Transgenic mouse model harboring the transcriptional fusion Ccl20-luciferase as a novel reporter of pro-inflammatory response

The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo.Laboratorio de Investigaciones del Sistema InmuneFacultad de Ciencias Exacta

Physical Activity and Screen Time in Adolescents and Their Nominated Friends

Background: The social transmission of obesity has been reported in adolescent social networks. However, the behavioral antecedents to obesity (physical activity [PA], screen time, and diet) are the factors that would actually be transmitted through these networks. To date, little is known about the social influences affecting an individual adolescent’s PA and screen time behaviors. Purpose: To determine the associations between an adolescent’s PA and screen time and his/her nominated friends’ PA and screen time. Methods: Data were obtained from EAT 2010 (Eating and Activity among Teens), a large cross-sectional study (n=2,126) conducted in 20 middle schools and high schools in Minneapolis/St. Paul, Minnesota, USA during the 2009-2010 academic year. Each participant (Ego) nominated up to six friends (Alters) from a school roster and data from those friends was also obtained as part of the school-based data collection procedures. PA and screen time were assessed with previously used and validated questionnaires. Generalized estimating equation models, stratified by gender, were used to assess associations between adolescents’ PA and screen time and their friends’ PA and screen time. Results: Females’ PA was associated with their male and female friends’ PA, including their female best friends (all p\u3c0.05). Males’ PA was associated with their female friends’ PA (p\u3c0.03). Females’ screen time was associated with their male and female friends’ screen time (p\u3c0.03), but not with their best friends. Males’ screen time was only associated with their female friends’ screen time (p=0.04). Conclusions: The associations between individual and friend PA and screen time, especially in females, indicate a need to consider these social relationships when investigating weight-related behaviors in adolescents. Longitudinal data using validated measures of PA and screen time, and analyzed using sophisticated modeling techniques are needed to better understand social influences on adolescent weight-related behaviors and inform future intervention efforts

Impacts of active school design on schooltime sedentary behavior and physical activity: A pilot natural experiment

Background Children spend a significant portion of their days in sedentary behavior (SB) and on average fail to engage in adequate physical activity (PA). The school built environment may influence SB and PA, but research is limited. This natural experiment evaluated whether an elementary school designed to promote movement impacted students\u27 school-time SB and PA. Methods Accelerometers measured SB and PA at pre and post time-points in an intervention group who moved to the new school (n = 21) and in a comparison group experiencing no school environmental change (n = 20). Difference-in-difference (DD) analysis examined SB and PA outcomes in these groups. Measures were also collected post-intervention from an independent, grade-matched group of students in the new school (n = 21). Results As expected, maturational increases in SB were observed. However, DD analysis estimated that the intervention attenuated increase in SB by 81.2 ± 11.4 minutes/day (p\u3c0.001), controlling for time in moderate to vigorous physical activity (MVPA). The intervention was also estimated to increase daily number of breaks from SB by 23.4 ± 2.6 (p \u3c .001) and to increase light physical activity (LPA) by 67.7 ± 10.7 minutes/day (p\u3c0.001). However, the intervention decreased MVPA by 10.3 ± 2.3 minutes/day (p\u3c0.001). Results of gradematched independent samples analysis were similar, with students in the new vs. old school spending 90.5 ± 16.1 fewer minutes/day in SB, taking 21.1 ± 2.7 more breaks from SB (p\u3c0.001), and spending 64.5 ± 14.8 more minutes in LPA (p\u3c0.001), controlling for time in MVPA. Students in the new school spent 13.1 ± 2.7 fewer minutes in MVPA (p\u3c0.001) than their counterparts in the old school. Conclusions This pilot study found that active school design had beneficial effects on SB and LPA, but not on MVPA. Mixed results point to a need for active classroom design strategies to mitigate SB, and quick access from classrooms to areas permissive of high-intensity activities to promote MVPA. Integrating active design with programs/policies to promote PA may yield greatest impact on PA of all intensities

The scavenger receptors SRA-1 and SREC-I cooperate with TLR2 in the recognition of the hepatitis C virus non-structural protein 3 by dendritic cells

Backgrounds & AimsThe hepatitis C virus NS3 protein is taken up by myeloid cells in a TLR2-independent manner and activates myeloid cells via TLR2. This study aimed to identify the endocytic receptor(s) involved in the uptake of NS3 by myeloid cells and its relation with TLR2. Methods Inhibitors and transfected cells were used to identify the nature of the NS3-binding receptors expressed by myeloid cells. The cooperation between scavenger receptors (SRs) and TLR2 in the NS3-mediated activation of myeloid cells was evaluated using inhibitors, cells from TLR2−/− mice, and confocal microscopy. The involvement of SRs in NS3 cross-presentation was evaluated in vitro using an NS3-specific human T-cell clone. Results We observed that SRs are the main binding structures for NS3 on myeloid cells and identified the SRs SRA-1 and SREC-I as endocytic receptors for NS3. Moreover, both SRs and TLR2 cooperate in NS3-induced myeloid cell activation. Conclusion This study highlights a central role for SRs in NS3 uptake and cross-presentation, and demonstrates a tightly orchestrated cooperation between signalling and endocytic innate receptors in NS3 recognition

Clostridium difficile ribotypes in Austria: a multicenter, hospital-based survey

A prospective, noninterventional survey was conducted among Clostridium difficile positive patients identified in the time period of July until October 2012 in 18 hospitals distributed across all nine Austrian provinces. Participating hospitals were asked to send stool samples or isolates from ten successive patients with C.difficile infection to the National Clostridium difficile Reference Laboratory at the Austrian Agency for Health and Food Safety for PCR-ribotyping and in vitro susceptibility testing. A total of 171 eligible patients were identified, including 73 patients with toxin-positive stool specimens and 98 patients from which C. difficile isolates were provided. Of the 159 patients with known age, 127 (74.3 %) were 65 years or older, the median age was 76 years (range: 9–97 years), and the male to female ratio 2.2. Among these patients, 73 % had health care-associated and 20 % community-acquired C. difficile infection (indeterminable 7 %). The all-cause, 30-day mortality was 8.8 % (15/171). Stool samples yielded 46 different PCR-ribotypes, of which ribotypes 027 (20 %), 014 (15.8 %), 053 (10.5 %), 078 (5.3 %), and 002 (4.7 %) were the five most prevalent. Ribotype 027 was found only in the provinces Vienna, Burgenland, and Lower Austria. Severe outcome of C. difficile infection was found to be associated with ribotype 053 (prevalence ratio: 3.04; 95 % CI: 1.24, 7.44), not with the so-called hypervirulent ribotypes 027 and 078. All 027 and 053 isolates exhibited in vitro resistance against moxifloxacin. Fluoroquinolone use in the health care setting must be considered as a factor favoring the spread of these fluoroquinolone resistant C. difficile clones

Pattern Transfer of Sub-10 nm Features via Tin-Containing Block Copolymers

Tin-containing block copolymers were investigated as materials for nanolithographic applications. Poly(4-trimethylstannylstyrene-block-styrene) (PSnS-PS) and poly(4-trimethylstannylstyrene-block-4-methoxystyrene) (PSnS-PMOST) synthesized by reversible addition–fragmentation chain transfer polymerization form lamellar domains with periodicities ranging from 18 to 34 nm. Thin film orientation control was achieved by thermal annealing between a neutral surface treatment and a top coat. Incorporation of tin into one block facilitates pattern transfer into SiO_2 via a two-step etch process utilizing oxidative and fluorine-based etch chemistries

Intragenic sequences in the trophectoderm harbour the greatest proportion of methylation errors in day 17 bovine conceptuses generated using assisted reproductive technologies

Abstract Background Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences. Results Of the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth. Conclusion By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos

Transgenic mouse model harboring the transcriptional fusion Ccl20-luciferase as a novel reporter of pro-inflammatory response

The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo.Laboratorio de Investigaciones del Sistema InmuneFacultad de Ciencias Exacta