No abstract

Modifications in chromatin, including DNA methylation and histone modification, are known to be important epigenetic determinants of gene transcription. DNA methylation levels fluctuate markedly in early mouse development. In preimplantation development, the mouse embryo undergoes active and passive genomic demethylation (3,15). This is restored at the time of implantation by the combined action of de novo and maintenance DNA methyltransferases (Dnmts). Studies of DNA methyltransferase 1-deficient (Dnmt1 Ϫ/Ϫ ) and Dnmt3a/ Dnmt3b-deficient (Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ]) mouse embryos have demonstrated that restoring DNA methylation is essential for development (13,19). Dnmt1 Ϫ/Ϫ and Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] embryos exhibit an early-lethal phenotype. At day 9.5 postcoitus, the embryos appear to have gastrulated but exhibit marked growth delay, having failed to turn or develop somites. In the presence of Dnmt1 deficiency, development is thought to fail because of cell death. Dnmt1-deficient embryoid bodies (EBs) aberrantly express Xist, down-regulate X-linked genes, and apoptose when induced to differentiate (20). Late-passage hypomethylated Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] embryonic stem (ES) cells are unable to form teratomas in vivo, but the cause of their differentiation failure has not been studied (4).Early embryonic development is characterized by high levels of Dnmt3a and Dnmt3b expression. These enzymes clearly have roles in initiating remethylation of the genome following preimplantation demethylation, but it is not known whether continued de novo methyltransferase activity is required for development once global remethylation has taken place. This was our reason for studying the differentiation of Dnmt[3a Ϫ/Ϫ , 3b Ϫ/Ϫ ] ES cells in vitro. These mutant ES cells were derived from fully methylated wild-type ES cells and would have been predicted to have retained most of their methylation because of the continued presence of the maintenance methyltransferase Dnmt1. In fact, while early-passage Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells are well methylated, DNA methylation levels fall progressively in culture (4). However, the rate of loss and the precise levels of methylation remaining have not been quantified. We have used a quantitative assay of DNA methylation to examine the effects of progressively decreasing genomic methylation levels on differentiation in vitro. Our studies reveal a clear but unexpected difference between the behaviors of hypomethylated Dnmt1 Ϫ/Ϫ and Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells in in vitro assays of differentiation. At very low levels of DNA methylation, Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells demonstrate an inability to initiate differentiation upon leukemia inhibitory factor (LIF) withdrawal, remaining viable and retaining markers characteristic of undifferentiated ES cells. MATERIALS AND METHODSES cell culture. ES cells were maintained on gelatin in a Glasgow modification of Eagle medium (Invitrogen) supplemented with 10% fetal calf serum, 100 M 2-mercaptoethanol, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 ...

Objective: To evaluate a peer-modelling and rewards-based intervention designed to increase children's fruit and vegetable consumption. Design: Over a 5-month period, children in an experimental and a control school were presented with fruit and vegetables at lunchtime. Children aged 5-7 y also received fruit at snacktime (mid-morning). The intervention was implemented in the experimental school and levels of fruit and vegetable consumption were measured at baseline, intervention and at 4-month follow-up. Setting: Two inner-city London primary schools. Subjects: In total, 749 children aged 5-11 y. Intervention: Over 16 days children watched video adventures featuring heroic peers (the Food Dudes) who enjoy eating fruit and vegetables, and received small rewards for eating these foods themselves. After 16 days there were no videos and the rewards became more intermittent. Main outcome measures: Consumption was measured (i) at lunchtime using a five-point observation scale; (ii) at snacktime using a weighed measure; (iii) at home using parental recall. Results: Compared to the control school, lunchtime consumption in the experimental school was substantially higher at intervention and follow-up than baseline (Po0.001), while snacktime consumption was higher at intervention than baseline (Po0.001). The lunchtime data showed particularly large increases among those who initially ate very little. There were also significant increases in fruit and vegetable consumption at home (Po0.05). Conclusions: The intervention was effective in bringing about substantial increases in children's consumption of fruit and vegetables.

The GAF can be rated reliably after minimal training. It provides a valid summary of symptoms and social functioning among schizophrenic patients provided they are not assessed when suffering from acute psychotic episodes.