High prevalence of fluoroquinolone-resistant Escherichia coli strains isolated from urine clinical samples.

BACKGROUND: Fluoroquinolone resistant Escherichia coli isolates have become an important challenge in healthcare settings in Iran. In this study, we have determined Fluoroquinolone resistant E. coli isolates (from both outpatients and inpatients) and evaluated mutations of gyrA and parC within the quinolone resistance-determining regions (QRDR) of these clinical isolates. MATERIALS AND METHODS: Clinical isolates were recovered from the urine sample of patients with urinary tract infections admitted at Alzahra hospital, Iran, between September and February 2013. We assessed antimicrobial susceptibility of all isolates and determined mutations in QRDR of gyrA and parC genes from 13 fluoroquinolone-resistant isolates by DNA sequencing. RESULTS: A total of 135 E. coli strains were obtained from 135 patients (91 outpatients and 44 inpatients). The resistance rate of fluoroquinolones (Ciprofloxacin, Norfloxacin and Ofloxacin) among our strains was 45.2%. Two E. coli isolates were shown just a single mutation, but other isolates possessed 2-5 mutations in gyrA and parC genes. Mutations in the QRDR regions of gyrA were at positions Ser83 and Asp87 and parC at positions Ser80, Glu84, Gly78. CONCLUSIONS: Ciprofloxacin is the most common antimicrobial agent used for treating urinary tract infections (UTIs) in healthcare settings in Iran. Accumulation of different substitutions in the QRDR regions of gyrA and parC confers high-level resistance of fluoroquinolones in clinical isolates.

Evaluation of a novel lentiviral vaccine expressing KMP11-HASPB fusion protein against Leishmania infantum in BALB/c mice.

Hydrophilic acylated surface protein B (HASPB) is an immunogenic Leishmania protein against which antibodies are produced in the sera of cutaneous and visceral Leishmaniasis (VL) patients. Kinetoplastid membrane protein 11 (KMP11) is another protein antigen of Leishmania which is reported as a promising candidate for vaccination of VL. It is a highly conserved surface protein present in all members of kinetoplastid family and is expressed in both promastigotes and amastigotes. In this study, the coding sequence of KMP11 and HASPB was cloned into a pCDH-cGFP lentiviral vector as a fusion protein. The gene expression was confirmed using RT-PCR and Western blot methods. After injection of the recombinant KMP11-HASPB-expressing lentiviruses to BALB/c mice, using ELISA technique, a significant increase in IFN-γ and IL-4 as well as IgG1 and IgG2a was observed compared to the control group. Furthermore, the number of parasites in the liver and spleen of vaccinated mice decreased significantly compared with the control group.

Antiproliferative effect of a synthetic aptamer mimicking androgen response elements in the LNCaP cell line.

Prostate cancer usually develops to a hormone-refractory state that is irresponsive to conventional therapeutic approaches. Therefore, new methods for treating aggressive prostate cancer are under development. Because of the importance of androgen receptors (ARs) in the development of the hormone-refractory state and AR mechanism of action, this study was designed. A single-stranded DNA as an aptamer was designed that could mimic the hormone response element (HRE). The LNCaP cells as an AR-rich model were divided into three sets of triplicate groups: the test group was transfected with Aptamer Mimicking HRE (AMH), Mock received only transfection reagents (mock) and a negative control. All three sets received 0, 10 and 100 nM of dehydroepiandrosterone (DHEA) separately. Data analysis showed hormone dependency of LNCaP cells in the negative control group upon treatment with 10 and 100 nM DHEA (compared with cells left untreated (P=0.001)). Transfection of AMH resulted in significant reduction of proliferation in the test group when compared with the negative control group with 10 (P=0.001) or 100 nM DHEA (P=0.02). AMH can form a hairpin structure at 37 °C and mimic the genomic HRE. Hence, it is capable of effectively competing with genomic HRE and interrupting the androgen signaling pathway in a prostate cancer cell line (LNCaP).

Using intron splicing trick for preferential gene expression in transduced cells: an approach for suicide gene therapy.

Suicide gene therapy is one of the most innovative approaches in which a potential toxic gene is delivered to the targeted cancer cell by different target delivery methods. We constructed a transfer vector to express green fluorescent protein (GFP) in transduced cells but not in packaging cells. We placed gfp under the control of the cytomegalovirus (CMV) promoter, which is positioned between the two long-terminal repeats in reverse direction. The intron-2 sequence of the human beta globin gene with two poly-A signals and several stop codons on the antisense strand was placed on the leading strand between the CMV promoter and gfp. For lentiviral production, the HEK293T and line were co-transfected with the PMD2G, psPAX2 and pLentiGFP-Ins2 plasmids. The HEK293T and line were transduced with this virus. PCR was performed for evaluation of intron splicing in transduced cells. The GFP expression was seen in 65% of the cells transduced. The PCR amplification of the genomic DNA of transduced cells confirmed the splicing of intron 2. The strategy is significant to accomplish our goal for preserving the packaging cells from the toxic gene expression during viral assembly and the resultant reduction in viral titration. Also it serves to address several other issues in the gene therapy.

Analysing two dinucleotide repeats of FVIII gene in Iranian population.

Using dinucleotide repeats for carrier detection and prenatal diagnosis of haemophilia A patients, led us to find different alleles and their frequencies in Iranian population. Polymerase chain reaction (PCR) amplification of two short tandem repeat (STR) loci of factor VIII (FVIII) gene was performed, and the PCR products were resolved on 10% native polyacrylamide gel, and samples were analysed with sequenced DNA markers made of PCR cloning of the dinucleotide FVIII gene fragments. Seven different alleles were observed for intron 13 STR, having 18-24 (CA) repeating units and five alleles for intron 22 STR having 24-28 repeating units of (CACT). Bands produced during dinucleotide study were defined in detail so this could improve the genotyping of heterozygotes and homozygotes. Conformational band produced were characterized to specify the dinucleotide pattern. Our results confirm the Hardy-Weinberg proportions of the heterozygosity rate of the 85 analysed individuals. The observed heterozygosity rate for intron 13 and 22 was 52% and 59% respectively. Our data also indicate that our population is closer to caucasians than to any other populations. Finding different dinucleotide repeat alleles and their frequencies has made it possible to identify carriers and provide prenatal diagnosis with more confidence. This allows antenatal diagnosis to be performed in the vast majority of carriers.

A novel single step double positive double negative selection strategy for beta-globin gene replacement.

beta-Thalassemias are a heterogeneous group of autosomal recessive disorders, characterized by reduced or absence of the beta-globin chain production by the affected alleles. Transplantation of genetically corrected autologous hematopoietic stem cell (HSC) is an attractive approach for treatment of these disorders. Gene targeting (homologous recombination) has many desirable features for gene therapy due to its ability to target the mutant genes and restore their normal expression. In the present study, a specific gene construct for beta-globin gene replacement was constructed consisting of: two homologous stems including, upstream and downstream regions of beta-globin gene, beta-globin gene lying between hygromycin and neomycin resistant genes as positive selection markers and thymidine kinase expression cassettes at both termini as negative selection marker. All segments were subcloned into pBGGT vector. The final plasmid was checked by sequencing and named as pFBGGT. Mammalian cell line COS-7 was transfected with linear plasmid by lipofection followed by positive and negative selection. DNA of the selected cells was analyzed by PCR and sequencing to confirm the occurrence of homologous recombination. In this novel strategy gene replacement was achieved in one step and by a single construct.