Use of KikGR a photoconvertible green-to-red fluorescent protein for cell labeling and lineage analysis in ES cells and mouse embryos

<p>Abstract</p> <p>Background</p> <p>The use of genetically-encoded fluorescent proteins has revolutionized the fields of cell and developmental biology and in doing so redefined our understanding of the dynamic morphogenetic processes that shape the embryo. With the advent of more accessible and sophisticated imaging technologies as well as an abundance of fluorescent proteins with different spectral characteristics, the dynamic processes taking place <it>in situ </it>in living cells and tissues can now be probed. Photomodulatable fluorescent proteins are one of the emerging classes of genetically-encoded fluorescent proteins.</p> <p>Results</p> <p>We have compared PA-GFP, PS-CFP2, Kaede and KikGR four readily available and commonly used photomodulatable fluorescent proteins for use in ES cells and mice. Our results suggest that the green-to-red photoconvertible fluorescent protein, Kikume Green-Red (KikGR), is most suitable for cell labeling and lineage studies in ES cells and mice because it is developmentally neutral, bright and undergoes rapid and complete photoconversion. We have generated transgenic ES cell lines and strains of mice exhibiting robust widespread expression of KikGR. By efficient photoconversion of KikGR we labeled subpopulations of ES cells in culture, and groups of cells within <it>ex utero </it>cultured mouse embryos. Red fluorescent photoconverted cells and their progeny could be followed for extended periods of time.</p> <p>Conclusion</p> <p>Transgenic ES cells and mice exhibiting widespread readily detectable expression of KikGR are indistinguishable from their wild type counterparts and are amenable to efficient photoconversion. They represent novel tools for non-invasive selective labeling specific cell populations and live imaging cell dynamics and cell fate. Genetically-encoded photomodulatable proteins such as KikGR represent emergent attractive alternatives to commonly used vital dyes, tissue grafts and genetic methods for investigating dynamic behaviors of individual cells, collective cell dynamics and fate mapping applications.</p

Genome Sequence of Fusobacterium nucleatum Subspecies Polymorphum — a Genetically Tractable Fusobacterium

Fusobacterium nucleatum is a prominent member of the oral microbiota and is a common cause of human infection. F. nucleatum includes five subspecies: polymorphum, nucleatum, vincentii, fusiforme, and animalis. F. nucleatum subsp. polymorphum ATCC 10953 has been well characterized phenotypically and, in contrast to previously sequenced strains, is amenable to gene transfer. We sequenced and annotated the 2,429,698 bp genome of F. nucleatum subsp. polymorphum ATCC 10953. Plasmid pFN3 from the strain was also sequenced and analyzed. When compared to the other two available fusobacterial genomes (F. nucleatum subsp. nucleatum, and F. nucleatum subsp. vincentii) 627 open reading frames unique to F. nucleatum subsp. polymorphum ATCC 10953 were identified. A large percentage of these mapped within one of 28 regions or islands containing five or more genes. Seventeen percent of the clustered proteins that demonstrated similarity were most similar to proteins from the clostridia, with others being most similar to proteins from other gram-positive organisms such as Bacillus and Streptococcus. A ten kilobase region homologous to the Salmonella typhimurium propanediol utilization locus was identified, as was a prophage and integrated conjugal plasmid. The genome contains five composite ribozyme/transposons, similar to the CdISt IStrons described in Clostridium difficile. IStrons are not present in the other fusobacterial genomes. These findings indicate that F. nucleatum subsp. polymorphum is proficient at horizontal gene transfer and that exchange with the Firmicutes, particularly the Clostridia, is common

OmpR controls Yersinia enterocolitica motility by positive regulation of flhDC expression

Flagella and invasin play important roles during the early stages of infection by the enteric pathogen Yersinia enterocolitica. Our previous study demonstrated that OmpR negatively regulates invasin gene expression at the transcriptional level. The present study focused on the role of OmpR in the regulation of flagella expression. Motility assays and microscopic observations revealed that an ompR mutant strain exhibits a non-motile phenotype due to the lack of flagella. An analysis of flhDC::lacZYA chromosomal fusions demonstrated a decrease in flhDC expression in ompR mutant cells, suggesting a role for OmpR in the positive control of flagellar master operon flhDC, which is in contrast to the negative role it plays in Escherichia coli. Moreover, high temperature or osmolarity and low pH decreased flhDC expression and OmpR was not required for the response to these factors. Evidence from an examination of the DNA binding properties of OmpR in vitro indicated that the mechanism by which OmpR regulates flhDC is direct. Electrophoretic mobility shift assays confirmed that OmpR binds specifically to the flhDC promoter region and suggested the presence of more than one OmpR-binding site. In addition, phosphorylation of OmpR by acetyl-P appeared to stimulate the binding abilities of OmpR. Together with the results of our previous studies revealing the negative role of OmpR in the regulation of invasin expression, these findings support a model in which invasion and motility might be reciprocally regulated by OmpR

A Micro RNA Processing Defect in Rapidly Progressing Idiopathic Pulmonary Fibrosis

BACKGROUND: Idiopathic pulmonary fibrosis exhibits differential progression from the time of diagnosis but the molecular basis for varying progression rates is poorly understood. The aim of the present study was to ascertain whether differential miRNA expression might provide one explanation for rapidly versus slowly progressing forms of IPF. METHODOLOGY AND PRINCIPAL FINDINGS: miRNA and mRNA were isolated from surgical lung biopsies from IPF patients with a clinically documented rapid or slow course of disease over the first year after diagnosis. A quantitative PCR miRNA array containing 88 of the most abundant miRNA in the human genome was used to profile lung biopsies from 9 patients with rapidly progressing IPF, 6 patients with slowly progressing IPF, and 10 normal lung biopsies. Using this approach, 11 miRNA were significantly increased and 36 were significantly decreased in rapid biopsies compared with normal biopsies. Slowly progressive biopsies exhibited 4 significantly increased miRNA and 36 significantly decreased miRNA compared with normal lung. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT). Five miRNA (miR-302c, miR-423-5p, miR-210, miR-376c, and miR-185) were significantly increased in rapid compared with slow IPF lung biopsies. Additional analyses of rapid biopsies and fibroblasts grown from the same biopsies revealed that the expression of AGO1 and AGO2 (essential components of the miRNA processing RISC complex) were lower compared with either slow or normal lung biopsies and fibroblasts. CONCLUSION: These findings suggest that the development and/or clinical progression of IPF might be the consequence of aberrant miRNA processing

Metabolic profile and psychological variables after bariatric surgery: association with weight outcomes

Purpose This study aims to examine associations between metabolic profile and psychological variables in post-bariatric patients and to investigate if metabolic and psychological variables, namely high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), glycated hemoglobin (HbA 1c), impulsivity, psychological distress, depressive and eating disorder symptoms are independently associated with percentage of excess weight loss (%EWL) after bariatric surgery.Methods One hundred and fifty bariatric patients (BMI = 33.04 +/- 5.8 kg/m(2)) who underwent to bariatric surgery for more than 28.63 +/- 4.9 months were assessed through a clinical interview, a set of self-report measures and venous blood samples. Pearson's correlations were used to assess correlations between %EWL, metabolic and psychological variables. Multiple linear regression was conducted to investigate which metabolic and psychological variables were independently associated with %EWL, while controlling for type of surgery.Results Higher TG blood levels were associated with higher disordered eating, psychological distress and depression scores. HDL-C was associated with higher depression scores. Both metabolic and psychological variables were associated with %EWL. Regression analyses showed that, controlling for type of surgery, higher % EWL is significantly and independently associated with less disordered eating symptoms and lower TG and HbA_1c blood concentrations (R-2 aj = 0.383, F (4, 82) = 14.34, p < 0.000).Conclusion An association between metabolic and psychological variables, particularly concerning TG blood levels, disordered eating and psychological distress/depression was found. Only higher levels of disordered eating, TG and HbA_1c showed and independent correlation with less weight loss. Targeting maladaptive eating behaviors may be a reasonable strategy to avoid weight regain and optimize health status post-operatively.This research was partially supported by Fundacao para a Ciencia e a Tecnologia/Foundation for Science and Technology through European Union COMPETE program Grant to Eva Conceicao (IF/01219/2014) and (PTDC/MHC-PCL/4974/2012), doctoral scholarship (SFRH/BD/104159/2014) to Ana Pinto-Bastos and doctoral scholarship (SFRH/BD/104182/2014) to Sofia Ramalho

Objectively measured patterns of sedentary time and physical activity in young adults of the Raine study cohort

Background: To provide a detailed description of young adults' sedentary time and physical activity. Methods: 384 young women and 389 young men aged 22.1±0.6 years, all participants in the 22 year old follow-up of the Raine Study pregnancy cohort, wore Actigraph GT3X+ monitors on the hip for 24 h/day over a one-week period for at least one 'valid' day (=10 h of waking wear time). Each minute epoch was classified as sedentary, light, moderate or vigorous intensity using 100 count and Freedson cut-points. Mixed models assessed hourly and daily variation; t-tests assessed gender differences. Results: The average (mean±SD) waking wear time was 15.0±1.6 h/day, of which 61.4±10.1 % was spent sedentary, 34.6±9.1 % in light-, 3.7±5.3 % in moderate- and, 0.3±0.6 % in vigorous-intensity activity. Average time spent in moderate to vigorous activity (MVPA) was 36.2±27.5 min/day. Relative to men, women had higher sedentary time, but also higher vigorous activity time. The 'usual' bout duration of sedentary time was 11.8±4.5 min in women and 11.7±5.2 min in men. By contrast, other activities were accumulated in shorter bout durations. There was large variation by hour of the day and by day of the week in both sedentary time and MVPA. Evenings and Sundays through Wednesdays tended to be particularly sedentary and/or inactive. Conclusion: For these young adults, much of the waking day was spent sedentary and many participants were physically inactive (low levels of MVPA). We provide novel evidence on the time for which activities were performed and on the time periods when young adults were more sedentary and/or less active. With high sedentary time and low MVPA, young adults may be at risk for the life-course sequelae of these behaviours

Breath biomarkers in idiopathic pulmonary fibrosis:A systematic review 11 Medical and Health Sciences

Background: Exhaled biomarkers may be related to disease processes in idiopathic pulmonary fibrosis (IPF) however their clinical role remains unclear. We performed a systematic review to investigate whether breath biomarkers discriminate between patients with IPF and healthy controls. We also assessed correlation with lung function, ability to distinguish diagnostic subgroups and change in response to treatment. Methods: MEDLINE, EMBASE and Web of Science databases were searched. Study selection was limited to adults with a diagnosis of IPF as per international guidelines. Results: Of 1014 studies screened, fourteen fulfilled selection criteria and included 257 IPF patients. Twenty individual biomarkers discriminated between IPF and controls and four showed correlation with lung function. Meta-analysis of three studies indicated mean (± SD) alveolar nitric oxide (CalvNO) levels were significantly higher in IPF (8.5 ± 5.5 ppb) than controls (4.4 ± 2.2 ppb). Markers of oxidative stress in exhaled breath condensate, such as hydrogen peroxide and 8-isoprostane, were also discriminatory. Two breathomic studies have isolated discriminative compounds using mass spectrometry. There was a lack of studies assessing relevant treatment and none assessed differences in diagnostic subgroups. Conclusions: Evidence suggests CalvNO is higher in IPF, although studies were limited by small sample size. Further breathomic work may identify biomarkers with diagnostic and prognostic potential