ABSTRACT
Objective: Dual inhibition of mitogen-activated protein kinase [MAPK] kinase [also known as MEK] and transforming growth factor beta [TGFbeta] type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells [ESC]. In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition
Materials and Methods: In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast [MEF] feeder cells in both R2i and serum conventional media. The isolated inner cell mass [ICM], ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real timepolymerase chain reaction [qRT-PCR] analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC
Results: qRT-PCR revealed a significantly higher expression of the pluripotency-related genes [Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and beta-Catenin] and lower expression of early differentiation genes [Gata6, Lefty2 and Cdx2] in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition
Conclusion: Our investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-beta signaling pathways in the first 5 days of ESC derivation
ABSTRACT
Selective deficiency of immunoglobulin A [IgA] is the most frequent primary hypogammaglobulinemia. As some IgA-deficient patients have IgA antibodies in their plasma which may cause anaphylactic reactions, blood centers usually maintain a list of IgA-deficient blood donors to prepare compatible blood components. In this study we determined the incidence of selective IgA deficiency [SIgAD] in normal adult Iranian population. 13022 normal Iranian blood donors were included in this study. The assay which we used was adapted to the manual pipetting system and ELISA reader was used for screening. Other classes of immunoglobulins [G, M], as well as secretory IgA and IgG subclasses were tested in IgA deficient cases by ELISA. SPSS was used for statistical analysis. Among 13022 studied cases, 11608 blood donors were males [89.14%] and 1414 were females [10.86%]. Their mean [ +/- SD] age and weight were 38.5 +/- 11 years and 82 +/- 12 Kg respectively. Twenty of the screened samples were found by means of ELISA to be IgA-deficient [less than 5mg/dl], [frequency; 1:651]. The data could indicate a compensation for IgA deficiency by serum IgM in one of our IgA deficient cases [Patient 5]. We observed a correlation between IgG3 and serum IgA in deficient cases [r=0.498, P=0.025]. Our results indicate that in present study the prevalence of S IgA D is in agreement with data from other Caucasians populations [from 1:300 to 1:700]. In conclusion, Selective IgA Deficiency could be almost asymptomatic in most cases in general population. Our study suggests that; due to high frequency of IgA deficiency in Iran, it seems necessary to measure IgA levels for every blood donor and blood recipient to find IgA deficient cases