16 research outputs found
Interaction of gelatin with polyenes modulates antifungal activity and biocompatibility of electrospun fiber mats
Topical application of antifungals does not have predictable or well-controlled release characteristics and requires reapplication to achieve therapeutic local concentration in a reasonable time period. In this article, the efficacy of five different US Food and Drug Administration-approved antifungal-loaded (amphotericin B, natamycin, terbinafine, fluconazole, and itraconazole) electrospun gelatin fiber mats were compared. Morphological studies show that incorporation of polyenes resulted in a two-fold increase in fiber diameter and the mats inhibit the growth of yeasts and filamentous fungal pathogens. Terbinafine-loaded mats were effective against three filamentous fungal species. Among the two azole antifungals compared, the itraconazole-loaded mat was potent against Aspergillus strains. However, activity loss was observed for fluconazole-loaded mats against all of the test organisms. The polyene-loaded mats displayed rapid candidacidal activities as well. Biophysical and rheological measurements indicate strong interactions between polyene antifungals and gelatin matrix. As a result, the polyenes stabilized the triple helical conformation of gelatin and the presence of gelatin decreased the hemolytic activity of polyenes. The polyene-loaded fiber mats were noncytotoxic to primary human corneal and sclera fibroblasts. The reduction of toxicity with complete retention of activity of the polyene antifungal-loaded gelatin fiber mats can provide new opportunities in the management of superficial skin infections
pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants
Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4th_FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4th_FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities
Branched Peptide, B2088, Disrupts the Supramolecular Organization of Lipopolysaccharides and Sensitizes the Gram-negative Bacteria
Dissecting the complexities of branched peptide-lipopolysaccharides (LPS) interactions provide rationale for the development of non-cytotoxic antibiotic adjuvants. Using various biophysical methods, we show that the branched peptide, B2088, binds to lipid A and disrupts the supramolecular organization of LPS. The disruption of outer membrane in an intact bacterium was demonstrated by fluorescence spectroscopy and checkerboard assays, the latter confirming strong to moderate synergism between B2088 and various classes of antibiotics. The potency of synergistic combinations of B2088 and antibiotics was further established by time-kill kinetics, mammalian cell culture infections model and in vivo model of bacterial keratitis. Importantly, B2088 did not show any cytotoxicity to corneal epithelial cells for at least 96 h continuous exposure or hemolytic activity even at 20 mg/ml. Peptide congeners containing norvaline, phenylalanine and tyrosine (instead of valine in B2088) displayed better synergism compared to other substitutions. We propose that high affinity and subsequent disruption of the supramolecular assembly of LPS by the branched peptides are vital for the development of non-cytotoxic antibiotic adjuvants that can enhance the accessibility of conventional antibiotics to the intracellular targets, decrease the antibiotic consumption and holds promise in averting antibiotic resistance.NRF (Natl Research Foundation, S’pore)ASTAR (Agency for Sci., Tech. and Research, S’pore)NMRC (Natl Medical Research Council, S’pore)Published versio
Electrostatic potential map on the adsorption of B4010 and B4010_R1A with bilayer.
<p>(A) POPC/POPE/POPS/Erg bilayer (B) B4010-adsorbed bilayer and (C) B4010_R1A-adsorbed bilayer. The negative and positive surfaces are labeled in red and blue, respectively whereas grey color indicates neutral surface.</p
MIC of synthetic linear and branched peptides against various yeasts and fungi.
a<p>Not determined.</p
Interaction of B4010 with model membrane.
<p>(A) Time course of calcein release from SUVs of PC:PE:PS:erg and PC:cholesterol. The peptide:lipid ratio is indicated in the graphs. (B)-(D) Snapshots illustrating the interaction between B4010 and mixed bilayer containing ergosterol. The acyl chains of the aggregated POPC (grey), POPE (cyan), POPS (pink) and ergosterol (orange) are presented in line form. The peptide backbone is shown in ribbon form. (E) Translocation of water molecules (green) from inner leaflet to the outer as a consequence of membrane perturbations caused by B4010.</p
Effects of various additives on candidacidal and membrane permeabilizing properties of B4010.
<p>(A) Effect of CCCP and NaN<sub>3</sub> on membrane potential. (B) Effect of various additives on (B) viability and (C) ATP release. (D) Effect of ion-channel inhibitors on membrane potential. The colored arrows indicate the time of addition of additives whereas the black arrows indicate B4010.</p
MIC of B4010 against <i>S. cerevisiae</i> mutants carrying altered sterol structure and composition.
a<p>Taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087730#pone.0087730-TeWelcher1" target="_blank">[29]</a>.</p
Antifungal properties of B4010 in the presence of metal ions and complex biological fluids.
<p>(A) MIC values determined in the presence of monovalent and divalent metal ions. The numbers above the bars indicate the determined MIC values. (B) Candidacidal properties of B4010 in the presence of metal ions. The concentration of B4010 was 5.5 µM. (C) Antifungal activity in the presence of trypsin. (D) Candidacidal activity of B4010 in the presence of 50% rabbit tear fluid.</p
Effect of B4010 on cytoplasmic membrane potential, membrane permeabilization and morphology of <i>C. albicans</i>.
<p>(A) SDS-PAGE showing lack of affinity of B4010 for insoluble chitin. (B) SYTOX Green uptake of <i>C. albicans</i> induced by varying concentration of B4010. (C) B4010-mediated membrane depolarization monitored by diSC<sub>3</sub>5 assay. (D) B4010-induced extracellular ATP release of <i>C. albicans</i>. The inset shows the kinetics of ATP release. B4010 concentration was 5.5 µM. (E) SEM of untreated <i>C. albicans</i>. Scale bar is 2 µm (inset scale bar = 200 nm). (F) SEM image of C. albicans treated with 5.5 µM B4010. Scale bar is 1 µm (inset scale bar = 100 nm).</p