Structures of common AHLs synthesized by <i>P aeruginosa.</i>

<p>Structures of the most common AHL quorum sensing compounds synthesized by <i>Pseudomonas aeruginosa</i> (C₄-HSL, and 3-oxo-C₁₂-HSL) and those used in this study (3-oxo-C₁₄-HSL, 3-oxo-C₁₂-HSL, COOH-3-oxo-C₁₂-HSL and 3-oxo-C₁₀-HSL).</p

A strong negative correlation is observed between peripheral mononuclear cell proliferation and AHL lipophilicity.

<p>The IC₅₀ for human peripheral mononuclear cell proliferation (PBMCs) isolated from three donors (* denotes n = 2) in the presence of AHLs of chain lengths between 8 and 14 carbons (values obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013522#pone.0013522-Chhabra1" target="_blank">[18]</a>) exhibit a strong negative correlation (Pearsons r = −0.895, <i>p</i> = 0.016) against the predicted octanol/water partition coefficient (LogP) for each compound (calculated using ACD/i-Lab LogP algorithm 12.0). n = 3, ±SEM. Projected LogP are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013522#pone-0013522-t001" target="_blank">Table 1</a>.</p

Binding profiles of the interactions of AHLs with Lymphocyte membranes.

<p>Binding profiles of 3-oxo-C₁₄-HSL (Δ, hyperbolic), 3-oxo-C₁₂-HSL (•, sigmoidal) and 3-oxo-C₁₀-HSL (×, neither) on titration to di-8-ANEPPS labeled T-Lymphocytes (40,000 cells/ml) at 37°C normalised to DMSO controls. Profiles were fitted to simple hyperbolic and sigmoidal binding models (equations 1 and 2) and F-Tests were used to determine the best fitting model. In each experiment n = 3, ±SEM.</p

Comparison of AHL octanol/water partition coefficients.

<p>Comparison of AHL octanol/water partition coefficients (LogP) values calculated from molecular structure using ACD/I-Lab LogP algorithm 12.0. Lipophilicity is observed to significantly increase as AHL chain length increases from C8 to C14 (r = 1.000, p<0.01, Spearman correlation). Addition of a carboxylic acid group to the 3-oxo-C12 HSL acyl chain results in a marked reduction in projected LogP. This compound was therefore expected to interact with membranes to a lesser degree than the major AHL released by <i>P. aeruginosa</i> 3-oxo-C12 HSL. LogP±95% confidence interval shown.</p

Effect of decreasing membrane dipole potential on membrane receptor conformation.

<p>Binding profiles of the P-glycoprotein ligand Saquinavir with T-Lymphocytes (40,000 cells/ml) after pre-treatment with 160 µM 3-oxo-C12 HSL (•) or an equivalent volume of DMSO (final concentration did not exceed 0.5%) (Δ) at 37°C. Data were normalized for saquinavir additions by titrating cells with equivalent volumes of DMSO (×, dashed lines). Profiles were fitted to simple hyperbolic and sigmoidal binding models (equations 1 and 2) and F-Tests were used to determine the best fitting model (sigmoidal for 3-oxo-C₁₂ HSL and DMSO pre-treatments). In each experiment n = 3, ±SEM.</p

Comparing the interactions of COOH-3-oxo-C₁₂ HSL and 3-oxo-C₁₂ HSL with artificial membrane systems and their effects on membrane dipole potential.

<p>Fluorescence difference spectra obtained by subtracting di-8-ANEPPS excitation spectra (λem = 590 nm) of PC(100%) [A] or PC(70%)Cholesterol(30%) [B] membrane vesicles (400 µM) from those obtained after these membranes were exposed to either 200 µM 3-oxo-C₁₂-HSL (dotted line, n = 5) or 200 µM COOH-3-oxo-C₁₂-HSL (solid black line). Before subtraction, each spectrum was normalized to the integrated areas so that the difference spectra would reflect only the spectral shifts. Each difference spectrum was then normalised to a DMSO control (dashed line). In all experiments the dye concentration was 10 µM and temperature was maintained at 37°C. [C] Binding profiles of 3-oxo-C₁₂ HSL (▴,•) and COOH-3-oxo-C₁₂ HSL (Δ,○) on titration to PC_100% (circles) or PC_70%Cholesterol_30% (triangles) di-8-anepps labeled liposomes (400 µM) at 37°C normalised to DMSO controls. Profiles were fitted to simple hyperbolic and sigmoidal binding models (equations 1 and 2) and extra sum of squares F-Tests were used to determine the best fitting in each case (3-oxo-C₁₂ HSL was hyperbolic while COOH-3-oxo-C₁₂ HSL fit poorly to both models). All experiments n = 3±SEM.</p

The interactions of AHLs with artificial membrane systems perturbs the membrane dipole potential.

<p>Fluorescence difference spectra obtained by subtracting di-8-ANEPPS excitation spectra (λem = 590 nm) of PC(100%) [A] or PC(70%)Cholesterol(30%) [B] membrane vesicles (400 µM) from those obtained after these membranes were exposed to the following QS molecules; 65 µM 3-oxo-C₁₄-HSL (thick dashed line), 200 µM 3-oxo-C₁₂-HSL (solid black line) and 200 µM 3-oxo-C₁₀-HSL (thin dashed and dotted line). Before subtraction, each spectrum was normalized to the integrated areas so that the difference spectra would reflect only the spectral shifts. Each difference spectrum was then normalised to a DMSO control (grey line) and a three point moving average applied to reduce noise. In all experiments the dye concentration was 10 µM and temperature was maintained at 37°C. [C] A dual wavelength ratiometric measurement of the dipole potential variation in di-8-ANEPPS. Additions of 22 µM 3-oxo-C_14-HSL or equivalent volumes of DMSO were made to 400 µM PC(100%). Samples were excited at 460 nm and 520 nm. Emission was read at 590 nm and the ratio <i>R</i>(460/520) was calculated (shown). All experiments n = 3.</p

Appendix A. Modeling the second definition of nonoverlapping chains.

Modeling the second definition of nonoverlapping chains

Complete database Bombus decline

The complete database, for GLOBAL DECLINE OF BUMBLEBEES IS PHYLOGENETICALLY STRUCTURED AND INVERSELY RELATED TO SPECIES RANGE SIZE AND PATHOGEN INCIDENCE, including potential variables associated with decline, described in the main text

Receiver operating characteristic curves for PHQ-9, HADS-D, and PHQ-9 categorical algorithm; CIS-R was the criterion standard.

<p>Receiver operating characteristic curves for PHQ-9, HADS-D, and PHQ-9 categorical algorithm; CIS-R was the criterion standard.</p