Iranian Biomedical Journal
Volume:19 Issue: 3, Jul 2015

In gene therapy, the use of RNA molecules as therapeutic agents has shown advantages over plasmid DNA, including higher levels of safety. However, transient nature of RNA has been a major obstacle in application of RNA in gene therapy. Here, we used the internal ribosomal entry site of encephalomyocarditis virus and the 3’ non-translated region of Poliovirus to design an enterovirus-like RNA for the expression of a reporter gene (enhanced green fluorescent protein) and a suicide gene (thymidine kinase of herpes simplex virus). The expression of these genes was evaluated by flow cytometry and cytotoxicity assay in human colorectal adenocarcinoma cell line (SW480). We then armed RNA molecules with a target sequence for hsa-miR-143 to regulate their expression by microRNA (miRNA) mimics. The results showed effective expression of both genes by Entrovirus-like RNA constructs. The data also showed that the restoration of hsa-miR-143 expression in SW480 leads to a significant translation repression of the introduced reporter and suicide genes. Collectively, our data suggest the potential use of Entrovirus-like RNA molecules in suicide gene therapy. Additionally, as a consequence of the possible downregulated miRNA expression in cancerous tissues, a decreased expression of gene therapy constructs armed with target sequences for such miRNA in cancer tissue is expected.

Serologic screening of gastric cancer (GC) by serum pepsinogens (sPG) levels and Helicobacter pylori (Hp) sero-status, though highly informative, has provided heterogeneous results. Here, we have evaluated the modifying effects of demographic factors on the risk impact of Hp sero-status/sPG levels in gastric cancer, with particular emphasis on age. A cross-sectional study was carried out on 1341 individuals (GC = 578, healthy = 763), who were stratified into two age groups: 35-59 years (middle-aged, n = 830) and ≥ 60 years (60 years-plus, n = 511). Demographic factors and serological states (Hp sero-staus and sPG levels) were recorded by subject interview and serum ELISAs, respectively. Covariate-specific odds ratios were calculated by multivariable logistic regression. Hp infection was consistently associated with increased sPGI and sPGII levels in the 60 year-plus, but not the middle-aged group. The joint examination of the variable states of the three serum biomarkers (Hp serology, sPGI, and sPGI/II ratio), in the 60 year-plus age group, demonstrated a stepwise escalation of risk from the single (sPGIlow; OR = 2.6), to double (sPGIlow/sPGI/IIlow; OR = 3.55, and Hppositive/sPGIlow; OR=5.0) and ultimately triple (Hppositive/PGIlow/PGI/IIlow; OR=10.48) positive states, in reference to the triple negatives. However, this pattern was not exhibited in the middle-aged subjects. Age was clearly identified as a modifying factor on the risk projection of the combined states of Hp serology and sPG levels in gastric cancer screening, reflected by the augmented (~10.5 fold) risk of GC in the triple positive (Hppositive/sPGIlow/sPGI/IIlow) 60 year-plus subjects, which was not evident in the middle-aged group.

Reduced susceptibility of Clostridium difficile to antibiotics is problematic in clinical settings. There is new evidence indicating the cotransfer of toxin-encoding genes and conjugative transposons encoding resistance to antibiotics among different C. difficile strains. To analyze this association, in the current study, we evaluated the frequency of toxigenic C. difficile among the strains with different multidrug-resistant (MDR) profiles in Iran. Antimicrobial susceptibility patterns and minimal inhibitory concentrations (MIC) of the isolates were determined against metronidazole, imipenem, ceftazidime, amikacin, and ciprofloxacin by agar dilution method. The association of the resistance profiles and toxigenicity of the strains were studied by PCR targeting tcdA and tcdB genes. Among 86 characterized strains, the highest and lowest resistance rates were related to ciprofloxacin (97%) and metronidazole (5%), respectively. The frequency of resistance to other antibiotics was as follow: imipenem (48%), ceftazidime (76%), and amikacin (76.5%). Among the resistant strains, double drug resistance and MDR phenotypes were detected in the frequencies of 10.4% and 66.2%, respectively. All of the metronidazole-resistant strains belonged to tcdA +/tcdB + genotype with triple or quintuple drug resistance phenotypes. MIC50 and MIC90 for this antibiotic was equally ≤ 8 μg/ml. These results proposed the association of tcdA +/tcdB + genotype of C. difficile and the emergence of resistance strains to broad-spectrum antibiotics and metronidazole.

Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran. Suspected patients (n = 165) were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012-2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b (Cyt b) markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed. Only L. major was identified in patients containing regular amastigotes'' shapes (oval or round) with a size of 2-4 µm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes (spindle, pear, or cigarette) were observed separately in non-classical wet lesions with more than 4 µm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation (χ 2 P > 0.05), including only one common haplotype (GenBank access no. EF413075). Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits.

Vascular calcification is an important stage in atherosclerosis. During this stage, vascular smooth muscle cells (VSMC) synthesize many osteogenic factors such as osteonectin (encoded by SPARC). Oxidative stress plays a critical role in atherosclerosis progression, and its accumulation in the vascular wall stimulates the development of atherosclerosis and vascular calcification. The osteonectin overexpression has been observed in the arterial wall during the course of atherosclerosis. However, the regulatory mechanism of oxidized low density lipoprotein (oxLDL)-mediated vascular calcification remains to be clarified. The aim of this study was to investigate the effect of oxLDL on the osteonectin gene expression through the Runx2 transcription factor. In this experimental study, VSMC were cultured in F-12K media and then treated with oxLDL. The expression of Runx2 and osteonectin genes was determined by real-time PCR method. Protein levels were investigated by the western blotting technique. The Runx2 gene was knocked down using siRNA in order to determine whether Runx2 regulates the osteonectin expression in VSMC induced by oxLDL. Then transfected cells were treated with oxLDL, and the expression levels of Runx2 and osteonectin were determined again. oxLDL was found to increase Runx2 and osteonectin gene expression (4.8 ± 0.47- and 9.2 ± 1.96-fold, respectively) after 48 h. Western blotting analysis confirmed the induced levels of Runx2 and osteonectin proteins. However, oxLDL-induced osteonectin expression was not observed to be blocked by Runx2 knockdown. The up-regulation of osteonectin by oxLDL is independent of Runx2, and it may be mediated by other transcription factors.

In previous studies, the neuroprotective effect of 17β-estradiol in diffuse traumatic brain injury has been shown. This study used ICI 182,780, a non-selective estrogen receptor antagonist, to test the hypothesis that the neuroprotective effect of 17β-estradiol in traumatic brain injury is mediated by the estrogen receptors. The ovariectomized rats were divided into eight groups. Brain injury was induced by Marmarou’s method. Estrogen was injected 30 minutes after traumatic brain injury, and ICI 182,780 was injected before traumatic brain injury and also before estrogen treatment. In one group only ICI 182,780 was injected. The brain water content and Evans blue dye content were measured 24 and 5 hours after traumatic brain injury, respectively. The neurologic outcomes and intracranial pressure were assessed before, 4, and 24 hours after traumatic brain injury. Brain water content and Evans blue content were less in estrogen-treated group comparison to vehicle group. ICI 182,780 eliminated the effects of estrogen on brain edema and brain blood barrier permeability. Intracranial pressure was increased significantly after trauma, and estrogen decreased intracranial pressure at 4 and 24 hours after traumatic brain injury in comparison to vehicle. This inhibitory effect was also eliminated by treatment with ICI182,780. ICI 182,780 also inhibited the estrogen induced increase in neurologic outcomes following traumatic brain injury. However, the use of ICI 182,780 alone had no neuroprotective effect after traumatic brain injury. The results suggest that classical estrogen receptors have probably a role in the neuroprotective function of estrogen following traumatic brain injury.

The serum concentration of high-density lipoprotein cholesterol (HDL-C) is one of the important heritable risk factors for cardiovascular disease and is a target for therapeutic intervention. In this study, we aimed to evaluate the effects of lecithin cholesterol acyltransferase (LCAT) gene polymorphism rs5923 on LCAT enzyme activity and serum HDL-C concentration. The study population was selected from consecutive individuals with HDL-C ≤ 5th percentile (n = 73) and extremely high HDL-C ≥ 95th percentile (n = 57) who had participated in the Tehran Lipid and Glucose Study. The rs5923 polymorphism was genotyped using direct sequencing. LCAT activity was measured by fluorometric assay kit, and lipid concentrations were measured using the enzymatic colorimetric method. The genotype frequencies were significantly different between the high HDL-C group (CC 94.7%, CT 5.3%) and the low HDL-C group (CC 83.6%, CT 16.4%) (P = 0.048). The T-allele frequencies in subjects with low and high HDL-C were 0.082 and 0.026, respectively (P = 0.16). The association of the single-nucleotide polymorphism rs5923 with low HDL-C was not statistically significant after adjustment for age, sex, and BMI (odd ratio = 2.65, 95% confidence interval = 0.32-21.5, P = 0.36, regression logistic analysis). Also, the effects of LCAT enzyme activity did not depend on the HDL-C level (P = 0.24). rs5923 polymorphism is not associated with low HDL-C levels in Iranian population

β-thalassemia is the most common monogenic disorder in human. The (CT) polymorphism at -158 upstream region of the γG-globin gene and pharmacological factors such as hydroxyurea have been reported to influence γ-globin gene expression and the severity of clinical symptoms of β-thalassemia. In the present study, 51 β-thalassemia intermediate patients were studied. Xmn1γG polymorphism genotype was determined using Tetra-Primer ARMS-PCR technique. Hemoglobin (Hb) and fetal hemoglobin (HbF) levels were determined by gel electrophoresis. Of 51 patients, 35 (68.6%) patients were heterozygous (CT) and 16 (31.4%) patients were homozygous (CC). Of 30 patients under treatment by hydroxyurea, 20 (66.7%) patients were heterozygous (CT) and 10 (33.3%) patients were homozygous (CC). Our results demonstrated that in the heterozygous (CT) genotype, the Hb (9.58 ± 1.25 gm/dl) and HbF (89.30 ± 21.87) levels were significantly higher in comparison with homozygous (CC) genotype (7.94 ± 1.34 gm/dl and 70.32 ± 40.56, respectively). Furthermore, we observed that after drug usage, the Hb and HbF levels in patients with heterozygous (CT) genotype (0.7 ± 1.26 gm/dl and 5.95±14.8, respectively) raised more in comparison with homozygous (CC) genotype (0.26 ± 1.43 gm/dl and 0.8±1.31, respectively). Hb and HbF levels in the patients carrying T allele are increased significantly, and they also response to hydroxyurea treatment.

The variable numbers of tandem-repeat (VNTR) alleles at the phenylalanine hydroxylase (PAH) gene have been used in carrier detection and prenatal diagnosis in phenylketonuria families. This study was carried out to analyze VNTR alleles at the PAH gene in Iranian Azeri Turkish population. In this study, 200 alleles from general population were studied by PCR. The frequencies of VNTR alleles were 45%, 46%, 2%, 3%, 1%, and 3% in studied group regarding 3, 8, 9, 11, 12, and 13 repeat copies, respectively. Statistically significant differences were not found between expected and observed frequencies of VNTR genotypes (P > 0.05). VNTR alleles with three and eight repeats were frequent, and the VNTR alleles with 13 repeats showed 3% frequency in the tested group. This study is the first report on tested population genetic structure using VNTR alleles at the PAH gene.