Zellbasierte Hochdurchsatzanalysen identifizieren eine zentrale, wachstumsregulierende Rolle von Cofilin-1 im humanen Pankreaskarzinom

Aufgrund der ungünstigen Prognose des humanen duktalen Adenokarzinoms des Pankreas ist es von großer Bedeutung, neue Zielgene zu identifizieren, die Ansatzstellen sowohl für neue Therapien als auch für eine frühere Diagnose bieten können. In der hier vorgestellten Arbeit wurden deshalb insgesamt 91 potentielle Kandidatengene funktionell charakterisiert, die in vorangegangenen Hochdurchsatz- Analysen in Karzinomgeweben eine differentielle Expression aufgewiesen hatten. Für diese Untersuchung wurde die Methode der „Reverse Transfection“ in unserem Labor etabliert, um die Experimente in einem parallelisierten Microarray-Verfahren durchzuführen. Es erfolgten Überexpressions- sowie Repressionsanalysen der Kandidatengene in verschiedenen transformierten und nicht-transformierten Zelllinien, in denen die subzelluläre Lokalisation der Genprodukte sowie ihr Einfluss auf zelluläre Funktionen wie Apoptose, Proliferation oder Differenzierung analysiert wurden. Die Evaluation dieser funktionellen Daten führte zu einer Auswahl von 10 Genen, die in den parallelisierten Experimenten reproduzierbare und auffällige Effekte zeigten. Eines dieser Kandidaten war das Gen Cofilin-1 (CFL1), welches zur ADF/Cofilin- Familie der aktin-bindenen Proteine gehört. Die „Reverse Transfection“-Experimente zeigten für CFL1 einen Wachstums-regulierenden Effekt, der im Pankreaskarzinom bisher unbeschrieben ist. In nachfolgenden, funktionellen Detailanalysen zeigte sich eine starke Überexpression von CFL1 im Pankreaskarzinom. RNAi-basierte Inaktivierungen von CFL1 in 4 Zelllinien führten zu einem signifikanten Rückgang des Zellwachstums, während apoptotische Kaskaden unbeeinflusst blieben. Des Weiteren konnte mittels Soft-Agar-Analysen eine signifikante Verlangsamung des substratunabhängigen Wachstums beobachtet werden. Durchfluss-zytometrische Analysen des Zellzyklus deuten auf eine Verlangsamung der G1/S-Phasen-Transition hin. In Migrationsanalysen konnte weiterhin eine signifikante Verlangsamung der Zellbewegungen in „Time Lapse“- und Wundheilungsexperimenten identifiziert werden, während die gerichtete Migration in Boyden-Kammer-Versuchen unbeeinflusst blieb. Auf Basis der vorliegenden Literaturdaten stellen dieser Ergebnisse den ersten Nachweis eines wachstumsregulierenden Effekts von CFL1 im Pankreaskarzinom dar

The effects of an adapted mental health literacy curriculum for secondary school students in Germany on mental health knowledge and help-seeking efficacy: results of a quasi-experimental pre-post evaluation study

BackgroundBecause the majority of mental illnesses develop early in life, effective preventative public mental health interventions are needed. Interventions fostering mental health literacy can be used to enhance personal resources and capacities to facilitate mental health care and thus, address help-seeking barriers. A Canadian mental health literacy school curriculum was adapted, piloted, and evaluated for the use in German schools. The study presents the intervention’s effects on mental health knowledge and help-seeking efficacy among 10th grade students in Germany.Methods10th grade students (aged 14–17 years old) from one secondary school participated in a pre- and post-intervention control group study. Both groups completed a questionnaire at two time points assessing mental health knowledge and help-seeking efficacy. Repeated measure analysis of variance (ANOVA) was employed to evaluate the intervention’s effects.ResultsData from 188 students was eligible for analysis. The analysis of the baseline data reveals a high comparability of the two groups in terms of demographics, and initial mental health knowledge and help-seeking efficacy scores. ANOVA results showed significant improvements for the intervention group having a large effect size for mental health knowledge (f = 0.574, p < 0.001, partial η2 = 0.25) and a medium effect size for help-seeking efficacy (f = 0.311, p < 0.001, partial η2 = 0.09).ConclusionThe first-time application and evaluation of an adapted mental health literacy school curriculum shows significant increases in mental health knowledge and help-seeking efficacy, two core dimensions of mental health literacy, among 10th grade students in Germany. Further studies are needed to confirm these results as well as have a more in-depth analysis on the interrelations of the different dimensions of mental health knowledge and help-seeking practices

Seminiferous tubule transfection in vitro to define post-meiotic gene regulation

The electronic version of this article is the complete one and can be found online at: http://www.rbej.com/content/7/1/67Background: Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation. Methods: Culture conditions were developed which allowed the continued incubation of isolated rat seminiferous tubules for up to 48 h without obvious cell death and loss of post-meiotic cells. Transfection of intact seminiferous tubules by microinjection and electroporation was optimized to achieve high expression efficiencies of control plasmids, using either fluorescent protein or luciferase as reporters, thereby allowing both morphological as well as quantitative assessment. Results: Successful transfection was achieved into all cell types except for mature spermatozoa. However, there appeared to be only limited cell-type specificity for the promoters used, even though these had appeared to be specific when used in transgenic animals. Conclusion: We have devised a methodology which allows relatively high throughput analysis of post-meiotic gene promoters into primary cells of intact seminiferous tubules. An apparent lack of cell-type specificity suggests that the gene fragments used do not contain sufficient targeting information, or that the transient episomal expression of the constructs does not encourage appropriate expression specificity. The results also highlight the doubtful interpretation of many studies using heterologous transfection systems to analyse post-meiotically expressed genes.Sandra Danner, Christiane Kirchhoff and Richard Ivel

An international cohort comparison of size effects on job growth

The contribution of different-sized businesses to job creation continues to attract policymakers’ attention; however, it has recently been recognised that conclusions about size were confounded with the effect of age. We probe the role of size, controlling for age, by comparing the cohorts of firms born in 1998 over their first decade of life, using variation across half a dozen northern European countries Austria, Finland, Germany, Norway, Sweden and the UK to pin down size effects. We find that a very small proportion of the smallest firms play a crucial role in accounting for cross-country differences in job growth. A closer analysis reveals that the initial size distribution and survival rates do not seem to explain job growth differences between countries, rather it is a small number of rapidly growing firms that are driving this result

Exploring the role of organizational policies and procedures in promoting research utilization in registered nurses

<p>Abstract</p> <p>Background</p> <p>Policies and procedures (P&Ps) have been suggested as one possible strategy for moving research evidence into practice among nursing staff in hospitals. Research in the area of P&Ps is limited, however. This paper explores: 1) nurses' use of eight specific research-based practices (RBPs) and RBP overall, 2) nurses' use and understanding of P&Ps, and 3) the role of P&Ps in promoting research utilization.</p> <p>Methods</p> <p>Staff nurses from the eight health regions governing acute care services across the Canadian province of Newfoundland and Labrador completed an anonymous questionnaire regarding their use of eight RBPs and associated P&Ps. Data were also obtained from authorities in six of the eight regions about existing relevant P&Ps. We used descriptive statistics and multivariate regression analysis to assess the relationship between key independent variables and self-reported use of RBP.</p> <p>Results</p> <p>Use of the eight RBPs ranged from 7.8% to 88.6%, depending on the practice. Nurses ranked P&P manuals as their number one source of practice knowledge. Most respondents (84.8%) reported that the main reason they consult the P&P manual is to confirm they are practicing according to agency rules. Multivariate regression analysis identified three significant predictors of being a user versus non-user of RBP overall: awareness, awareness by regular use, and persuasion. Six significant predictors of being a consistent versus less consistent user of RBP overall were also identified: perception of P&P existence, unit, nursing experience, personal experience as a source of practice knowledge, number of existing research-based P&Ps, and lack of time as a barrier to consulting P&P manuals.</p> <p>Conclusion</p> <p>Findings suggest that nurses use P&Ps to guide their practice. However, the mere existence of P&Ps is not sufficient to translate research into nursing practice. Individual and organizational factors related to nurses' understanding and use of P&Ps also play key roles. Thus, moving research evidence into practice will require careful interplay between the organization and the individual. P&Ps may be the interface through which this occurs.</p

Type 2 diabetes gene TCF7L2 polymorphism is not associated with fetal and postnatal growth in two birth cohort studies

Background: An inverse association between birth weight and the risk of developing type 2 diabetes (T2D) in adulthood has been reported. This association may be explained by common g

Elite Suppressor–Derived HIV-1 Envelope Glycoproteins Exhibit Reduced Entry Efficiency and Kinetics

Elite suppressors (ES) are a rare subset of HIV-1–infected individuals who are able to maintain HIV-1 viral loads below the limit of detection by ultra-sensitive clinical assays in the absence of antiretroviral therapy. Mechanism(s) responsible for this elite control are poorly understood but likely involve both host and viral factors. This study assesses ES plasma-derived envelope glycoprotein (env) fitness as a function of entry efficiency as a possible contributor to viral suppression. Fitness of virus entry was first evaluated using a novel inducible cell line with controlled surface expression levels of CD4 (receptor) and CCR5 (co-receptor). In the context of physiologic CCR5 and CD4 surface densities, ES envs exhibited significantly decreased entry efficiency relative to chronically infected viremic progressors. ES envs also demonstrated slow entry kinetics indicating the presence of virus with reduced entry fitness. Overall, ES env clones were less efficient at mediating entry than chronic progressor envs. Interestingly, acute infection envs exhibited an intermediate phenotypic pattern not distinctly different from ES or chronic progressor envs. These results imply that lower env fitness may be established early and may directly contribute to viral suppression in ES individuals

Split-Cre Complementation Indicates Coincident Activity of Different Genes In Vivo

Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive “split-Cre” fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting

Storylines: an alternative approach to representing uncertainty in physical aspects of climate change

As climate change research becomes increasingly applied, the need for actionable information is growing rapidly. A key aspect of this requirement is the representation of uncertainties. The conventional approach to representing uncertainty in physical aspects of climate change is probabilistic, based on ensembles of climate model simulations. In the face of deep uncertainties, the known limitations of this approach are becoming increasingly apparent. An alternative is thus emerging which may be called a ‘storyline’ approach. We define a storyline as a physically self-consistent unfolding of past events, or of plausible future events or pathways. No a priori probability of the storyline is assessed; emphasis is placed instead on understanding the driving factors involved, and the plausibility of those factors. We introduce a typology of four reasons for using storylines to represent uncertainty in physical aspects of climate change: (i) improving risk awareness by framing risk in an event-oriented rather than a probabilistic manner, which corresponds more directly to how people perceive and respond to risk; (ii) strengthening decision-making by allowing one to work backward from a particular vulnerability or decision point, combining climate change information with other relevant factors to address compound risk and develop appropriate stress tests; (iii) providing a physical basis for partitioning uncertainty, thereby allowing the use of more credible regional models in a conditioned manner and (iv) exploring the boundaries of plausibility, thereby guarding against false precision and surprise. Storylines also offer a powerful way of linking physical with human aspects of climate change

Identification of a BRCA2-Specific modifier locus at 6p24 related to breast cancer risk

Common genetic variants contribute to the observed variation in breast cancer risk for BRCA2 mutation carriers; those known to date have all been found through population-based genome-wide association studies (GWAS). To comprehensively identify breast cancer risk modifying loci for BRCA2 mutation carriers, we conducted a deep replication of an ongoing GWAS discovery study. Using the ranked P-values of the breast cancer associations with the imputed genotype of 1.4 M SNPs, 19,029 SNPs were selected and designed for inclusion on a custom Illumina array that included a total of 211,155 SNPs as part of a multi-consortial project. DNA samples from 3,881 breast cancer affected and 4,330 unaffected BRCA2 mutation carriers from 47 studies belonging to the Consortium of Investigators of Modifiers of BRCA1/2 were genotyped and available for analysis. We replicated previously reported breast cancer susceptibility alleles in these BRCA2 mutation carriers and for several regions (including FGFR2, MAP3K1, CDKN2A/B, and PTHLH) identified SNPs that have stronger evidence of association than those previously published. We also identified a novel susceptibility allele at 6p24 that was inversely associated with risk in BRCA2 mutation carriers (rs9348512; per allele HR = 0.85, 95% CI 0.80-0.90, P = 3.9×10−8). This SNP was not associated with breast cancer risk either in the general population or in BRCA1 mutation carriers. The locus lies within a region containing TFAP2A, which encodes a transcriptional activation protein that interacts with several tumor suppressor genes. This report identifies the first breast cancer risk locus specific to a BRCA2 mutation background. This comprehensive update of novel and previously reported breast cancer susceptibility loci contributes to the establishment of a panel of SNPs that modify breast cancer risk in BRCA2 mutation carriers. This panel may have clinical utility for women with BRCA2 mutations weighing options for medical prevention of breast cancer