A Simple PCR Method for Rapid Genotype Analysis of the TH-MYCN Transgenic Mouse

BACKGROUND: The TH-MYCN transgenic mouse is the most widely used murine model of human neuroblastoma, in which a human MYCN oncogene is targeted to neuroectodermal cells of developing mice under the influence of the rat tyrosine hydroxylase promoter. So far, homozygous transgenic mice have been identified by either Southern blot or quantitative real-time PCR. PRINCIPAL FINDINGS: To establish a simple and reliable genotyping method by conventional PCR, we confirmed the integration of the transgene in the TH-MYCN transgenic mouse by Southern blot and inverse PCR analyses. Our results showed that either five or six copies were found to be inserted in a head-to-tail tandem configuration at a single locus. The MYCN transgene/host DNA junction was sequenced and the integration site was identified at chromosome 18qE4. Finally, we succeeded in designing rapid, simple and reliable genotyping method by common PCR using primers flanking the integrated TH-MYCN transgene. CONCLUSION: We established a simple and reliable genotyping PCR method for determining the integration site of the TH-MYCN transgene that enables all possible genotypes to be distinguished within several hours. TH-MYCN mice are excellent model for human neuroblastoma study, thus our results will largely be useful for facilitating the pace of neuroblastoma study, including in the study of the tumourigenic process, and in the development of therapies to treat patients suffering from neuroblastoma

Spatial heterogeneity and peptide availability determine CTL killing efficiency in vivo

The rate at which a cytotoxic T lymphocyte (CTL) can survey for infected cells is a key ingredient of models of vertebrate immune responses to intracellular pathogens. Estimates have been obtained using in vivo cytotoxicity assays in which peptide-pulsed splenocytes are killed by CTL in the spleens of immunised mice. However the spleen is a heterogeneous environment and splenocytes comprise multiple cell types. Are some cell types intrinsically more susceptible to lysis than others? Quantitatively, what impacts are made by the spatial distribution of targets and effectors, and the level of peptide-MHC on the target cell surface? To address these questions we revisited the splenocyte killing assay, using CTL specific for an epitope of influenza virus. We found that at the cell population level T cell targets were killed more rapidly than B cells. Using modeling, quantitative imaging and in vitro killing assays we conclude that this difference in vivo likely reflects different migratory patterns of targets within the spleen and a heterogeneous distribution of CTL, with no detectable difference in the intrinsic susceptibilities of the two populations to lysis. Modeling of the stages involved in the detection and killing of peptide-pulsed targets in vitro revealed that peptide dose influenced the ability of CTL to form conjugates with targets but had no detectable effect on the probability that conjugation resulted in lysis, and that T cell targets took longer to lyse than B cells. We also infer that incomplete killing in vivo of cells pulsed with low doses of peptide may be due to a combination of heterogeneity in peptide uptake and the dissociation, but not internalisation, of peptide-MHC complexes. Our analyses demonstrate how population-averaged parameters in models of immune responses can be dissected to account for both spatial and cellular heterogeneity

Fungal Adenylyl Cyclase Acts As a Signal Sensor and Integrator and Plays a Central Role in Interaction with Bacteria

10.1371/journal.ppat.1003612PLoS Pathogens910

Escherichia coli induces apoptosis and proliferation of mammary cells

Mammary cell apoptosis and proliferation were assessed after injection of Escherichia coli into the left mammary quarters of six cows. Bacteriological analysis of foremilk samples revealed coliform infection in the injected quarters of four cows. Milk somatic cell counts increased in these quarters and peaked at 24 h after bacterial injection. Body temperature also increased, peaking at 12 h postinjection, The number of apoptotic cells was significantly higher in the mastitic tissue than in the uninfected control. Expression of Bax and interleukin-1 beta converting enzyme increased in the mastitic tissue at 24 h and 72 h postinfection, whereas Bcl-2 expression decreased at 24 h but did not differ significantly from the control at 72 h postinfection, Induction of matrix metalloproteinase-g, stromelysin-1 and urokinase-type plasminogen activator was also observed in the mastitic tissue. Moreover, cell proliferation increased in the infected tissue, These results demonstrate that Escherichia coli-induced mastitis promotes apoptosis and cell proliferation

Why do women not use antenatal services in low and middle income countries? A metasynthesis of qualitative studies

Background: Almost 50% of women in low & middle income countries (LMIC’s) don’t receive adequate antenatal care. Women’s views can offer important insights into this problem. Qualitative studies exploring inadequate use of antenatal services have been undertaken in a range of countries, but the findings are not easily transferable. We aimed to inform the development of future antenatal care programmes through a synthesis of findings in all relevant qualitative studies. Methods and Findings: Using a pre-determined search strategy, we identified robust qualitative studies reporting on the views and experiences of women in LMIC’s who received inadequate antenatal care. We used meta-ethnographic techniques to generate themes and a line of argument synthesis. We derived policy relevant hypotheses from the findings. We included 21 papers representing the views of more than 1230 women from 15 countries. Three key themes were identified: ‘Pregnancy as socially risky and physiologically healthy’; ‘Resource use and survival in conditions of extreme poverty’and ‘Not getting it right first time’. The line of argument synthesis describes a dissonance between programme design and cultural contexts that may restrict access and discourage return visits. We hypothesize that centralized, risk-focused antenatal care programmes may be at odds with the resources, beliefs and experiences of pregnant women who underuse antenatal services. Conclusions: Our findings suggest that there may be a mis-alignment between current antenatal provision and the social and cultural context of some women in LMIC’s. Antenatal care provision that is theoretically and contextually at odds with local contextual beliefs and experiences are likely to be underused, especially when attendance generates increased personal risks of lost family resource or physical danger during travel; when the promised care is not delivered due to resource constraints; and when women experience covert or overt abuse in care settings

Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli

BACKGROUND: Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library. RESULTS: Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport. CONCLUSION: Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the potential of functional clones interacting with host proteins to inhibit growth would make this approach most relevant for the study of prokaryotic genomes

Approaches to the evaluation of outbreak detection methods

BACKGROUND: An increasing number of methods are being developed for the early detection of infectious disease outbreaks which could be naturally occurring or as a result of bioterrorism; however, no standardised framework for examining the usefulness of various outbreak detection methods exists. To promote comparability between studies, it is essential that standardised methods are developed for the evaluation of outbreak detection methods. METHODS: This analysis aims to review approaches used to evaluate outbreak detection methods and provide a conceptual framework upon which recommendations for standardised evaluation methods can be based. We reviewed the recently published literature for reports which evaluated methods for the detection of infectious disease outbreaks in public health surveillance data. Evaluation methods identified in the recent literature were categorised according to the presence of common features to provide a conceptual basis within which to understand current approaches to evaluation. RESULTS: There was considerable variation in the approaches used for the evaluation of methods for the detection of outbreaks in public health surveillance data, and appeared to be no single approach of choice. Four main approaches were used to evaluate performance, and these were labelled the Descriptive, Derived, Epidemiological and Simulation approaches. Based on the approaches identified, we propose a basic framework for evaluation and recommend the use of multiple approaches to evaluation to enable a comprehensive and contextualised description of outbreak detection performance. CONCLUSION: The varied nature of performance evaluation demonstrated in this review supports the need for further development of evaluation methods to improve comparability between studies. Our findings indicate that no single approach can fulfil all evaluation requirements. We propose that the cornerstone approaches to evaluation identified provide key contributions to support internal and external validity and comparability of study findings, and suggest these be incorporated into future recommendations for performance assessment

Conceptualizing pathways linking women's empowerment and prematurity in developing countries.

BackgroundGlobally, prematurity is the leading cause of death in children under the age of 5. Many efforts have focused on clinical approaches to improve the survival of premature babies. There is a need, however, to explore psychosocial, sociocultural, economic, and other factors as potential mechanisms to reduce the burden of prematurity. Women's empowerment may be a catalyst for moving the needle in this direction. The goal of this paper is to examine links between women's empowerment and prematurity in developing settings. We propose a conceptual model that shows pathways by which women's empowerment can affect prematurity and review and summarize the literature supporting the relationships we posit. We also suggest future directions for research on women's empowerment and prematurity.MethodsThe key words we used for empowerment in the search were "empowerment," "women's status," "autonomy," and "decision-making," and for prematurity we used "preterm," "premature," and "prematurity." We did not use date, language, and regional restrictions. The search was done in PubMed, Population Information Online (POPLINE), and Web of Science. We selected intervening factors-factors that could potentially mediate the relationship between empowerment and prematurity-based on reviews of the risk factors and interventions to address prematurity and the determinants of those factors.ResultsThere is limited evidence supporting a direct link between women's empowerment and prematurity. However, there is evidence linking several dimensions of empowerment to factors known to be associated with prematurity and outcomes for premature babies. Our review of the literature shows that women's empowerment may reduce prematurity by (1) preventing early marriage and promoting family planning, which will delay age at first pregnancy and increase interpregnancy intervals; (2) improving women's nutritional status; (3) reducing domestic violence and other stressors to improve psychological health; and (4) improving access to and receipt of recommended health services during pregnancy and delivery to help prevent prematurity and improve survival of premature babies.ConclusionsWomen's empowerment is an important distal factor that affects prematurity through several intervening factors. Improving women's empowerment will help prevent prematurity and improve survival of preterm babies. Research to empirically show the links between women's empowerment and prematurity is however needed