Reactivation of a silenced H19 gene in human rhabdomyosarcoma by demethylation of DNA but not by histone hyperacetylation

BACKGROUND: The active copy of the imprinted gene H19 is turned off by inappropriate methylation in several pediatric tumors including Wilms' Tumour and embryonal rhabdomyosarcoma. H19 controls in cis the linked Insulin-like Growth Factor 2 (IGF2) gene, encoding an important growth factor. Recent work has suggested that methylation of a gene may lead to deacetylation of its associated histones and that silenced genes can be reactivated by increasing histone acetylation levels. RESULTS: Treatment of a rhabdomyosarcoma cell line which has a silent, methylated H19 gene with histone deacetylase (HDAC) inhibitors under conditions which gave maximal hyperacetylation of histone 4, both globally and at the H19 gene itself could not reactivate H19 or affect the active Insulin-like Growth Factor 2 (IGF2) gene, but caused clear up-regulation of the Tissue-type Plasminogen Activator (TPA) gene, a non-imprinted gene known to respond to changes in histone acetylation. In contrast, mild treatment of the cells with the methylation inhibitor 5-AzaC-2'-deoxycytidine (AzaC) on its own was able to reactivate H19. Combining AzaC treatment with HDAC inhibitors gave a reduced rather than enhanced reactivation. These findings were confirmed in mouse primary liver and kidney explants which maintain normal imprinting, where we also found that the silent Igf2 gene could not be reactivated by HDAC inhibitors. CONCLUSION: These results suggest that DNA methylation rather than histone acetylation is the primary determinant of silencing of H19 in rhabdomyosarcoma

OBTAINING THE SENSED TEMPERATURES FROM A DETAILED MODEL OF A WELDED THERMOCOUPLE

ABSTRACT When imbedded in dissimilar materials subject to large temperature gradients, thermocouples are known to yield erroneous (bias) temperature measurements. It has been established that the bias error may be accounted for with an appropriate computational model and the measured temperatures may be corrected with an appropriate kernel function. In this work, a thermocouple with a welded bead is considered. Early two-dimensional models considered the thermocouple to be a single wire with effective thermal properties. The model in the current investigation is threedimensional and represents the sensor as two wires, each with unique thermal properties. The welded bead is represented as a separate entity with properties distinct from those of the wires. The problem of determining what location in the threedimensional model corresponds to the measured temperature is considered. Earlier models have considered the sensed temperature to be the temperature at the tip of the twodimensional thermocouple or, in three-dimensional models, the temperature at the center of the volume of the welded bead. In the current work, a theory is set forth for identifying the location at which the temperature is sensed by a thermocouple. This theory is in line with traditional thermoelectric theory and is supported with experimental evaluation with thermal imaging as well as examination of thermocouples by scanning electron microscopy and energy dispersive X-ray analysis. The significance of accurate modeling of the sensed temperatures is demonstrated with a numerical experiment

\u3cem\u3eBorrelia burgdorferi\u3c/em\u3e SpoVG DNA- and RNA-Binding Protein Modulates the Physiology of the Lyme Disease Spirochete

The SpoVG protein of Borrelia burgdorferi, the Lyme disease spirochete, binds to specific sites of DNA and RNA. The bacterium regulates transcription of spoVG during the natural tick-mammal infectious cycle and in response to some changes in culture conditions. Bacterial levels of spoVG mRNA and SpoVG protein did not necessarily correlate, suggesting that posttranscriptional mechanisms also control protein levels. Consistent with this, SpoVG binds to its own mRNA, adjacent to the ribosome-binding site. SpoVG also binds to two DNA sites in the glpFKD operon and to two RNA sites in glpFKD mRNA; that operon encodes genes necessary for glycerol catabolism and is important for colonization in ticks. In addition, spirochetes engineered to dysregulate spoVG exhibited physiological alterations

Small store presence in Japan

The determinants of the development of small store presence in Japan are investigated using a fixed effects multinomial logit market share model. Large stores tend to have higher market shares in shop-types with increasing shares in consumer expenditures, increasing inventory turnover, and increasing diversification, but do not seem to be able to profit from scale economies in labour use

Statistical Inference of In Vivo Properties of Human DNA Methyltransferases from Double-Stranded Methylation Patterns

DNA methyltransferases establish methylation patterns in cells and transmit these patterns over cell generations, thereby influencing each cell's epigenetic states. Three primary DNA methyltransferases have been identified in mammals: DNMT1, DNMT3A and DNMT3B. Extensive in vitro studies have investigated key properties of these enzymes, namely their substrate specificity and processivity. Here we study these properties in vivo, by applying novel statistical analysis methods to double-stranded DNA methylation patterns collected using hairpin-bisulfite PCR. Our analysis fits a novel Hidden Markov Model (HMM) to the observed data, allowing for potential bisulfite conversion errors, and yields statistical estimates of parameters that quantify enzyme processivity and substrate specificity. We apply this model to methylation patterns established in vivo at three loci in humans: two densely methylated inactive X (Xi)-linked loci ( and ), and an autosomal locus (), where methylation densities are tissue-specific but moderate. We find strong evidence for a high level of processivity of DNMT1 at and , with the mean association tract length being a few hundred base pairs. Regardless of tissue types, methylation patterns at are dominated by DNMT1 maintenance events, similar to the two Xi-linked loci, but are insufficiently informative regarding processivity to draw any conclusions about processivity at that locus. At all three loci we find that DNMT1 shows a strong preference for adding methyl groups to hemi-methylated CpG sites over unmethylated sites. The data at all three loci also suggest low (possibly 0) association of the de novo methyltransferases, the DNMT3s, and are consequently uninformative about processivity or preference of these enzymes. We also extend our HMM to reanalyze published data on mouse DNMT1 activities in vitro. The results suggest shorter association tracts (and hence weaker processivity), and much longer non-association tracts than human DNMT1 in vivo