Functional study in NSE-Hu-Bcl-2 transgenic mice: a model for retinal diseases starting in Müller cells

In NSE-Hu-Bcl-2 transgenic mice, line 71, retina undergoes early postnatal degeneration linked to the prior death of Müller cells. The purpose of this study was to complete the characterization of this retinal dysfunction by using electroretinographic (ERG) recordings in both scotopic and photopic conditions. Here, we showed that both rod and cone systems were profoundly affected in NSE-Hu-Bcl-2 transgenic mice as soon as 15 postnatal days in accordance with histological study performed previousl

Potential Cross-Talk between Alternative and Classical NF-κB Pathways in Prostate Cancer Tissues as Measured by a Multi-Staining Immunofluorescence Co-Localization Assay

<div><p>Background</p><p>While the classical NF-κB/p65 pathway is known to be involved in prostate cancer progression and is associated with poor patient outcome, the role of the NF-κB /RelB alternative protein is not well defined. Here we analyzed the activation of both NF-κB pathways in prostate cancer tissues and correlate this activation with clinical features of the disease.</p><p>Methods</p><p>A multiple immunofluorescence technique was employed to concomitantly and quantitatively visualize the nuclear localization of p65 and RelB in 200 paraffin embedded samples. Epithelia were defined using appropriate fluorochrome markers and the resulting immunofluorescent signals were quantified with an automated scoring system.</p><p>Results</p><p>The nuclear frequency of p65 was found to be significantly increased in tumor tissues as compared with normal adjacent tissue, whereas the frequency for RelB was decreased (p < 0.001, Wilcoxon test). As previously reported, p65 nuclear frequency was associated with a risk of biochemical recurrence. Although, RelB nuclear frequency alone did not predict recurrence, the presence of activated RelB reduced the risk of recurrence associated with the activation of p65.</p><p>Conclusion</p><p>For the first time p65/RelB co-distribution was assessed in prostate cancer tissues and suggested a negative crosstalk between the two NF-κB pathways in prostate cancer progression.</p></div

Association between nuclear p65 and/or RelB and biochemical recurrence in prostate cancerpatients.

<p>Kaplan–Meier biochemical recurrence-free survival curves <b>A.</b> High (>20%) and low (<20%) frequency of nuclear p65 in the epithelia of cancer tissues. <b>B.</b> High (>5%) and low (<5%) frequency of nuclear RelB in the epithelia of cancer tissues. Significance (<i>p</i>) is indicated by log rank. <b>C.</b> Double nuclear epithelial staining of p65 and RelB. <b>D.</b> Epithelial and nuclear staining of p65 and RelB. The κB negative variable represents patients without positive tissue cores for p65 and RelB. The variable p65 alone or RelB alone represents patients positive for only nuclear p65 or nuclear RelB. The variable p65 + RelB are patients with presence of both nuclear subunits in the same core. <b>E.</b> Analysis of the epithelial proportion of nuclear p65 to RelB in tissue cores within both p65 and RelB staining. The ratio p65/RelB represents the frequency of p65 to the frequency of RelB. The variable p65 > RelB represents a ratio of at least 2 fold more p65 than RelB; p65-RelB represents a ratio between 0.50 and 2 fold, and RelB>p65 represents a ratio of at least 2 fold more RelB than p65.</p

Nuclear co-localization of p65 and RelB by a quadruple multiple staining approach.

<p><b>A.</b> Epithelial staining with CK18, CK19 and PSA defines the epithelial mask using orange fluorochromes (A546, Cy3). <b>B.</b> Identification of epithelial area (presented in grey for optimal contrast in subsequent analyses) as ROI #1 (region of interest #1). Identification of nuclei (grey surrounded by red tracing) as ROI#2 from pre-defined ROI#1. P65 and RelB fluorescence were subsequently evaluated separately in ROI#1 and #2. <b>C.</b> RelB staining with green fluorescent dye (A488) and p65 staining with red fluorescent dye (Cy5). Steps 1, 2 and 3 correspond to the fluorescence analysis process followed using Visiomorph DP software.</p

Example of immunofluorescence of p65 and RelB.

<p><b>A.</b> Magnification (40X) highlighting glandular structures in prostate cancer tissues. Merge: superimposed images of RelB (A488) in green, p65 (Cy5) in red and nuclei (DAPI) in blue, in normal adjacent (top) or cancer tissues (bottom). Arrows show stained nuclei. <b>B</b>. Comparative quantification from IHC (left) and IF (right) staining of p65. Graphs show the frequency distribution of nuclear p65 as evaluated visually (IHC) or automatically (IF). <b>C</b>. Frequency of nuclear p65 (left), RelB (middle) and double stained nuclei (right) in normal adjacent and tumor tissues. The comparison between adjacent normal and tumor cores was conducted using a Wilcoxon‘s test.</p

Univariate and multivariate Cox regression analysis.

<p>Cox regression models: significance (p < 0.05) and Hazard ratio (HR) are indicated. CI: Confidence interval. PSA: Prostate Specific Antigen. NF-κB variables (p65 and RelB): status in cancer tissues. Significance is considered at p < 0.5.</p><p>Univariate and multivariate Cox regression analysis.</p

Association between nuclear p65 and/or RelB and biochemical recurrence in prostate cancerpatients.

<p>Kaplan–Meier biochemical recurrence-free survival curves <b>A.</b> High (>20%) and low (<20%) frequency of nuclear p65 in the epithelia of cancer tissues. <b>B.</b> High (>5%) and low (<5%) frequency of nuclear RelB in the epithelia of cancer tissues. Significance (<i>p</i>) is indicated by log rank. <b>C.</b> Double nuclear epithelial staining of p65 and RelB. <b>D.</b> Epithelial and nuclear staining of p65 and RelB. The κB negative variable represents patients without positive tissue cores for p65 and RelB. The variable p65 alone or RelB alone represents patients positive for only nuclear p65 or nuclear RelB. The variable p65 + RelB are patients with presence of both nuclear subunits in the same core. <b>E.</b> Analysis of the epithelial proportion of nuclear p65 to RelB in tissue cores within both p65 and RelB staining. The ratio p65/RelB represents the frequency of p65 to the frequency of RelB. The variable p65 > RelB represents a ratio of at least 2 fold more p65 than RelB; p65-RelB represents a ratio between 0.50 and 2 fold, and RelB>p65 represents a ratio of at least 2 fold more RelB than p65.</p

Spearman correlation test between NF-κB and clinic-pathological parameters.

<p>Significant Spearman correlations are indicated in bold. Spearman correlations were considered significant at <i>p</i> < 0.05.</p><p>Spearman correlation test between NF-κB and clinic-pathological parameters.</p

Exopolysaccharide (EPS) synthesis by oenococcus oeni: from genes to phenotypes

Oenococcus oeni is the bacterial species which drives malolactic fermentation in wine. The analysis of 50 genomic sequences of O. oeni (14 already available and 36 newly sequenced ones) provided an inventory of the genes potentially involved in exopolysaccharide (EPS) biosynthesis. The loci identified are: two gene clusters named eps1 and eps2, three isolated glycoside-hydrolase genes named dsrO, dsrV and levO, and three isolated glycosyltransferase genes named gtf, it3, it4. The isolated genes were present or absent depending on the strain and the eps gene clusters composition diverged from one strain to another. The soluble and capsular EPS production capacity of several strains was examined after growth in different culture media and the EPS structure was determined. Genotype to phenotype correlations showed that several EPS biosynthetic pathways were active and complementary in O. oeni. Can be distinguished: (i) a Wzy -dependent synthetic pathway, allowing the production of heteropolysaccharides made of glucose, galactose and rhamnose, mainly in a capsular form, (ii) a glucan synthase pathway (Gtf), involved in β-glucan synthesis in a free and a cell-associated form, giving a ropy phenotype to growth media and (iii) homopolysaccharide synthesis from sucrose (α-glucan or β-fructan) by glycoside-hydrolases of the GH70 and GH68 families. The eps gene distribution on the phylogenetic tree was examined. Fifty out of 50 studied genomes possessed several genes dedicated to EPS metabolism. This suggests that these polymers are important for the adaptation of O. oeni to its specific ecological niche, wine and possibly contribute to the technological performance of malolactic starters