Semi-formal Models to Support Program Development: Autonomic Management within Component Based Parallel and Distributed Programming

Functional and non-functional concerns require different programming effort, different techniques and different methodologies when attempting to program efficient parallel/distributed applications. In this work we present a "programmer oriented" methodology based on formal tools that permits reasoning about parallel/distributed program development and refinement. The proposed methodology is semi-formal in that it does not require the exploitation of highly formal tools and techniques, while providing a palatable and effective support to programmers developing parallel/distributed applications, in particular when handling non-functional concerns.

DNA methyltransferase expression in the mouse germ line during periods of de novo methylation

DNA methyltransferase (DNMT) 3A and DNMT3B are both active de novo DNA methyltransferases required for development, whereas DNMT3L, which has no demonstrable methyltransferase activity, is required for methylation of imprinted genes in the oocyte. We show here that different mechanisms are used to restrict access by these proteins to their targets during germ cell development. Transcriptional control of the Dnmt3l promoter guarantees that message is low or absent except during periods of de novo activity. Use of an alternative promoter at the Dnmt3a locus produces the shorter Dnmt3a2 transcript in the germ line and postimplantation embryo only, whereas alternative splicing of the Dnmt3b transcript ensures that Dnmt3b1 is absent in the male prospermatogonia. Control of subcellular protein localization is a common theme for DNMT3A and DNMT3B, as proteins were seen in the nucleus only when methylation was occurring. These mechanisms converge to ensure that the only time that functional products from each locus are present in the germ cell nuclei is around embryonic day 17.5 in males and after birth in the growing oocytes in females.

Direct reprogramming of adult cells: avoiding the pluripotent state

The procedure of using mature, fully differentiated cells and inducing them toward other cell types while bypassing an intermediate pluripotent state is termed direct reprogramming. Avoiding the pluripotent stage during cellular conversions can be achieved either through ectopic expression of lineage-specific factors (transdifferentiation) or a direct reprogramming process that involves partial reprogramming toward the pluripotent stage. Latest advances in the field seek to alleviate concerns that include teratoma formation or retroviral usage when it comes to delivering reprogramming factors to cells. They also seek to improve efficacy and efficiency of cellular conversion, both in vitro and in vivo. The final products of this reprogramming approach could be then directly implemented in regenerative and personalized medicine.

MicroRNA-199b modulates vascular cell fate during iPS cell differentiation by targeting the notch ligand jagged1 and enhancing VEGF signaling

Aims: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalised medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation towards ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration.

Methods and Results: In this study we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen-coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR-199b is involved in EC differentiation. A step-wise increase in expression of miR-199 was detected during EC differentiation. Notably, miR-199b targeted the Notch ligand JAG1, resulting in VEGF transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR-199b-mediated inhibition of iPS cell differentiation towards SMCs. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR-199b also regulated VEGF expression and angiogenesis.

Conclusions: This study indicates a novel role for miR-199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses.

WS02.7 Initial and chronic MRSA infection in cystic fibrosis

Objectives Chronic MRSA infection, which affects approximately 26% of CF patients in the USA, is associated with declining lung function and poor outcomes (Dasenbrook, 2010). Anaerobic niches have been described within the CF lung, potentially influencing the virulence of MRSA. This study aims to compare initial and chronic CF MRSA isolates, following aerobic and anaerobic culture. Methods Isolates, obtained from CF sputum at first isolation [“early” (n = 10)] or up to 5 years later, during chronic infection [“late” (n = 15)] were cultured in aerobic and anaerobic conditions. Differences in virulence were compared using the Galleria mellonella infection model. Biofilm formation of each isolate was assessed following staining with crystal violet. Production of Δ-haemolysin (Δ-hly), a surrogate marker for expression of the virulence regulator agr, was determined by haemolysis assay. Results MRSA grown in anaerobic conditions had significantly increased virulence in the G. mellonella model (p = 0.007), increased biofilm formation (p = 0.006) and increased Δ-hly production (p<0.0001). No significant difference between Δ-hly production or biofilm formation were observed between early and late isolates; however late isolates were found to be more virulent in the G. mellonella model (p = 0.0002). Conclusion These results suggest that an anaerobic environment, as found in the CF lung, may increase virulence of MRSA and aid in the establishment of chronic infection. Further clinical studies are required to determine how these phenotypic changes are associated with transition to chronic infection and patient outcome.

Vaccinations and childhood type 1 diabetes mellitus: a meta-analysis of observational studies

Aims/hypothesis The aim of this study was to investigate the association between routine vaccinations and the risk of childhood type 1 diabetes mellitus by systematically reviewing the published literature and performing meta-analyses where possible.

Methods A comprehensive literature search was performed of MEDLINE and EMBASE to identify all studies that compared vaccination rates in children who subsequently developed type 1 diabetes mellitus and in control children. ORs and 95% CIs were obtained from published reports or derived from individual patient data and then combined using a random effects meta-analysis.

Results In total, 23 studies investigating 16 vaccinations met the inclusion criteria. Eleven of these contributed to meta-analyses which included data from between 359 and 11,828 childhood diabetes cases. Overall, there was no evidence to suggest an association between any of the childhood vaccinations investigated and type 1 diabetes mellitus. The pooled ORs ranged from 0.58 (95% CI 0.24, 1.40) for the measles, mumps and rubella (MMR) vaccination in five studies up to 1.04 (95% CI 0.94, 1.14) for the haemophilus influenza B (HiB) vaccination in 11 studies. Significant heterogeneity was present in most of the pooled analyses, but was markedly reduced when analyses were restricted to study reports with high methodology quality scores. Neither this restriction by quality nor the original authors’ adjustments for potential confounding made a substantial difference to the pooled ORs.

Conclusions/interpretation This study provides no evidence of an association between routine vaccinations and childhood type 1 diabetes.

Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus , is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the Msp I satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus , up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation.