The functions of azurin of Pseudomonas aeruginosa and human mammaglobin-A on proapoptotic and cell cycle regulatory genes expression in the MCF-7 breast cancer cell line.

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) difference after transfection. The expression of BAK, FAS, and BAX genes in transformed cells compare with non-transformed and transformed MCF-7 by pBudCE4.1 were increased statistically significant (p < 0.05) increases. Although, the increase of SURVIVIN and P53 expressions in transformed cells were not 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.

Biodecomposition of Phenanthrene and Pyrene by a Genetically Engineered Escherichia coli.

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<0.05). Moreover, catalase test was positive while the oxidase tests were negative. 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.

Frontier Therapeutics and Vaccine Strategies for SARS-CoV-2 (COVID-19): A Review.

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).

The concurrent effects of azurin and Mammaglobin-A genes in inhibition of breast cancer progression and immune system stimulation in cancerous BALB/c mice.

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 < 0.05). These results were supported by histological studies using H&E staining. The ELISA and q-real-time PCR techniques showed the range of IFN-γ, IL-12, IL-4, and IL-17A cytokines in the infused mice by recombinant vector alone or combined with nanoparticles compared to the healthy mice and infused animals by intact pBudCE4.1 were significantly increased (p < 0.05). Accordingly, the expression of the tumor markers CEA, Krt20, and Muc1 were significantly decreased in treated mice either by the sole recombinant vector or combined with nanoparticles (p < 0.05). These findings indicated that pBudCE4.1-azurin-MAM-A recombinant vector plays an essential role against the formation and expansion of breast tumors in the animal model. In addition, this recombinant vector is safe and has the proper ability to stimulate the immune system. In addition, the chitosan nanoparticle represents a promising adjuvant for DNA vaccine delivery, which improves the immune system stimulation and boosts the vaccine performance.

Valproic Acid Promotes Apoptosis and Cisplatin Sensitivity Through Downregulation of H19 Noncoding RNA in Ovarian A2780 Cells.

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.

Aflatoxin M1 Contamination Levels in Cheeses Sold in Isfahan Province, Iran.

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.

MicroRNA-183 Family in Inner Ear: Hair Cell Development and Deafness.

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.

Mutation Screening of Exons 7 and 13 of the TMC1 Gene in Autosomal Recessive Non-syndromic Hearing Loss (ARNSHL) in Iran.

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.

Evaluation of Microbial Contamination and Chemical Qualities of Cream-filled Pastries in Confectioneries of Chaharmahal Va Bakhtiari Province (Southwestern Iran).

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.

Small supernumerary marker chromosomes and their correlation with specific syndromes.

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.

Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines.

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.

Comparison of human dermal fibroblasts (HDFs) growth rate in culture media supplemented with or without basic fibroblast growth factor (bFGF).

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 < 0.05). The findings of the present study demonstrate that appropriate supplementation of culture media with growth factor like bFGF could enhance the proliferation and differentiation capacity of cells and improve cells growth rate. Similarly, fibroblast growth factors did not induce any chromosomal abnormality in cells. Furthermore, in HDFs cultured in bFGF supplemented media, the p27kip1 and cyclin D1 genes were up-regulated and suggesting an important role for bFGF in cell-cycle regulation and progression and fibroblast division stimulation. It also suggests that the effects of bFGF on different cell types with/or without production of bFGF or other regulation factors be investigated in future.

Molecular assay to fraud identification of meat products.

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.

Cytogentic analysis of human dermal fibroblasts (HDFs) in early and late passages using both karyotyping and comet assay techniques.

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 < 0.05). These findings indicate that single-strand breaks or DNA damage after many passages may have occurred in HDF cells. Our results demonstrate that only early passages of HDF cells maintain cytogenetic stability and are good candidates for gene reprogramming. In conclusion, karyotype testing alone can not be used for detection of all signs of cytogenetic abnormality and DNA damages of cells. So, for precise evaluation of DNA damage and cytogenetic instability of fibroblast cells comet assay and karyotype techniques could complement each other.