Additional file 3: Tables S2–S8. of Cyclic AMP signaling restricts activation and promotes maturation and antioxidant defenses in astrocytes

Lists of genes regulated in cAMP-treated cells and present in published astrocyte signatures. Genes that are regulated in cAMP-treated cells (genes within the “core enrichment” as defined by GSEA analysis), and that are reported to be regulated in previously published astrocyte signatures: developing (Table S2), mature (Table S3), cytokine-treated (Table S4), LPS (Table S5), MCAO (Table S6), in vivo (Table S7) and in vitro (Table S8) astrocytes. The lists represent the respective intersections in the Venn diagrams of Figure 4C. In GSEA analysis, genes were ranked on the basis of signal-to-noise ratio within the microarray data, starting with strongly upregulated and ending with strongly downregulated genes. The third column of the tables indicate the rank of the respective gene, i.e. low rank values indicate strong upregulation and high ones close to the maximal value of 21,492 indicate strong downregulation. (XLS 108 kb

Impact of wall potential on the fluid-wall interaction in a cylindrical capillary and a generalized Kelvin equation

In the present work a generalized Kelvin equation for a fluid confined in thick-walled cylindrical capillary is developed. This has been accomplished by including the potential energy function for interaction between a solid wall of a capillary and a confined fluid into the Kelvin equation. Using the Lennard-Jones 12-6 potential, an explicit form of the potential energy functions as expressed by hypergeometrical functions have been derived firstly, for the interaction between a solid wall and a test atom placed at an arbitrary point in a long open-end capillary, and thereafter for the body-body interaction between the solid wall and a confined Lennard-Jones fluid. Further, this generalized Kelvin equation has been applied to detailed description hysteresis phenomena in such capillaries. All numerical calculations have been carried out for the model argon-graphite system at 90 K

Additional file 5: Table S10. of Cyclic AMP signaling restricts activation and promotes maturation and antioxidant defenses in astrocytes

Antibodies used in this study. (XLSX 11 kb

miRNAs expression analysis.

<p>Real-time PCR was used to measure relative expression of miR-181a and miR-30c in skeletal muscle (A) and fibroblasts (B) from UCMD patients and in serum samples from UCMD, BM and DMD patients for miR-181a (C) and miR-30c (D). miRNA expression level was normalized against U6 miRNA. Results were calculated relative to control samples and are represented as mean and standard error.</p

Cell adhesion assay.

<p>Adherence of UCMD fibroblasts (n = 7) relative to control fibroblasts (n = 7) for fibronectin, vitronectin, laminin and collagen type I (experiments were performed in triplicate). To normalize adhesion values between experiments, we expressed the results as a ratio between the absorbance values for collagen type IV (which was the substrate that showed the smallest variability between individual cultures and experiments, data not shown) and each ECM protein, (student t-test * p < 0.05).</p

Co-variance analysis was used to plot the expression values of a selection of genes of interest in untreated patient cells (P), control cells (C) and patient cells treated with AA (P_AA).

<p>Median and ranges are represented. A. HLA-DRA, B. COL1A1, C. TNC, D. TNX.</p

Ingenuity Pathway Analysis.

<p>Graphic representation of the network “cell cycle, skeletal and muscular system development”. Nodes represent genes and lines show the relationship between genes. The intensity of the node color indicates the degree of the up-regulation (red) or down-regulation (green) of significant genes in the P-C comparison. Non-color nodes are added by the tool. For a detailed legend refer to <a href="http://ingenuity.force.com/ipa/articles/Feature_Description/Legend" target="_blank">http://ingenuity.force.com/ipa/articles/Feature_Description/Legend</a>.</p

Extracellular matrix gene correlation network.

<p>This network represents Pearson correlations (R >0.8, p-value<0.005) computed considering expression values of genes of interest according to the microarray data and using Cytoscape tool. We selected those genes on the ECM-receptor interaction KEGG pathway and COL6A genes. Continuous lines represent positive correlations and discontinuous lines negative ones. Those correlations that are significant in patients´cells only are represented in black lines. Orange lines represent those correlations that are present in both patients and control cells but are of different sign (positive or negative) whereas those that have the same sign are represented by lilac lines. Red lines around gene symbols represent significantly over-expressed genes and green lines those that were under-expressed in patients´ fibroblasts relative to control fibroblasts.</p

Integrin-α3 expression in collagen VI deficient fibroblasts.

<p>A. Immunofluorescence for integrin-α3 in confluent fibroblast cultures. B. Representative western blot analysis. The intensity of the bands corresponding to integrin-α3 (ITGA3) was quantified by densitometry using α-tubulin (A-TUB) as a loading control and expressed as arbitrary units relative to the control samples.</p

Gene Expression Profiling Identifies Molecular Pathways Associated with Collagen VI Deficiency and Provides Novel Therapeutic Targets

<div><p>Ullrich congenital muscular dystrophy (UCMD), caused by collagen VI deficiency, is a common congenital muscular dystrophy. At present, the role of collagen VI in muscle and the mechanism of disease are not fully understood. To address this we have applied microarrays to analyse the transcriptome of UCMD muscle and compare it to healthy muscle and other muscular dystrophies. We identified 389 genes which are differentially regulated in UCMD relative to controls. In addition, there were 718 genes differentially expressed between UCMD and dystrophin deficient muscle. In contrast, only 29 genes were altered relative to other congenital muscular dystrophies. Changes in gene expression were confirmed by real-time PCR. The set of regulated genes was analysed by Gene Ontology, KEGG pathways and Ingenuity Pathway analysis to reveal the molecular functions and gene networks associated with collagen VI defects. The most significantly regulated pathways were those involved in muscle regeneration, extracellular matrix remodelling and inflammation. We characterised the immune response in UCMD biopsies as being mainly mediated via M2 macrophages and the complement pathway indicating that anti-inflammatory treatment may be beneficial to UCMD as for other dystrophies. We studied the immunolocalisation of ECM components and found that biglycan, a collagen VI interacting proteoglycan, was reduced in the basal lamina of UCMD patients. We propose that biglycan reduction is secondary to collagen VI loss and that it may be contributing towards UCMD pathophysiology. Consequently, strategies aimed at over-expressing biglycan and restore the link between the muscle cell surface and the extracellular matrix should be considered. </p> </div