Mouse adipose tissue collection and processing for RNA analysis

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture

Effets du TGF-[Bêta]₁ sur l'interaction des fibroblastes cardiaques et des cellules vasculaires lisses avec une matrice extracellulaire à trois dimensions : implication des intégrines

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Role of the Renin-Angiotensin System in Healthy and Pathological Pregnancies

Introduction: Pregnancy is a physiological process that necessitates many cardiovascular and hemodynamic adaptations to ensure the survival of the foetus and well‐being of the mother. The renin‐angiotensin system (RAS) has been suggested as key player in many of these changes as it is critical for blood pressure control as well as fluid and salt homeostasis in the non‐pregnant state

Adipose tissue (P)RR regulates insulin sensitivity, fat mass and body weight

Objective We previously demonstrated that the handle-region peptide, a prorenin/renin receptor [(P)RR] blocker, reduces body weight and fat mass and may improve insulin sensitivity in high-fat fed mice. We hypothesized that knocking out the adipose tissue (P)RR gene would prevent weight gain and insulin resistance. Methods An adipose tissue-specific (P)RR knockout (KO) mouse was created by Cre-loxP technology using AP2-Cre recombinase mice. Because the (P)RR gene is located on the X chromosome, hemizygous males were complete KO and had a more pronounced phenotype on a normal diet (ND) diet compared to heterozygous KO females. Therefore, we challenged the female mice with a high-fat diet (HFD) to uncover certain phenotypes. Mice were maintained on either diet for 9 weeks. Results KO mice had lower body weights compared to wild-types (WT). Only hemizygous male KO mice presented with lower total fat mass, higher total lean mass as well as smaller adipocytes compared to WT mice. Although food intake was similar between genotypes, locomotor activity during the active period was increased in both male and female KO mice. Interestingly, only male KO mice had increased O2 consumption and CO2 production during the entire 24-hour period, suggesting an increased basal metabolic rate. Although glycemia during a glucose tolerance test was similar, KO males as well as HFD-fed females had lower plasma insulin and C-peptide levels compared to WT mice, suggesting improved insulin sensitivity. Remarkably, all KO animals exhibited higher circulating adiponectin levels, suggesting that this phenotype can occur even in the absence of a significant reduction in adipose tissue weight, as observed in females and, thus, may be a specific effect related to the (P)RR. Conclusions (P)RR may be an important therapeutic target for the treatment of obesity and its associated complications such as type 2 diabetes

In-situ observation and numerical modeling of contact creep and recovery on oriented semi-crystalline polymer surfaces

Semi-crystalline polymers are commonly used in industrial sectors where surfaces undergo many damages like scratches. In order to avoid as much as possible these surface damages, it is necessary to develop mechanical models able to predict such contact mechanical responses. The aim of this work is to study the effect of orientation, through stretching in the solid state, on the contact mechanics for semi-crystalline polymer surfaces. To that purpose, bulk polymers are uniaxially oriented at various stretching ratio, using hot two-mill rolling process, and are characterized by X-ray analysis. Contact creep and recovery tests were performed on these samples thanks to a home-made experimental device [1]. This apparatus allows an in-situ observation of the contact area during creep step on non-transparent materials. Then, the recovery of the residual imprint is quickly (few seconds after the unloading of the indenter) recorded by non-contact methods. This experiment gives access to the true contact geometry and provides valuable information about the early stage of viscoelastic recovery. Hence, the influence of surface orientation on the contact response was investigated on a model semi-crystalline polymer: HDPE to identify structural parameters that govern viscoelastic/viscoplastic behavior of the surface. The effects of strain levels and creep duration on viscoelastic behavior were also studied. As regards the latter parameter, it was shown that creep duration has no major effect on creep step. Nonetheless, for a same imposed strain, residual depths increase with longer creep duration, inducing, in some case, a permanent deformation of the surface. Regarding the effect of orientation, if the contact creep of the non-oriented surface with a spherical indentor displays a circular contact area, the same experiment performed with oriented semi-crystalline polymers shows an elliptical contact area. Two assumptions can be made to explain this contact shape: (a) the polymeric surface displays anisotropic mechanical properties or (b) the sample is isotropic but the contact takes place on a curved surface due to the rolling stage. In order to better understand the in-situ observations of the contact shape during the contact creep and recovery of the residual imprint, numerical modeling of the surface response was performed using MSC MARC® software. The aim is to reproduce, as closely as possible, the contact creep and recovery experiment. To that purpose an axisymmetric numerical model was created, considering the indentation sphere to be infinitely rigid. This model gives access to the true contact radius during the creep phase. First results seem to indicate that the elliptical shape of the contact area is rather govern by the anisotropic mechanical properties of the semi-crystalline polymer surfaces.   Acknowledgement: This research forms part of the research program of the Dutch Polymer Institute (DPI) project #783. The authors would like to acknowledge the funding support ""MARMA"" from Carnot MICA and HOLO3 (Alsace region) company for developing the instrument.   Reference: [1] : T. Chatel, C. Gauthier, H. Pelletier, V. Le Houérou, D. Favier and R. Schirrer. Journal of Physics D: Applied Physics, 2011, 44, 375-40

Gene-expression profiling of microdissected breast cancer microvasculature identifies distinct tumor vascular subtypes

INTRODUCTION: Angiogenesis represents a potential therapeutic target in breast cancer. However, responses to targeted antiangiogenic therapies have been reported to vary among patients. This suggests that the tumor vasculature may be heterogeneous and that an appropriate choice of treatment would require an understanding of these differences. METHODS: To investigate whether and how the breast tumor vasculature varies between individuals, we isolated tumor-associated and matched normal vasculature from 17 breast carcinomas by laser-capture microdissection, and generated gene-expression profiles. Because microvessel density has previously been associated with disease course, tumors with low (n = 9) or high (n = 8) microvessel density were selected for analysis to maximize heterogeneity for this feature. RESULTS: We identified differences between tumor and normal vasculature, and we describe two subtypes present within tumor vasculature. These subtypes exhibit distinct gene-expression signatures that reflect features including hallmarks of vessel maturity. Potential therapeutic targets (MET, ITGAV, and PDGFRβ) are differentially expressed between subtypes. Taking these subtypes into account has allowed us to derive a vascular signature associated with disease outcome. CONCLUSIONS: Our results further support a role for tumor microvasculature in determining disease progression. Overall, this study provides a deeper molecular understanding of the heterogeneity existing within the breast tumor vasculature and opens new avenues toward the improved design and targeting of antiangiogenic therapies

Faecal pharmacokinetics of orally administered vancomycin in patients with suspected Clostridium difficile infection

<p>Abstract</p> <p>Background</p> <p>Oral vancomycin (125 mg qid) is recommended as treatment of severe <it>Clostridium difficile </it>infection (CDI). Higher doses (250 or 500 mg qid) are sometimes recommended for patients with very severe CDI, without supporting clinical evidence. We wished to determine to what extent faecal levels of vancomycin vary according to diarrhoea severity and dosage, and whether it is rational to administer high-dose vancomycin to selected patients.</p> <p>Methods</p> <p>We recruited hospitalized adults suspected to have CDI for whom oral vancomycin (125, 250 or 500 mg qid) had been initiated. Faeces were collected up to 3 times/day and levels were measured with the AxSYM fluorescence polarization immunoassay.</p> <p>Results</p> <p>Fifteen patients (9 with confirmed CDI) were treated with oral vancomycin. Patients with ≥4 stools daily presented lower faecal vancomycin levels than those with a lower frequency. Higher doses of oral vancomycin (250 mg or 500 mg qid) led to consistently higher faecal levels (> 2000 mg/L), which were 3 orders of magnitude higher than the MIC₉₀of vancomycin against <it>C. difficile</it>. One patient receiving 125 mg qid had levels below 50 mg/L during the first day of treatment.</p> <p>Conclusions</p> <p>Faecal levels of vancomycin are proportional to the dosage administered and, even in patients with increased stool frequency, much higher than the MIC₉₀. Patients given the standard 125 mg qid dosage might have low faecal levels during the first day of treatment. A loading dose of 250 mg or 500 mg qid during the first 24-48 hours followed by the standard dosage should be evaluated in larger studies, since it might be less disruptive to the colonic flora and save unnecessary costs.</p

Identification of PDL-1 as a novel biomarker of sensitizer exposure in dendritic-like cells

The development of novel in vitro methods to assess risks of allergic sensitization are essential in reducing animal testing whilst maintaining consumer safety. The main research objectives of this study were to identify novel biomarkers to assess the sensitization predictability of chemicals. Phenotypic and cytokine responses of moDCs and MUTZ-3 cells were investigated following application of contact sensitizers; dinitrochlorobenzene (DNCB), cinnamaldehyde (Cin), eugenol (E), isoeugenol (IE), P-phenylenediamine (PPD) and non-sensitizers; salicyclic acid (SA) and sodium lauryl sulphate (SLS). CD86 was up-regulated on MUTZ-3 cells in response to DNCB, Cin and PPD, however, moDCs only modulated CD86 in response to DNCB and E. PDL-1 (Programmed death receptor ligand-1) proved a promising sensitization biomarker in MUTZ-3 cells where up-regulation occurred in response to DNCB, Cin, IE and PPD. Additionally, moDC-expressed PDL-1 was modulated in response to Cin, IE and E thus demonstrating improved sensitizer predictability when compared with CD86. MCP-1 and RANTES were identified as biomarkers of DNCB exposure but MCP-1 did not show any change in expression above controls for the other sensitizers investigated. However, RANTES was increased in MUTZ-3 cells by both DNCB and Cin. Our findings highlight novel biomarkers which, in MUTZ-3 cells, could be taken forward within a multiple biomarker in vitro assay ensuring strong and reliable predictability. © 2010 Elsevier Ltd

SUR QUELQUES CURIOSITÉS D'HISTOIRE NATURELLE DANS LES PERTUIS CHARENTAIS : FAUNE DES INVERTÉBRÉS MARINS

Eight invertebrate species, rediscovered, demographically expanding or newly observed are reported from the Pertuis Charentais Sea. They were sampled from intertidal rocky shores (Alpheus macrocheles, Aslia lefevrei, Epitonium clathrulatum and Haliotis tuberculata), intertidal sand flats (Africorchestia spinifera and Arcuatula senhousia) and subtidal bottoms (Aslia lefevrei and Rapana venosa). One species is pelagic (Lepas anatifera). Most of them are within their natural range. However, R. venosa, native to Southeast Asia, has been introduced in the Pertuis Charentais since the 2010s and its populations are currently expanding. The new northern limit of Africorchestia spinifera along the Atlantic coast is defined as the Ré Island. Phoresis of Crepidula fornicata on Carcinus maenas is noted but was already described in European waters whereas it is a hitherto undescribed and unexpected association with the gastropod R. venosa.Eight invertebrate species, rediscovered, demographically expanding or newly observed are reported from the Pertuis Charentais Sea. They were sampled from intertidal rocky shores (Alpheus macrocheles, Aslia lefevrei, Epitonium clathrulatum and Haliotis tuberculata), intertidal sand flats (Africorchestia spinifera and Arcuatula senhousia) and subtidal bottoms (Aslia lefevrei and Rapana venosa). One species is pelagic (Lepas anatifera). Most of them are within their natural range. However, R. venosa, native to Southeast Asia, has been introduced in the Pertuis Charentais since the 2010s and its populations are currently expanding. The new northern limit of Africorchestia spinifera along the Atlantic coast is defined as the Ré Island. Phoresis of Crepidula fornicata on Carcinus maenas is noted but was already described in European waters whereas it is a hitherto undescribed and unexpected association with the gastropod R. venosa

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead