Additional file 2: Table S2. of Identification of functional single nucleotide polymorphisms in the branchpoint site

List of primer sequences utilized in the study. (XLSX 10 kb

Antimicrobial Properties of an Immunomodulator - 15 kDa Human Granulysin

<div><p>Granulysin, a cationic protein expressed by human natural killer cells and cytotoxic T lymphocytes, is a mediator for drug-induced Stevens-Johnson syndrome and graft-versus-host disease. Some 15 kDa granulysin are processed into 9 kDa forms and sequestered in cytolytic granules, while others are constitutively secreted into body fluids. Both 9 and 15 kDa granulysin have been shown to be a serum marker for cell-mediated immunity. Furthermore, 15 kDa is able to activate monocyte differentiation. However, its antimicrobial properties have not been clearly addressed. Here, we report a novel method to prepare both the soluble 9 and 15 kDa granulysin and show that the 15 kDa form is more effective than the 9 kDa form in exerting specific antimicrobial activity against <i>Pseudomonas aeruginosa</i> within a range of few micromolars. We also show that the 15 kDa granulysin is able to hyperpolarize the membrane potential and increase membrane permeability of treated bacteria. Interestingly, the bactericidal activity and membrane permeability of the granulysins were markedly reduced at lower pH (pH 5.4) as a result of probable increase in hydrophobicity of the granulysins. Additionally, we’ve also shown the granulysin to inhibit biofilm formation by <i>P</i>. <i>aeruginosa</i>. These results suggest that the 15 kDa granulysin exhibits a novel mechanism in bacteria killing in a way that’s different from most antimicrobial peptides. Our novel granulysin preparation methodology will be useful for further study of action mechanisms of other antimicrobial, cytotoxic and immunomodulating properties in granulysin-mediated diseases.</p></div

Effect of 15 kDa granulysin on the biofilm formation of <i>Pseudomonas aeruginosa</i> PAO1.

<p>Microbes were grown in M63 minimal media in the presence of SMAP-29 and 15 kDa granulysin at 37°C for 24 hr and quantified by crystal violet staining and measured at the absorbance of 600 nm. Error bars represent the standard errors of the means. The asterisks indicate samples that are significantly different from control. (Student’s t-tests; p≤0.01).</p

Effect of cations and pH on the antimicrobial activity, membrane permeability against <i>Pseudomonas aeruginosa</i> PAO1 and secondary structure of 15 kDa granulysin.

<p>The antimicrobial activities of 2 μM 15 kDa granulysin were determined in the presence of NaCl (A) and MgCl₂ (B) at the concentrations indicated. Microbes were incubated with granulysin at different pH values and the remaining colony-forming units were determined (C). SYTOX^™ Green was added to monitor the change in bacterial membrane permeability in the presence of 1 μM granulysin at pH 5.4 and pH 7.4, respectively (D). Fluorescence emission spectra of 8-Anilino-1-naphthalenesulfonic acid (ANS) was excited at 370 nm and measured between 400 and 600 nm in the presence of 1 μM granulysin at pH 7.4 (E) and pH 5.4 (F). Arrow indicates the blue shift (470 nm) of emission spectra of ANS-granulysin hydrophobic complex. Data plotted are normalized by the fluorescence intensity of ANS alone at pH 7.4 or pH 5.4, respectively. NFI, normalized fluorescence intensity. Circular dichroism spectrum of 20 μM 15 kDa granulysin in 20 mM HEPES, 50 mM NaCl, pH 7.4 or pH 5.4 (G). M.E., molar ellipticity.</p

Morphology of <i>Pseudomonas aeruginosa</i> PAO1 after 15 kDa granulysin treatment.

<p>Approximately 1×10⁷ microbes were treated with 2 μM 15 kDa granulysin for 0 min (A-C) and 180 min (D-F) and observed by transmission electron microscopy (TEM).</p

Additional file 15: of An integrated analysis tool for analyzing hybridization intensities and genotypes using new-generation population-optimized human arrays

Sequence information of the primers used for qPCR. (XLSX 11 kb

Morphology of RBCs from cellulose- (A, D) and garlic-supplemented (B, E) diet groups.

<p>RBCs were stained with Giemsa (week 6) to show darkly-stained cells (arrows in Panel B). (A) Most RBCs of control samples stained intensely around the edges rather than in the middle, reflecting their bi-concave shape. Among the uniform-sized RBCs are many small platelets and a neutrophil with a multi-lobed nucleus. (B) Darkly-stained RBCs in garlic-fed samples are non-nucleated, round or ovoid-shaped cells (arrows in Panel B) with almost the same size as the RBCs in control samples. They have more intense colors with a dark blue tint. (C) The % difference of darkly-stained cells at each time point (mean±s.e.m., n = 3–4, Brilliant Cresyl blue staining) between the two diet groups (control:black bars; garlic:gray bars) is significant (student's <i>t</i> test: *, <i>p</i><0.05; **, <i>p</i><0.01) throughout the time course. (D and E) SEM of RBCs (5000×) from control (D) and garlic-fed (E) mice (week 17). RBCs of garlic-fed samples contain more round-shaped cells (arrows in E) and those with a smaller dent at center. Scale bars: 100 µm (Panels A and B) or 5 µm (Panels D and E).</p

Evidence that CO treatment promotes increased erythropoiesis by an erythropoietin-independent mechanism.

<p>(A) Red blood cell indices of B6 mice on garlic-supplemented or garlic-free (cellulose-supplemented) diets with or without the treatment of CO at week 38: red blood cell concentration (RBC count, 10¹²/L), hemoglobin concentration (HGB, g/L), hematocrit (HCT, %), mean corpuscular volume (MCV, fL), mean corpuscular hemoglobin concentration (MCHC, g/L). Statistics: +, Air-Cellulose (AC) <i>vs</i>. Air-Garlic (AG); ‡, CO-Cellulose (COC)<i>vs</i>. CO-Garlic (COG); * CO-Cellulose (COC) <i>vs</i>. Air-Cellulose (AC); #, Air-Garlic (AG) <i>vs</i>. CO-Garlic (COG). One symbol, <i>p</i><0.05; 2 symbols, <i>p</i><0.01; 3 symbols, <i>p</i><0.005; 4 symbols, <i>p</i><0.001; 6 symbols, <i>p</i><0.0001 (student’s <i>t</i> test); n = 7–10. The arrangements for each block of bars are (left to right): thick cross-hatched bars, cellulose diet and treated with CO (COC); filled bars, cellulose diet and treated with air (AC); open bars, garlic diet and treated with air (AG); thin cross-hatched bars, garlic diet and treated with CO (COG). (B) Plasma erythropoietin (EPO) levels (ng/mL), n = 4 for each set of samples at different point of the time course. Symbols are the same as those in Panel A. The pairwise comparisons between AC and COG are: week 7, <i>p</i><0.013; week 15, <i>p</i><0.003; week 23, <i>p</i> = 0.065; week 27, <i>p</i> = 0.084; week 38, <i>p</i><0.007 (student's <i>t</i> test).</p

Histochemical phenotypes of spleens of C57BL/6J (n = 6) mice on cellulose-(A, C, E) and garlic-supplemented (B, D, F) diets (week 15).

<p>Spleen sections were shown with no staining (A, B) to reveal bilirubin, with Prussian blue stain for Fe²⁺(C, D), and with Gomori stain for macrophages <i>via</i> acid phosphatase and counter-stained for nuclei with methyl green (E, F). (G) Time course (n = 4) of Fe²⁺ accumulation in the spleens due to dietary garlic (4%). (H) Dose-response (n = 6) of dietary garlic in spleen sections. Scale bars represent 200 µm (A–F). (Statistics: *, <i>p</i><0.05; ***, <i>p</i><0.005, student's <i>t</i> test). Black bars: control; gray bars: garlic diet.</p

QPCR analyses of selected genes in mouse bone marrow (n = 3).

<p>All data were normalized aganist Week 3 data (1.00) from control mice --- Numbers are mean±sd<b>. Bold numbers</b>: <i>P</i><0.05 (student's t-test).</p