No abstract

Azoles are the most commonly used class of antifungal drugs, yet where they localize within fungal cells and how they are imported remain poorly understood. Azole antifungals target lanosterol 14α-demethylase, a cytochrome P450, encoded by ERG11 in Candida albicans, the most prevalent fungal pathogen. We report the synthesis of fluorescent probes that permit visualization of antifungal azoles within live cells. Probe 1 is a dansyl dye-conjugated azole, and probe 2 is a Cy5-conjugated azole. Docking computations indicated that each of the probes can occupy the active site of the target cytochrome P450. Like the azole drug fluconazole, probe 1 is not effective against a mutant that lacks the target cytochrome P450. In contrast, the azole drug ketoconazole and probe 2 retained some antifungal activity against mutants lacking the target cytochrome P450, implying that both act against more than one target. Both fluorescent azole probes colocalized with the mitochondria, as determined by fluorescence microscopy with MitoTracker dye. Thus, these fluorescent probes are useful molecular tools that can lead to detailed information about the activity and localization of the important azole class of antifungal drugs.

The underlying physics governing the diffusion of a tracer particle in a viscoelastic material is a topic of some dispute. The long-term memory in the mechanical response of such materials should induce diffusive motion with a memory kernel, such as fractional Brownian motion (fBM). This is the reason that microrheology is able to provide the shear modulus of polymer networks. Surprisingly, the diffusion of a tracer particle in a network of a purified protein, actin, was found to conform to the continuous time random walk type (CTRW). We set out to resolve this discrepancy by studying the tracer particle diffusion using two different tracer particle sizes, in actin networks of different mesh sizes. We find that the ratio of tracer particle size to the characteristic length scale of a bio-polymer network plays a crucial role in determining the type of diffusion it performs. We find that the diffusion of the tracer particles has features of fBm when the particle is large compared to the mesh size, of normal diffusion when the particle is much smaller than the mesh size, and of the CTRW in between these two limits. Based on our findings, we propose and verify numerically a new model for the motion of the tracer in all regimes. Our model suggests that diffusion in actin networks consists of fBm of the tracer particle coupled with caging events with power-law distributed escape times.

Supramolecular gels often become destabilizedb y the transition of the gelator into amore stable crystalline phase, but often the long timescale and sporadic localization of the crystalline phase preclude ap ersistent observation of this process.W epresent apentapeptide gel-crystal phase transition amenable for continuous visualization and quantification by common microscopic methods,a llowing the extraction of kinetics and visualization of the dynamics of the transition. Using optical microscopya nd microrheology,w es howt hat the transition is as poradic event in whichg el dissolution is associated with microcrystalline growth that follows as igmoidal rate profile.The two phases are based on b-sheets of similar yet distinct configuration. We also demonstrate that the transition kinetics and crystal morphology can be modulated by extrinsic factors,i ncluding temperature,s olvent composition, and mechanical perturbation. This work introduces an accessible model system and methodology for studying phase transitions in supramolecular gels.

Actin is one of the most studied cytoskeleton proteins showing a very rich span of structures and functions. For example, Adenosine triphosphate (ATP)-assisted polymerization of actin is used to push...