Molecular identification, phylogenetic analysis and antifungal susceptibility patterns of Aspergillusnidulans complex and Aspergillusterreus complex isolated from clinical specimens.

OBJECTIVE: Aspergillus sections Terrei and Nidulantes are the less common causes of invasive aspergillosis and pulmonary aspergillosis (PA) in immunocompromised patients when compared to A. fumigatus and A. flavus. Identifying these fungi as the infectious agent is crucial because of the resistance to amphotericin B (AMB) and increased lethality. The aim of this study was to identify the molecular status, evaluate the genetic diversity and examine the antifungal susceptibility profile of the uncommon Aspergillus species. Forty-five uncommon Aspergillus species were identified based on the microscopic and macroscopic criteria. Then, the molecular identification was performed using the sequencing beta tubulin (benA) gene. In vitro antifungal susceptibility to amphotericin B (AMB), itraconazole (ITC), ravuconazole (RAV), voriconazole (VRC), caspofungin (CFG) isavuconazole (ISA) and posaconazole (POS) test was performed according to the CLSI M38-A2 guidelines. RESULTS: A. terreus was the most species detected, followed by A. nidulans, A. latus, A.ochraceus, and A. citrinoterreus, respectively. The analysis of the benA gene showed the presence of 12 distinct genotypes among the A. terreus isolates. The other species did not show any intraspecies variation. CFG exhibited the lowest MEC50/MIC50 (0.007µg/mL), followed by POS (0.125µg/mL), VRC, ITC, ISA (0.25µg/mL), RAV (0.5µg/mL), and AMB (8µg/mL). Among all the isolates, only 15.5% (7/45) were susceptible to AMB. CONCLUSION: Antifungal susceptibility pattern of the uncommon Aspergillus species is useful to improve patient management and increase knowledge concerning the local epidemiology. Moreover, this information is necessary when an outbreak dealing with drug-resistant infections occurs.

Comparison of in vitro antifungal activity of novel triazoles with available antifungal agents against dermatophyte species caused tinea pedis.

OBJECTIVE: Dermatophytes are a group of keratinophilic fungi that invade and infect the keratinized tissues and cause dermatophytosis. We investigated effectiveness of novel triazole (luliconazole and lanaconazole) in comparison with available antifungal agents against dermatophyte species isolated from patients with tinea pedis. MATERIAL AND METHODS: A total of 60 dermatophytes species were isolated from the patients with tinea pedis. Identification of species was done by DNA sequencing of the ITS1-5.8S rDNA-ITS2 rDNA region. In vitro antifungal susceptibility testing with luliconazole and lanaconazole and available antifungal agent was done in accordance with the Clinical and Laboratory Standards Institute, M38-A2 document. RESULTS: In all investigated isolates, luliconazole had the lowest minimum inhibitory concentration (MIC) (MIC range=0.0005-0.004µg/mL), while fluconazole (MIC range=0.4-64µg/mL) had the highest MICs. Geometric mean MIC was the lowest for luliconazole (0.0008µg/mL), followed by lanoconazole (0.003µg/mL), terbinafine (0.019µg/mL), itraconazole (0.085 µg/mL), ketoconazole (0.089µg/mL), econazole (0.097µg/mL), griseofulvin (0.351 µg/mL), voriconazole (0.583µg/mL) and fluconazole (11.58µg/mL). CONCLUSION: The novel triazoles showed potent activity against dermatophytes and promising candidates for the treatment of tinea pedis caused by Trichophyton and Epidermophyton species. However, further studies are warranted to determine the clinical implications of these investigations.

Association of rs2620381 polymorphism in miR-627 and gastric cancer.

Background: Gastric cancer is a complicated malignancy whose aetiology is not well characterized. Single nucleotide polymorphisms (SNPs), some located within microRNA genes, are linked with gastric cancer. We hypothesized a link between SNP rs2620381 (A > C) in miR-627 and gastric cancer.Material and methods: We recruited 280 healthy controls and 240 gastric cancer patients. Genotyping was conducted by allele-specific polymerase chain reaction. In addition, in silico analyses were carried out via databases and web tools including miRBase, dbSNP, RNAfold, MiRNASNP V2.0, miRWalk V2.0, miRTarBase, and miRmap.Results: Any C genotype in rs2620381 was linked to gastric cancer: CC vs. AA: OR/95% CI 2.67 (1.17-6.09), p = 0.01, CC+AC vs. AA: OR/95% CI 1.66 (1.12-2.46), p = 0.01, CC vs. AC+AA: OR/95% CI 2.44 (1.07-5.54), p = 0.03. The minor allele C of miR-627 was linked with gastric cancer compared with A allele (OR/95%CI 1.88 (1.30-2.73), p = 0.0008). There were no links between age, sex, tumour type, distant metastasis, and tumour stages and the miR-627 polymorphism in gastric cancer patients.Conclusion: Presence of the C SNP in miR-627 rs2620381 in linked with gastric cancer, and may be important in pathogenesis.

miR-559 polymorphism rs58450758 is linked to breast cancer.

Background: MicroRNAs (miRNAs) participate in gene regulation and the control of cancer-related mechanisms such as apoptosis, invasion and differentiation. Single nucleotide polymorphisms (SNP) of the miRNA encoding genes may influence the development of cancer. We hypothesized a link between miR-559 SNP rs58450758 and breast cancer.Materials & methods: Bioinformatics analyses were performed to predict the miR-559 target genes and the effect of the rs58450758 SNP on the stem-loop structure. A total of 129 breast cancer cases and 153 controls were genotyped using PCR-RFLP.Results: The recessive genotype (TT) was more common among breast cancer patients (23.3%) than among controls (2%). The non-dominant genotypes (CT+TT) were associated with breast cancer in patients (OR 3.62; 95% CI, 1.95-6.69; p < 0.0001). Bioinformatics analyses suggested that rs58450758 changes miR-559 secondary structure and forms new DICER sites in the pre-miRNA.Conclusion: The miR-559 rs58450758 variant is linked to breast cancer.

Molecular genetic diagnosis of Glanzmann syndrome in Iranian population; reporting novel and recurrent mutations.

BACKGROUND: Glanzmann thrombasthenia (GT) is a rare autosomal recessive abnormality of platelet aggregation with quantitative and/or qualitative abnormality of αIIbß3 integrin. The αIIbß3 is a platelet fibrinogen receptor, which is required for platelet aggregation, firm adhesion, and also spreading. The disease is more prevalent in the populations with a higher rate of consanguineous marriages as in some Middle Eastern populations including Iraq, Jordan, and Iran. Different types of mutations in ITGA2B and ITGB3 genes have been previously reported to cause the disease. RESULT: In this study, 16 patients with the clinical diagnosis of GT were studied. Direct sequencing of the exons and exon-intron boundaries of the above genes revealed mutations in 14 patients (detection rate: 87.5%). Briefly, out of fifteen types of identified mutations, 14 were novel. Seven mutations in the ITGB3 gene included 4 missense [c.2T > C, c.155 G > T, c. 538 G > A, c.1990 G > T], one nonsense mutation [c.1303 G > T], a small deletion [c.1656_1658delCTC] and a deletion of one nucleotide [c.401delA]. Mutations in the ITGA2B were 8 different mutations consisting 2 missense [c.286 T > A, c.842 C > T], 2 deletions [c.1899 del T, c.189-319_236del], an insertion [c.1071_1072insG] and one splice site mutations [c.409-3 C > G], one synonymous mutation that might alter the normal splicing process [c.1392 A > G] and a nonsense mutation [c.1555 C > T]. The causative mutation in 2 patients remained unknown. Using long-range PCR and sequencing, we found a rather large deletion. The break point of this deletion covers 319 nt from the last part of the first intron and 48 nt from the beginning of the second exon of ITGA2B gene. The deletion was also detected in two unrelated patients with the same ethnicity. In addition, in silico analyses of novel mutations were performed. CONCLUSION: There was no recurrent mutation in the studied population. This may be due to either small sample size or the heterogeneity of the studied population.

Aberrant methylation of miR-125b1 in gastric cancer: A case-control study.

Gastric cancer (GC) is a complex heterogeneous process and the molecular mechanisms underlying its initiation or propagation are still not very well characterized. Aberrant gene expression are key features of cancer. DNA methylation in a promoter region is an important epigenetic mechanism for the gene silencing. Here, the impact of DNA methylation in regulating the expression of miR-125b1 is explored. A total of 285 genetically unrelated subjects including 175 healthy controls and a total of 110 GC patients participated in this study. we performed nested methylation-specific polymerase chain reaction (MS-PCR) to evaluate methylation pattern of miR-125b1 promoter and quantitative real-time polymerase chain reaction (qRT-PCR) to determine the RNA expression changes in GC and normal tissues. The frequency of methylated allele was 24.5% in GC cases but only 10% in normal tissues. Statistically significant correlation between CpG dinucleotide methylation of miR-125b1 promoter and increased risk of gastric adenocarcinoma was observed (OR=2.57; 95%CI 1.60-4.13; P=0.0001). In addition, miR-125b1 promoter methylation correlated with tumor location and stages. miR-125b1 expression was much higher in normal tissue compared to cancerous tissue. However, methylation status of the miR-125b1 promoter was not correlated with miR-125b1 expression in cancerous specimens (p<0.05). In conclusion, this is a first report of miR-125b1 promoter methylation in GC. More research is needed to fully elucidate the underlying molecular mechanisms of GC susceptibility.

Effects of miR-27a, miR-196a2 and miR-146a polymorphisms on the risk of breast cancer.

Background microRNAs (miRNAs) are potentially involved in many physiopathological processes, including regulation of cell growth, differentiation, apoptosis and cancer. Single nucleotide polymorphisms of the genes encoding miRNAs can alter their expression and may influence cancer risks. This case-control study explored the relationship between three microRNA polymorphisms (miR-27a, miR-196a2 and miR -146a) and breast cancer (BC). Methods A total of 353 breast cancer cases and 353 controls were genotyped for miR-27a (rs895819), miR-196a2 (rs11614913) and miR -146a (rs2910164). The miR-27a and miR-146a variants were discriminated using a PCR-restriction fragment length polymorphism method, while miR-196a2 were analysed by tetra-primers amplification refractory mutation system PCR. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to estimate associations. Results The CC homozygous genotype of miR-146a (rs2910164) was seen in 45(12.7%) patients with breast cancer and 18(5.1%) controls (OR 4.09 [95%CI 2.19-7.67] p < 0.001). The minor allele G of miR-27a was associated with a decreased risk of breast cancer (OR 0.24 [95% CI 0.14-0.42] p < 0.001). The miR-196a2 (rs11614913) was not related to breast cancer (p > 0.05). Conclusion Our data indicate that miR-146a (rs2910164) and miR-27a (rs895819) variants contribute to breast cancer. Further studies in larger populations including other genetic and environmental factors are required to achieve a definitive conclusion.

Association of leukemia inhibitory factor gene polymorphism and in vitro fertilization outcome in a population in northern Iran.

Several studies have been demonstrated that endometrial leukemia inhibitory factor (LIF) is important in embryo implantation. LIF is a secreted glycoprotein with a variety of biological functions including stimulation of cell proliferation, differentiation and survival that are all essential for blastocyete development and implantation. The LIF receptor activates several signaling pathways in diverse cell types, including Jak/STAT, MAPK and PI3-kinase pathways in the endometrium of fertile woman. It has been suggested that the initial lower expression of LIF in proliferative phase may be one of the causes for multiple failure of implantation. The aim of this study was to evaluate the association between maternal genotype of SNP 3951C/T LIF and in vitro fertilization and embryo transfer (IVF-ET) outcome in infertile women. This case-control study was comprised of infertile patients (n=70) and women having one healthy child as controls (n=73). Genotyping for SNP-3951C/T was performed by PCR/RFLP. Allele and genotype distribution did not differ significantly between patients and controls (P>0.05). The LIF genotype frequencies amongst the 70 cases were C/C=40%, C/T=52.8% and T/T=7.2%; the C and T allele frequencies were 66% and 34%, respectively. The LIF genotype frequencies amongst the 73 controls were C/C=45.20%, C/T=50.70% and T/T=4.1%; the C and T allele frequencies were 70% and 30%, respectively. In conclusion, the results of this study indicate that SNP 3951C/T of LIF may not be associated with IVF-ET outcome in this population. Although more studies should be considered with larger number of patients and control subjects to confirm our results.

[The GG genotype of the HSPA1B gene is associated with increased risk of glaucoma in northern Iran].

Glaucoma is a heterogeneous eye disease characterized by optic nerve atrophy and visual field defects. The disease damages the retinal ganglion cells (RGC) and their functional axons. Heat shock proteins 70 (HSP70) are molecular chaperons that could have a protective effect in the development of glaucoma. Polymorphisms of HSP70 may alter protein function or expression and are associated with the susceptibility to glaucoma. The purpose of this study was to investigate whether the HSPA1B 1267A/G (rs1061581) and HSPA1L 2437T/C (rs2227956) variants contribute to glaucoma susceptibility. Genomic DNA samples from 169 patients with glaucoma and 178 healthy controls were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Here we show that the presence of HSPA1B 1267GG genotype significantly increases the risk of glaucoma (OR = 3.16, 95% CI = 1.45-6.89, p = 0.003). The prevalence of HSPA1L 2437T/C genotypes in patients and controls did not differ significantly (p = 0.31, χ^(2) = 2.32). However, large population based studies are required for further evaluation and confirmation of our finding.

[Mitochondrial DNA deletion Δ4977 in peptic ulcer disease].

Reactive oxygen species (ROS) play a critical role in peptic ulcer disease (PUD). Due to the high rate of ROS production and limited capacity for DNA repair within mitochondria, mtDNA is susceptible to oxidative damage and mutations. mtDNA deletion Δ4977 is one of the most common deletion events identified in mitochondria. We examined the association of 4977-bp mtDNA deletion with PUD. Genotypes were determined in bioptic samples of 150 PUD patients and 190 controls. The 4977-bp mtDNA deletion was found more frequently among patients with PUD (52%) than among controls (22.63%). The strong association between the mtDNA 4977-bp deletion and PUD was confirmed (OR = 3.7; 95% CI, 2.32-5.91; P = 0.0001). The 4977-bp deletion in mitochondrial DNA may be a risk factor for PUD, or may reflect an increase in oxidative stress that commonly accompanies underlying PUD disease. Larger population-based studies are needed to uncover the possible causal relationship between this deletion and peptic ulcer disease.

Administration of vitamin D3 induces CNPase and myelin oligodendrocyte glycoprotein expression in the cerebral cortex of the murine model of cuprizone-induced demyelination.

In the central nervous system (CNS) the main proteins of myelin are proteolipid protein (PLP), myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and CNPase. Myelin oligodendrocyte glycoprotein is a minor component of the myelin sheath, but is an important autoantigen linked to the pathogenesis of multiple sclerosis (MS). CNPase is expressed exclusively by oligodendrocytes in the CNS, and the appearance of CNPase seems to be one of the earliest events of oligodendrocyte differentiation and myelination. In this study the effects of vitamin D on total protein concentration, CNPase and MOG expression in the cerebral cortex of the murine model of cuprizone-induced demyelination was investigated. The mice were treated by cuprizone for five weeks in order to induce demyelination. The mice were then divided into 3 groups. The first group was injected intraperitoneally (IP) with vitamin D diluted in olive oil in the amount of 5 µg/kg/daily body weight. The second group (SHAM) was injected IP with olive oil and the third group was left without any injection as the control group (n = 11 for each group). After five weeks the mice were killed and the cerebral cortex was collected and the expression of CNPase and MOG was studied by Western blot. Total protein concentration in the vitamin D injected, SHAM and control groups were 0.918 ± 0.003, 0917 ± 0.004 and 0.916 ± 0.004 g/l, respectively (p > 0.05). However, a significant increase in the MOG and CNPase expression was seen in vitamin D injected group as compared to SHAM and control groups. It is concluded that vitamin D plays a role in the process of remyelination by increasing MOG and CNPase expression in the cortex.

Thenar muscles H reflex in patients with fibromyalgia: A case control study.

OBJECTIVE: To delineate H-reflex parameters and specify the diagnostic accuracy measures of thenar muscle H-reflex in Fibromyalgia (FM). METHODS: The study was a cross sectional study performed on 30 subjects with FM and 30 healthy volunteers in two major referral hospitals. We recorded the number of obtainable thenar H-reflexes and their minimum latency, threshold and amplitude in each group. RESULTS: There was a significantly more chance to elicit the H-reflex in patients with FM. H reflex threshold and minimum latency were lower in FM group but no significant difference was shown for H wave amplitude. According to our study, thenar H-reflex has 46.7% sensitivity, 86.7% specificity and 66.7% diagnostic accuracy to detect FM. It also has moderate predictive values and positive likelihood ratio but low negative likelihood ratio. CONCLUSION: Higher rate of thenar muscle H-reflex in fibromyalgia can be interpreted as a confirmatory finding to central sensitization theory for this disorder. Obtaining H-reflex from thenar muscles could be a helpful diagnostic tool for fibromyalgia that increases the confidence in diagnosis. Although it is a weak tool for screening because of low sensitivity, it has a relatively high specificity.