on a monist/holistic ontology and phenomenological epistemology. Finally, theanalysis identified a number of theoretical associations, including health, physical activity and academic performance.Conclusions: Current literature contains different representations of the physical literacy construct. The costs and benefits of adopting an exclusive approach versus pluralism are considered. Recommendations for both researchers and practitioners focus on identifying and clearly articulating the definitions, philosophical assumptions and expected outcomes prior to evaluating the effectiveness of this emerging concept

Transcript profiling of candidate genes in testis of pigs exhibiting large differences in androstenone levels

<p>Abstract</p> <p>Background</p> <p>Boar taint is an unpleasant odor and flavor of the meat and occurs in a high proportion of uncastrated male pigs. Androstenone, a steroid produced in testis and acting as a sex pheromone regulating reproductive function in female pigs, is one of the main compounds responsible for boar taint. The primary goal of the present investigation was to determine the differential gene expression of selected candidate genes related to levels of androstenone in pigs.</p> <p>Results</p> <p>Altogether 2560 boars from the Norwegian Landrace and Duroc populations were included in this study. Testicle samples from the 192 boars with most extreme high or low levels of androstenone in fat were used for RNA extraction, and 15 candidate genes were selected and analyzed by real-competitive PCR analysis. The genes Cytochrome P450 c17 (<it>CYP17A1</it>), Steroidogenic acute regulatory protein (<it>STAR</it>), Aldo-keto reductase family 1 member C4 (<it>AKR1C4</it>), Short-chain dehydrogenase/reductase family member 4 (<it>DHRS4</it>), Ferritin light polypeptide (<it>FTL</it>), Sulfotransferase family 2A, dehydroepiandrosterone-preferring member 1 (<it>SULT2A1</it>), Cytochrome P450 subfamily XIA polypeptide 1 (<it>CYP11A1</it>), Cytochrome b5 (<it>CYB5A</it>), and 17-beta-Hydroxysteroid dehydrogenase IV (<it>HSD17B4</it>) were all found to be significantly (P < 0.05) up-regulated in high androstenone boars in both Duroc and Landrace. Furthermore, Cytochrome P450 c19A2 (<it>CYP19A2</it>) was down-regulated and progesterone receptor membrane component 1 (<it>PGRMC1</it>) was up-regulated in high-androstenone Duroc boars only, while <it>CYP21 </it>was significantly down-regulated (2.5) in high-androstenone Landrace only. The genes Nuclear Receptor co-activator 4 (<it>NCOA4</it>), Sphingomyrlin phosphodiesterase 1 (<it>SMPD1</it>) and 3β-hydroxysteroid dehydrogenase (<it>HSD3B</it>) were not significantly differentially expressed in any breeds. Additionally, association studies were performed for the genes with one or more detected SNPs. Association between SNP and androstenone level was observed in <it>CYB5A </it>only, suggesting cis-regulation of the differential transcription in this gene.</p> <p>Conclusion</p> <p>A large pig material of highly extreme androstenone levels is investigated. The current study contributes to the knowledge about which genes that is differentially expressed regard to the levels of androstenone in pigs. Results in this paper suggest that several genes are important in the regulation of androstenone level in boars and warrant further evaluation of the above mentioned candidate genes, including analyses in different breeds, identification of causal mutations and possible gene interactions.</p

Integrated metatranscriptomic and metagenomic analyses of stratified microbial assemblages in the open ocean

As part of an ongoing survey of microbial community gene expression in the ocean, we sequenced and compared ~38 Mbp of community transcriptomes and ~157 Mbp of community genomes from four bacterioplankton samples, along a defined depth profile at Station ALOHA in North Pacific subtropical gyre (NPSG). Taxonomic analysis suggested that the samples were dominated by three taxa: Prochlorales, Consistiales and Cenarchaeales, which comprised 36–69% and 29–63% of the annotated sequences in the four DNA and four cDNA libraries, respectively. The relative abundance of these taxonomic groups was sometimes very different in the DNA and cDNA libraries, suggesting differential relative transcriptional activities per cell. For example, the 125 m sample genomic library was dominated by Pelagibacter (~36% of sequence reads), which contributed fewer sequences to the community transcriptome (~11%). Functional characterization of highly expressed genes suggested taxon-specific contributions to specific biogeochemical processes. Examples included Roseobacter relatives involved in aerobic anoxygenic phototrophy at 75 m, and an unexpected contribution of low abundance Crenarchaea to ammonia oxidation at 125 m. Read recruitment using reference microbial genomes indicated depth-specific partitioning of coexisting microbial populations, highlighted by a transcriptionally active high-light-like Prochlorococcus population in the bottom of the photic zone. Additionally, nutrient-uptake genes dominated Pelagibacter transcripts, with apparent enrichment for certain transporter types (for example, the C4-dicarboxylate transport system) over others (for example, phosphate transporters). In total, the data support the utility of coupled DNA and cDNA analyses for describing taxonomic and functional attributes of microbial communities in their natural habitats.Gordon and Betty Moore FoundationUnited States. Dept. of EnergyNational Science Foundation (U.S.) (Science and Technology Center Award EF0424599

Genome-Wide Functional Profiling Identifies Genes and Processes Important for Zinc-Limited Growth of Saccharomyces cerevisiae

Zinc is an essential nutrient because it is a required cofactor for many enzymes and transcription factors. To discover genes and processes in yeast that are required for growth when zinc is limiting, we used genome-wide functional profiling. Mixed pools of ∼4,600 deletion mutants were inoculated into zinc-replete and zinc-limiting media. These cells were grown for several generations, and the prevalence of each mutant in the pool was then determined by microarray analysis. As a result, we identified more than 400 different genes required for optimal growth under zinc-limiting conditions. Among these were several targets of the Zap1 zinc-responsive transcription factor. Their importance is consistent with their up-regulation by Zap1 in low zinc. We also identified genes that implicate Zap1-independent processes as important. These include endoplasmic reticulum function, oxidative stress resistance, vesicular trafficking, peroxisome biogenesis, and chromatin modification. Our studies also indicated the critical role of macroautophagy in low zinc growth. Finally, as a result of our analysis, we discovered a previously unknown role for the ICE2 gene in maintaining ER zinc homeostasis. Thus, functional profiling has provided many new insights into genes and processes that are needed for cells to thrive under the stress of zinc deficiency

Genetic Interaction Maps in Escherichia coli Reveal Functional Crosstalk among Cell Envelope Biogenesis Pathways

As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among >235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target