Acanthamoeba keratitis (AK) is a rare cornea disease caused by species of the Acanthamoeba genus. The antifungal voriconazole blocks the ergosterol synthesis in the protozoan membrane and is active against the cysts and trophozoites of Acanthamoeba spp. Due to the low stability of voriconazole, its options for eye drops are scarce. This study aimed to investigate the stability of the biological activity of voriconazole against two strains of Acanthamoeba castellanii and one clinical isolate from a patient with AK. To evaluate the stability of the biological activity of voriconazole, strains of A. castellanii (ATCC 50492) were exposed to different periods and voriconazole concentrations stored at 4 °C for 7, 15, and 30 days. The cytotoxicity assays were performed using SIRC (ATCC CCL-60™) cell line. The results indicated the amoebicidal effect of voriconazole against Acanthamoeba spp. within 24 h and 48 h of exposure, and the voriconazole solution was stable and retained antiamoebic activity when stored at 4 °C for up to 30 days. In the cytotoxicity test, the result demonstrated low cytotoxicity of the drug to the corneal rabbit cell line. However, there is a need to carry out further synergistic effects with other antiamoebic drugs and then in vivo experiments in the AK animal model.
O‐GlcNAc (O‐linked β‐N‐acetylglucosamine) is a dynamic intracellular post‐translational modification (PTM) that regulates several cellular processes. This PTM is catalyzed by the covalent attachment of GlcNAc on residues of serine/threonine in proteins by the enzyme O‐GlcNAc transferase (OGT), while its removal is performed by O‐GlcNAcase (OGA). This PTM is limited by the concentration of UDP‐GlcNAc, the end‐product from the hexosamine biosynthetic pathway (HBP) that, together with OGT and OGA levels and activity, will regulate protein O‐GlcNAcylation. Although O‐GlcNAc has been reported in all studied metazoans, functional analysis during development in Hexapoda is restricted to the model organism Drosophila melanogaster. In this study, the role of O‐GlcNAc during development was investigated in an emerging model system, the beetle Tribolium castaneum. Comparison of the transcriptional profile of the HBP rate‐limiting step enzyme glutamine‐6‐phosphate amidotransferase (GFAT) during developmental stages showed higher levels during larval stages. Interestingly, OGT is modulated during embryonic development with an increase of mRNA levels during gastrulation while OGA remained unaltered in all stages. In situ hybridization analysis revealed an embryonic limited ubiquitous pattern of OGA during egg development, while GFAT is expressed in both embryonic and extra‐embryonic cells. Finally, parental knock down of OGT by RNA interference (RNAi) shows physiological impairment of oocyte maturation and a decrease in egg laying and embryonic survival, while OGA knockdown did not lead to physiologically and phenotypic significant changes. Altogehter, these results provide an important characterization of the O‐GlcNAc machinery in the most diverse order of insects, and suggest a critical role of O‐GlcNAcylation in a stage‐specific manner during Tribolium castaneum development.
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