Design, Construction and Cloning of Truncated ORF2 and tPAsp-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector

Background: Hepatitis E Virus (HEV) is the causative agent of enterically transmitted acute hepatitis and has high mortality rate of up to 30% among pregnant women. Therefore, development of a novel vaccine is a desirable goal. Objectives: The aim of this study was to construct tPAsp-PADRE-truncated open reading frame 2 (ORF2) and truncated ORF2 DNA plasmid, which can assist future studies with the preparation of an effective vaccine against Hepatitis E Virus. Materials and Methods: A synthetic codon-optimized gene cassette encoding tPAsp-PADRE-truncated ORF2 protein was designed, constructed and analyzed by some bioinformatics software. Furthermore, a codon-optimized truncated ORF2 gene was amplified by the polymerase chain reaction (PCR), with a specific primer from the previous construct. The constructs were sub-cloned in the pVAX1 expression vector and finally expressed in eukaryotic cells. Results: Sequence analysis and bioinformatics studies of the codon-optimized gene cassette revealed that codon adaptation index (CAI), GC content, and frequency of optimal codon usage (Fop) value were improved, and performance of the secretory signal was confirmed. Cloning and sub-cloning of the tPAsp-PADRE-truncated ORF2 gene cassette and truncated ORF2 gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pVAX-tPAsp-PADRE-truncated ORF2 (aa 112-660) and pVAX-truncated ORF2 (aa 112-660). The expression of truncated ORF2 protein in eukaryotic cells was approved by an Immunofluorescence assay (IFA) and the reverse transcriptase polymerase chain reaction (RT-PCR) method. Conclusions: The results of this study demonstrated that the tPAsp-PADRE-truncated ORF2 gene cassette and the truncated ORF2 gene in recombinant plasmids are successfully expressed in eukaryotic cells. The immunogenicity of the two recombinant plasmids with different formulations will be evaluated as a novel DNA vaccine in future investigations

Explaining the continuum of social participation among older adults in Singapore: from 'closed doors' to active ageing in multi-ethnic community settings.

OBJECTIVES: This study aims to identify and explain the continuum in which older people in Singapore participate in community and social life, highlighting the influence of culture and policy context on social participation. METHODS: Using an ethnographic approach in a neighbourhood (n=109), we conducted focus groups with older adults of different ethnicities, exploring experiences of social participation. Next, participants took 50 photographs relating to 'lives of elders', showcasing the socio-ecological context that influenced social participation. Lastly, go-along interviews were conducted in various precincts with community leaders. RESULTS: A continuum of social participation emerged among older adults, ranging from (1) marginalization and exclusion, to (2) 'comfort-zoning' alone (3) seeking consistent social interactions, (4) expansion of social network, and (5) giving back to society. Seeking consistent social interactions was shaped by a preference for cultural grouping and ethnic values, but also a desire for emotional safety. Attitudes about expanding one's social network depended on the psychosocial adjustment of the older person to the prospect of gossip and 'trouble' of managing social relations. Despite the societal desirability of an active ageing lifestyle, cultural scripts emphasizing family meant older adults organized participation in social and community life, around family responsibilities. Institutionalizing family reliance in Singapore's welfare approach penalized lower-income older adults with little family support from accessing subsidies, and left some living on the margins. DISCUSSION: To promote inclusiveness, ageing programs should address preferences for social participation, overcoming barriers at the individual, ethnic culture and policy level

Disorganization of the splenic microanatomy in ageing mice

The precise mechanisms responsible for immunosenescence still remain to be determined, however, considering the evidence that disruption of the organization of primary and secondary lymphoid organs results in immunodeficiency, we propose that this could be involved in the decline of immune responses with age. Therefore, we investigated the integrity of the splenic microarchitecture in mice of increasing age and its reorganization following immune challenge in young and old mice. Several differences in the anatomy of the spleen with age in both the immune and stromal cells were observed. There is an age‐related increase in the overall size of the white pulp, which occurs primarily within the T‐cell zone and is mirrored by the enlargement of the T‐cell stromal area, concurrent to the distinct boundary between T cells and B cells becoming less defined in older mice. In conjunction, there appears to be a loss of marginal zone macrophages, which is accompanied by an accumulation of fibroblasts in the spleens from older animals. Furthermore, whereas the reorganization of the white pulp is resolved after several days following antigenic challenge in young animals, it remains perturbed in older subjects. All these age‐related changes within the spleen could potentially contribute to the age‐dependent deficiencies in functional immunity

The theoretical and empirical basis of a BioPsychoSocial (BPS) risk screener for detection of older people's health related needs, planning of community programs, and targeted care interventions

Background This study introduces the conceptual basis and operational measure, ofBioPyschoSocial (BPS) healthand related risk to better understand how well older people are managing and to screen for risk status. The BPS Risk Screener is constructed to detectvulnerabilityat older ages, and seeks to measure dynamic processes that place equal emphasis on Psycho-emotional and Socio-interpersonal risks, as Bio-functional ones. We validate the proposed measure and describe its application to programming. Methods We undertook a quantitative cross-sectional, psychometric study withn = 1325 older Singaporeans, aged 60 and over. We adapted the EASYCare 2010 and Lubben Social Network Scale questionnaires to help determine the BPS domains using factor analysis from which we derive the BPS Risk Screener items. We then confirm its structure, and test the scoring system. The score is initially validated against self-reported general health then modelled against: number of falls; cognitive impairment; longstanding diseases; and further tested against service utilization (linked administrative data). Results Three B, P and S clusters are defined and identified and a BPSmanaging score(‘doing’ well, or ‘some’, ‘many’, and ‘overwhelming problems’) calculated such that the risk of problematic additive BPS effects, what we term health‘loads’, are accounted for. Thirty-five items (factor loadings over 0.5) clustered into three distinct B, P, S domains and were found to be independently associated with self-reported health: B: 1.99 (1.64 to 2.41), P: 1.59 (1.28 to 1.98), S: 1.33 (1.10 to 1.60). The fit improved when combined into the managing score 2.33 (1.92 to 2.83, < 0.01). The score was associated with mounting risk for all outcomes. Conclusions BPS domain structures, and the novel scoring system capturing dynamic BPS additive effects, which can combine to engender vulnerability, are validated through this analysis. The resulting tool helps render clients’ risk status and related intervention needs transparent. Given its explicit and empirically supported attention to P and S risks, which have the potential to be more malleable than B ones, especially in the older old, this tool is designed to be change sensitive

Normal levels of p27Xic1 are necessary for somite segmentation and determining pronephric organ size

The Xenopus laevis cyclin dependent kinase inhibitor p27Xic1 has been shown to be involved in exit from the cell cycle and differentiation of cells into a quiescent state in the nervous system, muscle tissue, heart and retina. We show that p27Xic1 is expressed in the developing kidney in the nephrostomal regions. Using over-expression and morpholino oligonucleotide (MO) knock-down approaches we show normal levels of p27Xic1 regulate pronephros organ size by regulating cell cycle exit. Knock-down of p27Xic1 expression using a MO prevented myogenesis, as previously reported; an effect that subsequently inhibits pronephrogenesis. Furthermore, we show that normal levels of p27Xic1 are required for somite segmentation also through its cell cycle control function. Finally, we provide evidence to suggest correct paraxial mesoderm segmentation is not necessary for pronephric induction in the intermediate mesoderm. These results indicate novel developmental roles for p27Xic1, and reveal its differentiation function is not universally utilised in all developing tissues

Low oxygen tension primes aortic endothelial cells to the reparative effect of tissue-protective cytokines

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared to 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis

Development and quantitative analyses of a universal rRNA-subtraction protocol for microbial metatranscriptomics

Metatranscriptomes generated by pyrosequencing hold significant potential for describing functional processes in complex microbial communities. Meeting this potential requires protocols that maximize mRNA recovery by reducing the relative abundance of ribosomal RNA, as well as systematic comparisons to identify methodological artifacts and test for reproducibility across data sets. Here, we implement a protocol for subtractive hybridization of bacterial rRNA (16S and 23S) that uses sample-specific probes and is applicable across diverse environmental samples. To test this method, rRNA-subtracted and unsubtracted transcriptomes were sequenced (454 FLX technology) from bacterioplankton communities at two depths in the oligotrophic open ocean, yielding 10 data sets representing ~350 Mbp. Subtractive hybridization reduced bacterial rRNA transcript abundance by 40–58%, increasing recovery of non-rRNA sequences up to fourfold (from 12% to 20% of total sequences to 40–49%). In testing this method, we established criteria for detecting sequences replicated artificially via pyrosequencing errors and identified such replicates as a significant component (6–39%) of total pyrosequencing reads. Following replicate removal, statistical comparisons of reference genes (identified via BLASTX to NCBI-nr) between technical replicates and between rRNA-subtracted and unsubtracted samples showed low levels of differential transcript abundance (<0.2% of reference genes). However, gene overlap between data sets was remarkably low, with no two data sets (including duplicate runs from the same pyrosequencing library template) sharing greater than 17% of unique reference genes. These results indicate that pyrosequencing captures a small subset of total mRNA diversity and underscores the importance of reliable rRNA subtraction procedures to enhance sequencing coverage across the functional transcript pool.Agouron InstituteGordon and Betty Moore FoundationUnited States. Dept. of Energy. Office of ScienceNational Science Foundation (U.S.) (NSF Science and Technology Center Award EF0424599

Regulation of neutrophil senescence by microRNAs

Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease

Subcoronary versus supracoronary aortic stenosis. an experimental evaluation

<p>Abstract</p> <p>Background</p> <p>Valvular aortic stenosis is the most common cause of left ventricular hypertrophy due to gradually increasing pressure work. As the stenosis develop the left ventricular hypertrophy may lead to congestive heart failure, increased risk of perioperative complications and also increased risk of sudden death. A functional porcine model imitating the pathophysiological nature of valvular aortic stenosis is very much sought after in order to study the geometrical and pathophysiological changes of the left ventricle, timing of surgery and also pharmacological therapy in this patient group.</p> <p>Earlier we developed a porcine model for aortic stenosis based on supracoronary aortic banding, this model may not completely imitate the pathophysiological changes that occurs when valvular aortic stenosis is present including the coronary blood flow. It would therefore be desirable to optimize this model according to the localization of the stenosis.</p> <p>Methods</p> <p>In 20 kg pigs subcoronary (n = 8), supracoronary aortic banding (n = 8) or sham operation (n = 4) was preformed via a left lateral thoracotomy. The primary endpoint was left ventricular wall thickness; secondary endpoints were heart/body weight ratio and the systolic/diastolic blood flow ratio in the left anterior descending coronary. Statistical evaluation by oneway anova and unpaired t-test.</p> <p>Results</p> <p>Sub- and supracoronary banding induce an equal degree of left ventricular hypertrophy compared with the control group. The coronary blood flow ratio was slightly but not significantly higher in the supracoronary group (ratio = 0.45) compared with the two other groups (subcoronary ratio = 0.36, control ratio = 0.34).</p> <p>Conclusions</p> <p>A human pathophysiologically compatible porcine model for valvular aortic stenosis was developed by performing subcoronary aortic banding. Sub- and supracoronary aortic banding induce an equal degree of left ventricular hypertrophy. This model may be valid for experimental investigations of aortic valve stenosis but studies of left ventricular hypertrophy can be studied equally well by graduated constriction of the ascending aorta.</p