Additional file 1: Figure S1. of Immune modulation of CD4+CD25+ regulatory T cells by zoledronic acid

The effect of ZA on CD4+ lymphocyte proliferation. (A) Isolated lymphocytes were labeled with CFSE, cultured in medium with or without 10 μM ZA and sensitized with anti-CD3 and anti-CD28 antibodies. (B) CD4+ lymphocyte proliferation curves were measured based on the percentage of cells with decreased fluorescence as compared to non-proliferating cells (0.4% at day 1). Data represent the mean values ± SEM and results from three independent experiments are shown. (TIF 134 kb

Additional file 2: Figure S2. of Immune modulation of CD4+CD25+ regulatory T cells by zoledronic acid

The effect of ZA on Treg cells apoptosis. (A) Treg cells were treated with and without ZA (10, 50 and 100 μM) for 24 h. Apoptosis was measured by Annexin V‑FITC/PI staining and flow cytometry. (B) Quantitative analysis of Treg cells by flow cytometry revealed no effect of ZA on Treg cell apoptosis. (TIF 131 kb

Hydrophobic residues are critical for the helix-forming, hemolytic and bactericidal activities of amphipathic antimicrobial peptide TP4

<div><p>Antimicrobial peptides are important components of the host innate defense mechanism against invading pathogens, especially for drug-resistant bacteria. In addition to bactericidal activity, the 25 residue peptide TP4 isolated from Nile tilapia also stimulates cell proliferation and regulates the innate immune system in mice. In this report, TP4 hyperpolarized and depolarized the membrane potential of <i>Pseudomonas aeruginosa</i> at sub-lethal and lethal concentrations. It also inhibited and eradicated biofilm formation. The <i>in vitro</i> binding of TP4 to bacterial outer membrane target protein, OprI, was markedly enhanced by a membrane-like surfactant sarkosyl and lipopolysaccharide, which converted TP4 into an α-helix. The solution structure of TP4 in dodecylphosphocholine was solved by NMR analyses. It contained a typical α-helix at residues Phe10-Arg22 and a distorted helical segment at Ile6-Phe10, as well as a hydrophobic core at the N-terminus and a cationic patch at the C-terminus. Residues Ile16, Leu19 and Ile20 in the hydrophobic face of the main helix were critical for the integrity of amphipathic structure, other hydrophobic residues played important roles in hemolytic and bactericidal activities. A model for the assembly of helical TP4 embedded in sarkosyl vesicle is proposed. This study may provide valuable insight for engineering AMPs to have potent bactericidal activity but low hemolytic activity.</p></div

NMR spectra and NOE connectivity of TP4 bound to DPC micelles.

<p>(A) The fingerprint (the upper panel) and amide-amide (the lower panel) regions of NOESY spectrum recorded at 150 ms mixing time for TP4 in DPC micelles at pH 3.5 and 318 K. Sequential resonance assignments of TP4 are labeled at the positions of NH-C_αH and NH-NH cross-peaks. (B) Summary of NOE connectivity, amide proton exchange, and temperature coefficients for TP4. Opened and filled circles in the exchange row show that the exchangeable amide protons are still visible after 6 hr and 12 hr in D2O. The temperature coefficient values greater than -4.5 ppb/K are shown as filled diamonds. The thickness of the line is relative to the intensity of the NOE as indicated in the bottom.</p

OprI-binding ability of TP4-derived peptides.

<p><b>(A)</b> Competition for OprI binding by TP4-derived peptides. Recombinant OprI (12μg, 48μg each) was pre-incubated with free mutated peptide before binding to biotinylated-TP4 which was immobilized on Streptavidin-conjugated gel. The gel pellet was analyzed by non-reducing SDS-PAGE/Coomassie blue staining. rOprI, recombinant OprI; S.t, Streptavidin. (B) Solubility of TP4-derived peptide. TP4-derived peptides (6 μg each) dissolved in 1x sarkosyl buffer (300 μl) were incubated at 37°C for 10 min, then spun at 14,000 x <i>g</i> for 15 min and analyzed by SDS-PAGE/Coomassie blue staining. C, control without treatment; P, precipitation.</p

MIC and MBC of TP4 mutants against microbes (μg/ml).

<p>MIC and MBC of TP4 mutants against microbes (μg/ml).</p

Hydrophobicity and solubility of TP4 in the presence of sarkosyl and LPS.

<p>(A) ANS emission spectrum of TP4 in solution containing sarkosyl. TP4 (4 μg) was dissolved in 200 μl of 10 mM sodium phosphate, pH7.4, 0.1 M NaCl, containing various concentrations of sarkosyl (0.075%, w/v, designated as 1x). (B) ANS emission spectrum of TP4 in solution containing LPS. TP4 (4 μg) was dissolved in 200 μl of 10 mM sodium phosphate, pH7.4, containing various amounts of LPS as indicated. ANS was added stepwise to the final concentrations as indicated (lines 1 to 6 at 0, 10, 20, 30, 40 and 50 μM, respectively). Arrows indicate the emission maximum at 470 nm or 520 nm for bound- and free-form ANS, respectively. (C, D) Solubility of TP4 in solution containing either sarkosyl or LPS. TP4 (4 μg) dissolved in 200 μl of respective solution containing sarkosyl or LPS at concentration as indicated was spun at 14,000 x <i>g</i> for 15 min and analyzed by SDS-PAGE/Coomassie blue staining.</p

Amino acid sequences of TP3 and TP4-derived peptides.

<p>Dashed line indicates the residue identical to that of wild type TP4.</p

Sarkosyl-Induced Helical Structure of an Antimicrobial Peptide GW-Q6 Plays an Essential Role in the Binding of Surface Receptor OprI in <i>Pseudomonas aeruginosa</i>

<div><p>The emergence of antibiotic-resistant microbial strains has become a public health issue and there is an urgent need to develop new anti-infective molecules. Although natural antimicrobial peptides (AMPs) can exert bactericidal activities, they have not shown clinical efficacy. The limitations of native peptides may be overcome with rational design and synthesis. Here, we provide evidence that the bactericidal activity of a synthetic peptide, GW-Q6, against <i>Pseudomonas aeruginosa</i> is mediated through outer membrane protein OprI. Hyperpolarization/depolarization of membrane potential and increase of membrane permeability were observed after GW-Q6 treatment. Helical structure as well as hydrophobicity was induced by an amphipathic surfactant, sarkosyl, for binding to OprI and possible to membrane. NMR studies demonstrated GW-Q6 is an amphipathic α-helical structure in DPC micelles. The paramagnetic relaxation enhancement (PRE) approach revealed that GW-Q6 orients its α-helix segment (K7-K17) into DPC micelles. Additionally, this α-helix segment is critical for membrane permeabilization and antimicrobial activity. Moreover, residues K3, K7, and K14 could be critical for helical formation and membrane binding while residues Y19 and W20 for directing the C-terminus of the peptide to the surface of micelle. Taken together, our study provides mechanistic insights into the mode of action of the GW-Q6 peptide and suggests its applicability in modifying and developing potent AMPs as therapeutic agents.</p></div

The schematic diagram shows the experimental procedures of the 2-DE-based proteomic analysis in this study.

<p>Three rabbit cell types (fibroblast, f-rES, and p-rES cells) were used for soluble protein extraction and analysis. The expression levels of protein spots were compared among all cell types.</p