Relation between sperm protamine transcripts with global sperm DNA methylation and sperm DNA methyltransferases mRNA in men with severe sperm abnormalities

This study aimed to evaluate the relationship between mRNA expression of DNA methyltransferases (DNMTs) such as DNMT1, DNMT3A and DNMT3B mRNA and sperm global DNA methylation with protamine transcripts in the sperm from men with severe sperm abnormalities. Sperm from each semen sample were isolated using a standard gradient isolation procedure by layering 1 mL of 40 (v/v) density gradient medium over 1 mL of 80 (v/v). A total of 30 oligoasthenoteratozoospermic ejaculates (OAT) and 30 normozoospermic ejaculates as controls were compared using real-time quantitative reverse transcriptase polymerase chain reaction for mRNA expression of DNMT1, 3A, 3B, protamine1 (P1) and protamine2 (P2). The enzyme-linked immunosorbent assay was used to detect global DNA methylation in sperm. A p-value of <0.05 was considered statistically significant. In OAT ejaculates, the increased level of DNMT3A, 3B mRNA, sperm global methylation, P1 plus P2 mRNA and decrease of P1�P2 ratio were significantly different. Also the content of protamine transcript was not correlated with sperm parameters. The increased total protamine transcript levels were associated with increased mRNA methyltransferases. The increase of DNMT1 may lead to an increased level of global methylation. © 2019, © 2019 The British Fertility Society

Increased Expression of MiR-27a and MiR-24-2 in Esophageal Squamous Cell Carcinoma

Purpose: Esophageal squamous cell carcinoma (ESCC) is one of the predominant types of esophageal cancer with poor prognosis which shows high prevalence in eastern countries. Studying microRNAs that were considered for their capabilities such as tissue-specific expression and involvement in different cell features may be informative in the field of diagnostic and prognostic tumor markers. The expression levels of miR-27a and miR-24-2 have been reported to be dysregulated in various cancers and contribute in tumorigenesis and progression; thus, evaluating their expressional behavior and its association with tumor states alteration in ESCC could potentially be helpful. Methods: The study was conducted on 30 fresh specimens including tumor and normal counterparts� tissues of ESCC. After the extraction of total RNA, complementary DNA synthesis was performed by the use of linear specific primers. Eventually, real-time polymerase chain reaction was carried out for the measurement of microRNAs expression level. Results: According to the obtained data, miR 27a and miR-24-2 were significantly upregulated (~2.5 fold, p < 0.05) in tumor specimens compared with their normal adjacent tissue; Moreover, upregulation of miR-27a and 24-2 showed cooperative relationship while analyzed. However, there was no correlation between clinicopathological features and microRNAs upregulation. Conclusions: The results of this study show that miR-27a and miR-24-2 cooperatively upregulate in ESCC and suggest that these microRNAs can be introduced as a candidate for further study in the field of screening and prognostic biomarkers. © 2019, Springer Science+Business Media, LLC, part of Springer Nature

Association of IL-17 and IL-23 follicular fluid concentrations and gene expression profile in cumulus cells from infertile women at risk for ovarian hyperstimulation syndrome

This study determined the association between the levels of interleukin (IL)-17 and IL-23 in follicular fluid (FF), as well as their mRNA levels in cumulus cells from infertile women at risk for ovarian hyperstimulation syndrome (OHSS). In this case-controlled study, the control group (n = 40) was infertile women whose partners had male factor infertility, whereas the case group (n = 40) was infertile women at risk of OHSS. IL-17 and IL-23 concentrations in FF were measured using an enzyme-linked immunosorbent assay method, whereas the mRNA expression levels of IL-17 and IL-23 of cumulus cells were determined using RT-PCR. Significantly higher levels of IL-17 were seen in the case group (p = 0.04), whereas there was no significant difference in IL-23 concentrations between the two groups (p = 0.3). The mRNA levels of IL-17 and IL-23 showed no significant differences. In the case group, there was a positive significant correlation between the IL-23 concentration in FF and the oocyte maturation rates (p = 0.01). In the case group, the number of follicles, MII oocytes, immature oocytes, fertilized oocytes and number of embryos were significantly higher than the control group (p < 0.05). Our findings showed that the mRNA expressions of IL-17 and IL-23 were similar in the two groups, and IL-17 was increased in the case group. © 2019 The British Fertility Society

A r c h i v e o f S I D Iranian Chemical Society Molecular Cloning, Expression and Sequence Analysis of DNA Polymerase I from an Iranian Thermophilic Bacterium, Bacillus sp. G (2006)

Thermostable DNA polymerases are widely used in DNA amplification reactions such as the Polymerase Chain Reaction (PCR), requiring the activity of the enzymes at high temperatures. The aim of the present study was to assess the potential biotechnological capabilities of Iranian thermostable DNA polymerases. To this end, we cloned the gene encoding a DNA polymerase from a novel thermophilic eubacterium, Bacillus sp. G (2006). Phylogentic analysis of this gene revealed that the new isolate belongs to the genera Bacillus. Sequence analysis of the fragment produced by degenerate primers also showed that it consists of 2,631 bp encoding an 876 amino acid protein, and subsequent amino acid sequence analysis of this DNA polymerase showed that it belongs to family A-type DNA polymerases. The expression vector pET28a (+) was chosen for expression of the gene fragment in the mesophilic host bacterium E. coli BL21. This expression vector has some advantages such as attachment of a Poly-His tag to the N-terminus of the protein for the ease of purification and a powerful promoter of lac-Z induced by IPTG. The band corresponding to the protein product was observed in the molecular weight range of about 100KDa on the SDS-PAGE gel after heat and Ni +2 -NTA column chromatography. Using the dot blot technique, the polymerase activity of the enzyme was qualitatively confirmed at 70 °C. Therefore, it is suggested that optimizations of this activity could make this enzyme appropriate for PCR processes in future