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

Staphylococcus aureus enterotoxin b down-regulates the expression of transforming growth factor-beta (TGF-β) signaling transducers in human glioblastoma

Background: It has been revealed that Staphylococcus aureus enterotoxin B (SEB) may feature anti-cancer and anti-metastatic advantages due to its ability to modify cell immunity processes and signaling pathways. Glioblastoma is one of the most aggressive human cancers; it has a high mortality nature, which makes it an attractive area for the development of novel therapies. Objectives: We examined whether the SEB could exert its growth inhibitory effects on glioblastoma cells partially through the manipulation of a key tumor growth factor termed transforming growth factor-beta (TGF-β). Materials and Methods: A human primary glioblastoma cell line, U87, was treated with different concentrations of SEB. The cell quantity was measured by the MTT assay at different exposure times. For molecular assessments, total ribonucleic acid (RNA) was extracted from either non-treated or SEB-treated cells. Subsequently, the gene expression of TGF-β transducers, smad2/3, at the messenger RNA (mRNA) level, was analyzed via a quantitative real-time polymerase chain reaction (qPCR) using the SYBR Green method. Significant differences between cell viability and gene expression levels were determined (Prism 5.0 software) using a one-way analysis of variance (ANOVA) test. Results: We reported that SEB could effectively down-regulate smad2/3 expression in glioblastoma cells at concentrations as quantity as 1 µg/mL and 2 µg/mL (P < 0.05 and P < 0.01, respectively). The SEB concentrations effective at regulating smad2/3 expression were correlated with those used to inhibit the proliferation of glioblastoma cells. Our results also showed that SEB was able to decrease smad2/3 expression at the mRNA level in a concentration- and time-dependent manner. Conclusions: We suggested that SEB could represent an agent that can significantly decrease smad2/3 expression in glioblastoma cells, leading to moderate TGF-β growth signaling and the reduction of tumor cell proliferation. © 2016, Ahvaz Jundishapur University of Medical Sciences

Overexpression of ubiquitin and amino acid permease genes in association with antimony resistance in Leishmania tropica field isolates

The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime®) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance. © 2013, Korean Society for Parasitology and Tropical Medicine

Mutation analysis of VSX1 and SOD1 in Iranian patients with keratoconus

Purpose: To evaluate mutations in the visual system homeobox gene 1 (VSX1) and superoxide dismutase 1 (SOD1) genes with keratoconus (KTCN), direct sequencing was performed in an Iranian population. Methods: One hundred and twelve autosomal dominant KTCN patients and fifty-two unaffected individuals from twentysix Iranian families, as well as one hundred healthy people as controls were enrolled. Genomic DNA was extracted from whole blood sample. Then to study the possible linkage between KTCN and six known loci linkage analysis was performed using 12 short tandem repeat (STR) markers. Also, the entire coding region and intron-exon boundaries of VSX1 and SOD1 were amplified by the PCR technique in each proband. Subsequently, PCR products were subjected to direct sequencing. Co-segregation analysis of the identified mutation was conducted in the family members. An Amplification Refractory Mutation System PCR (ARMS-PCR) was additionally employed for detection of the identified mutation in healthy controls. Results: Linkage analysis of aforementioned loci did not detect evidence for linkage to KTCN. Direct PCR sequencing revealed two single nucleotide polymorphisms (SNPs; g.1502T>G and g.9683C>T), as well as two missense mutations that have been previously reported (R166W and H244R) in VSX1. We also found three undescribed SNPs (g.4886G>A, g.4990C>G, and g.9061T>A) in SOD1. The R166W and H244R mutations were co-segregated in affected family members but not in those that were unaffected. Moreover, the ARMS-PCR strategy did not detect the identified mutations in controls. Conclusions: Our data suggest a significant association between KTCN patients and VSX1 genetic alterations (p.R166W and p.H244R). Although our findings support VSX1 as a plausible candidate gene responsible for keratoconus, other chromosomal loci and genes could be involved in KTCN development. Taken together, our results suggest that p.R166W and p.H244R could have possible pathogenic influences on KTCN

Screening of prognostic factors using multiplex RT-PCR technique on different leukemic cell lines

&quot;nBackground: Leukemia is one of the most common pediatric malignancies. T-cell Acute Lymphoblastic Leukemia (T-ALL) accounts for 15% of hematopoetic cancers. It has been well understood that identification of genetic alterations associated with leukemias is very critical. The molecular genetic techniques have promoted the identification of leukemia-associated genetic changes that may characterize the most accurate predictors of clinical outcome. These considerations reinforce the requirement for rapid identification of the abnormalities. &quot;nMethods: Multiplex RT-PCR, a highly sensitive and specific method applied to screen simultaneously three most frequent transcription factors, TLX1/HOX11, TLX3/HOX11L2 and TAL1/SCL which are associated with T-cell Acute Lymphoblastic Leukemia (T-ALL). &quot;nResults: We describe here our efforts to establish a multiplex RT-PCR analysis system that facilitates the detection of HPB-ALL and K562 cell lines, respectively. &quot;nConclusion: The multiplex RT-PCR technique is a sensitive, valuable and cost-effective diagnostic tool which could improve our ability to accurately and rapidly risk-stratification of patients with childhood T-ALL. In order to perform multiplex RT-PCR technique researchers do not need bone marrow samples and they can employ this method using peripheral blood samples. Therefore, the status of treatment could be followed by assessment of the level of mRNA expression of oncogenic transcriptional factor using peripheral blood sample. Use of this procedure not only provides the best results in short term for specialist, but also clinicians could have opportunities to choose suitable treatment strategies with decrement of drug side effects

Lack of association between the C677T single nucleotide polymorphism of the MTHFR gene and glaucoma in Iranian patients

Glaucoma is a major cause of blindness worldwide. A single nucleotide polymorphism of the MTHFR gene (C677T) has been associated with susceptibility to this disease, although this is controversial in the last decade. In this study, the possible association between the MTHFR C677T polymorphism and the risk of developing primary open angle (POAG) and pseudoexfoliation glaucoma (PEXG) was investigated. For this, a prospective study consisting of 73 POAG, 85 PEXG and 90 matched controls was undertaken in an Iranian population. Genomic DNA was extracted from whole blood. Genotyping of all individuals for the MTHFR C677T polymorphism was conducted using the PCR-RFLP technique. Our findings revealed no significant association between the MTHFR C677T polymorphism in POAG and PEXG compared with controls. Consistent with several other studies, our analysis suggests that the MTHFR C677T polymorphism is unlikely to be a factor contributing to the risk of developing specific forms of glaucoma

Potential functions of the human homeobox TGIFLX/Y genes in normal and abnormal development

Homeobox genes encode transcription factors that play important roles in the developmental and normal cellular processes in all metazoans. TGIFLX/Y (TGIFLX and TGIFLY) are members of the homeobox superfamily of genes. Their expression is specifically detected in human adult testis but their functions remain to be investigated. Identification of relevant target genes should make a key contribution to a complete understanding of the mechanisms by which TGIFLX/Y functions in both normal and abnormal developmental processes. In this review, we provide an overview of recent studies on different aspects of TGIFLX/Y with a focus on the current state of research about their roles in tumorigenesis and azoospermia

Association of TGIFLX/Y mRNA expression with prostate cancer

Prostate cancer is the most common type of solid tumor and a leading cause of cancer-related death of men living in the developed world. In recent years, the molecular mechanisms involved in prostate cancer development and/or progression have been intensely studied and several genes have been identified. TGIFLX/Y (TGIFLX and TGIFLY) are members of the homeobox superfamily of genes whose function(s) is unknown. To investigate TGIFLX/Y mRNA expression in prostate cancer, we studied two different types of clinical samples, namely 60 prostate tumors and 15 cases of benign prostate hyperplasia (BPH), by RT-PCR. Our results revealed that most prostate tumors (73.5%) express at least one of these genes, although different patterns of TGIFLX/Y mRNA expression were observed. In some tumor samples the expression of both genes was detected, while in others no expression of either gene was observed. Notably, there was a significant correlation between expression of both TGIFLX and TGIFLY and a Gleason score of >or=6 (P = 0.038). By contrast, expression of TGIFLX/Y mRNA in BPH samples could not be detected. These results suggest an association of TGIFLX/Y expression with the progression of prostate cancer

Performance comparison of numerical inversion methods for Laplace and Hankel integral transforms in engineering problems

Different methods for the numerical evaluations of the inverse Laplace and inverse of joint Laplace–Hankel integral transforms are applied to solve a wide range of initial-boundary value problems often arising in engineering and applied mathematics. The aim of the paper is to present a performance comparison among different numerical methods when they are applied to transformed functions related to actual engineering problems found in the literature. Most of our selected test functions have been found in the solution of boundary value problems of applied mechanics such as those related to transient responses of isotropic and transversely isotropic half-space to concentrated impulse or those related to viscoelastic wave motion in layered media. These classes of test functions are frequently encountered in similar problems such as those in boundary element or boundary integral equations, theoretical seismology, soil–structure-interaction in time domain and so on. Therefore, their behavior with different numerical inversion algorithms could make a useful guide to a precise choice of more suitable inversion method to be used in similar problems. Some different methods are also investigated in detail and compared for the inversion of the joint Hankel–Laplace transforms, where more sophisticated integrand functions are encountered. It is shown that Durbin, Crump, D’Amore, Fixed-Talbot, Gaver–Whyn–Rho (GWR), and Direct Integration methods have excellent performance and produce good results when applied to the same problems. On the contrary, Gaver–Stehfest and Piessens methods furnish results not very reliable for almost all classes of transformed functions and they seem good only for “simple” transformed functions. Particularly the performance of GWR algorithm is very good even for transformed functions with infinite number of singularities, where the other methods fail. In addition, in case of double integral transforms, only the Fixed-Talbot, Durbin and Weeks methods are recommended