{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T12:24:28Z","timestamp":1787142268945,"version":"build-2736575974"},"reference-count":34,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T00:00:00Z","timestamp":1787097600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100005357","name":"Slovak Research and Development Agency","doi-asserted-by":"publisher","award":["APVV-24-0167"],"award-info":[{"award-number":["APVV-24-0167"]}],"id":[{"id":"10.13039\/501100005357","id-type":"DOI","asserted-by":"publisher"}]},{"award":["APVV-24-0167"],"award-info":[{"award-number":["APVV-24-0167"]}],"id":[{"id":"https:\/\/ror.org\/037nx0e70","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Scientific Grant Agency of the Ministry of Education, Research, Development and Youth of the Slovak Republic","award":["1\/0307\/26"],"award-info":[{"award-number":["1\/0307\/26"]}]},{"award":["1\/0307\/26"],"award-info":[{"award-number":["1\/0307\/26"]}],"id":[{"id":"https:\/\/ror.org\/02z80ct15","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>Background\/Objectives: Azole resistance in fungal pathogens is a growing clinical and environmental concern. However, short-term cellular responses during the first hours of azole exposure remain insufficiently characterised, particularly in filamentous fungi. This study used Neurospora crassa as a genetically tractable model to investigate early cellular responses to azole stress. Methods: Exponentially growing mycelia were exposed to conidium-derived MIC80 reference concentrations of fluconazole, voriconazole, ravuconazole, and ketoconazole. Early responses were assessed by monitoring radial growth, intracellular glycerol, H2DCF-DA microscopy, antioxidant enzyme activities, Calcofluor White staining, and RT-qPCR analysis of genes associated with osmoregulation, sterol homeostasis, oxidative stress, and cell-wall remodelling. Results: Azole exposure was associated with reduced net post-transfer radial growth and rapid treatment- and time-dependent changes in intracellular glycerol. Induction of hog1 together with treatment-dependent changes in gpd1 and glycerol accumulation was consistent with an early osmoregulatory response. Sterol-homeostasis genes showed selective feedback regulation, and several cell-wall-remodelling genes underwent treatment-dependent transcriptional changes. Qualitative H2DCF-DA microscopy showed limited oxidant-associated fluorescence during azole challenge. Catalase activity remained close to control levels, whereas representative SOD-activity experiments showed larger fold changes after voriconazole and ravuconazole exposure. Representative Calcofluor White micrographs showed irregular septation and localised regions of enhanced cell-wall-associated staining. Conclusions:\u00a0Neurospora crassa shows multiple concurrent early responses to azole stress involving osmotic adjustment, sterol-pathway feedback, antioxidant responses, and cell-wall remodelling. These findings provide a framework for future studies examining how such stress responses contribute to recovery, tolerance, or longer-term adaptation.<\/jats:p>","DOI":"10.3390\/antibiotics15080811","type":"journal-article","created":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T11:04:12Z","timestamp":1787137452000},"page":"811","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Early Cellular Responses of Neurospora crassa to Azole Stress: Osmotic Adjustment, Redox Buffering, Sterol-Pathway Feedback, and Cell Wall Remodelling"],"prefix":"10.3390","volume":"15","author":[{"given":"Tom\u00e1\u0161","family":"Pag\u00e1\u010d","sequence":"first","affiliation":[{"name":"Institute of Biochemistry and Microbiology, Faculty of Food and Chemical Technology, Slovak University of Technology in Bratislava, Radlinsk\u00e9ho 9, 812 37 Bratislava, Slovakia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J\u00e1n","family":"V\u00edgla\u0161","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Microbiology, Faculty of Food and Chemical Technology, Slovak University of Technology in Bratislava, Radlinsk\u00e9ho 9, 812 37 Bratislava, Slovakia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9110-5526","authenticated-orcid":false,"given":"Petra","family":"Olejn\u00edkov\u00e1","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Microbiology, Faculty of Food and Chemical Technology, Slovak University of Technology in Bratislava, Radlinsk\u00e9ho 9, 812 37 Bratislava, Slovakia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e00449-20","DOI":"10.1128\/mBio.00449-20","article-title":"Threats posed by the fungal kingdom to humans, wildlife, and agriculture","volume":"11","author":"Fisher","year":"2020","journal-title":"mBio"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e428","DOI":"10.1016\/S1473-3099(23)00692-8","article-title":"Global incidence and mortality of severe fungal disease","volume":"24","author":"Denning","year":"2024","journal-title":"Lancet Infect. 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