Pathobiology Research
Year:2015 | Volume:18 | Issue:1|Papers Count:8

Although more than 98% of the human genome is transcribed, most of these transcripts are not translated into proteins. Rather, they are considered as non-coding RNAs.MicroRNAs (miRNAs) are very short non-coding RNAs approximately 22 nucleotides in length which regulate many key processes of cells such as growth, proliferation, differentiation, cell cycle, apoptosis (programmed cell death) and metabolism. On the other hand, it is known that these small regulatory molecules are involved in many human diseases such as different cancers and cardiovascular disorders. Therefore, discovery and functional characterization of novel miRNAs is a prominent achievement. Low abundance and spatiotemporal expression of these mediator molecules make their discovery difficult by conventional methods. Therefore, bioinformatics software have been designed for the prediction of stem-loop structures capable of producing miRNA precursors in the human genome. On the other hand, there are several bioinformatics tools for prediction of miRNA target genes. Prediction of miRNA target genes helps to characterize the function of a miRNA. In this paper, we have reviewed some of the common efficient bioinformatics tools and experimental approaches used for prediction and identification of the miRNA genes and their target genes.

Objective: Generation 5 poly (amidoamine) dendrimers are promising multipotent gene delivery vectors that provide favorable DNA condensation properties; however, their high toxicity limits their applications. Toxicity of PAMAM dendrimers depends on their type, generation and applied dosage in a way that lower generations (lower than G5 dendrimers) and anionic dendrimers have lower toxicity than higher generations and cationic dendrimers. The aim of this study is to evaluate the effect of PEGylation on toxicity of G5 PAMAM dendrimers.Methods: In this study, to improve their characteristics as gene delivery carriers, G5 PAMAM dendrimers were conjugated to polyethylene glycol molecules (PEG, molecular weight 3500) at three different molar ratios of 10, 20 and 30. Also the number of conjugated PEG chains was quantified using TNBSA and Ellman assays. The effect of different degrees of PEGylation on cytotoxicity and transfection efficiency of modified PAMAM dendrimers toward BT-474 and MCF-10A cell lines were assessed.Results: Compared to unconjugated, PEG conjugated PAMAM dendrimers had lower in vitro cytotoxicity, particularly at higher PEG to PAMAM molar ratios. Among all prepared PEG-PAMAM dendrimers, G5 PAMAM dendrimers that conjugated to PEG at a molar ratio of 10.1 had the highest in vitro transfection rate in both cell lines.Conclusion: Our results showed that these PEG-conjugated PAMAM dendrimers possess a great potential for in vitro gene delivery.

Objective: Organophosphorus (OPs) compounds are widely used in many pesticides, insecticides and chemical nerve agents. These compounds are hazardous for humans and the environment. Organophosphate hydrolase (OPH) is a homodimeric protein initially isolated fromPseudomonas diminuta MG and Flavobacterium species. This enzyme is able to degrade a broad spectrum of toxic OPs compounds. Using immobilized OPH commonly presents a variety of advantages versus the free form of the enzyme.Advantages include an increase in stability, cost reduction by simple recovery and reutilization of the enzyme, quick and easy separation of the reactant and product in the reaction medium.Methods: Plasmid pET-26b (+) was used to generate the OPH protein under the control of the T7lac promoter.E. coli BL21 (DE3) pLysS was used as the host for expression of the OPH enzyme. Recombinant OPH was secreted into the extracellular medium and the purified enzyme was immobilized on the surface ofBacillus subtilis spores by the adsorption method, for the first time.Results: Approximately 42% to 45% enzymatic activity was determined to be associated with spores. Optimal pH and temperature of the enzyme were not altered by the presence of the spores. Thermo and pH stabilities of the immobilized enzyme was higher than the free form of the enzyme.Conclusion: Bacillus subtilis spores are safe for humans and the environment. Therefore this system can be considered an environmentally friendly biocatalyst for degradation of OPs.

Objective: Hexavalent chromium [Cr (VI)] compounds are well-known environmental contaminants generated from industrial processes. Several studies have reported the harmful effects of Cr (VI) on different organs, however, little is known about neurotoxic effects of Cr (VI). The aim of this study is to investigate the toxic effects of Cr (VI) on PC12 cells.Methods: PC12 cells were cultured following standard protocol and exposed to various concentrations (1-100 mM) of potassium dichromate (K2Cr2O7) for 24, 48 and 72 h. After exposure, cell viability was measured by the MTT assy. Also following exposure, production of reactive oxygen species (ROS) and lipid peroxidation were measured.Results: Potassium dichromate induced significant cell death in PC12 cells. The IC50 values for cytotoxicity were 22.02 for 24 h, 1.88 for 48 h, and 1.85 for 72 h of exposure.Significant differences between IC50 for 24 h of exposure compared to 48 and 72 h of exposure were observed (p<0.05). ROS production and lipid peroxidation significantly increased in the Cr (VI) treated groups compared to the control group (p<0.05).Conclusion: The results indicated that Cr (VI) induced dose and time dependent cytotoxicity in PC12 cells which indicated neurotoxic effects of Cr (VI). Mechanisms of Cr (VI) induced toxicity have not been fully determined, however increased production of ROS and lipid peroxidation in Cr (VI) treated groups demonstrated that oxidative stress might be involved in neurotoxicity of Cr (VI).

Objective: MicroRNAs (miRNAs) are single-stranded small RNAs 18-25 nucleotides in length that regulate gene expression through translational inhibition and mRNA cleavage.Aberrant expression of miRNAs contribute to several diseases. This has increased interest in profiling the expressions of these molecules. Real-time quantitative PCR (RQ-PCR) is a sensitive, quantitative technique for gene expression assessment. To correct for systematic variables such as the amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalized to an endogenous control gene which is stably-expressed across the test sample set. To avoid occurring further error in the quantification of gene expression data, it is necessary that candidate endogenous controls be validated in the samples of interest. In this study the expression of miRNA-16 and small nuclear RNA (snRNA) -U6 in hepatocellular carcinoma (HCC) cell lines under dendrosomal curcumin treatment were evaluated to identify appropriate endogenous controls for dendrosomal curcumin-related miRNA expression assays.Methods: HCC cell lines were treated with dendrosomal curcumin. Dendrosomal curcumin entry into HepG2 and HuH-7 cells was assessed by fluorescent microscopy images. RNA was extracted and cDNA, after polyA polymerization, was synthesised.Then, we performed gene expression assays using RQ-PCR.Results: In this treatment condition, miRNA-16 for HepG2, snRNA-U6 and the combined miRNA-16 and snRNA-U6 for HuH-7 were suitable endogenous controls.Conclusion: These genes are appropriate endogenous controls for miRNA expression assays in HCC cell lines under treatment with dendrosomal curcumin. There are stable, non-significant expression changes of these genes.

Objective: Different cryoprotectants are used for cryopreservation of ovarian tissue in patients at risk of infertility. Ethylene glycol (EG), dimethyl sulfoxide (DMSO) and propanediol (PROH) have been chosen as the basic permeable cryoprotectants due to their decreased glass-formation characteristics compared to other cryoprotectants. In the present study, the effects of two different vitrification methods on whole mouse ovarian tissue by the use of a novel staining method (trypan blue) has been evaluated.Methods: Ovaries of 8 day-old NMRI mice were isolated and divided among the control, vitrification 1 (Vit1) and vitrification 2 (Vit2) groups. The Vit1 solution was composed ofα-MEM+20% FBS+15% EG+15% DMSO. The Vit 2 solution was composed of α-MEM+15% FBS+20% EG+20% PROH. Vit1 and Vit2 procedures were performed at 4˚C and room temperature, respectively. Warming was performed in α-MEM+20% FBS supplemented with 1M sucrose in the Vit1 group and α-MEM+15% FBS with descending concentrations of sucrose (1, 0.5, 0.25 M) in the Vit2 group. Control and vitrified warmed ovaries were put in α-MEM supplemented by 0.4% trypan blue for 20 min, and then stained ovaries were fixed in Bouin’s fixative, serially sectioned in paraffin wax and finally quantitatively evaluated under a light microscope.Results: The highest percentage of primordial follicles was observed in the control group.There was a significant difference between the control and Vit1 groups, and between the Vit1 and Vit2 groups (p<0.05). No significant difference was observed in primary and preantral follicles between the control and vitrification groups.Conclusion: Vitrification with EG and PROH are more suitable for preservation of follicle reserves in ovaries. Trypan blue staining is a faster and easier method for evaluation of ovarian tissue.

Objective: Exercise activity could be an amendment to polycystic ovary syndrome (PCOS). However the acute and chronic effects of various exercise intensities on serum leptin levels are ambiguous. This study investigates the acute and chronic responses of various intensities of exercise on serum leptin levels and weights of female rats with PCOS.Methods: In this semi-empirical study, 80 adult Wistar rats (185±22 gr) after PCOS induction were divided into two groups. Group 1 participated in an exercise program at an intensity of 50%-55% maximal oxygen consumption (20 m/min), 70%-75% maximal oxygen consumption (28 m/min) and 80%-85% maximal oxygen consumption (34 m/min). Group 2 participated in an eight-week training program, three days a week for 60 minutes. One-way analysis of variance test (ANOVA) was used to compare differences between groups. Significance was p<0.05.Results: In the acute training group, there was no change in weight in the sub-groups of group 1. In group 2, training reduced in the medium intensity 2 compared to the PCOS control 2 groups. Serum leptin levels did not respond to one session of exercise at various intensities in group 1 subgroups. Leptin levels significantly reduced in the medium intensity 2 group compared to the PCOS control 2 (p=0.044) group.Conclusion: One exercise session does not seem to significantly affect serum leptin levels. Exercise training at medium intensity probably can reduce leptin levels and weight in subjects as a non-pharmaceutical alternative in PCOS patients.

Objective: Obesity increases production of the extracellular matrix (ECM) role in pathological cardiovascular damage. Regular exercise and the use of medicinal plants, particularly celery, in damage to be effective. The aim of this study is to investigate the changes in matrix metalloproteinases 2, 9 (MMP 2, 9) and tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) with synchronized exercise training and herbal supplementation with celery in overweight women.Methods: We randomly divided 28 overweight women into four groups: exercise, supplement, exercise–supplement, and control. Pilates training was performed for three sessions per week, for 60 minutes per session. Celery was administered at a dose of 3900 g per day in 3 capsules as a supplement. Blood sampling was performed before and 48 h after the last intervention. The analysis was performed by a paired t-test and one way analysis of variance (p£0.05).Results: After eight weeks, the levels of MMP-2, MMP-9 and body weight decreased and TIMP-1 increased in the exercise, supplement, and exercise–supplement groups (p<0.05).A significant difference was observed between the groups.Conclusion: The results showed that Pilates training and celery each, separately, had positive effects on MMP-2, MMP-9 and TIMP-1 in overweight women. However, the simultaneous effect of exercise and supplementation led to better efficiency.