Structure and properties of EC5.

<p>A. Edmundson helical wheel presentation of 12-mer EC5. Hydrophobic residues are represented by diamonds and positive charge as pentagons. The most hydrophobic residue is green, with the amount of green decreasing proportionally to the hydrophobicity with least hydrophobic being yellow. B. Ribbon and surface representation of EC5. Ribbon model shows α-helical peptide as the conformation. Green-most hydrophobic. Secondary structure of the peptide was determined and viewed using PYMOLv0.99.</p

Hemolytic activity of EC5.

<p>1% suspension of chicken RBCs were made with PBS. 100 µl and 50 µl of this suspension were incubated with different concentrations of the peptide in 96 well microtiter plates. Results were read visually.</p

AMP- bacterial membrane interaction studied by molecular dynamic simulations.

<p>EC5 was simulated with a POPE∶POPG membrane bilayer model using the Cluspro 2.0 and Hex protein docking server. PDB files generated were visualized using PYMOLv0.99.</p

Effect of EC5 on different bacteria.

<p>A. Plasma, B. Platelets. Blood matrices were spiked with bacteria and incubated with different concentrations of EC5. Growth was monitored after 2 h by plating them onto NA plates.</p

MIC of EC5 against bacteria (µg/ml).

<p>MIC of EC5 against bacteria (µg/ml).</p

Mechanism of action of EC5 against <i>E. coli</i> and <i>P. aeruginosa</i>.

<p>A. Outer-membrane permeabilization mediated by EC5 as assessed by NPN uptake. Effect of EC5 and Polymyxin B on NPN fluorescence. Value on y axis is the maximum fluorescence upon NPN uptake by the bacteria. B. EC5-induced permeability of bacterial cells studied using Syto9 and PI staining. Peptide treated cells had increased membrane permeability as seen by increase in red fluorescence whereas live or untreated cells showed increase in green fluorescence. C. Cytoplasmic membrane depolarization using the fluorescent dye diSC₃-5. Corresponding values on y axis represents maximum intensity upon release of the dye mediated by EC5 plotted against time (min). D. EC5 mediated inhibition of ATP synthesis. ATP concentration was measured after the addition of EC5 and polymyxin B at various concentrations and the luminescence units measured.</p

Binding efficiency of EC5 to different bacteria.

<p>A. ELISA based assay– 96 well microtiter plates were coated with six bacteria and incubated with the biotinylated peptide and the binding was detected using strepatavidin–HRP and developed using TMB substrate. (**–p<0.001, *–p<0.05). B. Fluorometry based assay– binding of EC5 to different bacteria was detected using streptavidin-conjugated Q dots. Results are presented as Mean±SD. (** – p<0.0001, *– p<0.001). Bac – Bacteria without peptide, Pep – Peptide without bacteria were used as controls.</p

Fold change in hsa-miR 1246 and F8 mRNA levels in HA patients compared to normal donors.

<p>(<b>A</b>) The fold change in has-miR 1246 was determined by RT-PCR. Total RNA was isolated from the blood of 5 control (CON) donors and 15 HA patients (HA). There is a significant increase in the has-miR 1246 levels in samples from HA patients compared to controls. (<b>B</b>) The fold change in <i>F8</i> mRNA was determined by RT-PCR. Total RNA was isolated from the blood of 5 control (CON) donors and 15 HA patients (HA). There is a significant decrease in the <i>F8</i> mRNA levles in samples from HA patients compared to controls.</p

Principal component analysis of significantly differentially expressed ncRNAs from 3-class ANOVA analysis and 3-class SAM analysis.

<p><b>(A)</b> 3-class ANOVA analysis. Principal component analysis of samples based on 88 significantly differentially expressed ncRNAs from 3-class ANOVA analysis (p-value < 0.01) reduces the dimentionality of the data and shows the clear separation between the hemophilia A patients with inhibitor development (red), the hemophilia A patients without inhibitor development (yellow), and the controls (turquoise). <b>(B)</b> 3-class SAM analysis. Principal component analysis of samples based on 11 significantly differentially expressed ncRNA probes from 3-class SAM analysis (FDR < 5%) reveals the distinct separation between the hemophilia A patients with inhibitor development (red), the hemophilia A patients without inhibitor development (yellow), and the controls (turquoise).</p

Hierarchical clustering and principal component analyses of significantly differentially expressed ncRNAs from ANOVA analysis.

<p>(A) Unsupervised hierarchical clustering analysis of 100 significantly differentially expressed ncRNAs between all hemophilia A patients (yellow and orange bars) and controls (red bar) shows the distinct ncRNA expression pattern of the two group (p-value < 0.01). Each row represents a subject, whereas each column is a ncRNA probe. Blue color in the heat map indicates down-regulation, whereas red indicates up-regulation. (B) Principal component analysis of samples on the basis of the 100 significantly differentially expressed ncRNAs identified by the ANOVA analysis described above. Yellow spheres represent HA samples, whereas turquoise spheres represent unaffected controls.</p