Lipin 1 is a recently discovered multifunctional protein involved in the metabolism of lipids, while PPARgamma is involved in adipocyte differentiation, and regulation of lipid metabolism. Up to now, ...LPIN1 and PPARG gene polymorphisms have been associated with type 2 diabetes, metabolic syndrome, and central obesity. In this study, we hypothesized that genetic variants within LPIN1 and PPARG genes were associated with traits of metabolic syndrome. Correlation between biochemical parameters (including but not limited to, glucose, HbA1c, insulin levels, HDL and LDL cholesterol, triglycerides, serum proteins, liver enzymes) and frequency of polymorphisms in LPIN1 (rs11693809 and rs2716610) and PPARG gene (rs10865710, rs3856806 and rs1801282), was tested in this study.
The study included 70 patients diagnosed with metabolic syndrome and type 2 diabetes. Two polymorphisms of LPIN1 gene (rs11693809 and rs2716610), and three polymorphisms of PPARG gene (rs10865710, rs385806 and rs1801282) were analyzed by real time PCR and conventional PCR-RFLP methods.
Our analysis revealed correlation between insulin levels and rs11693809 LPIN1 polymorphism in diabetic patients. Also the results of this study showed an association of rs10865710 and rs385806 polymorphism of PPARG with HDL cholesterol and LDL plus total cholesterol levels, respectively.
These data reflect an association of analyzed PPARG and LPIN1 gene polymorphisms with values of insulin, HDL, LDL and total cholesterol witch indicates an important role of these genes in lipid metabolism and pathogenesis of type 2 diabetes and metabolic syndrome.
Plasma/serum microRNAs (miRNAs) are proposed to have a role in the development of osteoporosis, and their levels can be affected by physical activity. Therefore, the aim of this study was to analyze ...differences in plasma miRNAs levels in postmenopausal women and to simultaneously find novel extracellular miRNAs that are associated with variations in physical activities.
We measured hsa-miR-148a-3p, hsa-miR-125b, hsa-miR-21-5p, hsa-miR-93-5p, and hsa-miR-30d-5p in a cohort of 149 postmenopausal women divided into subgroups based on the amount of physical activity by using quantitative polymerase chain reaction (qPCR). Further, the correlation of plasma microRNAs with sit-to-stand performance was examined. DIANA web server was used to find which Kyoto Encyclopedia of Genes and Genomes pathways (P < 0.05) were regulated by miRNAs associated with physical activity and sit-to-stand performance.
Plasma levels of hsa-miR-30d-5p were significantly (P = 0.015) lower in participants with higher physical activity, and a borderline trend for hsa-miR-21-5p (P = 0.06) was noticed. Higher plasma levels of hsa-miR-21-5p (P = 0.01) and hsa-miR-93-5p (P = 0.04) were observed in the group with reduced sit-to-stand performance. The two miRNAs seem to primarily regulate fatty acid metabolism-related processes.
Our results suggest that levels of selected cell-free miRNAs in postmenopausal women are associated with different physical activity regimes.
This is the first study performed in population from Bosnia & Herzegovina (BH), in which we analysed a significance of genetic variations in drug-metabolising enzyme, cytochrome P450 (CYP), in ...pathogenesis of Type 2 diabetes. We have determined allele frequencies for CYP2C9*2, CYP2C19*2, and CYP2D6*4 in diabetic patients and nondiabetic controls. Genomic DNA was extracted from blood samples collected from 37 diabetic and 44 nondiabetic subjects. A real-time polymerase chain reaction was used for the detection of specific CYP polymorphisms, with the application of the specific TaqMan® SNP genotyping tests (Applied Biosystems). Interestingly, results from this study have demonstrated that frequencies of CYP2C19*2 and CYP2D6*4 variants were in line, while frequency of CYP2C9*2 polymorphism seemed to be lower in this sample of BH population as compared to the Caucasians genotype data. Furthermore, no significant difference in allele frequencies for CYP2C9*2, CYP2C19*2, and CYP2D6*4 was demonstrated between diabetic and nondiabetic subjects. Thus, results form this study seem to indicate no relationship between CYP2C9, CYP2C19, and CYP2D6 genotype and diabetes susceptibility in Bosnian population. This in part may reflect a limited study population included in our study and would require larger cohorts to reveal potential relationships between analysed CYP genetic variants and diabetes risk. In addition, it would be pertinent to further explore possible effects of CYP genetic variations on therapeutic and adverse outcomes of oral antidiabetics, which might be the key in optimising therapy for individual patient with Type 2 diabetes.
Gilbert's syndrome is one of the most common metabolic syndromes in the human population characterised by mild unconjugated hyperbilirubinemia resulting from reduced activity of the bilirubin ...conjugating enzyme UDP-glucuronosyltransferase (UGT1A1). Although Gilbert's syndrome is usually quite benign UGT1A1(TA)n genotyping is important in exclusion of more serious causes of hyperbilirubinemia and since it has significant implications for personalised medicine. The aim of our study was to develop plasmid based reference materials which could be used for UGT1A1(TA)n genotyping.
Plasmids were generated using recombinant DNA technology and their number of repeats as well as the entire sequence verified by Sanger sequencing. Their suitability as reference materials was tested using sizing by capillary electrophoresis and denaturing high performance liquid chromatography.
Plasmids containing all four different alleles (TA)5, (TA)6, (TA)7 and (TA)8 that are present in the human population as well as a plasmid with (TA)4 repeats were successfully generated.
Prepared plasmid reference materials allow the creation of all possible UGT1A1(TA)n polymorphism genotypes and can serve as an efficient substitute for the human genomic DNA reference material in routine genotyping and in the development of new genotyping tests.
•We developed a new plasmid reference material for UGT1A1(TA)n genotyping.•Plasmid reference material contains all four different alleles known to be present in the human population.•Plasmid reference material contains enough sequence upstream and downstream of polymorphic site for novel method development.•A new fragment analysis method for UGT1A1(TA)n genotyping was developed.
The accuracy of techniques such as microarrays, reverse transcription polymerase chain reaction, and whole transcriptome shotgun sequencing is critically dependent on RNA quality. We have repeatedly ...observed extensive RNA degradation following trypsinization, a routine procedure used to dissociate adherent tissue culture cells prior to RNA extraction. This study investigated the cause of this degradation and identifies an alternative procedure that enables extraction of intact high-quality RNA. Trypsinization and several alternative procedures were used to dissociate a range of different cell lines prior to RNA extraction. The contribution of exogenous ribonucleases or induction of endogenous ribonucleases by trypsin reagent proteases to RNA degradation was examined. Trypsinization resulted in a complete degradation of RNA regardless of cell line type, differentiation stage, or passage number. This occurred when intact RNA was incubated directly with trypsin and was not suppressed by inhibiting trypsin’s protease activity. Prevention of degradation by sodium hypochlorite treatment of trypsin reagent identified the presence of ribonucleases in trypsin derived from animal pancreas. Consistent extraction of high-quality RNA requires the use of direct cell lysis with a phenol guanidine-based reagent or an animal origin-free protease-based dissociation agent if enzymatic detachment prior to RNA extraction cannot be avoided.
Differences in the frequency of distribution of the cytochrome P450 (CYP) allelic variants have been demonstrated between distinct ethnic groups, contributing to observed interindividual variation in ...drug response. In this study we determined, for the first time, prevalence of the common allelic variants of the polymorphic CYP enzymes, CYP3A4*1B and CYP3A5*3, in the population of Bosnia and Herzegovina (BH).
Genomic DNA was extracted from blood samples collected from 140 unrelated subjects. A real-time PCR was used for the detection of CYP polymorphisms, with the application of the specific TaqMan SNP Genotyping Assay (Applied Biosystems) for CYP3A5*3, while CYP3A4*1B was genotyped by high-resolution melting analysis.
Our results have shown that the distribution of CYP3A4*1B and CYP3A5*3 alleles was in line with the data reported in European Caucasians. We confirmed that CYP3A4*1B mutant allele is rare in Caucasians, being present in only 5.1% individuals. However, CYP3A5*3 polymorphism was found to be predominant in the Bosnian population with an incidence of 94%, similarly to other European populations tested so far. Interestingly, we have demonstrated a strong linkage disequilibrium between CYP3A5*3 and CYP3A4*1B alleles. No significant difference in allele frequencies for CYP3A4*1B and CYP3A5*3 has been shown between male and female subjects participating in our study.
Our data demonstrated the high prevalence of CYP3A5*3 allele in Bosnian population, indicating significance of analysis of CYP3A5 and CYP3A4 polymorphisms and corresponding allele frequencies in specific ethnic groups. Importantly, results of this study may lead to translation of pharmacogenetics and individualized therapeutic approach in current clinical practices in BH.
Abstract
Purpose of the study: Sufficient oxygen supply to bone tissue is essential for normal bone development and efficient bone repair. Hypoxia and hypoxia-inducible factor 1 (HIF1 ) signaling ...pathway have been shown to exhibit profound effects on proliferation, differentiation as well as gene and protein expression in osteoblasts, osteoclasts and mesenchymal stem cells; however, as epigenetic mechanisms also perform an important regulatory role in these cells, our aim was to elucidate whether hypoxia mimetic deferoxamine could influence epigenetic mechanisms in bone cells by modulating the gene expression levels of chromatin-modifying enzymes.
Materials and methods: Osteoblast cell line HOS was exposed to deferoxamine, a widely used hypoxia mimetic, and expression profile of 40 genes associated with histone acetylation, deacetylation and DNA methylation was determined using quantitative real time polymerase chain reaction (qPCR) array followed by individual qPCR analyses. In addition, genes associated with hypoxia response, RANK/RANKL/OPG system, WNT/ -catenin signaling pathway and oxidative stress were also analyzed.
Results: We observed induced expression of histone deacetylase 9 (HDAC9) and suppressed expression of K(lysine) acetyltransferase 5 (KAT5) and DNA methyltransferase 3A (DNMT3A) demonstrating for the first time that expression of genes encoding chromatin-modifying enzymes could be influenced by hypoxia mimetic in HOS cells.
Conclusions: Based on our results we can conclude that hypoxia mimetic deferoxamine influences expression of histone acetylation- and DNA methylation-associated genes in osteoblasts and that further studies of hypoxia-induced epigenetic changes in bone cells should be undertaken.
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