Evaluation of the antifungal effect of chlorogenic acid against strains of Candida spp. resistant to fluconazole: apoptosis induction and in silico analysis of the possible mechanisms of action.

Introduction. Candida spp. are commensal fungal pathogens of humans, but when there is an imbalance in the microbiota, or weak host immunity, these yeasts can become pathogenic, generating high medical costs.Gap Statement. With the increase in resistance to conventional antifungals, the development of new therapeutic strategies is necessary. This study evaluated the in vitro antifungal activity of chlorogenic acid against fluconazole-resistant strains of Candida spp. Mechanism of action through flow cytometry and in silico analyses, as well as molecular docking assays with ALS3 and SAP5, important proteins in the pathogenesis of Candida albicans associated with the adhesion process and biofilm formation.Results. The chlorogenic acid showed in vitro antifungal activity against the strains tested, causing reduced cell viability, increased potential for mitochondrial depolarization and production of reactive oxygen species, DNA fragmentation and phosphatidylserine externalization, indicating an apoptotic process. Concerning the analysis through docking, the complexes formed between chlorogenic acid and the targets Thymidylate Kinase, CYP51, 1Yeast Cytochrome BC1 Complex e Exo-B-(1,3)-glucanase demonstrated more favourable binding energy. In addition, chlorogenic acid presented significant interactions with the ALS3 active site residues of C. albicans, important in the adhesion process and resistance to fluconazole. Regarding molecular docking with SAP5, no significant interactions were found between chlorogenic acid and the active site of the enzyme.Conclusion. We concluded that chlorogenic acid has potential use as an adjuvant in antifungal therapies, due to its anti-Candida activity and ability to interact with important drug targets.

Synergistic effects of ketamine and azole derivatives on Candida spp. resistance to fluconazole.

The emergence of Candida spp. with resistance to antifungal molecules, mainly the azole class, is an increasing complication in hospitals around the globe. Aim: In the present research, we evaluated the synergistic effects of ketamine with two azole derivatives, itraconazole and fluconazole, on strains of Candida spp. to fluconazole. Materials & methods: The drug synergy was evaluated by quantifying the fractional inhibitory concentration index and by fluorescence microscopy and flow cytometry techniques. Results: Our achievements showed a synergistic effect between ketamine in addition to the two antifungal agents (fluconazole and itraconazole) against planktonic cells and biofilms of Candida spp. Conclusion: This combination promoted alteration of membrane integrity, generation of reactive oxygen species, damage to and DNA and externalization of phosphatidylserine.

A mechanistic approach to the in-vitro resistance modulating effects of fluoxetine against meticillin resistant Staphylococcus aureus strains.

Emergence of methicilin resistant Staphylococcus aureus (MRSA) strains is a major cause of infirmity worldwide and has limited our therapeutic options against these pathogens. In this regard, the search for candidates with an antimicrobial activity, with a greater efficacy and a lower toxicity, is necessary. As a result, there is greater need to search for resistance modifying agents which, in combination with existing drugs, will restore the efficacy of these drugs. The antibacterial effect of fluoxetine was determined by a broth microdilution method (the M07-A9 method of the Clinical and Laboratory Standard Institute) and flow cytometry techniques in which the probable mechanism of action of the compound was also assessed. The isolates used in the study belonged to the Laboratory of Bioprospecting of Antimicrobial Molecules (LABIMAN) of the Federal University of Ceará. After 24 h, Methicillin-resistant Sthaphylococcus aureus (MRSA) strains showed fluoxetine MICs equal to 64 µg/mL and 128 µg/mL, respectively. Cytometric analysis showed that treatment with fluoxetine caused alterations to the integrity of the plasma membranes and DNA damage, which led to cell death, probably by apoptosis.

Transient Expression of Functional Glucocerebrosidase for Treatment of Gaucher's Disease in the Goat Mammary Gland.

Gaucher disease (GD) is an orphan disease characterized by the lack or incapacity of glucocerebrosidase (hGCase) to properly process glucosylceramide, resulting in its accumulation in vital structures of the human body. Enzyme replacement therapy supplies hGCase to GD patients with a high-cost recombinant enzyme produced in vitro in mammalian or plant cell culture. In this study, we produced hGCase through the direct injection of recombinant adenovirus in the mammary gland of a non-transgenic goat. The enzyme was secreted in the milk during six days at a level up to 111.1 ± 8.1 mg/L, as identified by mass spectrometry, showing high in vitro activity. The milk-produced hGCase presented a mass correspondent to the intermediary high-mannose glycosylated protein, which could facilitate its delivery to macrophages through the macrophage mannose receptor. Further studies are underway to determine the in vivo delivery capacity of milk-hGCase, but results from this study paves the way toward the generation of transgenic goats constitutively expressing hGCase in the milk.