Correlation between Methylation and Expression Level of P15 and P16 Genes during Differentiation of Cord Blood Stem Cells into Erythroid Lineage Mediated by Erythropoietin

Background: Several influential factors such as transcription factors and intracellular signaling components are involved in differentiation of stem cells into a specific lineage. P15 and p16 proteins are among these factors. Accumulating evidences has introduced the epigenetic as a master regulator of these factors during lineage specification. The main objective of this study is to determine the correlation between the expression level and methylation pattern of P15 and P16 genes in erythroid lineage after in vitro differentiation by erythropoietin (EPO).Materials and Methods: The purified and expanded CD34+ cord blood stem cells were differentiated into erythroid lineage in the presence of EPO. DNA was isolated from both cord blood stem cells and differentiated cells. The Real-Time PCR performed using cDNA and the isolated DNA was used in methylation Specific PCR (MSP) reaction for methylation pattern analysis in both pre and post differentiation stages.Results: The study demonstrated that P15 and P16 genes have partial methylation after erythroid differentiation by EPO. The Expression of P15 gene was higher after differentiation and the expression of P16 gene had a slightly decreased level in post differentiation stage.Conclusion: Significant increase in P15 gene expression after differentiation to erythroid lineage, suggests the remarkable efficacy of this gene in erythroid function. According to upregulation of P15 gene after differentiation despite unchanged methylation status and slight down regulation of P16 gene with slight hyper-methylation of the gene it can be suggested that although the methylation can affects the expression level of P16 gene, the P15 gene is not affected by this mechanism during erythroid differentiation mediated by EPO

Gene Expression Status and Methylation Pattern in Promoter of P15INK4b and P16INK4a in Cord Blood CD34+ Stem Cells

Objective(s): Stem cell differentiation into different cell lineages depends upon several factors, cell cycle control elements and intracellular signaling elements, including P15INK4b and P16INK4a genes. Epigenetics may be regarded as a control mechanism which is affected by these factors with respect to their promoter structure.   Materials and Methods: The CD34 + cord blood stem cells were purified, isolated and then expanded. The undifferentiated day genome was isolated from part of the cultured cells, and the seventh day differentiated genome was isolated from the other part after differentiation to erythroid lineage. The procedure was followed by a separate Real-Time PCR for the two genes using the obtained cDNA. The processed DNA of the former stages was used for MSP (Methylation Specific PCR) reaction. Finally, pre- and post differentiation results were compared. Results: After performing MSP for each gene, it became clear that P15INK4b gene has undergone methylation and expression in predifferentiation stage. In addition, its status has not been changed after differentiation. P15INK4b gene expression was reduced after the differentiation. The other gene, P16INK4a, showed no predifferentiation methylation. Itwas completely expressed methylated and underwent reduced expression after differentiation. Conclusion : Specific predifferentiation expression of P15INK4b and P16INK4a genes along with reduction in their expression after erythroid differentiation indicated animportant role for these two genes in biology of CD34+ cells in primary stages and before differentiation. In addition, both genes are capable of epigenetic modifications due to the structure of their promoters

Neonatal Infections: a 5-Year Analysis in a Neonatal Care Unit in North East of Iran

Background: Neonatal infections are one of the major causes of death in Iran. Since identifying the risk factors, types, site, bacterial causes, and case fatality rate of an infection can be effective in selecting preventive and therapeutic methods, and appropriate supportive measures, this study aimed to investigate the aforementioned factors in the neonatal intensive care unit (NICU) of Ghaem Hospital in Mashhad- Iran during a 5-year period.Materials and Methods: This cross-sectional study was conducted from Jan 2010 to Jun 2016 on 221 infants diagnosed with infections (positive blood, cerebrospinal fluid, or urine cultures, and radiographic evidence of lung infection as well as laboratory and clinical evidence of infection). Data collection tools consisted of a researcher-made questionnaire including maternal and neonatal characteristics and clinical and laboratory evaluation. Moreover, the infants were followed up until hospital discharge or death. Data were analyzed using SPSS-16.Results: The incidence of neonatal infection was 11.6%. About 70% of the infants were born preterm and 52% of the infected infants were born by cesarean. The most common pathogens of sepsis were gram-negative bacteria (55%), coagulase-negative staphylococci (35%) and other gram-positive bacteria (10%). There were three main causes of infection of central nervous system (CNS): Klebsiella (66%), Escherichia coli (17%), and Acinetobacter (17%). Infant mortality rate due to infection was 28.1%. The causes of death included meningitis (60%), sepsis (27%), and UTI (16%).Conclusion: According to our study, the prevalence of infection and mortality rate in our ward is higher compared to developed countries. The most common cause of infections was gram-negative bacteria, but coagulase-negative staphylococci become more prevalent and needs more attention