Effect of GAs on germination of <i>Aspergillus fumigatus</i> spores.

<p>(A) A conidiospore suspension was incubated in RPMI medium at 37°C with (40 µg/ml) or without GAs for 15 h under static condition in microtiter wells. Percentage of germination was calculated at least from nine different fields from triplicate wells. A spore is considered germinated when the length of the germ tube is twice or more the size of a spore. Scale bars = 25 µm. (B) Table showing the impact of GAs on <i>A. fumigatus</i> spore germination and germ tube lengths. The lengths of germ tubes were measured by using µScope software (µScope Essential) and shown ± SD.</p

Fungal strains used in this study.

<p>Fungal strains used in this study.</p

Effect of a <i>Gymnema sylvestre</i> fraction (#194) on <i>Candida albicans</i> yeast-to-hypha conversion, growth and viability.

<p>(A) Stationary-phase <i>C. albicans</i> yeast cells grown in YNB medium were resuspended (1×10⁵ cfu/ml) in RPMI 1640 medium +50 mM glucose (buffered with HEPES 50 mM, pH 7.3) containing equal volume of DMSO (-194) or in the presence of fraction #194 and incubated in microtiter plates at 37°C with gentle shaking for 16 h. Cells were viewed under microscope and photographed. (B) Growth of <i>C. albicans</i> in the presence or absence of fraction #194 (but with equal volume of DMSO). Yeast cells were incubated in YPD liquid medium at 30°C in microtiter wells without shaking for the indicated times and growth of cells was determined by measuring absorbance (OD₆₃₀). Experiments were repeated at least twice each with triplicates. Error bars indicate standard deviations (SD). (C) Viability of cells exposed to vehicle control or fraction #194 in RPMI medium at 37°C were determined by removing aliquots of cell suspensions at t = 8 h of growth, vortexing for 30 seconds at top speed and diluting them ten fold serially before spotting 5 µl on YPD agar plates. Plates were incubated at 30°C for 16 h and then photographed.</p

Purification and identification of gymnemic acids (GAs).

<p>(A) Solvent extracted and semi-purified GAs were fractionated on preparative HPLC (Sunfire C₁₈ 5 µm, 250×10 mm) using an isocratic mobile phase (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074189#s2" target="_blank">Materials and Methods</a>” for details). Fractions with major peaks were collected using an automated fraction collector, vacuum dried and assayed for inhibition of <i>C. albicans</i> yeast-to-hypha conversion. Individual fractions (F2, F5, F7 and F8) were evaluated for purity and molecular weight analyses using analytical HPLC-ELSD-DAD-MS, ESIMS, HRESIMS, ¹H NMR and ¹³C NMR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074189#pone.0074189.s001" target="_blank">Figures S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074189#pone.0074189.s020" target="_blank">S20</a>). (B) The four gymnemic acids (<b>1–4</b>) were identified using mass and NMR data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074189#pone.0074189.s001" target="_blank">Figures S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074189#pone.0074189.s020" target="_blank">S20</a> for details of GA species) according to Liu <i>et. al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074189#pone.0074189-Liu1" target="_blank">[35]</a> and Yoshikawa <i>et. al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074189#pone.0074189-Yoshikawa1" target="_blank">[36]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074189#pone.0074189-Yoshikawa2" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074189#pone.0074189-Yoshikawa3" target="_blank">[38]</a>. The general structure of GA, methylbutyroyl and tigloyl are shown.</p

GAs-mediated conversion of <i>C. albicans</i> hyphae into yeast cells.

<p>(A) Four hours old hyphae of <i>C. albicans</i> were incubated in hyphal growth promoting medium (RPMI) at 37°C with or without GAs in microtiter wells under static condition. At the indicated post incubation time intervals (+2, +5, +8 and +11 h), conversion of hyphae into yeasts was monitored using an inverted microscope. Scale bars = 25 µm. (B) Percentage of released yeast cells from hyphae due to GAs exposure, at least from 3 different wells, were counted at each time points. Error bars indicate standard deviation. (C) Live/dead assay of yeast cells using propidium iodide (PI) stain was performed with the cells generated from GAs-exposed hyphae. An aliquot of cells from +11 h sample was stained with PI and viewed under fluorescence (FLU) microscope (Zeiss) with red filter. Corresponding DIC images were also recorded. As a positive control, yeast cells were killed by exposing them to 100% ethanol for 5 minutes and washed twice with sterile water to remove ethanol. Cells were stained with PI in parallel with test samples. Scale bar = 10 µm. (D) Viability of cells from 11 h post GAs-treated samples (duplicates) were two fold-serially diluted and 5 µl from each dilution (1 to 4) were spotted on YPD agar plate. Growth of cells was assessed after 16 h incubation at 30°C. Sparse growth of colonies can be seen from the 4^th diluted samples.</p

Effect of GAs on <i>C. albicans</i> infection in <i>Caenorhabditis elegans</i> and mammalian cells.

<p>(A) Rescue of <i>C. albicans</i> infected <i>C. elegans</i> from death by GAs. Larvae of <i>C. elegans</i> fed with yeast cells of <i>C. albicans</i> were incubated in RPMI medium with or without GAs (40 µg/ml) in a 96 well microtiter plate and incubated at 30°C for 2–4 days. Arrow in the top left panel (-GAs) shows the growth of <i>C. albicans</i> hyphae from the dead worms while addition of GAs (+GAs) prevent growth of hyphae from the worms’ body and hence worms survival (top right panel). Small round structures in the background are <i>C. albicans</i> yeast cells. Inset of <i>C. elegans</i> from GAs treated well shows confocal microscopic image of <i>C. elegans</i> containing <i>C. albicans</i> yeast cells in the worm’s gut (arrow). Scale bar (inset) = 10 µm. Bar graph at lower left panel indicates the % worms surviving after 4 days of exposure to GAs or to AMB. Survival of worms was determined by their movements and absence of hyphal growth from worms using microscope. Error bars indicate SD from the averages of 3 independent experiments. (B) GAs are non hemolytic and nontoxic to mammalian cells. Hemolytic assay was performed on tryptic soy agar plate containing human red blood cells (hRBC, 5%) (left side). A diagrammatic representation with sample identity is shown on the right side. Different fractions containing GAs [<i>G. sylvestre</i> extract, GE 1 mg/ml, 4 µl; fraction #194 (4 µl); and purified GAs (40 µg/ml, 4 µl)] were diluted in PBS and spotted on hRBC-agar. Positive controls including actively growing <i>Staphylococcus aureus</i> cells (2 µl) or PBS containing Triton X-100 (1%) (Tri-X) were also spotted on the blood agar medium as controls. Plates were incubated for 24–48 h at 37°C and the results were recorded by image capture. White clear halos around spots indicate hemolytic activity. GAs are not toxic to mammalian kidney epithelial cells (far right sector). Napthaquinone (NAP, 50 µg/ml) killed the kidney epithelial cells whereas solvent control (-GAs), amphotericin B (+AMB) or test compounds (+GAs; 40 µg/ml) did not. Scale bar = 10 µm.</p

Gymnemic acids inhibit hyphal formation and extension by <i>C. albicans</i>.

<p>Effect of the addition of a mixture of GAs (GAs, 40 µg/ml) on the yeast-to-hypha conversion and filamentation induced by liquid, solid RPMI or in liquid YPD in the presence of 10% fetal bovine serum at 37°C in microtiter wells. Scale bars = 25 µm.</p

Inhibition of <i>C. albicans</i> yeast-to-hypha transition by individual GAs.

<p><i>C. albicans</i> yeast cells were incubated in hyphae inducing medium (RPMI) in microtiter wells without shaking at 37°C with the indicated GA for 16 h. Each GA was solubilized in 75% methanol and added to the yeast cell suspension at final concentrations of 60 µg/ml. The final concentration of solvent was <5%. Cells were monitored under microscope using 10x×63x objective (Zeiss) and images were recorded. Solvent control contains equal volume of 75% methanol. Arrows show vesicle like structures in yeast cells. Scale bars = 5 µm.</p

Coupling the <i>Torpedo</i> Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of <i>Torpedo</i> electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to <i>Torpedo</i>-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated <i>Torpedo</i> nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of <i>Torpedo</i> electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility