A protocol for visualizing active cathepsin K in osteoclasts with a quenched-fluorescence-activity-based probe.

Herein, we provide a protocol for visualizing active osteoclast cathepsin K (CatK) with the quenched-fluorescent-activity-based probe qTJK17. We describe steps for isolating peripheral blood mononuclear cells, their differentiation into osteoclasts, and TRAP staining using an acid phosphatase leukocyte kit. We then detail visualization of active CatK. The probe qTJK17 includes a reactive group, acyloxymethylketone, that binds to the CatK active site, recognition sequence, and fluorescence donor-acceptor pair. This protocol can determine the exact localization of active CatK in osteoclasts. For complete details on the use and execution of this protocol, please refer to Janiszewski et al. (2023).1.

Lipid composition and cell surface hydrophobicity of Candida albicans influence the efficacy of fluconazole-gentamicin treatment.

Adherence of the fungus, Candida albicans, to biotic (e.g. human tissues) and abiotic (e.g. catheters) surfaces can lead to emergence of opportunistic infections in humans. The process of adhesion and further biofilm development depends, in part, on cell surface hydrophobicity (CSH). In this study, we compared the resistance of C. albicans strains with different CSH to the most commonly prescribed antifungal drug, fluconazole, and the newly described synergistic combination, fluconazole and gentamicin. The hydrophobic strain was more resistant to fluconazole due to, among others, overexpression of the ERG11 gene encoding the fluconazole target protein (CYP51A1, Erg11p), which leads to overproduction of ergosterol in this strain. Additionally, the hydrophobic strain displayed high efflux activity of the multidrug resistance Cdr1 pump due to high expression of the CDR1 gene. On the other hand, the hydrophobic C. albicans strain was more susceptible to fluconazole-gentamicin combination because of its different effect on lipid content in the two strains. The combination resulted in ergosterol depletion with subsequent Cdr1p mislocalization and loss of activity in the hydrophobic strain. We propose that C. albicans strains with different CSH may possess altered lipid metabolism and consequently may differ in their response to treatment.