Analysis of CAV2-miR-29a interaction in humans and pigs by means of reporter gene assays.

<p>The interaction between miR-29a and human as well as porcine CAV2 was verified by means of reporter gene assays using HeLa and IPEC-J2 cells, respectively. Identified target sites between miR-29a and human (panel A) as well as porcine CAV2 (panel B) were analysed using RNAhybrid. Relative luciferase activity (Luc <i>_Gaussia</i>: Luc <i>_Cypridina</i>) was determined respective to nonsense miRNA mimics as well as mutagenised seeds (red letters) serving as controls. The columns show means of normalised luciferase activity each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (*: P<0.05; **: P<0.01; ***: P<0.001, paired t test).</p

Western Blot analysis of CAV2 and CDC42 after intestinal <i>Salmonella</i> infection.

<p>Panels A–C show immuno detection of CAV2, CDC42 and GAPDH on Western Blots at 3 h, 3 d and 28 d p.i. (S: <i>Salmonella</i> infected; C: non-infected control; SP: <i>Salmonella</i> infected and co-treated with probiotics). Panel B exemplifies detection of respective proteins in pooled ileal samples from individually infected animals while panels A and C show relative mean protein expression (CAV2:GAPDH and CDC42:GAPDH) calculated from quadruplicate Western Blots and error bars show the standard deviation (*: P<0.05, Mann-Whitney U test).</p

Predicted regulation of focal adhesion and actin cytoskeleton by microRNAs during intestinal <i>Salmonella</i> infection.

<p>The figure shows the first regulative concept of focal adhesion and actin cytoskeleton pathways in intestinal <i>Salmonella</i> infection of mammals. Interactions between miRNAs and mRNAs are based on the microarray study described above and were proved by RNAhybrid analysis as shown by calculated P values (*: P<0.05; **: P<0.01; ***: P<0.001).</p

Expression analysis of miR-29a and selected targets by means of RT-qPCR.

<p>The Tukey box plots show relative mean expression of individual samples each measured in triplicates while outliers are shown as black dots (S: <i>Salmonella</i> infected; C: non-infected control; SP: <i>Salmonella</i> infected and co-treated with probiotics). Asterisks indicate statistical significance according to Mann-Whitney U test (*: P<0.05; **: P<0.01; ***: P<0.001). Panels A, B, C, D, E, F and G show the expression of miR-29a, AKT3, BAIAP2, COL4A1, VCL, CAV2 and CDC42 at 3 h, 3 d and 28 d p.i., respectively.</p

Heatmaps of exemplified mRNA and miRNA expression data clustered after microarray analysis.

<p>Columns (A–D) represent temporal expression of ileal samples collected from <i>Salmonella</i> infected (S), <i>Salmonella</i> infected and co-treated with probiotics (SP) and non-infected controls (C) at 3 h, 3 d and 28 d p.i. Colours represent log 2 ratios of the respective samples versus the common reference according to the scales shown below. Samples represent a pool of at least five infection experiments. An averaged trace of the expression profile (± SD) is integrated as a white graph. Gene clusters showing significantly differential expressions in corresponding time points p.i. were identified by the analysis of variance and Kruskal-Wallis and Dunn's post test (*: P<0.05; **: P<0.01; ***: P<0.001). Panel A and B exemplify two clusters of identified protein coding genes. Colours indicate genes involved in similar pathways (orange: pathogenic <i>E. coli</i> infections and regulation of actin cytoskeleton; green: immune response related pathways; blue: focal adhesion; gray: oxidative phosphorylation; purple: ribosome). Panel C and D illustrate miRNAs being induced after infection or showed balanced expression, respectively.</p

Immuno detection of CAV2 and CDC42 after RNAi and overexpression experiments in HT-29 and IPEC-J2 intestinal cells.

<p>Panel A exemplifies the expression of CAV2 as well as CDC42 after RNAi (nonsense controls, anti-miR-29a, miR-29a and siRNA CAV2) and transient overexpression of CAV2 (pMIREX0 as a mock control and pMIREXCAV2) in HT-29 cells. Panel B shows immunocyto detection of CAV2 and CDC42 after transfection (according to panel A) in intestinal cell lines of both investigated species human (HT-29) and pig (IPEC-J2). Scales are indicated by white bars (10 µm). Overview images are included in the bottom right corner.</p

Glycine-Terminated Dendritic Amphiphiles for Nonviral Gene Delivery

Development of nonviral vectors for the successful application of gene therapy through siRNA/DNA transfection of cells is still a great challenge in current research., In the present study, we have developed multivalent polyglycerol dendron based amphiphiles with well-defined molecular structures that express controlled glycine arrays on their surfaces. The structure–activity relationships with respect to the siRNA complexation, toxicity, and transfection profiles were studied with synthesized amphiphilic polycations. Our findings revealed that a second-generation amphiphilic dendrimer (G2-octaamine, <b>4</b>) that has eight amine groups on its surface and a hydrophobic C-18 alkyl chain at the core of the dendron, acts as an efficient vector to deliver siRNA and achieve potent gene silencing by investigating the knockdown of luciferase and GAPDH gene activity in HeLa cells. Interestingly, the amphiphilic vector is nontoxic even at higher ratio of N/P 100. To the best of our knowledge this is the first example of successful in vitro siRNA transfection using dendritic amphiphiles. We believe that this supramolecular complex may serve as a new promising alternative for nonviral siRNA delivery systems and will be investigated for in vivo siRNA delivery in the future