Research in Molecular Medicine
Volume:2 Issue: 3, Aug 2014

Despite the introduction of new antifungal agents, resistances to antifungal therapy continue to increase and outcome of invasive fungal infections treatment is frequently suboptimal. A large amount of the recent effort in antifungal drug discovery has focused on a limited set of targets with functions known or expected to be important for fungal viability and virulence. A variety of techniques can be used to identify fungal genes of interest. Gene expression profiling, RNA mediated gene silencing and insertional mutagenesis are three main molecular genetics technologies used to identify and validate antifungal drug targets. The term RNA interference (RNAi) refers to a cellular process by which a sequence-specific double-stranded RNA (dsRNA) inhibits the expression of a gene. This mechanism is strongly conserved in eukaryotes and has been documented to be existed in different fungal species such as Candida albicans, Aspergillus nidulans and Penicillium marneffei. Many vital and virulence genes have been successfully knocked down using RNAi technology. RNAi can be regarded as a promising approach for discovery of new gene targets for the design of fungus-specific antifungal agents. Here we discuss about a novel approach and its application in designing new molecular antifungal targets.

The pathogenesis of inflammatory bowel disease is complex and multifactorial. Studies have led to the current concept that Toll-like receptors represent key mediators of innate host defense in the intestine, and they are involved in maintaining mucosal as well as commensal homeostasis. We studied the possibility of simvastatin and antagonist of receptors of interleukin-1 for pharmacological correction of acute ileitis in rats with a focus on the expression intensity studies of TLR2 TLR4 with lymphocytes of small intestine. Experiments were carried out on male Wistar rats aged 5–7 months (body mass 260–285 g). Rats were divided into four experimental groups: group 1; control, group 2; rats with indomethacin-induced ileitis, group 3; rats given simvastatin (20 mg/kg, for 5 days, subcutaneously), group 4; rats given antagonist of receptors of interleukin-1 (3 mg/kg, for 5 days, subcutaneously). The TLR2 and TLR4 immunopositive lymphocytes were determined using a direct immunofluorescence technique with using a monoclonal rat antibody. We established that development of ileitis was accompanied with the change of amount of TLR2+ and TLR4+ lymphocytes and the density of TLR2, TLR4 in immunopositive cells. Drug administration during the development of experimental pathology was accompanied by changes in the expression of TLR2, TLR4 and their density on lymphocytes. Simvastatin and antagonist of receptors of interleukin-1 seemed to be beneficial in indomethacin-induced rat ileitis model through modulate TLR2 and TLR4 expression with lymphocytes of small intestine

Brucellosis is a major bacterial zoonoses of global importance caused by Brucella spps. FCγRIIA receptor plays a central role in phagocytosis of IgG2-opsonized bacteria. FCγRIIA exhibits allelic polymorphisms with different capacities for binding IgG2 and phagocytosis. Cells expressing FcγRIIa-H131, bind more efficiently to complexed IgG2 than those expressing the FcγRIIa-R131 variant. The purpose of this study was to evaluate the association of FCγRIIA polymorphisms with susceptibility to or severity of brucellosis. In this study we evaluated FCγRIIA polymorphisms (R/R131, R/H131, H/H131) in 67 patients with brucellosis and 67 age, sex and geographical matched healthy volunteers. FCγRIIA genotyping was performed by using a sequence-specific primer polymerase chain reaction (SSP-PCR). Comparison of the FCγRIIA genotypes distribution in patients with brucellosis and controls showed a higher frequency in FCγRIIA-R/R131 homozygosity in patients than controls (47.8% vs. 28.4%). Logistic regression analysis showed that there is a significant correlation between R/R131 genotype and brucellosis (OR=2.3, 95% CI=1.3-4.2, P=0.04). Although the frequency of the FCγRIIA-R/R131 was higher in patients with chronic brucellosis compared with acute brucellosis, we did not find any statistically significant differences (53.8% vs. 46.3%, P=0.65). The result of this study showed that the homozygous genotype of FCγRIIA-R/R131 in patient with brucellosis may be associated with susceptibility to brucellosis as a genetic risk factor.

Due to the predisposing conditions in patients with cystic fibrosis (CF) caused by defective mucociliary clearance facilitates of colonization and invasion with Candida species has dramatically increased. Traditional methods for identification problems are imminent and are time-consuming. Therefore, molecular techniques utilizing amplification of target DNA provide quick and precise methods for diagnosis and identification of Candida species. Therefor, the aim of current study was identification of the most medically common isolated Candida species from the air way of CF patients by PCR-RFLP and amplification of HWP1 gene. A total of 42 CF patients presenting symptoms referred to pediatric respiratory diseases research center were screened for the presence of Candida spp. The isolates initially were phenotypically identified and confirmed by molecular approaches based on restriction fragment length polymorphism (PCR-RFLP) for discrimination of C. albicans of non albicans and amplification of HWP1 gene for discrimination of C. albicans from C. dubliniensis and C. africana was conducted. Results show that C. albicans was the most frequently isolated species (83.8%) followed by non-albicans included C. parapsilosis (7.1%), C. glabrata (3.2%), and C. tropicalis (3.2%). The restriction patterns of each Candida species were perfectly specific. Since MspI is not able to discriminate between three morphological related species, C. albicans, C. dubliniensis and C. africana, we used PCR amplification of hwp1 gene, which (7.1%) species from C. albicans identified as C. dubliniensis, however C. africana strains were not found. The present study found that C. albicans as predominant species isolated from the CF patients. It can be concluded that molecular diagnostic methods are reliable and would be useful for identification of medically important Candida species in clinical samples. Therefore considerable attention has been paid to prevention and treatment of microbial growth, which has resulted in improvement of patient management.

Phytoestrogens, a group of plant-derived polyphenolic compounds have recently come into considerable attention due to the increasing information on their potential adverse effects in human health. Some of phytoestrogens show estrogenic activity that may be carcinogenic for human. In the present study here, we investigated the transcriptional effects of variety of phytoestrogens on the bovine oxytocin and the thymidine kinase-ERE promoter by estrogen receptor α in MDA-MB 231 breast cancer cell line. Cells were seeded for transfections into 12- well plates at a density of 100000 cells per well and were transfected with a total of 3 μg of plasmid DNA using calcium phosphate coprecipitation. Estrogen and some phytoestrogens (naringenin, 8-prenyl-naringenin and 6-(1, 1-dimethylallyl) naringenin were used for stimulation of transfected cells. Findings of our study clearly demonstrated the subtype-selective activation of estrogen receptor (ER)α and (ER)β by the phytoestrogen naringenin (activating estrogen receptor β) and its substituted forms 8-prenyl-naringenin and 6-(1, 1-dimethylallyl) naringenin (activating estrogen receptor α), on the ERE-controlled promoter as well as on the oxytocin gene promoter. The study revealed that some phytoestrogens show estrogenic activity by classical or non-classical mechanisms as well as exhibit estrogenic activity by undetermined mechanisms in transfected MDA-MB 231 cell line.

The association between Methylene tetrahydrofolate reductase polymorphism and Coronary Artery diseases risk has been both confirmed and refuted in a number of published studies. The aim of this study was to investigate whether genetic polymorphisms of MTHFR (C677T, A1298C) contributed to the development of myocardial infarction (MI). The present case-control study consisted of 54 patients with a history of MI and 54 genders matched normal controls. The SNPs genotypes were determined using polymerase chain reaction followed by restriction fragment length polymorphism method. No significant association of the MTHFR A1298C with the risk of MI was observed. However, the allele frequencies of C677T SNP differed significantly among cases and controls (0.83 vs. 0.30). A strong positive relationship between the TT genotype and the risk of MI supported with a significant P value < 0.001 (OR= 11.87, 95% CI: 4.7- 29.9, p < 0.001). The results of present study show the importance of C677T SNP as a potential biomarker for screening susceptible cases to MI.

The diagnosis of toxoplasmosis is most critical in pregnant women who acquire infection during gestation and also in fetuses and newborns who are congenitally infected. This study described the performance of molecular and confirmatory serologic testing for toxoplasma infection in the tissues of human spontaneous aborted fetuses and their mother's blood. 87 random samples from the tissues of body of spontaneous aborted fetuses (less than 14 weeks) in a separate container of preservative solution were collected from the delivery room of the university maternity hospital, Arak- Iran, during autumn 2012 to 2013. In the ward, 3 ml of blood sample of their mothers were collected and the sera were separated and analyzed by ELISA method for the detection of specific IgG. DNA extraction from the tissues of fetuses was performed and stored until use. The PCR reaction was performed by a pair of primers. PCR products were analyzed by electrophoresis and stained with safe stain. It is necessary to mention first that the written consent was obtained from their mothers and after recovery, a demographic questionnaire was completed. Most of the mothers were 20-29 years of age and the correlation between the location of residence, contact with cats and eating undercooked foods, not significant. Serological tests on the sera of 87 mothers for anti-Toxoplasma IgG showed 39.08% positive results. The results of PCR amplification showed that none of the 87 samples from aborted fetuses were infected with Toxoplasma. gondii Not observed any evidence of Toxoplasmosis in aborted fetuses, and it appears that Toxoplasma was not the cause of spontaneous abortion in this area of Iran but considering the importance of the infection during pregnancy, the control measurements during pregnancy is required.