Evaluation of mRNA expression levels of cyp51a and mdr1, candidate genes for voriconazole resistance in Aspergillus flavus

Background: Voriconazole Resistance (VRC-R) in Aspergillus flavus isolates impacts the management of aspergillosis, since azoles are the first choice for prophylaxis and therapy. However, to the best of our knowledge, the mechanisms underlying voriconazole resistance are poorly understood. Objectives: The present study was designed to evaluate mRNA expression levels of cyp51A and mdr1 genes in voriconazole resistant A. flavus by a Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technique. Materials and Methods: Five A. flavus isolates with resistance to VRC were examined by a RT-PCR approach. Results: Four out of five isolates revealed cyp51A and mdr1 mRNA overexpression. Interestingly, the isolate, which was negative for cyp51A and mdr1 mRNA expression showed a high voriconazole Minimum Inhibitory Concentration (MIC). Furthermore, a computational-based analysis predicted that voriconazole resistance could be mediated through cooperation with a network protein interaction. Conclusions: Our experimental and in silico findings may provide new insight in the complex molecular pathways of drug resistance and also could assist design an efficient therapeutic strategy for aspergillosis treatment. © 2015 Ahvaz Jundishapur University of Medical Sciences

Expression of efflux pumps and fatty acid activator one genes in azole resistant Candida glabrata isolated from immunocompromised patients

Acquired azole resistance in opportunistic fungi causes severe clinical problems in immunosuppressed individuals. This study investigated the molecular mechanisms of azole resistance in clinical isolates of Candida glabrata. Six unmatched strains were obtained from an epidemiological survey of candidiasis in immunocompromised hosts that included azole and amphotericin B susceptible and azole resistant clinical isolates. Candida glabrata CBS 138 was used as reference strain. Antifungal susceptibility testing of clinical isolates was evaluated using Clinical and Laboratory Standards Institute (CLSI) methods. Complementary DNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technology, semiquantitative RT-PCR, and sequencing were employed for identification of potential genes involved in azole resistance. Candida glabrata Candida drug resistance 1 (CgCDR1) and Candida glabrata Candida drug resistance 2 (CgCDR2) genes, which encode for multidrug transporters, were found to be upregulated in azole-resistant isolates (�2-fold). Fatty acid activator 1 (FAA1) gene, belonging to Acyl-CoA synthetases, showed expression in resistant isolates �2-fold that of the susceptible isolates and the reference strain. This study revealed overexpression of the CgCDR1, CgCDR2, and FAA1 genes affecting biological pathways, small hydrophobic compounds transport, and lipid metabolism in the resistant clinical C.glabrata isolates. © 2016 Tehran University of Medical Sciences. All rights reserved

Identification of Azole Resistance Markers in Clinical Isolates of Candida tropicalis Using cDNA-AFLP Method

Background: Global reports have highlighted the increasing prevalence of Candida tropicalis infections as well as organism's drug resistance. This study aimed at identifying azole resistance markers in clinical isolates of C. tropicalis, which will be a great resource for developing new drugs. Methods: Two susceptible and resistant isolates of C. tropicalis were recovered from an epidemiological investigation of candidiasis in immunocompromised patients. C. tropicalis ATCC 750 was used as reference strain. Antifungal susceptibility to fluconazole and itraconazole was determined using Clinical and Laboratory Standards Institute (CLSI) method. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technology and real-time reverse-transcriptase (RT) PCR were used for identification of potential genes involved in azole resistance of C. tropicalis clinical isolates. Results: Five genes encoding the following enzymes were identified as superoxide dismutase (SOD) implicated in antioxidant defense, ornithine aminotransferase (OAT), acetyl ornithine aminotransferase (ACOAT), adenosylmethionine-8-amino-7-oxononanoate aminotransferase (DAPA AT), and 4-aminobutyrate aminotransferase (ABAT)-belonging to pyridoxal phosphate (PLP) dependent enzymes and acting in an important physiological role in many fungal-cell cycles. Real-time RT-PCR confirmed mRNA level of the aforementioned genes. Conclusion: Our findings showed that factors such as PLP-dependent enzymes and SOD might be implicated in drug resistance in C. tropicalis clinical isolate. Therefore, further studies are required to explore the accurate biological functions of the mentioned genes that would be helpful for effective drug development. © 2016 Wiley Periodicals, Inc

Study of the distribution of Malassezia species in patients with pityriasis versicolor and healthy individuals in Tehran, Iran

BACKGROUND: Pityriasis versicolor is a superficial infection of the stratum corneum which caused by a group of yeasts formerly named pityrosporium. The taxonomy of these lipophilic yeasts has recently been modified and includes seven species referred as Malassezia. The aim of this study is to compare the distribution of Malassezia species isolated from pityriasis versicolor lesions and those isolated from healthy skins. METHODS: Differentiation of all malassezia species performed using morphological features and physiological test including catalase reaction, Tween assimilation test and splitting of esculin. RESULTS: In pityriasis versicolor lesions, the most frequently isolated species was M. globosa (53.3%), followed by M. furfur (25.3%), M. sympodialis(9.3%), M. obtusa (8.1%) and M. slooffiae (4.0%). The most frequently isolated species in the skin of healthy individuals were M. globosa, M. sympodialis, M. furfur, M. sloofiae and M. restricta which respectively made up 41.7%, 25.0%, 23.3%, 6.7% and 3.3% of the isolated species. CONCLUSIONS: According to our data, M. globosa was the most prevalent species in the skin of healthy individuals which recovered only in the yeast form. However, the Mycelial form of M. globosa was isolated as the dominant species from pityriasis versicolor lesions. Therefore, the role of predisposing factors in the conversion of this yeast to mycelium and its subsequent involvement in pityriasis versicolor pathogenicity should be considered

Molecular Pathways and Druggable Targets in Head and Neck Squamous Cell Carcinoma

Head and neck cancers are a heterogeneous group of neoplasms, affecting an ever increasing global population. Despite advances in diagnostic technology and surgical approaches to manage these conditions, survival rates have only marginally improved and this has occurred mainly in developed countries. Some improvements in survival, however, have been a result of new management and treatment approaches made possible because of our ever-increasing understanding of the molecular pathways triggered in head and neck oncogenesis, and the growing understanding of the abundant heterogeneity of this group of cancers. Some important pathways are common to other solid tumours, but their impact on reducing the burden of head and neck disease has been less than impressive. Other less known and little-explored pathways may hold the key to the development of potential druggable targets. The extensive work carried out over the last decade, mostly utilising next generation sequencing has opened up the development of many novel approaches to head and neck cancer treatment. This paper explores our current understanding of the molecular pathways of this group of tumours and outlines associated druggable targets which are deployed as therapeutic approaches in head and neck oncology with the ultimate aim of improving patient outcomes and controlling the personal and economic burden of head and neck cancer

Patterns of differentially expressed genes in oral mucosal lesions visualised under autofluorescence (VELscope™)

Objectives: We aimed to elucidate the molecular pathways associated with fluorescence properties of oral potentially malignant disorders (OPMD) visualised under direct tissue autofluorescence (VELscope™). Materials and Methods: Forty-two oral mucosal biopsies correlated with clinical fluorescence characteristics were categorised based on histopathological diagnosis. Four oral squamous cell carcinoma (OSCC), 15 oral epithelial dysplasia (OED), nine oral lichen planus (OLP) and 14 oral epithelial hyperplasia (OEH) presenting with three fluorescence patterns including retained fluorescence (RF), loss of fluorescence (LAF) with blanching (LB) and LAF with no blanching (LNB) were assessed. Relative gene expression was measured through RNA sequencing. Results: Although each lesion type had a specific set of histology-related differentially expressed genes (DEGs), all tested samples shared a number of DEGs, and we could not identify a discriminatory component between histological groups. Gene ontology enrichment revealed LAF in OEH was mostly due to changes in inflammation, cell cycle regulation and apoptosis, while in OED was due to inflammation, angiogenesis and extracellular matrix remodelling. Inflammatory reactions were associated with diascopic fluorescence (DF) for both OEH and OED. Conclusion: Uncovering the molecular mechanisms underlying LAF and DF may lead to reduction in the number of false-positive and false-negative findings and improve the efficacy and utility of VELscope™

Next generation sequencing applications in head and neck oncology

Head and neck cancer remains a major medical problem with significant morbidity, mortality and quality oflife issues. Over the recent past there hasbeen an increase in incidence, a shift in etiological factors, a growing proportion oftumours in younger cohorts, and a greater realisation of the heterogeneity of this group of tumours particularly within head and neck squamous cell carcinomas. The arrival of high-throughputmassively parallel sequencing technologies in diagnostic laboratories heralds an opportunity for uncovering driver mutations in head and neck cancer, understanding of disease stratification, personalisation of treatment strategies within the framework of genomic medicine, and discovery of potential druggable targets for disease-specific treatment. Next-generation sequencing (NGS) is a powerful tooland has the potential to transform the reactive and treatment-based nature of cancer care, to actively predict the riskfor disease and aim to prevent it. The underlying goal of NGS application is to achieve the concept of “genome-informed personalised medicine”. An important factor in harnessing NGS technologiesin personalised management of head and neckoncology lies in thefeedback between scientists and cliniciansinvolved in cancer care A genuine diagnosisand appropriate aetiology-matched treatmentis only possible if our decisions are basedon both the genotype and phenotype of our patients