فصلنامه تازه های بیوتکنولوژی سلولی مولکولی
پیاپی 25 (زمستان 1395)

Aim and Urinary tract infection is one of the most prevalent diseases in human. Unfortunately, the indiscriminate use of antibiotics leads to resistance in bacteria gradually. The aims of this study were to determine the prevalence and distribution of antibiotic resistance to tetracycline resistance genes in uropathogenic Escherichia coli isolated.
Material and In this study, 210 urine samples were collected during four months. Antibiotic susceptibility testing was performed as recommended by the Clinical Laboratory Standards Institute (CLSI) using disk diffusion method. All isolates were then examined for presence of the tetA and tetB genes.
Of the 210 urine samples with UTI collected from hospitals, the 150 isolates of E. coli were isolated. Most of antibiotic resistance was related to tetracycline (68%) and least of antibiotic resistance was related to chloramphenicol (8.66%). Among isolates (86.27%), were tetA gene and in 83 isolates (81.37%) tetB gene was detected. 74 (72.45%) isolates were positive for both genes, and 5 (4.90%) isolates were negative for them.
Resistance to tetracycline and other antibiotics, and the presence of tetA and tetB genes in UPEC strains are alarming signs in our area. The current study strongly recommends restricted the consumption of antibiotics including tetracycline. Further studies should be conducted in order to find out the extent of the problem in other areas.

Aim and Alarming increase in fungal infections, especially in immunocompromised patients against the infectious agents and the vast advances in diagnostic methods of such diseases, causing extensive investigations for the diagnosis of fungal infections with more modern methods in different groups of patients. Our main objective is to evaluate and compare methods of laboratory diagnosis of smear-culture-molecular in fungal infections (dermatophytes, Aspergillus, Mucor) in clinical samples.
since August 2014 to January 2015, 420 cases of suspected fungal infections, from the Razi Hospital, Baqiyatallah hospital and Massoud laboratory were collected. Smear preparation of the samples was carried out and on the basis of these findings, type of lesion and diagnosed by specialists, samples are grouped. Culture and molecular methods (PCR and PCR-Sequencing) at the same time was performed.
Of the 420 samples analyzed by direct smear, 15 (5/3%) of Aspergillus, 64 samples (15/2%) of dermatophytes and zero sample of Mucor were identified. In 15 specimens of Aspergillus, 12 samples (80%) consisted with positive cultures and 3 cases (20%) were negative and based on Nested PCR results, all of 15 samples were positive. Of 64 samples of dermatophytes, 40 cases (62/5%) were positive by culture method and 16 cases (25%) were negative. 6 samples (3/9%) consisted with saprophytic contamination and 2 samples (12/3%) showed Aspergillus growth at the injection site, respectively. Based on Nested PCR method, 62 samples (8/96%) showed positive result.
The results of this study indicate that the PCR, with respect to its high sensitivity, bearings good capabilities for detection of fungal disease in the early stages of infection. Since the rapid and specific diagnosis of fungal infections, especially with regard to the genera and species of the fungal organisms, provide the possibility of proper and adequate applying of antifungal therapy protocols. Thus in this context, appropriate performance of rapid, specific and sensetive techniques would be necessary and inevitable.

Aim and Successful in vitro embryo production extremely depends on the components of culture media during in vitro maturation. With ingredients like Anitol, Fennel has properties similar to estrogen. The aim of this study was to investigate the effect of Fennel essence on nuclear maturation of bovine oocyte.
In order to in vitro maturation off oocytes, Cumulus-oocyte complexes (No. 772) from follicles 2–8 mm in diameter were aspirated and cultured in M199 maturation media containing 10% FBS, 10% bovine follicular fluid, 5 µg/ml FSH, 1 µg/ml oestradiol- 17β, 0.81mM sodium pyruvate and 50 µg/ml gentamicin sulfate. Maturation culture media supplemented with 0, 5, 10, 20, 30 and 50 mg/ml of Fennel essence and were cultured in a CO2 incubator containing 5% CO2 and 95% humidity in air at 38.5ºC for 24 h.
Results showed that the amount of maturation of oocytes under 5, 10, 20 and 30 mg/ml of Fennel essence were not significantly different based on expansion of cumulus cells and polar body extrusion in compared with control (P>0.05). However, 50 mg/ml of Fennel essence significantly decreased expansion of cumulus cells and polar body extrusion in compared to other groups (P From the results of current study we can conclude that, Fennel essence, based on cumulus expansion and polar body extrusion, unable to improve nuclear maturation of bovine oocytes. In order to investigate the effect of Fennel essence on nucleus and cytoplasmic maturation of bovine oocytes, it is suggested to use in vitro fertilization.

Aim & Multiple Sclerosis is one of the most common inflammatory central nervous system diseases. In patients, there is a defect in apoptotic pathway making T auto reactive cells non-apoptotic and entering to blood stream. Among the genes related to this disease, there is a gene named CD95L involved in apoptosis pathway.
Material & In this survey, we included 30 MS patients and 30 healthy controls which were matched by sex and age. Blood was taken from cases and controls with consent. Next, total RNA was extracted and cDNA was synthesized. Then, by TaqMan Real-time PCR technique, the expression level of CD95L and HGPRT genes was evaluated. The results obtained were analyzed by Linreg and Rest softwares.
In this study, CD95L expression in MS patients did not show any significant decrease compared to healthy controls (p Value: 0.197). In addition, in comparison of CD95L expression level between men and women no significant decrease was seen (p Value (M): 0.142, p Value (F): 0.156).
In this study, no significant difference of CD95L gene expression between multiple sclerosis patients and healthy controls was seen.

Aim and Multiple sclerosis (MS) is an autoimmune demyelinating disease. Many different gene association studies (GAS) have been used some single nucleotide polymorphisms (SNPs) in interleukin 7 receptor alpha (IL7Rα) gene as MS disease susceptibility, mainly in populations in western countries. There have been few reports of association between IL7Ra gene and MS in Asian population. SNPrs11567685 is in promoter region and probably involved in IL7Rα gene expression.
A total of 100 unrelated patients and 100 unrelated ethnically matched healthy controls were studied in Khuzestan province. The genotype and allele frequencies of this SNP measured between MS cases and controls by using PCR-RFLP. Cases and controls were analyzed for Hardy-Weinberg equilibrium and significance differences by using chi-square test.
Our population was at Hardy-Weinberg equilibrium and significant association was found in SP MS (Secondary Progressive) and SP MS (Secondary Progressive Primary Progressive) in Allelic and Genotypic frequencies at SNPrs11567685. Also, significant associations were found in Male and Female patients in MS subtypes.
It was indicated in this study that Promoter region of IL7Rα gene has significant effect on MS disease. So, this gene region can be used as a biomarker in early diagnoses of MS.

Aim & Frankincense is a herbal product that has diverse therapeutic effects. Beta-boswellic acid, the main ingredient of frankincense, is poorly soluble in water and its bioavailability is very low. The aim of present study was to evaluate the effect of loading of beta-boswellic acid in dendrosomal nanoparticles on its bioavailability and cell uptake by MTT assay.
For MTT assay, B65 cells were treated with different doses of nave beta-boswellic acid or dendrosomal beta-boswelic acid for 24, 48 and 72 hours. After adding MTT, colorimetery was done using ELISA reader and IC50 was then calculated. The data was analyzed with One Way Anova program using SPSS v.16 software.
The IC50 for nave beta-boswellic acid and dendrosomal beta-boswellic acid was 88/09 and 58/42 µM in 24h, 58/37 and 44/87 µM in 48h and 21/09 and 16/69 µM in 72h respectively.
The results showed that loading of beta-boswellic acid in dendrosomal nanoparticles increases the cell toxicity effect of beta-boswellic acid. This observation might be due to the increased cell uptake of beta-boswellic acid in the presence of dendrosomal nanoparticles.

Aim and Considering the widespread application of silver nanoparticles in daily life, the side effects to humans, it is growing increasingly. But the effects of silver nanoparticle on the stage of embryonic development are not fully understood. This study investigates the potential teratogenic effects of silver nanoparticles on the developmental stages of fetal rats.
silver nanoparticle concentrations of 0, 125, 750 and 1500 mg / kg / day orally (gavage) administrated from days sixth to nineteenth pregnancy. The embryos harvested via cesarean on day nineteenth gestation. Weight and CR (Crown-Rump) embryos separately were measured. The CR measured by Image j software. The length of the tail, the limbs, fingers and toes, edema, eyes and nose during all the embryos were examined
According to data obtained, we could not find any significant different in the length of the tail, the limbs, fingers, edema, eyes and nose between the experiments and control groups. The weight and CR of experiments embryos were increased in compare with control groups and shows significant different except that the mean the 125 mg / kg / day grout that did not show significant different.
Based on these results, we can conclude that silver nanoparticles can effect on embryo development stages. These changing are dose dependent and can increase the weight and CR of rat embryo at 19th day gestation.

Aims and AmpC β-lactamases hydrolyze penicillins, cephalosporins and cephamycins and resist inhibition by clavulanate, sulbactam and tazobactam. In this study, has been paid to prevalence of AmpC beta-lactamase enzymes produced by Escherichia coli isolates from various hospitals of Kermanshah city.
Material and A total of 100 clinical isolates of Escherichia coli were collected from the city of Kermanshah and after identification screened by Disk diffusion agar method and combined disk for production AmpC beta-lactamase. The isolates that were phenotypic have features these enzymes tested by Multiplex PCR. Also resistance of ESBLs producing isolates to ciprofloxacin, amikacin, meropenem and azteronam was measured by Microdilution broth for determination MIC (Minimal inhibitor concentration).
In this study, 43% of clinical isolates were Ampc beta-lactamase producers, based on phenotypic. After most review by Multiplex PCR showed 44.1% of them has Ampc beta-lactamase gene.
Prevalence of AmpC beta-lactamase due to ineffective prescription of the third of generation cephalosporins has become a major challenge for the treatment of disease. Phenotypic tests and Molecular methods such as PCR the detection of beta-lactamases enzymes can be very useful. Although this study has shown a high prevalence of AmpC beta-lactamase the city of Kermanshah, which is major cause of the self use of antibiotics or lack of antibiogram before beginning treatment. Lack of knowledge about phenotypic and molecular methods can provoke the development of resistance of AmpC beta-lactamase.

Aim & Drug delivery, and using of nanocarriers like pegylated nanoliposomal cisplatin are known as one of the effective method for cancer treatment. In this study, various nanoparticle formulations of cisplatin were prepared and efficacy of the prepared formulations were evaluated in vitro environment.
Reverse phase evaporation technique was used to obtain the pegylated nanoliposomal cisplatin. Cytotoxicity of pegylated nanoliposomal cisplatin were determined in A2780CP ovarian cancer cell lines by using MTT assay. Loading percentage of cisplatin was obtained by inductively coupled plasma spectroscopy.
The mean size, and zeta potential of nanodrug formulations were obtained 100 ± 2.2 nm, and -18.1 ±1.5 mV , respectively. Cytotoxicity of free drug and nanodrug (IC50 values) were estimated 76.6 ± 3.1 µg/mL , and 46.6 ± 2.3 µg/mL in A2780CP ovarian cancer cell lines, respectively.
Results of this study showed that liposomal nanoparticles could be considered as a suitable nanocarrier for cisplatin. It showed that cytotoxicity of nanodrug was increased significantly in comparison to free drug.

Aim and Urtica dioica L. is used as medicinal plants to cure some disease such as; digestives disease or declined blood sugar and hypertension. It is known that by the increasing of population the confirmed need of pharmacy industries to medicinal plants as well as, the importance of their effective materials in nutritional adornment and hygienic are caused them to deserve special attention and research at the point of cultivation, production and consumption. In this research sample plants were collected central area of Mazandaran province was studied in aspect of genetic variation.
In order to evaluate the genetic diversity, the genomic DNA was extracted by using modified CTAB protocol. The evaluation of quality and quantity of DNA was examined with agaros gel electrophoresis. Six RAPD markers have been used for PCR analysis.
Out of 155 RAPD bands, 68/38% were polymorphism. The data were analyzed with NTSYS programs. The dandrogram was drawn based on UPGMA.
Investigation the genetic variation of this plant with RAPD indicated that this marker is suitable to determine the polymorphic loci and to estimate the genetic distance.

Aim and Uncomplicated urinary tract infections (UTIs) are among the most common infectious diseases in human. Escherichia coli is the causal pathogen in UTIs. For treatment of UTIs, fluoroquinolones are used. fluoroqinolones-resistant E. coli was showed by alterations in the genes that encode for the quinolone target subunits.
one hundred isolates of the urinary samples were collected. E. coli isolates were identified by biochemical tests. The minimal inhibitory concentration (MIC) of fluoroquinolones were determined for isolates. DNA was extracted and PCR amplification was carried out for the specific regions of gyrA and parC genes. Then amplified regions were sequenced.
rom 100 samples, 70 samples were E. coli, 30 samples were Kellebsiella. Evaluating MICs for 70 E. coli isolates to fluoroquinolones, it was found that 45 (64.28%) were resistant to quinolones. Sequencing of gyrA and parC specific subunits has revealed five single mutations in the gyrA gene and two single mutations in parC gene.
Cunclusion: Fluroquinolones are potent antibacterial agents for treatment of UTIs. Mutations of DNA gyrase and topoisomerase IV associate with the fluoroquinolones resistance in E. coli. Results have indicated a relationship between MICs of the quinolones for E. coli isolates with mutations in gyrA and parC genes. This study also signifies an association between mutations in gyrA and parC genes in E. coli isolates with fluoroquinolone resistance.

Aim & Iron oxide nanoparticles as an element, inducing contrast in nuclear magnetic resonance (MRI). Nevertheless, effects of nanoparticles on human health have not yet been fully investigated. In this study, using chitosan coating iron oxide nanoparticles on the hematological toxicity is investigated.
A total of 60 adult female Wistar rats were divided into 10 groups of 6 rats. Concentrations of 50, 100 and 150 mg of chitosan per kg body weight nanoparticles coated which chitosan, were injected intraperitoneally into of rats. Then, hematological parameters (WBC, RBC, PLT and MCV) were measured.
Results were analyzed using SPSS 20 software (One way ANOVA). The average number of blood cells (WBC and RBC) in the groups treated with higher concentrations of 50 mg/kg body weight was significantly decreased as compared to the control animals. There was no fatality among the animals during the experiment.
According to the results, it can be concluded that short-term use of iron oxide nanoparticles coated with chitosan does not induce specific toxicity in the body of experimental animals.

Aim and Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are two forms of non-melanoma skin cancer. Recent research indicated that Merkel cell polyomavirus (MCPyV) is associated with Merkel cell Carcinoma (MCC). So, the aim of current study was presence of MCPyV in the tumor tissue and role of large T antigen (LT Ag) mutations that associated with MCPyV oncogenic properties in LTAg region in positive samples that referred to Razi Hospital in Tehran within 2015-2016.
In this cross-sectional study, 55 tissue samples containing: 30 BCC, 10 SCC and 15 individual healthy samples were collected and genomic DNA was extracted. To finding positive samples, quantitative Real-Time PCR was done and the full-length of Large T antigen region was amplified and sequenced directly for detection of probable mutations.
MCPyV genome was detected only 10% of BCC samples. There is no correlation between sex (P-Value=0.33), age (P-Value=0.5), or stage of cancer (P-Value=0.25) and the presence of MCPyV genome in our populations of study.
Viral infections are one of the factors in causing cancer. Although, several studies were reported that frame shift mutation at the N-terminus of LT Ag region is associated with tumorigenesis of the virus, but in current study, no any mutations causing stop codons or frame shifting were observed. As our knowledge this is the first study presenting data on the prevalence of MCPyV in non-melanoma skin cancer from Iranian patients. However, further studies with more samples are necessary.

Aim and Cardiomyocytes derived from human embryonic stem cells (hESCs) could be useful in restoring heart function after myocardial infarction or in heart failure. The hESCs can differentiate in vitro into spontaneously contracting cardiomyocytes and produce a model to investigate the early developmental stages of this system. After removing of cells from their feeder layer, hESCs create embryoid bodies (EB). Plating of EB result in developing areas of beating cells.
After stem cell culture, cardiomyocyte’s mRNA extracted in different time after differentiation. The expression pattern CD-34, OCT-4 and Brachyury were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR).
There was an enhanced expression of as CD-34 from day 21 in EB in suspension. The OCT-4 gene expression was in 5-day-old EB and Brachyury expression was significantly increased by day 21.
hESCs might be useful as an effective model system for understanding the developmental processes and functioning of the human heart.