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Azurin protein of Pseudomonas aeruginosa is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular immune. The purpose of the present study was to investigate the effects of simultaneous expression of azurin and human MAM-A genes on the mRNA expression level of apoptosis-related and cell cycle genes in MCF-7 breast cancer cell line. The recombinant or empty plasmids were separately transferred into MCF-7 cells using Lipofectamine reagent. Flow cytometry was done to detect cell death and apoptosis. The expression of azurin and MAM-A genes were evaluated by IF assay, RT-PCR and western blot methods. Finally, apoptosis-related and cell cycle genes expression was examined in transformed and non-transformed MCF-7 cells by qPCR method. The successful expression of azurin and MAM-A genes in the MCF-7 cell were confirmed by RT-PCR, IF and western blotting. The apoptosis assay was showed a statistically significant (p 0.05). Co-expression of azurin and MAM-A genes could induce apoptosis and necrosis in human MCF-7 breast cancer cells by up-regulation of BAK, FAS, and BAX genes. In future researches, it must be better the immune stimulation of pBudCE4.1-azurin-MAM-A recombinant vector in animal models and therapeutic approaches will be evaluated.

Background: Genetically engineered microorganisms (GEMs) can be used for bioremediation of the biological pollutants into nonhazardous or less-hazardous substances, at lower cost. Polycyclic aromatic hydrocarbons (PAHs) are one of these contaminants that associated with a risk of human cancer development. Genetically engineered E. coli that encoded catechol 2,3- dioxygenase (C230) was created and investigated its ability to biodecomposition of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) measurement. We revised patents documents relating to the use of GEMs for bioremediation. This approach have already been done in others studies although using other genes codifying for same catechol degradation approach. Objective: In this study, we investigated biodecomposition of phenanthrene and pyrene by a genetically engineered Escherichia coli. Methods: Briefly, following the cloning of C230 gene (nahH) into pUC18 vector and transformation into E. coli Top10F, the complementary tests, including catalase, oxidase and PCR were used as on isolated bacteria from spiked soil. Results: The results of HPLC measurement showed that in spiked soil containing engineered E. coli, biodegradation of phenanthrene and pyrene comparing to autoclaved soil that inoculated by wild type of E. coli and normal soil group with natural microbial flora, were statistically significant (p Conclusion: These findings indicated that genetically manipulated E. coli can provide an effective clean-up process on PAH compounds and it is useful for bioremediation of environmental pollution with petrochemical products.

Background and aims: Breast cancer is one of the most common types of malignancy in women with morbidity and mortality (15.0%) in the world. The antitumor activity of azurin protein produced by Pseudomonas aeruginosa has been described before. Mammaglobin-A (MAM-A) protein is especially expressed in 40%-80% of breast cancer types and this protein is a very specific molecular marker for stimulating the immune system. Accordingly, this study investigated the effects of pBudCE4.1-azurin-MAM-A recombinant vector on the induction of the immune system in laboratory mice by the real-time polymerase chain reaction (PCR) method.Methods: The pBudCE4.1-azurin-MAM-A recombinant and empty vectors were purchased and then separately transformed into Escherichia coli for multiplying. Next, each plasmid was extracted and the accuracy of transformation was confirmed by the PCR. These recombinant and empty (control) vectors were separately infused into the thigh muscle of the animals and the healthy group was infused with phosphatebuffered saline. The infusion sites, blood specimens, as well as the serum of the animals were collected and examined by serological and molecular tests.Results: Molecular and serological studies showed that the serum and expression levels of IL-2, IL-6, IL-7, and IL-10 in infused mice with pBudCE4.1-azurin-MAM-A recombinant vector significantly increased compared to healthy animals and injected mice with an empty vector (PConclusion: In general, the findings revealed that the pBudCE4.1-azurin-MAM-A recombinant vector can stimulate the immune system of the mouse by an increase in the expression levels of IL-2, IL-6, IL-7, and IL-10. Thus, it would be better to examine the effects of this recombinant vector as a DNA vaccine on the prevention and treatment of breast cancer.

Background and aims : Human immunodeficiency virus type 1 (HIV-1) based-lentivirus vector is one of the most promising viral vectors for gene delivery in different cell lines including gastric cell lines. Therefore, the aim of this study was to produce a lentivirus vector for transduction and expression of green fluorescent protein (GFP) in human gastric cancer cell line, AGS. Materials and Methods: In this piece of work, Escherichia coli HB101 was transformed with plasmids psPAX2, pTD, and pMD2.G, following the purification of which their DNA was extracted along with their quantity and quality evaluated to be used in the next experiments. Subsequently, to produce the vector, the packaging cells were transfected with the plasmids and the vector containing supernatant was collected and purified using ultracentrifuge. ELISA was used to confirm the construction of the vector. Fluorescent microscopy and flow cytometry were used to check the expression of GFP in the cell line and to calculate the percentage of GFP expression, respectively. Results: In this study, the results of ELISA confirmed the construction of the plasmid used in this study. AGS cells were infected with viruses produced to detect the viral activity and GFP expression was evaluated by fluorescence microscopy and flow cytometry after 72 hours. Based on the results of flow cytometry, GFP was expressed in over 90% of transduced AGS cells. Conclusion: The results of this study showed that lentiviral vector is a highly efficient vector for expression of GFP gene in AGS cell line.

COVID-19 is considered as the third human coronavirus and has a high potential for transmission. Fast public health interventions through antibodies, anti-virals or novel vaccine strategies to control the virus and disease transmission have been extremely followed. SARS-CoV-2 shares about 79% genomic similarity with SARS-CoV and approximately 50% with MERS-CoV. Based on these similarities, prior knowledge in treating SARS-CoV and MERS-CoV can be used as the basis of majority of the alternatives for controlling SARS-CoV-2. Immunotherapy is an effective strategy for clinical treatment of infectious diseases such as SARS-CoV-2. Passive antibody therapy, which decreases the virus replication and disease severity, is assessed as an effective therapeutic approach to control SARS-CoV-2 epidemics. The close similarity between SARS-CoV-2 genome with the SARS-CoV genome caused both coronaviruses to bind to the same angiotensin-converting enzyme 2 (ACE2) receptors that found in the human lung. There are several strategies to develop SARS-CoV-2 vaccines, which the majority of them are based on those developed previously for SARS-CoV. The interaction between the spike (S) protein of SARS-CoV-2 and ACE2 on the host cell surface leads to the initiation of SARS-CoV-2 infection. S protein, which is the main inducer of neutralizing antibodies, has been targeted by most of these strategies. Vaccines that induce an immune response against the S protein to inhibit its binding with the host ACE2 receptor, can be considered as effective vaccines against SARS-CoV-2. Here, we aimed to review frontier therapeutics and vaccination strategies for SARS-CoV-2 (COVID-19).

Azurin protein of Pseudomonas aeruginosa is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular immune. The purpose of the present study was to investigate the effects of simultaneous expression of azurin and human MAM-A genes on the mRNA expression level of apoptosis-related and cell cycle genes in MCF-7 breast cancer cell line. The recombinant or empty plasmids were separately transferred into MCF-7 cells using Lipofectamine reagent. Flow cytometry was done to detect cell death and apoptosis. The expression of azurin and MAM-A genes were evaluated by IF assay, RT-PCR and western blot methods. Finally, apoptosis-related and cell cycle genes expression was examined in transformed and non-transformed MCF-7 cells by qPCR method. The successful expression of azurin and MAM-A genes in the MCF-7 cell were confirmed by RT-PCR, IF and western blotting. The apoptosis assay was showed a statistically significant (p 0.05). Co-expression of azurin and MAM-A genes could induce apoptosis and necrosis in human MCF-7 breast cancer cells by up-regulation of BAK, FAS, and BAX genes. In future researches, it must be better the immune stimulation of pBudCE4.1-azurin-MAM-A recombinant vector in animal models and therapeutic approaches will be evaluated.

Methods: A large pore amine functionalized MSN (MSNs-NH2) is described here to facilitate delivery of clarithromycin (CLM) as an antibacterial drug and enhance the efficacy against Gram positive and Gram negative bacterial samples. Prepared particles were characterized by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), nitrogen adsorption/desorption isotherms, Fourier Transform Infrared (FT-IR) spectroscopy and X-ray Diffraction (XRD) spectroscopy. The antimicrobial activity was evaluated by agar well diffusion and broth dilution methods. Therefore, the biodistribution of FITC-MSNs was investigated by measure the NIR intensity fluorescent of fluorescent images from whole animal and dissected organs of NMRI mice. Results: The results showed that the CLM loaded MSNs-NH2 (CLM/MSNs-NH2) were successfully prepared having good payload and pH-sensitive drug release kinetics. The antimicrobial investigation against Staphylococcus aureus and Escherichia coli was showed better performance of antimicrobial activity of these nanoparticles. In vivo and ex vivo fluorescent imaging investigation on NMRI mice were shown that FITC-MSNs-NH2 accumulated in the liver and kidney and notably in lung tissue. Conclusion: The CLM/MSNs-NH2 exhibited higher antimicrobial activity and enhanced the possibility of microbial infection therapy especially at respiratory infections

Cisplatin resistance is one of the main limitations in the treatment of ovarian cancer, which is partly mediated by long noncoding RNAs (lncRNAs). H19 is a lncRNA involving in cisplatin resistance in cancers. Valproic acid (VPA) is a commonly used drug for clinical treatment of seizure disorders. In addition, this drug may display its effects through regulation of noncoding RNAs controlling gene expression. The aim of the present study was the investigation of VPA treatment effect on H19 expression in ovarian cancer cells and also the relation of the H19 levels with apoptosis and cisplatin resistance. Briefly, treatment with VPA not only led to significant increase in apoptosis rate, but also increased the cisplatin sensitivity of A2780/CP cells. We found that following VPA treatment, the expression of H19 and EZH2 decreased, but the expression of p21 and PTEN increased significantly. To investigate the involvement of H19 in VPA-induced apoptosis and cisplatin sensitivity, H19 was inhibited by a specific siRNA. Our results demonstrate that H19 knockdown by siRNA induced apoptosis and sensitized the A2780/CP cells to cisplatin-induced cytotoxicity. These data indicated that VPA negatively regulates the expression of H19 in ovarian cancer cells, which subsequently leads to apoptosis induction, cell proliferation inhibition, and overwhelming to cisplatin resistance. The implication of H19→EZH2→p21/PTEN pathway by VPA treatment suggests that we could repurpose an old drug, valproic acid, as an effective drug for treatment of ovarian cancer in the future.

Background Atoh1 has a pivotal effect on hair cell differentiation and viability. Due to the essential role of this gene in the development and differentiation of inner ear hair cells, this study investigated the mutations in the Atoh1 (also known as Hath1 in humans) gene. Methods In this study, 40 families with deafness living in Chaharmahal va Bakhtiari, Isfahan, and Khuzestan provinces were selected from genetic clinics in Iran. The genomic DNA was extracted from the blood samples of 40 deaf patients, and the Atoh1 gene was studied by PCR amplification followed by sequencing. A High-Resolution Melting Analysis (HRM) method was established to evaluate the melting point of natural and mutant PCR products. The observed allelic changes in the Atoh1 gene were further analyzed in-silico. The ATOH1 protein structure and function were analyzed by Gromax software. Results HRM method revealed a higher melting point for a mutant PCR product compared to the natural sequence; this clear difference could help further rapid, easy, and low-cost detection in other samples. This mutant sample belonged to a patient with syndromic hearing loss who exhibited two missense mutations (c.605A>G and c.397G>A) in the Atoh1 gene. Possible structural and functional outcomes due to the mutation on the ATOH1 protein were predicted and explained by molecular dynamics simulation methods using Gromax software. Conclusion Because of the vital developmental roles, mutations in the ATOH1 gene are mostly lethal and rare. The observation of tolerable mutations in this protein may open doors for a better physiological understanding of the importance of different motifs in this protein.

Objectives Aflatoxin M1 (AFM1)-contaminated dairy products pose serious human health risks, causing liver and renal failure if consumed. They are also related to decreased milk and egg production in infected animals. This study investigated the AFM1 contamination levels in cheeses sold in Isfahan province, Iran, by enzyme-linked immunosorbent assay (ELISA). Methods A total of 100 white cheese samples were randomly collected from supermarkets in Isfahan province and after extraction using dichloromethane were prepared for the ELISA. Results Of the 100 samples, 52 (52%) were contaminated by AFM1, at levels ranging from 50.2 to 424.4 ng/kg. The remaining 48% of the samples had undetectable AFM1 levels (< 50 ng/kg). Based on the standard limit set by the European Commission and Iran, 8% (8/100) of the AFM1-positive samples (with concentrations between 250.2 and 424.4 ng/kg) had levels higher than the permissible value of 250 ng/kg. Conclusion Although the percentage of cheese samples in Isfahan province with AFM1 levels exceeding the national permissible limit was low, the examination of cheeses and the milk used for their production is nevertheless important for ensuring public health. Furthermore, optimum storage conditions of animal feed should be ensured, and livestock nutrition must be monitored for the presence of AFM1 and other aflatoxins.

Trichomonas vaginalis is a causative agent of vaginitis in female and urethritis in men. It is primarily transmitted by sexually route. It is known that each geographical area has its own set of Trichomonas vaginalis strain. Parasite strains in each region have its specific characterizations and different strains of the parasite are able to cause various diseases with the acuity and severity. The aim of this study was to determine the genotyping of Trichomonas vaginalis strains in the Shahrekord city (Chaharmahal Va Bakhtiari province, southwest Iran). A total of 1725 vaginal samples were taken from clinically suspected women for Trichomonas vaginalis infection and 21 specimens were diagnosed as positive by direct smear wet mount and culture repeated passage of the parasite in the modified TYI-S-33 medium. The genomic DNA was extracted from each sample and the nested polymerase chain reaction was applied using specific oligonucleotide primers for actin gene amplification. Finally, the restriction fragment length polymorphism using RsaI, MseI, and HindII restriction enzymes were done on PCR products for genotyping. PCR-RFLP analysis of 21 positive cases (1.22%) was showing the most frequent genotype was H (8 cases), followed by G (4 cases), E (3 cases), and P (2 cases). N and I genotypes were detected in each 1 case. Also, there was 2 cases mix (E and H) genotype. The findings of the present work were showed 7 different genetic strains in isolated Trichomonas vaginalis from symptomatic and asymptomatic women in Shahrekord city. In this study high level of H genotype in referred women in Shahrekord city was observed and H, G, E, and I genotypes were may be related to burning and itching as well as H, P, and mix genotypes were associated with malodorous discharge with pelvic pain in this region of Iran. For a suggestion, it would be better in further studies the accurate determination of genetic diversity of this parasite done in Chaharmahal Va Bakhtiari province and other parts of the country.

Objectives High consumption of bakery products such as cream-filled pastries may cause serious health risks and food poisoning to humans. Therefore, investigation of the microbial and chemical qualities of bakery products containing cream is necessary. The purpose of the present study was to investigate the chemical qualities and microbial contaminations of cream-filled pastries collected from confectioneries located in six cities in Chaharmahal Va Bakhtiari province (Southwestern Iran). Methods Microbial tests and chemical characteristics (fat and acidity level) were done on 228 cream-filled pastries samples that were collected randomly from various confectioneries. Results After microbial tests, it was found that 33.33% of all samples were contaminated by microbial agents. The microbial tests showed that Shahrekord (10.09%) and Broujen (9.21%) cities had high levels of contamination and in Koohrang (1.31%) it was low compared with the other four cities. High contamination of coliforms (61.84%), staphylococci (48.68%), and yeast (27.63%) were observed in almost all samples. The chemical analysis showed maximum amounts of fat content and titratable acidity in cream-filled pastry samples obtained from Lordegan and Shahrekord cities, respectively. Conclusion The findings of the present work demonstrated that the microbial contamination and chemical quality of cream-filled pastries produced in confectionaries of Chaharmahal Va Bakhtiari province were not in acceptable ranges. These problems may be related to fecal contamination of cream samples or lack of hygiene by handlers and it is necessary to observe the standards of hygiene and to develop safe food handling techniques and aseptic pastry manufacturing systems in some confectioneries of Chaharmahal Va Bakhtiari province.

Genetically engineered microorganisms (GEMs) can be used for bioremediation of the biological pollutants into nonhazardous or less-hazardous substances, at lower cost. Polycyclic aromatic hydrocarbons (PAHs) are one of these contaminants that associated with a risk of human cancer development. Genetically engineered E. coli that encoded catechol 2,3- dioxygenase (C230) was created and investigated its ability to biodecomposition of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) measurement. We revised patents documents relating to the use of GEMs for bioremediation. This approach have already been done in others studies although using other genes codifying for same catechol degradation approach.In this study, we investigated biodecomposition of phenanthrene and pyrene by a genetically engineered Escherichia coli.Briefly, following the cloning of C230 gene (nahH) into pUC18 vector and transformation into E. coli Top10F, the complementary tests, including catalase, oxidase and PCR were used as on isolated bacteria from spiked soil.The results of HPLC measurement showed that in spiked soil containing engineered E. coli, biodegradation of phenanthrene and pyrene comparing to autoclaved soil that inoculated by wild type of E. coli and normal soil group with natural microbial flora, were statistically significant (p0.05). Moreover, catalase test was positive while the oxidase tests were negative.These findings indicated that genetically manipulated E. coli can provide an effective clean-up process on PAH compounds and it is useful for bioremediation of environmental pollution with petrochemical products.

In the present study, the simultaneous application of azurin gene of P. aeruginosa and MAM-A antigen on the induction of immune responses against breast cancer tumors was investigated in BALB/c mice. The pBudCE4.1-azurin-MAM-A recombinant vector was generated and prepared at a large scale. This recombinant vector alone or combined with chitosan nanoparticles was infused into the hip muscle of animals. Animals were divided into the “prevention” and “therapy” categories. The animals of prevention category were first, immunized by a recombinant vector and then exposed to chemical cancer inducers; while the animals in the therapy category were first treated with chemical compounds and then infused by a recombinant plasmid. The tumor tissues, infusion sites, and blood specimens were collected and examined by serological, molecular, and histological tests. The breast tumor incidence in the infused animals by recombinant plasmid alone or combined with nanoparticles (in both prevention and therapy categories) compared with infused mice by empty pBudCE4.1 vector was significantly decreased (p

In the present study, the simultaneous application of azurin gene of P. aeruginosa and MAM-A antigen on the induction of immune responses against breast cancer tumors was investigated in BALB/c mice. The pBudCE4.1-azurin-MAM-A recombinant vector was generated and prepared at a large scale. This recombinant vector alone or combined with chitosan nanoparticles was infused into the hip muscle of animals. Animals were divided into the “prevention” and “therapy” categories. The animals of prevention category were first, immunized by a recombinant vector and then exposed to chemical cancer inducers; while the animals in the therapy category were first treated with chemical compounds and then infused by a recombinant plasmid. The tumor tissues, infusion sites, and blood specimens were collected and examined by serological, molecular, and histological tests. The breast tumor incidence in the infused animals by recombinant plasmid alone or combined with nanoparticles (in both prevention and therapy categories) compared with infused mice by empty pBudCE4.1 vector was significantly decreased (p

In recent years antibiotic-resistant bacteria including those producing extended-spectrum β-lactamases increase, which could affect their pathogencity and lead to many disease in both humans and animals. Enterobacteriaceae commonly express plasmid-encoded β-lactamases family like TEM-1 gene, which encodes resistance to penicillin in Escherichia coli. Insects, especially cockroaches, are important vectors to spread these bacteria in human living environmment. The present study was performed to evaluate the frequency of extended spectrum β-lactamase producing gene TEM-1 and the antimicrobial susceptibility patterns of Escherichia coli strains isolated from cockroaches in Iran using antibiogram and molecular techniques. In this study, 110 cockroaches (Periplaneta americana) were collected from residential environments in Chahrmahal Va Bakhtiari province (Southwest Iran) and after bacterial culture on specific media the antimicrobial susceptibility patterns of isolated Escherichia coli strains from cockroaches were evaluated by disk diffusion test and molecular assay. The amplification of TEM-1 gene by PCR revealed a 296 bp fragment on gel electrophoresis. The results of the present research showed that Escherichia coli infection was detected in 84 samples (76.36%) and 11 samples had the TEM-1 gene (10%). The antibiotic susceptibility pattern using the disk diffusion test indicated that most Escherichia coli isolates from cockroaches were sensitive to imipenem (100%) and amikacin (87.8%), while resistance to aztreonam, ceftazidime, and ceftriaxone was 88.1%, 80.5%, and 78.2%, respectively. These findings showed that cockroaches are important agents for transmission of Enterobacteriaceae and that extended-spectrum β-lactamases producing genes like TEM-1 by Escherichia coli could increase the gastrointestinal infections and antibiotic resistance. So, antibacterial drugs such as aztreonam, ceftazidime, and ceftriaxone must be used with caution, while imipenem and amikacin antibiotics are helpful to reduce Escherichia coli infection caused by cockroaches. © 2016, Bulgarian Journal of Veterinary Medicine. All rights reserved.

Bioremediation use to promote degradation and/or removal of contaminants into nonhazardous or less-hazardous substances from the environment using microbial metabolic ability. Pseudomonas spp. is one of saprotrophic soil bacterium and can be used for biodegradation of polycyclic aromatic hydrocarbons (PAHs) but this activity in most species is weak. Phenanthrene and pyrene could associate with a risk of human cancer development in exposed individuals. The aim of the present study was application of genetically engineered P. putida that produce dioxygenase for degradation of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) method. The nahH gene that encoded catechol 2,3-dioxygenase (C23O) was cloned into pUC18 and pUC18-nahH recombinant vector was generated and transformed into wild P. putida, successfully. The genetically modified and wild types of P. putida were inoculated in soil and pilot plan was prepared. Finally, degradation of phenanthrene and pyrene by this bacterium in spiked soil were evaluated using HPLC measurement technique. The results were showed elimination of these PAH compounds in spiked soil by engineered P. putida comparing to dishes containing natural soil with normal microbial flora and inoculated autoclaved soil by wild type of P. putida were statistically significant (p0.05) but it was few impact on this process (more than 2%). Additional and verification tests including catalase, oxidase and PCR on isolated bacteria from spiked soil were indicated that engineered P. putida was alive and functional as well as it can affect on phenanthrene and pyrene degradation via nahH gene producing. These findings indicated that genetically engineered P. putida generated in this work via producing C23O enzyme can useful and practical for biodegradation of phenanthrene and pyrene as well as petroleum compounds in polluted environments.

Iranian white cheese is one of the most important kinds of cheese produced in large scale with high consumption in the country. This dairy product transmits bacterial pathogens like Salmonella spp. Antibiotic resistant Salmonella are widespread in the world. This study was performed to evaluate the frequency of antimicrobial-resistant Salmonella enteritidis and related genes isolated from traditional and industrial Iranian white cheeses. A total of 200 traditional and industrial Iranian white cheeses were collected within Chaharmahal Va Bakhtiari province (southwest Iran). After culturing on specific media using standard bacterial tests the Salmonella sp. was isolated. For specific detection of S. enteritidis from other Salmonella strains sefA gene was studied. Finally, the antibiotic susceptibility patterns were investigated. Results showed that 17 % of cheese samples were contaminated by Salmonella and 5.5 % of specimens by S. enteritidis. The frequencies of resistance genes including tetA, tetB, tetC, cat3, and floR in isolated S. enteritidis were 36.4, 54.5, 81.8, 54.5, and 36.4 %, respectively. All isolated S. enteritidis were susceptible to ciprofloxacin, cefotaxime, and ceftazidime (100 %). In addition, most of them were resistance to chloramphenicol (64 %) and susceptible to gentamicin (98 %). The Salmonella contamination was more frequent in traditional Iranian white cheeses (11.5 %) as compared to industrial (5.5 %) samples (p

In the present study, the influence of phenanthrene and pyrene on cytogenetic stability of human dermal fibroblasts using alkaline comet assay was evaluated. The cells were isolated from foreskin samples obtained from healthy male infants and in low passages (P 1–3) were exposed to various concentrations (0.0900, 0.0625, 0.0320, and 0.0066 ppm) of phenanthrene and pyrene. Then, alkaline comet assay was performed to investigate the influence of these compounds on DNA damage and cytogenetic stability of human dermal fibroblasts. In the present work H2O2 treatment was used as a positive control of comet assay to create DNA damages. The analysis of alkaline comet assay parameters by CaspLab software showed the tail DNA% in high concentration (0.0900 ppm) of phenanthrene and pyrene in the exposed cells got increased as compared to normal cells, while head DNA% decreased. Also, similar to positive control (H2O2), DNA damage with long tail comet was observed in high concentration of these compounds as compared to normal cells. The comparison of comet assay parameters specially head DNA% and tail DNA% between each concentrations of phenanthrene and pyrene with other groups (healthy cells and H2O2 treatment) by ANOVA and post hoc Tukey test showed that these parameters were more significantly different (p < 0.05). These results indicated that phenanthrene and pyrene even in low dosage are dangerous and can increase the DNA damage and affect on cytogenetic stability of dermal fibroblasts. These findings suggested that phenanthrene and pyrene could increase the related diseases and risk of skin cancer in exposed individuals.

The efficient induction of embryonic stem cells into hematopoietic stem cells (HSCs) requires understanding the pro or inhibitory regulatory networks, which are involved in these pathways. Transforming growth factor β family is a super family of growth factors such as TGF-β1, TGF-β2, TGF-β3, and some other cytokines. Previous works indicate that some of TGF-β subfamilies have negative regulatory effect during hematopoietic differentiation and HSC proliferation. The inhibitory effects of TGF-β1 in early stage of differentiation of endothelial and hematopoietic cells have been shown. Therefore, in the present study, in order to evaluate the promotion of hematopoietic differentiation, the activities of TGF-β regulatory factor were reduced through knockdown of TGF-βR2 expression. The ESCs were transduced by Lentivirus carrying SiTGF-βR2 and its scramble separately. Green fluorescent protein (GFP) was used as a marker to monitor gene expression. Six days post transduction GFP+ colonies were used for EB generation and the next day, the same size EBs were transferred in differentiation media involved in defining cytokines. The expression level of hematopoietic-specific genes was evaluated in 0, 7, and 15-days of differentiation. The real-time PCR analysis in 15 day EBs showed 2–3-folds increase in SiTGF-βR2 as compared with the scramble. Therefore, this finding may be confirmed if TGF receptor silencing can reduce the inhibitory effect of TGF-β during early hematopoietic differentiation.

The virulence factors such as shiga-like toxin (Stx) and immunogenic P39 protein in Escherichia coli and Brucella melitensis are related to disease of digestive system in human worldwide. In the present study the stx2 and p39 genes were cloned into expression plasmid pEEF1D-FLAG (pcDNA 3.1+) as a divalent DNA vaccine candidate. The Enterohemorrhagic E. coli ATCC 3081 and smooth virulent B. melitensis strain M5 were obtained and cultured on specific media. Bacterial DNA was extracted from colonies and was used for p39 and stx2 genes amplification by PCR. The amplified products on 2% agarose gel electrophoresis were revealed 285 and 1220 bp fragments for stx2 and p39 genes, respectively. Each amplified genes were T/A cloned into pGEM-T easy vector and pGEM-T-stx2 and pGEM-T-p39 were produced. The stx2 and p39 genes were sub-cloned in linearized expression vector (pcDNA 3.1+) using HindIII, XhoI and XbaI restriction enzymes and pCDNA3-stx2-p39 was generated. This final construct was confirmed by PCR and enzymes digestion. The results were showed stx2 and p39 genes were sub-cloned, successfully into pcDNA 3.1+ to generate pcDNA 3.1+-stx2-p39 recombinant vector. According to these findings novel recombinant pcDNA 3.1+-stx2- p39 construct that was produced in this study could be useful as DNA vaccine candidate in animal models against shiga-like toxin producing E. coli and virulence B. melitensis strains in future studies.

Bacterial infections are one of the important agents in the creation of gallstones in the gallbladder. In recent years the spread of antibiotic-resistant bacteria such as extended-spectrum ?-lactamases (ESBL) is increasing and of concern in hospitalized patients worldwide. The purpose of this study was to investigate the antibiotic susceptibility patterns of isolated bacteria from the bile specimens of patients with chronic and acute cholecystitis who had been operated by single-incision laparoscopic cholecystectomy (SILC) in Isfahan (Iran) 2 using an antibiogram susceptibility test and molecular technique. The bile fluids of 91 patients were obtained from the Al-Zahra hospital and were cultured on specific media for the isolation of Gram-negative and positive bacteria and the disk diffusion test was done to determine the antibiotic susceptibility patterns of isolated bacteria. Finally, bacterial DNA was extracted from the bile samples and polymerase chain reaction (PCR) was performed to investigate extended-spectrum ?-lactamases genes. The bacteria Escherichia coli, Klebsiella pneumoniae, Proteus spp. and Staphylococcus aureus were detected in bile specimens cultured with high frequency, and the results showed that biliary infection increased with aging in patients with gallstone disease operated by SILC. The results showed a high frequency of ESBL genes including TEM, SHV, and CTX-M in isolated bacteria (especially Escherichia coli and Klebsiella spp.). Thus, evaluating the antibiotic susceptibility patterns and screening of ESBLs bacteria in patients with gallstones are essential. Prescribing suitable drugs, designing good strategies, and informing the medical community could decrease bile infection and antibiotic-resistant bacteria in clinical centers and hospitals.

Background: It is well known that bioremediation or using microorganism enzymes plays an important role in decomposition and changing of oil pollutants and PAH compounds into nonhazardous or less-hazardous substances. The genetically manipulated bacteria like Pseudomonas sp. can enhancement the natural biodegradation activity of polycyclic aromatic hydrocarbons (PAHs) in polluted cities. Pseudomonas putida (P. putida) is a saprotrophic soil bacterium that is found throughout various environments such as soil and freshwater environments. Oil and petroleum industries are important exposure of PAHs in environment that strongly associated with the development of human cancers. Objectives: In the present work, P. putida was genetically manipulated for the biodegradation of oil in spiked soil and its activity was measured using the high-performance liquid chromatography (HPLC) method. Methods: The catechol 2,3-dioxygenase (C23O) encoded gene (nahH) was cloned into pUC18 for generating pUC18-nahH. This recombinant vector was transferred into P. putida, successfully, and both wild type and genetically modified P. putida were inoculated in spiked soil with oil for pilot plan preparation. Then, biodegradation activity of this bacterium on the elimination of phenanthrene and pyrene as an oil indicator in spiked soil were evaluated by the HPLC measurement technique. Results: The results showed the biodegradation of these PAH compounds in oil-spiked soil by genetically manipulated P. putida in both groups (containing autoclaved soil and dish containing natural soil) comparing to the inoculated group by wild type of P. putida were statistically significant (P < 0.05). Finally, confirmatory tests (catalase, oxidase, and PCR) were accomplished on isolated bacteria from spiked soil and were indicated that engineered P. putida was alive and functional by producing the C23O enzyme for biodegradation activity of oil. Conclusions: These findings demonstrated a degradable strain of P. putida that was generated in this study by genetic engineering can be useful to assess biodegradation of PAHs compounds in oil and petrochemical pollutions.

Stem cells are undifferentiated and self-renewal cells which could be obtained from the body or artificially derived from an adult somatic cell by forced expression of specific genes. In recent years stem cells are widely used in laboratory for tissue engineering and therapeutic applications. There are different factors and conditions that affect the stem cell culture such as feeder layers, atmosphere, kind of medium, growth factors, passages number, and conditional media with animal or human sources. Optimization of stem cell culture for medical approaches and regenerative medicine is important. Therefore, in the present study, the effect of these factors and agents on optimization of stem cell culture has been discussed. This review study showed that optimization of feeder layer, atmosphere, and using supplemented media with essential growth factors could help in maintaining the stem cells in undifferentiated state in vitro. The present study indicated that optimization of stem cell culture depends on the kind of each cell type and using stem cells in low passage number could decrease chromosomal abnormalities and DNA damages. For inhibiting the stem cell contamination by feeder cell lines, culture of these cells on feeder free systems like Matrigel matrix in conditioned media supplemented with essential growth factors is useful. Also, for eradicating immune system responses and reducing the risk of animal pathogen transfer, culture of stem cells on human feeder in optimized media is suitable for therapeutic approaches and regenerative medicine.

Human dermal fibroblasts (HDFs) are a potential source of somatic cells for genetic manipulation and tissue engineering. Confirmation of cytogenetic stability of these cells is an essential step for cell nuclear transfer and generation of a suitable and functional induced pluripotent stem cells line. HDF cells were isolated and cultured from human foreskin samples. Cytogenetic stability of these cells was evaluated in early (3–4) and late (10–15) passages using karyotype test and alkaline comet assay techniques. HDF cells in early and late passages showed normal karyotype but by comet assay abnormality and DNA damages in late passages of HDFs were observed. Also, the parameters of alkaline comet assay in early passages of HDFs compared with late passages and positive control groups more significantly were different (p

Background: Non-syndromic hearing loss (NSHL) is the most common birth defect and occurs in approximately 1/1,000 newborns. NSHL is a heterogeneous trait and can arise due to both genetic and environmental factors. Mutations of the transmembrane channel-like 1 (TMC1) gene cause non-syndromic deafness in humans and mice. Objectives: The aim of the present study was to investigate the association of TMC1 gene mutations of the locus DFNB7/11 in exons 7 and 13 in a cohort of 100 patients with hearing loss in Iran using polymerase chain reaction–single-stranded conformation polymorphism (PCR-SSCP), heteroduplex analysis (HA), and DNA sequencing. Patients and Methods: In this experimental study, the blood samples of 100 NSHL patients were collected from 10 provinces in Iran. These patients had a mean age of 16.5 ± 2.01 years and 74.15% of their parents had consanguinity. DNA was extracted from specimens and mutations of exons 7 and 13 of the TMC1 gene were investigated using PCR-SSCP. All samples were checked via HA reaction and suspected specimens with shift bands were subjected to DNA sequencing for investigation of any gene variation. Results: In this study, no mutation was found in the two exons of TMC1 gene. It was concluded from these results that mutations of the TMC1 gene’s special exons 7 and 13 have a low contribution in patients and are not great of clinical importance in these Iranian provinces. Conclusions: More studies are needed to investigate the relationship between other parts of this gene with hearing loss in different populations through the country. More research could clarify the role of this gene and its relation with deafness and provide essential information for the prevention and management of auditory disorders caused by genetic factors in the Iranian population.

miRNAs are essential factors of an extensively conserved post-transcriptional process controlling gene expression at mRNA level. Varoius biological processes such as growth and differentiation are regulated by miRNAs. Web of Science and PubMed databases were searched using the Endnote software for the publications about the role miRNA-183 family in inner ear: hair cell development and deafness published from 2000 to 2016. A triplet of these miRNAs particularly the miR-183 family is highly expressed in vertebrate hair cells, as with some of the peripheral neurosensory cells. Point mutations in one member of this family, miR-96, underlie DFNA50 autosomal deafness in humans and lead to abnormal hair cell development and survival in mice. In zebrafish, overexpression of the miR-183 family induces extra and ectopic hair cells, while knockdown decreases the number of hair cell. The miR-183 family (miR-183, miR-96 and miR-182) is expressed abundantly in some types of sensory cell in the eye, nose and inner ear. In the inner ear, mechanosensory hair cells have a robust expression level. Despite much similarity of these miRs sequences, small differences lead to distinct targeting of messenger RNAs targets. In the near future, miRNAs are likely to be explored as potential therapeutic agents to repair or regenerate hair cells, cell reprogramming and regenerative medicine applications in animal models because they can simultaneously down-regulate dozens or even hundreds of transcripts.

Tp53 is an important tumor suppressor gene, which induces cell growth arrest or apoptosis when subjected to cytotoxic stimuli. Association has been reported between various cancers and p53 codon 72 polymorphism. Our objective was to investigate the possible association between p53 at codon 72 for Arg/Arg, Arg/Pro and Pro/Pro allele polymorphisms in blood samples from 187 prostate cancer patients and 185 controls in southwest Iran by nested-polymerase chain reaction (PCR) of p53 exon 4 and digestion with BstUI restriction enzyme and the DNA fragments were then resolved by electrophoresis in 2% agarose gel. The frequencies of Arg/Arg, Arg/Pro and Pro/Pro genotypes were 39.57% (74/187), 52.41% (98/187) and 8.02% (15/187), respectively, in the cases with prostate cancer, and 27.03% (50/185), 60% (111/185) and 12.97% (24/185), respectively in the healthy controls. Statistically, analyzed and combined results showed there was a significant difference in the frequency of the Arg/Arg genotype and Arg allele between prostate cancer cases and control (p>0.001). These findings suggest that p53 Arg/Arg genotype could be a risk factor for the development of prostate cancer among patients in southwest Iran. Key words: Prostate cancer, suppressor gene (p53) codon 72, polymorphism, Iran.

The tumor suppressor gene p53 play general role in cell cycle control, the initiation of apoptosis, and in deoxyribonucleic acid (DNA) repair. The human p53 gene is mutated and accumulated in more than 50% of cancers. Codon 72 exon 4 polymorphism (Arg72Pro) of the p53 gene has been implicated in cancer risk. Our objective was to investigate the possible association between p53 Arg72Pro polymorphism and susceptibility to breast cancer among Iranian population. The p53 Arg72Pro genotypes were determined by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis in 135 breast cancer cases and 140 controls. The PCR products were digested with BstUI restriction enzyme and the DNA fragments were then resolved by electrophoresis in 2% agarose gel. Out of the 135 breast cancer samples, 70 (51.85%) samples were heterozygous (Arg/Pro), 52 (38.52%) samples homozygous for arginine (Arg/Arg) and 13 (9.63%) samples homozygous for proline (Pro/Pro). The frequencies of the three p53 genotypes; Arg/Pro, Arg/Arg and Pro/Pro in controls were 58.57, 25.72 and 15.71%, respectively. Hemozygosity for Arg/Arg of p53 codon 72 is potentially one of the genetic risk factors for breast cancer. The p53 codon 72 Arg/Arg polymorphism may be used as a stratification marker in screening individuals at a high risk of breast cancer. Key words: Breast cancer, p53 codon 72, Polymorphism, Iran.

Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells artificially derived from an adult somatic cell (typically human fibroblast) by forced expression of specific genes. In recent years, different feeders like inactivated mouse embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and feeder free system have commonly been used for supporting the culture of stem cells in undifferentiated state. In the present work, the culture of hiPSCs and their characterizations on BD Matrigel (feeder-and serum-free system), MEF and HDF feeders using cell culture methods and molecular techniques were evaluated and compared. The isolated HDFs from foreskin samples were reprogrammed to hiPSCs using gene delivery system. Then, the pluripotency ability of hiPSCs cultured on each layer was determined by teratoma formation and immunohistochemical staining. After EBs generation the expression level of three germ layers genes were evaluated by Q-real-time PCR. Also, the cytogenetic stability of hiPSCs cultured on each condition was analyzed by karyotyping and comet assay. Then, the presence of pluripotency antigens were confirmed by Immunocytochemistry (ICC) test and alkaline phosphatase staining. This study were showed culturing of hiPSCs on BD Matrigel, MEF and HDF feeders had normal morphology and could maintain in undifferentiated state for prolonged expansion. The hiPSCs cultured in each system had normal karyotype without any chromosomal abnormalities and the DNA lesions were not observed by comet assay. Moreover, up-regulation in three germ layers genes in cultured hiPSCs on each layer (same to ESCs) compare to normal HDFs were observed (p < 0.05). The findings of the present work were showed in stem cells culturing especially hiPSCs both MEF and HDF feeders as well as feeder free system like Matrigel are proper despite benefits and disadvantages. Although, MEFs is suitable for supporting of stem cell culturing but it can animal pathogens transferring and inducing immune response. Furthermore, HDFs have homologous source with hiPSCs and can be used as feeder instead of MEF but in therapeutic approaches the cells contamination is a problem. So, this study were suggested feeder free culturing of hiPSCs on Matrigel in supplemented media (without using MEF conditioned medium) resolves these problems and could prepare easy applications of hiPSCs in therapeutic approaches of regenerative medicine such as stem-cell therapy and somatic cell nuclear in further researches.

Basic fibroblast growth factor (bFGF or FGF-2) is a member of the FGF family secreted by different kinds of cells like HDFs and it is an important nutritional factor for cell growth and differentiation. The HDFs release bFGF in culture media at very low. The present study aims to investigate the HDFs growth rate in culture media supplemented either with or without bFGF. In brief, HDFs were isolated from human foreskin sample and were cultured in vitro in media containing bFGF and lack of this factor. The cells growth rate was calculated by trypan blue. The karyotyping was performed using G-banding to investigate the chromosomal abnormality of HDFs in both groups. Total RNA of each groups were extracted and cDNA samples were synthesized then, real-time Q-PCR was used to measure the expression level of p27kip1 and cyclin D1 genes normalized to internal control gene (GAPDH). The karyotype analysis showed that HDFs cultured in media or without bFGF had normal karyotype (46 chromosomes, XY) and chromosomal abnormalities were not observed. The cell growth rates in both groups were normal with proliferated exponentially but the slope of growth curve in HDFs cultured in media containing bFGF was increased. Karyotyp test showed that bFGF does not affect on cytogenetic stability of cells. The survey of p27kip1 and cyclin D1 genes by real-time Q-PCR showed that the expression level of these genes were up-regulated when adding bFGF in culture media (p

Brucella melitensis is a Gram negative coccobacillus bacterium from the Brucellaceae family. Brucellosis is an infectious zoonotic disease affecting most species of domestic animals, but sheep and goats, particularly milking breeds, are the most susceptible. Cattle may occasionally be affected and the disease may appear in pigs. vacB gene is one important gene of B. melitensis that encoded a protein (RNase R) and in B. melitensis and Brucella abortus has no impact on bacterial virulence. The aim of the present study was to construct a novel recombinant vector as B. melitensis vacB gene knockouts candidate. B. melitensis were collected from Microbiology laboratory of Islamic Azad University of Shahrekord Branch and cultured into Brucella agar. Genomic deoxyribonucleic acid (DNA) was extracted and the polymerase chain reaction (PCR) were performed using designed primers for amplification of upstream and downstream regions of vacB gene of B. melitensis and kan gene of pET28 vector. Then, amplified fragments were cloned using T/A cloning technique and the construction was transformed into competent Escherichia coli Top10F' strain in LB media. The final construction was confirmed by digestion with XhoI, KpnI, XbaI, and BamHI restriction enzymes. PCR amplified products for flanking regions of vacB gene and kan gene on 1% agarose gel revealed 609, 595, and 870 bp, fragments, respectively. The results showed that vacB gene and kan gene were cloned in E. coli successfully. Sub-cloning of all fragment into pET-32 vector were done successfully and pET-32-UpKan-Down recombinant vector was generated. The findings of this study showed that the designed novel recombinant construct (pET-32-Up-Kan-Down recombinant plasmid) is useful for genetic engineering and for manipulate of vacB gene from B. melitensis.

The human mitochondrial DNA is a 16569 bp closed, circular molecule that was sequenced and mapped. The D-loop of mitochondrial DNA is a major control site for mtDNA expression and it is important in maternal inheritance. This region in different population is vary and contains essential transcription and replication elements and it could be used for detection of mother inheritance, and human evolution. The aim of present study was to determine the D-loop region polymorphism in Bakhtiarian population in southwest Iran. The blood samples of 168 healthy people of Bakhtiarian population in Iran were collected and total DNA was extracted and then, mtDNA D-loop region was amplified by PCR using specific primers. Restriction fragment length polymorphism (RFLP) was analyzed in PCR products by Hae III and Alu I restriction endonuclease. Amplified fragments for mtDNA D-loop region on 1% agarose gel revealed a fragment of about 1024 bp. The results of this study showed 5 restriction patterns for Hae III enzyme (with 1 heteroplasmy) and 2 restriction patterns for Alu I enzyme (with 2 heteroplasmies) in Bakhtiarian population. The findings showed a low level of genetic polymorphism in D-loop region and it is related to high kinship marriages and low range of migration in Bakhtiarian population. Keywords : mtDNA, D-loop region, RFLP, Polymorphism, Bakhtiarin population, Iran

Detection of species fraud in meat products is important for consumer protection and food industries. A molecular technique such as PCR method for detection of beef, sheep, pork, chicken, donkey, and horse meats in food products was established. The purpose of this study was to identification of fraud and adulteration in industrial meat products by PCR-RFLP assay in Iran. In present study, 224 meat products include 68 sausages, 48 frankfurters, 55 hamburgers, 33 hams and 20 cold cut meats were collected from different companies and food markets in Iran. Genomic DNA was extracted and PCR was performed for gene amplification of meat species using specific oligonucleotid primers. Raw meat samples are served as the positive control. For differentiation between donkey’s and horse’s meat, the mitochondrial DNA segment (cytochrome-b gene) was amplified and products were digested with AluI restriction enzyme. Results showed that 6 of 68 fermented sausages (8.82%), 4 of 48 frankfurters (8.33%), 4 of 55 hamburgers (7.27%), 2 of 33 hams (6.6%), and 1 of 20 cold cut meat (5%) were found to contain Haram (unlawful or prohibited) meat. These results indicate that 7.58% of the total samples were not containing Halal (lawful or permitted) meat and have another meat. These findings showed that molecular methods such as PCR and PCR-RFLP are potentially reliable techniques for detection of meat type in meat products for Halal authentication.

A small supernumerary marker chromosome (sSMC) is a structurally abnormal chromosome. It is an additional chromosome smaller than one chromosome most often lacking a distinct banding pattern and is rarely identifiable by conventional banding cytogenetic analysis. The origin and composition of an sSMC is recognizable by molecular cytogenetic analysis. These sSMCs are seen in different shapes, including the ring, centric minute, and inverted duplication shapes. The effects of sSMCs on the phenotype depend on factors such as size, genetic content, and the level of the mosaicism. The presence of an sSMC causes partial tris- or tetrasomy, and 70% of the sSMC carriers are clinically normal, while 30% are abnormal in some way. In 70% of the cases the sSMC is de novo, in 20% it is inherited from the mother, and in 10% it is inherited from the father. An sSMC can be causative for specific syndromes such as Emanuel, Pallister-Killian, or cat eye syndromes. There may be more specific sSMC-related syndromes, which may be identified by further investigation. These 10 syndromes can be useful for genetic counseling after further study.