The effect of microRNA-375 overexpression, an inhibitor of Helicobacter pylori-induced carcinogenesis, on lncRNA SOX2OT

Background: Helicobacter pylori is a major human pathogenic bacterium in gastric mucosa. Although the association between gastric cancer and H. pylori has been well-established, the molecular mechanisms underlying H. pylori-induced carcinogenesis are still under investigation. MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression at the posttranscriptional level. Recently, studies have revealed that miRNAs are involved in immune response and host cell response to bacteria. Also, microRNA-375 (miR-375) is a key regulator of epithelial properties that are necessary for securing epithelium-immune system crosstalk. It has been recently reported that miR-375 acts as an inhibitor of H. pylori-induced gastric carcinogenesis. There are few reports on miRNA-mediated targeting long noncoding RNAs (lncRNAs). Objectives: This study aimed to examine the possible effect of miR-375 as an inhibitor of H. pylori-induced carcinogenesis on the expression of lncRNA SOX2 overlapping transcript (SOX2OT) and SOX2, a master regulator of pluripotency of cancer stem cells. Materials and Methods: In a model cell line, NT-2 was transfected with the constructed expression vector pEGFP-C1 contained miR- 375. The RNA isolations and cDNA synthesis were performed after 48 hours of transformation. Expression of miR-375 and SOX2OT and SOX2 were quantified using real-time polymerase chain reaction and compared with control cells transfected with pEGFP-C1-Mock clone. Cell cycle modification was also compared after transfections using the flow cytometry analysis. Results: Following ectopic expression of miR-375, SOX2OT and SOX2 expression analysis revealed a significant decrease in their expression level (P < 0.05) in NT-2 cells compared to the control. Cell cycle analysis following ectopic expression of miR-375 in the NT-2 cells using propidium iodine staining revealed significant extension in sub-G1 cell cycle. Conclusions: This is the first report to show down-regulation of SOX2OT and SOX2 following induced expression of miR-375. This findingmaysuggest expression regulation potential between different classes of ncRNAs, for example between miR-375andSOX2OT. This data not only extends our understanding of possible ncRNA interactions in cancers but also may open novel investigation lines towards elucidation of molecular mechanisms controlling H. pylori inflammation and carcinogenesis. © 2016, Ahvaz Jundishapur University of Medical Sciences

Arthroconidia production in Trichophyton rubrum and a new ex vivo model of onychomycosis

The dermatophyte fungus Trichophyton rubrum often produces arthroconidia in vivo, and these cells are thought to be involved in pathogenesis, and, in shed skin scales, to act as a source of infection. The purpose of this study was (i) to examine the environmental and iatrogenic factors which affect arthroconidiation in T. rubrum in vitro, (ii) to look at arthroconidia formation in a large number of clinical isolates of T. rubrum and (iii) to develop a new model for the study of arthroconidia formation in nail tissue. Arthroconidia production was studied in T. rubrum grown on a number of media and under conditions of varying pH, temperature and CO2 concentration. The effect of the presence of antifungals and steroids on arthroconidia formation was also examined. Nail powder from the healthy toenails of volunteers was used as a substrate for arthroconidial production. On Sabouraud dextrose agar in the presence of 10 CO2 plus air, arthroconidial formation occurred optimally at 37 °C and pH 7.5, and was maximal at 10 days. Most isolates of T. rubrum showed a similar level of arthroconidial production, and only two out of 50 strains were unable to produce arthroconidia. Subinhibitory levels of some antifungals and betamethasone resulted in the stimulation of arthroconidia formation. Arthroconidial production in ground nail material also occurred under the same optimal conditions, but took longer to reach maximal levels (14 days). These in vitro and ex vivo results provide a useful basis for the understanding of arthroconidium formation in vivo in infected tissues such as nails. © 2006 SGM

Determination of changes in the expression of MIR-212 and EGFR genes in clinical samples from areas infected with trichophyton rubrum compared with non-infected areas

Background: Skin infections with dermatophytes (dermatophytosis) are common human fungal infections, the most common cause of which is Trichophyton rubrum. Antimicrobial peptides (AMPs), which have potential anti-microbial effects, are affected by epidermal growth factor receptor (EGFR) gene. Elevated expression of this gene in skin cells activates AMPs and prevents cloning of dermatophytes in keratinocytes. However, mRNA of EGFR gene is muted with increased expression of microRNAs (miRNAs), in particular miR-212. Therefore, EGFR inhibition may have a negative impact on AMP localization in dermatophyte-infected keratinocytes. Objectives: This study aimed to determine the changes in the expression of miR-212 and EGFR genes in cutaneous tissues affected by T. rubrum compared to their healthy margins. Methods: The number of samples in this study was estimated to be 72. The fungus was cultivated on Sabouraud Dextrose Agar medium. Isolation and optimization of total RNA and synthesis and optimization of cDNA for the EGFR and miR-212 genes were performed. Amplification of these target genes was performed using real-time polymerase chain reaction (RT-PCR). In data aggregation and analysis, changes in expression of the target genes were calculated via 2-∆∆Ct ratio. P value was considered &lt; 0.05. Results: In samples infected with T. rubrum, miR-212 significantly reduced the expression of EGFR gene, and in these samples, the expression of miR-212 gene was eight times higher compared to the expression of the EGFR gene. In control samples, the expression of miR-212 was much lower than that of the EGFR gene. Conclusions: By enhancing the expression of miR-212 in this study, the function of EGFR gene mRNA was turned off, leading to the reduction of AMPs, and in turn, the colonization of T. rubrum and creation of dermatophytosis on the skin. © 2018, Author(s)

Use of Rolling Circle Amplification to Rapidly Identify Species of Cladophialophora Potentially Causing Human Infection

The genus Cladophialophora comprises etiologic agents of disease in immunocompetent patients, ranging from mild cutaneous colonization to cerebral encephalitis, in addition to saprobic species. Due to the high degree of phenotypic similarity between closely related species of the genus, identification problems are imminent. In the present study, we described rapid and sensitive rolling circle amplification (RCA) method based on species-specific padlock probes targeted for the internal transcribed spacer regions of rDNA. ITS regions of 12 Cladophialophora species were sequenced, and subsequently, 10 specific padlock probes were designed for the detection of single nucleotide polymorphisms. The majority of circularizable padlock probes were designed based on single nucleotide polymorphisms (SNPs), while for C. bantiana, C. immunda and C. devriesii were characterized by two or more nucleotides. Individual species-specific probes correctly identified in all ten Cladophialophora species correctly by visualization on 1.2 % agarose gels used to verify specificity of probe-template binding; no cross-reactivity was observed. Simplicity, sensitivity, robustness and low costs provide RCA a distinct place among isothermal techniques for DNA diagnostics. However, restriction and specificity and sensitivity should be lowered and increased, respectively, to be useful for a wide variety of clinical applications

Molecular Characterization of Highly Susceptible Candida africana from Vulvovaginal Candidiasis

Phylogenetic studies highlight Candidaafricana as an atypical variant within Candidaalbicans species complex which is dominantly recovered from vaginal specimens. This study aimed to characterize C. africana isolates from patients with vulvovaginal candidiasis (VVC) by molecular methods and in vitro susceptibilities. One hundred and fifty-six (48.44 ) Candida strains were collected from 322 patients diagnosed with VVC. Of these, 114 (73.07 ) were germ tube positive and presented green color on the chromogenic medium, thus classified as C. albicans species complex. One hundred and nine (95.61 ) out of 114 isolates were identified as C. albicans, while five (4.38 ) isolates were identical with C. africana based on hwp1 PCR. C. africana appeared to be highly susceptible to the tested antifungals. For all strains of C. africana, fluconazole MIC was 2-log2-dilution steps less active than amphotericin B, which in turn was 2-log2-dilution steps and 3-log2-dilution steps less active than other azoles and echinocandin agents, respectively. In conclusion, among the C. albicans species complex, C. albicans predominantly and C. africana rarely occur in vaginal mucosa. Due to limited information on molecular epidemiology of this novel yeast, more studies using molecular methods are needed to elucidate the inter- and intraspecific genomic variations of C. africana isolates. © 2015, Springer Science+Business Media Dordrecht

Systemic infection with Candida albicans in breast tumor bearing mice: Cytokines dysregulation and induction of regulatory T cells

Objective: The effect of candidemia on immunologic parameters in breast tumor bearing patients is not well studied. Here, we hypothesised that candidemia in the tumor background may change the outcome of immunologic parameters and tumor condition. Method: Mice were divided into four groups, including normal, tumor, Candida infected (only Candidiasis) and tumor/Candidiasis groups. Tumor changes were recorded daily after tumor transplantation and induction of candidemia. Splenocytes of mice were harvested, cultured, and stimulated with PHA; afterwards, IL-4, IL-10, IFN-γ, TNF-α and TGF-β cytokines were assessed using ELISA kits. We also evaluated the population of CD4 + CD25 + Foxp3 + regulatory T cells in the tumor infiltrated and splenocytes. Results: The results showed that infection with C. albicans decreased the IFN-γ/IL-4 ratio in tumor/candidiasis and candidiasis groups versus their non-infected controls. IL-10, TGF-β and TNF-α levels increased in the candidiasis group. In addition, Candidemia led to an increase in the Treg population in tumor microenvironment and splenocytes of experimental groups compared with non-infected controls. Finally, candidemia increased tumor growth of tumor/Candidiasis group compared with the tumor group. Conclusion: It seems that systemic infection with C. albicans could not only induce regulatory T cells but also result in dysregulation of cytokine network and thereby facilitate tumor growth. © 2018 Elsevier Masson SA