Search

Abstract Exosomes play a role in tissue/organ development and differentiation. Retinoic acid induces differentiation of P19 cells (UD-P19) to P19 neurons (P19N) that behave like cortical neurons and express characteristic neuronal genes such as NMDA receptor subunits. Here we report P19N exosome-mediated differentiation of UD-P19 to P19N. Both UD-P19 and P19N released exosomes with characteristic exosome morphology, size, and common protein markers. P19N internalized significantly higher number of Dil-P19N exosomes as compared to UD-P19 with accumulation in the perinuclear region. Continuous exposure of UD-P19 to P19N exosomes for six days induced formation of small-sized embryoid bodies that differentiated into MAP2-/GluN2B-positive neurons recapitulating RA-induction of neurogenesis. Incubation with UD-P19 exosomes for six days did not affect UD-P19. Small RNA-seq identified enrichment of P19N exosomes with pro-neurogenic non-coding RNAs (ncRNAs) such as miR-9, let-7, MALAT1 and depleted with ncRNAs involved in maintenance of stem cell characteristics. UD-P19 exosomes were rich with ncRNAs required for maintenance of stemness. P19N exosomes provide an alternative method to genetic modifications for cellular differentiation of neurons. Our novel findings on exosomes-mediated differentiation of UD-P19 to P19 neurons provide tools to study pathways directing neuron development/differentiation and develop novel therapeutic strategies in neuroscience. Graphical Abstract

The increasing incidence of hospital-acquired infections caused by drug-resistant pathogens, host toxicity, the poor efficacy of drugs and high treatment costs has drawn attention to the potential of natural products as antifungals in mucocutaneous infections and combinational therapies. Moreover, cellular and subcellular targets for these compounds may provide better options for the development of novel antifungal therapies. Eugenol, methyl eugenol and estragole are phenylpropanoids found in essential oil. They are known to possess pharmacological properties including antimicrobial activity. Induction of oxidative stress characterized by elevated levels of free radicals and an impaired antioxidant defence system is implicated as a possible mechanism of cell death. An insight into the mechanism of action was gained by propidium iodide cell sorting and oxidative stress response to test compounds in Candida albicans. The extent of lipid peroxidation (LPO) of cytoplasmic membranes was estimated to confirm a state of oxidative stress. Activity levels of primary defence enzymes and glutathione were thus further determined. Whereas these compounds cause fungal cell death by disrupting membrane integrity at minimum inhibitory concentrations (MIC), sub-MIC doses of these compounds significantly impair the defence system in C. albicans. The study has implications for understanding microbial cell death caused by essential oil components eliciting oxidative stress in Candida. The formation of membrane lesions by these phenylpropanoids thus appears to be the result of free radical cascade-mediated LPO.

Natural isopropyl cresols have been reported to have antifungal activity. This work is an attempt to examine thymol and carvacrol against 111 fluconazole-sensitive and -resistant Candida isolates. Insight into the mechanism of action was elucidated by flow cytometric analysis, confocal imaging and ergosterol biosynthesis studies. The susceptibility tests for the test compounds were carried out in terms of minimum inhibitory concentrations (MICs), disc diffusion assays and time-kill curves against all Candida isolates by employing standard protocols. Propidium iodide (PI) cell sorting has been investigated by flow cytometric analysis and confocal imaging. Haemolytic activity on human erythrocytes was studied to exclude the possibility of further associated cytotoxicity. Both compounds were found to be effective to varying extents against all isolates, including the resistant strains. In contrast to the fungistatic nature of fluconazole, our compounds were found to exhibit fungicidal nature. Significant impairment of ergosterol biosynthesis was pronouncedly induced by the test entities. Negligible cytoxicity was observed for the same compounds. Furthermore, it was observed that the positional difference of the hydroxyl group in carvacrol slightly changes its antifungal activity. Carvacrol and thymol show strong fungicidal effect against all of the Candida isolates. The mechanisms of action of these natural isopropyl cresols appear to originate from the inhibition of ergosterol biosynthesis and the disruption of membrane integrity.

The increasing incidence of drug-resistant pathogens and host toxicity of existing antifungals attracts attention toward the efficacy of natural products as antifungals in mucocutaneous infections and combinational therapies. The composition and antifungal activity of the essential oil obtained from Ocimum sanctum (OSEO) was studied. On GC-MS analysis, OSEO showed a high content of methyl chavicol (44.63%) and linalool (21.84%). Antifungal activity of OSEO and its two main constituents was determined against sixty clinical and five standard laboratory isolates of Candida. OSEO, methyl chavicol and linalool showed inhibitory activity toward all tested strains. The mechanism of their fungicidal action was assessed by studying their effect on the plasma membrane using flow cytometry, confocal imaging and determination of the levels of ergosterol, a fungal-specific sterol. Propidium iodide rapidly penetrated a majority of yeast cells when they were treated with OSEO concentrations just above MIC, implying that fungicidal activity resulted from extensive lesions of the plasma membrane. OSEO and its components also caused a considerable reduction in the amount of ergosterol. The present study indicates that OSEO, methyl chavicol and linalool have significant antifungal activity against Candida, including azole-resistant strains, advocating further investigation for clinical applications in the treatment of fungal infections.