Epigenetics refers to the study of mechanisms able to influence gene expression in a stable and potentially heritable manner without altering the DNA sequence. These mechanisms include posttranslational histone modifications, miRNA-mediated post-transcriptional regulation and DNA methylation. The accumulation of molecular errors over time resulting, at least partly, in the alteration of normal epigenetic patterns is being widely associated with aging. Epigenetic processes are also considered important mechanisms through which environmental and stochastic stressors promote numerous pathologies in humans. It is, therefore, reasonable to expect that several complex multi-factorial late-onset disorders, like osteoporosis and osteoarthritis, could have a strong epigenetic component. The focal point of all skeletal pathologies is the deregulation of bone remodeling, mediated by bone-forming osteoblasts and boneresorbing osteoclasts. In order to keep both processes in balance, the activity, differentiation and apoptosis of both cell types have to be tightly regulated. In particular, the differentiation of osteoblasts and osteoclasts is accompanied by profound changes in gene expression. It has been shown that histone deacetylation and DNA methylation negatively regulate the expression of several genes associated with different stages of osteoblast differentiation; however, several miRNAs promote osteoblastogenesis. Furthermore, inactivating mutations in the miRNA coding regions could be associated with the pathogenesis of osteoporosis. The aim of this review is to highlight the role of epigenetic mechanisms in bone remodeling and bone homeostasis, so as to implicate their diagnostic and therapeutic potential in skeletal diseases.
COBISS.SI-ID: 3604593
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 trypsinʼs 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.
COBISS.SI-ID: 3690865
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.
COBISS.SI-ID: 3644273