JOURNAL OF CELL AND MOLECULAR RESEARCH
Year:2021 | Volume:13 | Issue:1|Papers Count:11

To achieve a reliable and persistent expression, the transgene should be precisely integrated into the genome safe harbor (GSH) loci. Little attention has been paid to find the safe harbor loci of the chicken (Gallus gallus domesticus) genome. Identification and characterization of GSH loci that allow the persistent and reliable expression of knock-in genes could be a major area of interest within the field of transgenic technology and is central to the development of transgenic livestock. Randomly integrated transgenes might encounter position effects and epigenetic silencing, so unstable phenotypes, as well as unreliable and unpredictable expression of the knock-in transgene could occur. In contrast to random gene insertion, site-specific gene targeting provides a superior strategy that exploits homologous recombination to insert a transgene of interest into a pre-determined locus. In this study, based on bioinformatics, gene expression atlas, and Hi-C analyses, the GSH region was predicted in the chicken genome between DRG1 and EIF4ENIF1 genes. To do so, we introduce a fast and easy-to-use pipeline that allows the prediction of orthologue GSH loci in all organisms, especially chickens. In addition, the procedure to design Cas9/gRNA expression and targeting vectors for targeting these predicted GSH regions is described in detail.

The p53 is a tumor suppressor protein that plays an essential role in controlling the cell cycle. In addition, vascular endothelial growth factor (VEGF) is one of the most strong and specific angiogenic factors. The main objective of this study was to evaluate the impact of p53 and VEGF-C gene expression in the neoplastic and normal mammary glands of canines as an animal model. Eleven benign and malignant and five normal specimens were collected. After RNA extraction and cDNA synthesis, relative quantification of p53 and VEGF-C genes was accomplished by real-time quantitative PCR (RT-qPCR), in which β-actin was used as a reference gene. The relative mRNA expression of the p53 and VEGF-C genes was analyzed by GLM procedure of SAS software v9. 2. The results indicated that the VEGF-C and p53 mRNA expression in neoplastic specimens was over-and down-expressed, respectively, compared with normal specimens. The p53 mRNA expression was significantly negatively associated with VEGF-C (~4 fold) in neoplastic specimens (P <0. 01). These findings emphasized that simultaneous evaluation of p53 and VEGF-C expression can be used as tumor biomarkers for the early diagnosis of malignancy in canines. Furthermore, RT-qPCR is a rapid and sensitive method for monitoring and investigating suspicious canines at the early stage of malignancy and may provide an alternative explanation for deregulated p53 signaling in breast cancer.

With the increased usage of nitrate fertilizers, the removal of their stable ionic and water-soluble end products is a challenge for human health. Several physicochemical methods have been examined for nitrate removal of water, but biological treatments are mostly preferred due to a higher efficiency and lower cost. To remove nitrogen from water, we investigated the potential of nitrate-reducing halophilic and halotolerant bacteria. A total of 50 strains from different saline and hypersaline environments of Iran, including the Incheboron wetland, Aran-Bidgol salt-lake, and Urmia endorheic salt-lake, were screened for nitrate reductase production. Among investigated bacteria, 60% and 19% of strains obtained from Urmia lake, and Incheboron wetland produced nitrate reductases, respectively. The nitrate reductase coding genes narG, and napA were analyzed in all strains with confirmed nitrate-reducing capacity. The napA gene was successfully amplified from a gram-negative halophilic strain, and the narG gene was detected in ten halophilic strains. Among nitrate-reducing isolates with the narG gene expression, the Kocuria rosea strain R3A34 showed the highest nitrate reductase production level. This strain was selected to optimize for its denitrifying activity. Results showed that 32° C, pH 7. 0, NaCl 8% (w/v), and mannitol (as a carbon source) provide the optimal environmental conditions for the efficient production of nitrate reductase by the Kocuria rosea strain R3A34. As these are compatible with wastewaters conditions, this bacterium can be a proper candidate for bioremediation of wastewaters from nitrate pollutants.

One of the newest diagnostic methods and treatment of cancer is to design new drugs. It is now possible to design a drug with desired properties in theory and evaluate its therapeutic effects through bioinformatics tools. Among the studied drugs, those based on cytokine genes, which increase the body's immunity against cancer, are of great interest. Cytokines are small proteins that play an essential role in cell signaling and can affect the function and behavior of surrounding cells. CCL21 chemokine is one of the cytokines that possess antitumor properties has the potential for chemoattraction of T lymphocytes and dendritic cells. Interleukin 1 beta (IL1β ) is a cytokine involving different cellular activities such as the activation of neutrophils, B-Cells, and T-Cells. In the present study, we designed a drug-based cytokine gene to activate T cells and B cells by inserting defined CCL21 epitope and IL1β peptide sequences into a protein construct. Molecular dynamics simulation was performed in Linux space using Gromex software. Results of RMSD, RMSF, and the radius of gyration obtained from the simulation showed the stability of both proteins, which indicated that there are no significant conformational differences between the commercial CCL21 and recombinant form. The interaction of synthetic construct and human CCL21 with the CCR7 receptor was also investigated by HADDOCK software. Obtained results showed no differences between these proteins, and recombinant protein has the same structural and conformational characteristics as human commercial CCL21.

Royal jelly (RJ) as a traditional medicinal agent has a variety of pharmacological benefits. In the present study, the effects of the royal jelly were investigated on the urinary bladder cancer cell line 5637 (HTB-9). MTT assay was performed to determine the percent of cell viability at different concentrations of the royal jelly. Moreover, in vitro wound-healing assay was applied to investigate the effects of RJ on cell migration. The activities and gene expression levels of matrix metalloproteinase 2 and 9 were assessed by zymography and Real time PCR, respectively. It was confirmed that royal jelly (RJ) at the concentration of 0. 7 mg/ml exerted a significant inhibitory effect on HTB 5637 cells and reduced cell viability to 72% in comparison to the control cultures (P-value<0. 009) during the first 72h of treatment. Furthermore, royal jelly Significantly decreased the bladder cancerous cell migration capacity, and induced a significant decrease in the transcriptional level of the MMP9 after 72h (50% of the controls; Pvalue<0. 049). However, R. J. S did not impose any effect on the expression level and activity of matrix metalloproteinase 2. The results indicated the potential of RJ as a promising natural anti-proliferative and antimetastatic drug.

Primordial germ cells (PGCs) are precursors of mature gametes, which transmit genetic information to the next generation. Due to the importance of PGCs in many fields, including developmental biology, genome editing, transgenesis, and conservation of avian genetic resources, various research aspects have focused on the cultivation of PGCs. Despite considerable progress in the establishment of specified culture media for the expansion of PGCs, a well-defined PGC culture medium has not yet been developed. This might be due to the complexity of the nutritional requirements of PGCs in the culture. Besides the nutritional needs, including vitamins, amino acids, salts, carbohydrates, and growth factors, a particular source of energy must be provided to sustain growth and viability. Glutamine is a major energy source for cultured cells, commonly added in cell culture media at higher concentrations than other amino acids. However, glutamine is very labile and rapidly degrades in solutions such as culture media. This generates ammonia as a by-product, which is toxic to the cultured cells and can affect cell viability and protein glycosylation. Therefore, the stability of glutamine in culture conditions is another concern for the longterm culture of PGCs. Here, we study the effect of glutamine stability on PGC culture using glutamine and GlutaMax (a commercial stabilized dipeptide form of glutamine). We found that the addition of GlutaMax in the medium promotes PGC proliferation. This effect might be exerted by minimizing production of toxic ammonia that results in maximizing cell performance and media stability.

This study aimed to investigate the cytotoxicity of a probiotic mixture on human breast cancer cell lines. To prepare the mixture, local probiotic bacteria were cultured, and the lyophilized supernatant was applied for downstream experiments. The antioxidant activity, total phenol content (TPC), and fatty acid composition of the bacterial supernatant (BS) were also measured. The possible cytotoxic/anti-proliferative effect of the probiotic mixture was accessed on both breast cancer cell lines at different concentrations using MTT assay. Furthermore, the apoptosis-inducing effects of the same mixture was studied by DAPI staining. The highest level of antioxidant activity and total phenol content (TPC) were detected for the BS at 3200 µ g/ml. According to the GC– MS analysis, linoleic acid (37. 40 %) and oleic acid (26. 93 %) were identifi ed as the major fatty acids of the BS. The MTT assay showed that the BS has anti-proliferative effects on MDA-MB-231 and MCF-7 cells in a time-and dose-dependent manner (IC50: 3200 μ g/ml). The apoptosis-inducing effects of the mixture was confirmed in both cell lines through morphological analyses of the cells’ nucleoli, and the formation of apoptotic bodies. According to these experiments, cytotoxic effects and apoptosis-inducing potential were confirmed for the BS against two human breast cancer cell lines, including MDA-MB-231, and MCF-7. Hence, it could be considered as a suitable anticancer agent.

Connexin-43 (Cx-43) plays axial roles in the propagation of action potentials and contractile coupling in the heart. Down-regulation of Cx-43 in the heart is associated with arrhythmia, dilated cardiomyopathy, and heart failure. To date, no studies have examined the effects of androgen deprivation therapy (ADT)-induced hypogonadism on the expression of Cx-43 in the heart. This study investigated the effects of testosterone deprivation and its replacement with testosterone enanthate on the expression of Cx-43 mRNA and the muscle-specific miRNAs miR-206 and miR1, as two potential regulators of the Cx-43 protein expression in the ventricular tissue. Accordingly, 21 male Wistar rats were divided into three groups: Ι ) Normal control, П ) ORX-S: castrated rats serving as animal models for ADT and receiving the sesame oil as a solvent of testosterone enanthate for ten weeks, and Ш ) ORX-T: these animals were castrated, receiving testosterone enanthate (25 mg/kg) for ten weeks. The relative expression of Cx-43 mRNA, miR206, and miR-1 was determined by qRT-PCR. Cx-43 mRNA was found to be decreased in the ORX-S group. The Cx-43 mRNA was up-regulated after the administration of testosterone enanthate. There were no significant changes in miR-206, and miR-1 levels in the ORX-S and ORX-T groups compared to the controls. Our results indicated that testosterone should be regarded as an important factor in the regulation of Cx-43 mRNA expression in the heart, and testosterone deprivation may down-regulate the Cx-43 mRNA expression; however, it doesn’ t alter miR-1 and miR206 levels. These results suggest that ADT-induced hypogonadism may put males at risk for cardiac dysfunctions.

The use of medicinal plants in the treatment of diseases has a long history dating back to the presence of humans on earth. Cancer has almost been an incurable disease, and among the various cancers, breast cancer is the most common type of cancer among women and imposes an enormous burden on patients. Although medical and surgical solutions have been proposed for the disease, it has not been successful enough to treat the disease in many patients. In recent years, more studies have been done on the effects of medicinal plants on breast cancer, and scientists are trying to find the exact mechanisms of action for these plants to find effective ways for controlling cancer cell growth. This article focuses on P53 and MDM2, two very important genes involved in regulation of cell growth and proliferation both in cancer and normal tissue, and we also gathered the list of natural compounds targeting the MDM2-p53 pathway. Our results provide a list of plant families that can influence this pathway and have great potential in designing treatments against cancers that encompass deregulation of the MDM2-p53 pathway.

One of the most prevalent bacterial infections, urinary tract infection (UTI), affects millions of people yearly worldwide. To control the increasing antibiotic-resistant infections, it is essential to introduce alternative approaches such as phage therapy. In this study, isolation, purification, and enrichment of eight lytic bacteriophages, which are active against antibiotic-resistant Escherichia coli strains from human urinary tract infections, were carried out. Molecular analysis of the bacteriophages was performed using two endonuclease enzymes (EcoRV and XbaI). Then, two of eight isolated bacteriophages with the highest host range were further characterized to determine their morphology, one-step growth, latent period, burst size, and stability under different environmental conditions. Allbacteriophage isolates (n=8) showed genome variation as it was evidenced by the enzyme digestion process (EcoRV). Both phages with the broadest host ranges (PEcMa2/17 and PEcMa3/17) showed an efficient lytic activity against five bacterial isolates. Electron microscopy confirmed that selected phages belong to Siphoviridae and Myoviridae families. The latent period of both propagated phages was determined as 15min. The burst size was estimated to be 100pfu/ml and 120pfu/ml in PEcMa2/17 and PEcMa3/17, respectively. Both phages showed more than 50% stability at 37º C and lower investigated temperatures, and they were survived efficiently in pH=7. It was while their genome properties were different. The introduced bacteriophages showed high stability and strong antibacterial potential against Escherichia coli strains from UTIs. As candidates for phage therapy, more characterization steps, such as molecular analysis and experimental assays are needed before the therapeutic application.

Wound healing is a complex biological process in which many molecules, including microRNA molecules, play an essential role in its regulation. It is well-established that reducing miR-155 expression can accelerate wound healing. This study investigated the effect of using nanoparticles loaded with vitamin C on miR-133, collagen I, and III expressions. In this study, first, nanoparticles of albumin protein were produced and then loaded with vitamin C. 3T3 mouse fibroblast cells were affected by these nanoparticles, and cell behavior was investigated to evaluate the toxicity and appropriate doses. In addition, the expression of collagen I and III genes was studied. The results showed that nanoparticles containing vitamin C in 20 µ g/ml concentration had a positive effect on collagen I and III expressions compared to the control group. Moreover, we observed a decrease in the expression of miR-133 in comparison to the control group. Therefore, according to the results of this study, it can be argued that nanoparticles containing vitamin C can significantly decrease the expression of the miR-133 gene and lead to collagen I and III gene overexpression in fibroblasts cells, which is directly effective in wound healing.