Background: There is increasing evidence indicating an incidence of infertility and also the risk of endometrial cancers among smokers. However, the mechanism underlying nicotine adverse effect on female reproduction remains unclear. Growing evidence has suggested that environmental exposures such as nicotine could modulate the epigenome. No study has yet been published to evaluate the direct effect of nicotine on the epigenome profiling of human endometrial stromal cells (HESC). Herein, we decided to examine the direct effects of nicotine on global genomic DNA methylation status and DNA methyl-transferases (DNMTs) gene expression in HESC. HESC were treated with different doses of nicotine (0 or control, 10 − 11 , 10 − 8 and 10 − 6) M for 24 h and their genomic global DNA methylation and gene expression of DNMTs (DNMT1, DNMT3A, and DNMT3B) were investigated using ELISA and real-time PCR, respectively. Results: Nicotine treatments reduced the average level of DNMTs gene expression by 90, 79, and 73.4% in 10 − 11 , 10 − 8 and 10 − 6 M of nicotine treated cells as compared to control cells, respectively (p < 0.05). Also, 10 − 8 and 10 − 6 M of nicotine concentrations effectively reduced the amounts of 5-methylated cytosine (5-mC) by 1.09 and 1.87% compared to control cells, respectively (p < 0.05). The 5-mC percentages were positively correlated with the relative cellular DNMTs expression in HESC as verified by the Pearson correlation test. Conclusion: An interesting possibility raised by the current study is that the reduced genomic global DNA methylation level in HESC may be partly due to the suppression of DNMTs gene expression caused by nicotine in these cells.
Purpose: GnRH-DFF40 (gonadotropin releasing hormone - DNA fragmentation factor 40) is a humanized recombinant immunotoxin and serves as a prospective candidate for targeted therapy of gonadotropin releasing hormone receptor (GnRHR) overexpressing malignancies. However, its production in Escherichia coli in a soluble and functional form still remains a challenge. Here we introduce two successful and reproducible conditions for production and purification of "difficult-to-express" GnRH-DFF40 protein. Methods: A synthetic codon optimized GnRH-DFF40 fusion gene was cloned in pET28a plasmid. Two methods including high cell density IPTG induction (HCDI) and autoinduction method (AIM) with a focus on obtaining high cell density have been investigated to enhance the protein production in (E. coli). Moreover, to obtain higher protein production several factors in the AIM method including carbon sources, incubation time and temperature, plasmid stability and double colony selection, were optimized. Results: Remarkable amounts of soluble GnRH-DFF40 protein were achieved by both methods. Cell density and protein yields in AIM was about 1.5 fold higher than that what obtained using HCDI. Initial screening showed that 25ºC is better to achieve higher protein production in both methods. pH alterations in AIM were maintained in a more constant level at 25ºC and 37ºC temperatures without any detrimental effects on cell growth during protein production phase up to 21 hours after incubation. Plasmid stability during growth and expression induction phase was maintained at a high level of 98% and 96% for AIM and HCDI methods, respectively. After parameter optimization and double colony selection in AIM, a very high yield of recombinant protein was achieved (528.3 mg/L). Conclusion: With the optimization of these high cell density expression methods, reproducible manifold enhancement of soluble protein yields can be achieved for "difficult-to-express" GnRH-DFF40 compared to conventional expression methods.
Background: The intracellular signaling pathways stimulated by CD44/hyaluronic acid (HA) interaction play a central role in the invasion and migration of cancer cells. Epithelial-mesenchyme transition (EMT) is an important factor in cancer metastasis and migration, which can be stimulated by the snail transcription factor. Previous studies showed cells that were subjected to snail-induced EMT, characterized by a CD44high/CD24low phenotype, expressed at their surface. Objectives: The aim of this study was to assess the inhibitory effect of CD44/HA interaction on the snail expression and invasive behavior of aggressive breast cancer cell line with a high CD44 expression in 2D and 3D culture. The cell surface binding capacity of the selected aptamer was evaluated via flow cytometry assay. Methods: To test our hypothesis, we disrupted the CD44/HA interaction by DNA aptamer, which specifically binds to the Hyaluronic Acid Binding Domain (HABD) of CD44. Then, expression level of snail mRNA was evaluated in MDA-MB 231 cells, cultured in 2D and 3D conditions by real-time PCR. Furthermore, invasive behavior was evaluated, using wound healing assay. Results: The results of this study showed that CD44 aptamer reduced snail expression and invasive behavior in MDA-MB 231 cell line. In addition, our result indicated that cells cultured in 3D were more sensitive to the aptamer in comparison to those cultured in the 2D model. Conclusions: The inhibition of CD44-HA interaction, using aptamer, negatively regulates the CD44 function in aggressive breast cancer cell line with the high level of CD44 expression.
Background: Neurodegenerative diseases are often the consequence of alterations in structures and functions of the Central Nervous System [CNS] in patients. Despite obtaining massive genomic information concerning the molecular basis of these diseases and since the neurological disorders are multifactorial, causal connections between pathological pathways at molecular level and CNS disorders development have remained obscure and need to be elucidated to a great extent. Objective: Animal models serve as accessible and valuable tools for understanding and discovering the roles of causative factors in the development of neurodegenerative disorders and finding appropriate treatments. Contrary to rodents and other small animals, large animals especially non-human primates [NHPs] are remarkably alike humans; hence, they establish suitable models for recapitulating the main human’s neuropathological manifestations that may not be seen in rodent models. Also, they serve as useful models to discover effective therapeutic targets for neurodegenerative disorders due to their similarity to humans in terms of physiology, evolutionary distance, anatomy, and behavior. Method: In this review, we recommend different strategies based on the CRISPR-Cas9 system for generating animal models of human neurodegenerative disorders and explain in vivo CRISPR-Cas9 delivery procedures of that are applied to disease models for therapeutic purposes. Results: With the emergence of CRISPR/Cas9 as a modern specific gene-editing technology in the field of genetic engineering, genetic modification procedures such as gene knock-in and knock-out have become increasingly easier compared to traditional gene targeting techniques. Unlike the old techniques, this versatile technology can efficiently generate transgenic large animal models without need to complicate lab instruments. Hence, these animals can accurately replicate the signs of neurodegenerative disorders. Conclusion: Preclinical applications of CRISPR/Cas9 gene-editing technology supply a unique opportunity to establish animal models of neurodegenerative disorders with high accuracy and facilitate perspectives for breakthroughs in the research on the nervous system disease therapy and drug discovery. Furthermore, the useful outcomes of CRISPR applications in various clinical phases are hopeful for their translation to the clinic in a short time.
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