Then the coated ITO glass was evaporated under vacuum for 2 h. The following procedure was used in succession: a square frame made of silicon served as a thickness (2 mm) spacer between the lipid-coated

glass and normal glass. The AZD6244 chamber was filled with 10 mM HEPES buffer (pH 7.2) through a hole in the silicon spacer. Immediately, the application of 1.7 V (peak-to-peak, sine wave) and 10 Hz to the ITO electrodes was carried out using a sweep function generator (Protek, Sweep Function Generator 9205C) for 2 h. GUVs from the ITO glass were then detached under conditions of 4 V (peak-to-peak, sine wave) and 4 Hz for 10 min. The peptides (at the MIC) were treated and changes of a single GUV were observed using an inverted fluorescence phase-contrast microscope (Leica, DFC420C) (Angelova & Dimitrov, 1986; Angelova et al., 1992; Lee & Lee, 2009). In this study, the antifungal effects of papiliocin were investigated to suggest the potential of the peptide as a novel antifungal peptide, by comparing it with melittin (Table 1), which was derived from the venom of honey bee Apis mellifera. Melittin is a representative membrane-active AMP, helping researchers to understand lipid–protein interactions at the molecular level, and is also known to Venetoclax have powerful antimicrobial and hemolytic activities (Habermann, 1972; Tosteson et al., 1985; Dempsey, 1990).

The antifungal activity of papiliocin against human fungal pathogens was first examined. AMPs have been considered to exhibit cell selectivity (Matsuzaki, 2009). This means that they selectively kill pathogenic microorganisms without being significantly toxic to human cells. This Thiamine-diphosphate kinase concept, which coincides with roles of AMPs in innate immunity, arises from a plethora of observations showing that AMPs are nonhemolytic at concentrations well above their MICs against various

microorganisms (Matsuzaki, 2009). A cytotoxicity assay showed that papiliocin exerted antifungal activities against human pathogenic fungal strains, including yeasts and filamentous fungi, with MIC values in the 5–20 μM range, whereas for melittin, MIC values in the 1.25–5 μM range were determined (Table 2). Furthermore, in a previous study, papiliocin did not cause hemolysis of human erythrocytes, at any of the tested concentrations (Kim et al., 2010). Therefore, these results suggest that papiliocin has the potential to be considered as a novel antibiotic peptide for treating fungal diseases in humans, with potent antifungal activity without toxicity to human red blood cells. As antifungal agents could display static or cidal patterns of activity (Lewis, 2007), a time-kill kinetic assay was carried out using C. albicans to elucidate the pattern of activity of papiliocin. Candida albicans is an important pathogen in humans and is versatile as a pathogen.