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Background: The emergence of tolerance to antifungal agents in Candida albicans complicates the treatment of fungal infections. Understanding the mechanisms underlying this tolerance is crucial for developing effective therapeutic strategies. Objective: This study aims to elucidate the genetic and molecular basis of ketoconazole tolerance in C. albicans, focusing on the roles of chromosomal aneuploidy, Hsp90, and calcineurin. Methods: The wild-type C. albicans strain SC5314 was exposed to increasing concentrations of ketoconazole (0.015-32 µg/mL) to select for tolerant adaptors. Disk diffusion and spot assays were used to assess tolerance. Whole-genome sequencing identified chromosomal changes in the adaptors. The roles of Hsp90 and calcineurin in maintaining and developing ketoconazole tolerance were investigated using specific inhibitors and knockout strains. Results: Adaptors exhibited tolerance to ketoconazole concentrations up to 16 µg/mL, a significant increase from the parent strain's inhibition at 0.015 µg/mL. All tolerant adaptors showed amplification of chromosome R, with 29 adaptors having trisomy and one having tetrasomy. This aneuploidy was unstable, reverting to euploidy and losing tolerance in drug-free conditions. Both Hsp90 and calcineurin were essential for maintaining and developing ketoconazole tolerance. Inhibition of these proteins resulted in loss of tolerance. The efflux gene CDR1 was not required for the development of tolerance. Chromosome R trisomy and tetrasomy induce cross-tolerance to other azole antifungal agents, including clotrimazole and miconazole, but not to other antifungal classes, such as echinocandins and pyrimidines, exemplified by caspofungin and 5-flucytosine. Conclusion: Ketoconazole tolerance in C. albicans is mediated by chromosomal aneuploidy, specifically chromosome R amplification, and requires Hsp90 and calcineurin. These findings highlight potential targets for therapeutic intervention to combat antifungal tolerance and improve treatment outcomes.
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Antifungal resistance and antifungal tolerance are two distinct terms that describe different cellular responses to drugs. Antifungal resistance describes the ability of a fungus to grow above the minimal inhibitory concentration (MIC) of a drug. Antifungal tolerance describes the ability of drug susceptible strains to grow slowly at inhibitory drug concentrations. Recent studies indicate antifungal resistance and tolerance have distinct evolutionary trajectories. Superficial candidiasis bothers millions of people yearly. Miconazole has been used for topical treatment of yeast infections for over 40 years. Yet, fungal resistance to miconazole remains relatively low. Here we found different clinical isolates of Candida albicans had different profile of tolerance to miconazole, and the tolerance was modulated by physiological factors including temperature and medium composition. Exposure of non-tolerant strains with different genetic backgrounds to miconazole mainly induced development of tolerance, not resistance, and the tolerance was mainly due to whole chromosomal or segmental amplification of chromosome R. The efflux gene CDR1 was required for maintenance of tolerance in wild type strains but not required for gain of aneuploidy-mediated tolerance. Heat shock protein Hsp90 and calcineurin were essential for maintenance as well as gain of tolerance. Our study indicates development of aneuploidy-mediated tolerance, not resistance, is the predominant mechanism of rapid adaptation to miconazole in C. albicans, and the clinical relevance of tolerance deserves further investigations.
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Aneuploidia , Antifúngicos , Calcineurina , Candida albicans , Farmacorresistencia Fúngica , Proteínas Fúngicas , Proteínas HSP90 de Choque Térmico , Miconazol , Pruebas de Sensibilidad Microbiana , Miconazol/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/genética , Candida albicans/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Antifúngicos/farmacología , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Calcineurina/metabolismo , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Candidiasis/microbiología , Candidiasis/tratamiento farmacológico , Tolerancia a MedicamentosRESUMEN
Cervical cancer (CC) is the most common malignant tumor of female reproductive system. MiR-4319 has been identified as an anti-oncogene in various cancers. In the present study, role of miR-4319 in CC was identified. Colony formation, flow cytometer, wound healing, and transwell assays were used to detect CC cell proliferation, apoptosis, migration, and invasion. The expression of miR-4319 was decreased in clinical CC tissues and CC cell lines. Upregulation of miR-4319 suppressed cell viability, proliferation, migration, and invasion, and induced cell apoptosis in CC cells. Moreover, tuftelin 1 (TUFT1) was verified as a direct target of miR-4319, as confirmed by dual-luciferase reporter assay. Additionally, TUFT1 expression was remarkably increased in clinical CC tissues and CC cell lines and was negatively associated with miR-4319 expression. Furthermore, overexpression of TUFT1 partially restored the effects of miR-4319 mimic on cell viability, proliferation, migration, invasion, and cell apoptosis in CC cells. To conclude, miR-4319 played an anti-cancer role in the occurrence and development of CC, which might be achieved by targeting TUFT1.
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Apoptosis , Movimiento Celular , Proliferación Celular , Proteínas del Esmalte Dental , Regulación Neoplásica de la Expresión Génica , MicroARNs , Neoplasias del Cuello Uterino , Femenino , Humanos , Apoptosis/genética , Secuencia de Bases , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Supervivencia Celular/genética , Proteínas del Esmalte Dental/genética , Proteínas del Esmalte Dental/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Invasividad Neoplásica , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/metabolismoRESUMEN
IMPORTANCE: Echinocandins are the newest antifungal drugs and are first-line treatment option for life-threatening systemic infections. Due to lack of consensus regarding what temperature should be used when evaluating susceptibility of yeasts to echinocandins, typically either 30°C, 35°C, or 37°C is used. However, the impact of temperature on antifungal efficacy of echinocandins is unexplored. In the current study, we demonstrated that Candida albicans laboratory strain SC5314 was more susceptible to caspofungin at 37°C than at 30°C. We also found that calcineurin was required for temperature-modulated caspofungin susceptibility. Surprisingly, the altered caspofungin susceptibility was not due to differential expression of some canonical genes such as FKS, CHS, or CHT genes. The molecular mechanism of temperature-modulated caspofungin susceptibility is undetermined and deserves further investigations.
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Antifúngicos , Candida albicans , Caspofungina/farmacología , Antifúngicos/uso terapéutico , Calcineurina/genética , Calcineurina/metabolismo , Temperatura , Equinocandinas/farmacología , Equinocandinas/uso terapéutico , Pruebas de Sensibilidad Microbiana , Farmacorresistencia FúngicaRESUMEN
Introduction: Candida albicans is a prevalent opportunistic human fungal pathogen. However, there are currently very few antifungal treatments available. Inositol phosphoryl ceramide synthase is an essential and fungal-specific protein that also provides a novel and promising antifungal target. Aureobasidin A is a widely used inhibitor of inositol phosphoryl ceramide synthase, however the mechanism of resistance to aureobasidin A is largely unknown in pathogenic fungi. Methods: Here we investigated how C. albicans adapted to low and high concentrations of aureobasidin A. Results and discussions: We identified trisomy of chromosome 1 as the predominant mechanism of rapid adaptation. Resistance to aureobasidin A was unstable because of the inherent instability of aneuploids. Importantly, chromosome 1 trisomy simultaneously regulated genes which were associated with aureobasidin A resistance that are on this aneuploid chromosome as well as on other chromosomes. Furthermore, the pleiotropic effect of aneuploidy caused altered resistance not only to aureobasidin A but also to other antifungal drugs including caspofungin and 5-flucytosine. We posit aneuploidy provides a rapid and reversible mechanism of development of drug resistance and cross resistance in C. albicans.
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Blood species identification of human and animals has attracted much attention in the areas of customs inspection and forensic science. The combination of vibrational spectroscopy and machine learning has been proven to be feasible and effective for this purpose. However, the popularization of this technology needs instrument which is compact, robust and more suitable for field application. Besides the quantity of the blood sample should be as little as possible. In this study, we proposed a system using echelle Raman spectrometer combined with surface enhanced Raman spectroscopy (SERS), which protocol combines the advantages of broadband and high resolution of echelle Raman spectrometer with the advantages of high SERS spectral sensitivity. The SERS spectra of 26 species including human were collected with echelle Raman spectrometer, and the convolutional neural network was used for species identification, with an accuracy rate of over 94%. The feasibility, validity and reliability of the combination of echelle Raman spectrometer and SERS for blood species identification were realized.
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Ciencias Forenses , Espectrometría Raman , Animales , Humanos , Reproducibilidad de los Resultados , Espectrometría Raman/métodosRESUMEN
Expansion microscopy has enabled super resolution imaging of biological samples. The accurate measurement of expansion factor and distortion typically requires locating and imaging the same region of interest in the sample before and after expansion, which is often time-consuming to achieve. Here we introduce a convenient method for relocation by utilizing isolated porcine glomeruli as landmarks during expansion. Following heat denaturation and proteinase K digestion protocols, the glomeruli exhibit expansion factor of 3.5 to 4 (only 7%-16% less expanded than the hydrogel), and 1% to 2% of relative distortion. Due to its appropriate size of 100 to 300 µm, the location of the glomerulus in the sample are visible to eyes, while its detailed shape only requires bright field microscopy. For expansion factors ranging from 3 to 10, the region in the vicinity of the glomerulus can be easily re-identified, and sometimes allows quantification of expansion factor and distortion under bright field without fluorescent labels.
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Hidrogeles , Microscopía , Animales , PorcinosRESUMEN
BACKGROUND: MicroRNAs are endogenous small noncoding RNAs, which play a critical role in regulating various biological and pathologic processes. Furthermore, miR-301a has been detected to be overly expressed in tumorigenic progression of ovarian cancer. However, the effects of miR-301a on ovarian cancer are still unclear. OBJECTIVE: The objective of this study is to investigate the molecular mechanisms of miR-301a in epithelial ovarian cancer cells. METHODS: The miR-301a expression in ovarian cancer cells was detected. Then, cell proliferation, cell cycle, and apoptosis of the miR-301a-mimic-transfected ovarian cancer cells were determined, as well as the effects of the miR-301a mimic on the PTEN/phosphoinositide 3-kinase (PI3K) signaling pathway were explored. RESULTS: We found that the miR-301a expression levels were markedly upregulated in ovarian cancer tissues and cells, and upregulation of miR-301a-promoted cell viability and proliferation. Our results also showed that the miR-301a-mimic accelerated cell cycle progression of ovarian cancer cells by targeting the CDK4/Cyclin-D1 pathway but not the CDK2/Cyclin-E pathway. Moreover, transfection of the miR-301a mimic into ovarian cancer cells could decrease the PTEN expression while increasing the PI3K and Akt phosphorylation, as compared with the miR-301a inhibitor group and the negative control group. CONCLUSION: Therefore, miR-301a should be an oncogene in ovarian cancer, and overexpression of miR-301a promoted proliferation of ovarian cancer cells by modulating the PTEN/PI3K/Akt signaling pathway.
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Apoptosis/fisiología , Proliferación Celular/fisiología , MicroARNs/fisiología , Neoplasias Ováricas/patología , Transducción de Señal/fisiología , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias Ováricas/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , TransfecciónRESUMEN
BACKGROUND: To establish a novel delivery system of RGD-conjugated resveratrol human serum albumin (HAS) nanoparticles in ovarian cancer therapy. METHODS: The nanoparticles system was characterized for physicochemical properties, the stability in the serum and in vitro release. The comparison between RVT injection, HSA-RVT NPs and RGD-HSA-RVT NPs regarding tissue distributions and pharmacokinetics was also carried out using mice as the animal models. RESULTS: The results showed that RGD-HSA-RVT NPs were characterized of small particle size about 128.2 nm and negative zeta potential about -21.42 mV, and drug controlled to release slowly on a biphasic pattern. Compared with control groups, RGD-HSA-RVT NPs showed the higher cellular uptake and cell inhibition rates. In vivo data showed that RGD-HSA-RVT NPs have good tumor enrichment characteristics and a significant difference in tumor inhibition, compared with the control group. CONCLUSION: RGD-conjugated resveratrol HSA nanoparticles are an ideal drug delivery system, which can play a role in the treatment of ovarian cancer.
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Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos , Nanopartículas/química , Oligopéptidos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Resveratrol/farmacología , Albúmina Sérica Humana/química , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/química , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Oligopéptidos/química , Neoplasias Ováricas/patología , Resveratrol/químicaRESUMEN
Long non-coding RNAs (lncRNAs), HOTAIR has been reported to be upregulated in cervical cancer development and progression. However, SNPs (single nucleotide polymorphisms) in the lncRNAs and their associations with cervical cancer susceptibility have not been reported. In the current study, we hypothesized that SNPs within the lncRNA HOTAIR may influence the risk of cervical cancer. We performed a case-control study including 510 cervical cancer patients (cases) and 713 cancer-free individuals (controls) to investigate the association between three haplotype-tagging SNPs (rs920778, rs1899663 and rs4759314) in the lncRNA HOTAIR and the risk of cervical cancer. We found a strong association between the SNP rs920778 in the intronic enhancer of the HOTAIR and cervical cancer (P<10-4). Moreover, the cervical cancer patients with homozygous TT genotype were significantly associated with tumor-node-metastasis (TNM) stage. In vitro assays with allele-specific reporter constructs indicated that the reporter constructs bearing rs920778T allele conferred elevated reporter gene transcriptional activity when compared to the reporter constructs containing rs920778C allele. Furthermore, HOTAIR expression was higher in cervical cancer tissues than that in corresponding normal tissues, and the high expression was associated with the risk-associated allele T. In summary, our studies provide strong functional evidence that functional SNP rs920778 regulates HOTAIR expression, and may ultimately influence the predisposition for cervical cancer.
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Predisposición Genética a la Enfermedad , ARN Largo no Codificante/genética , Neoplasias del Cuello Uterino/genética , Alelos , Estudios de Casos y Controles , Línea Celular Tumoral , China , Elementos de Facilitación Genéticos , Femenino , Humanos , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
PURPOSE: To investigate the expression of p15(INK4b), p16(INK4a) and p21(Waf1/Cip1) in specimens from cases of normal cervical epithelium (NCE), cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma (SCC), and to evaluate whether there is evidence implicating oncogene-induced senescence (OIS) in cervical squamous cell cancer development. METHODS: The immunohistochemical expression of p15(INK4b), p16(INK4a) and p21(Waf1/Cip1) were investigated in formalin-fixed paraffin-embedded specimens from 19 NCE, 51 CIN and 21 SCC cases, respectively. Comparisons among different groups for each marker were performed with Chi-square test. RESULTS: The expression of p15(INK4b), p16(INK4a) and p21(Waf1/Cip1) were significantly higher in both CIN and SCC compared to NCE. Furthermore, the expression of p15(INK4b) and p21(Waf1/Cip1) was significantly higher in CIN Ð compared to CIN Ð, and these expressions were statistically higher in CIN Ш compared to CIN Ð, respectively. The p16(INK4a) expression was significantly higher in CIN Ш compared to CIN Ð. CONCLUSIONS: The results suggested that the senescence programs mediated by p15(INK4b), p16(INK4a) and p21(Waf1/Cip1) were activated during the stage of CIN and SCC, and demonstrated that senescence may play important role in preventing from NCE to SCC.