2 research outputs found
NanoDCFH-DA: A Silica-based Nanostructured Fluorogenic Probe for the Detection of Reactive Oxygen Species
A biocompatible fluorescent nanoprobe for the detection of reactive oxygen species in biological systems has been designed, synthesized and characterized, circumventing some of the limitations of the molecular probe diacetyl 2',7'-dichlorodihydrofluorescein (DCFH-DA). It has been synthetized the nanoparticulate form of DCFH-DA by covalently attaching the widely used fluorescent probe DCFH-DA to a mesoporous silica nanoparticle through a linker. The reactivity of nanoDCFH-DA has been tested toward several reactive oxygen species. In addition, it has been proved to slow down DCFH-DA reaction with molecular oxygen and it hampers from interactions with proteins. As a final piece of evidence, in vitro studies showed that the nanoprobe is internalized by HeLa cancer cells, thus being capable of detecting intracellularly generated reactive oxygen species. To sum up, it can be stated that nanoDCFH-DA overcomes two major problems of free DCFH-DA, namely oxidation of the probe by air and interaction with proteins in biological systems. This "nano" approach has thus proven useful to extend the utility of an existing and valuable fluorescent probe to complex biological systems.status: publishe
Stochastic modeling reveals kinetic heterogeneity in post-replication DNA methylation.
DNA methylation is a heritable epigenetic modification that plays an essential role in mammalian development. Genomic methylation patterns are dynamically maintained, with DNA methyltransferases mediating inheritance of methyl marks onto nascent DNA over cycles of replication. A recently developed experimental technique employing immunoprecipitation of bromodeoxyuridine labeled nascent DNA followed by bisulfite sequencing (Repli-BS) measures post-replication temporal evolution of cytosine methylation, thus enabling genome-wide monitoring of methylation maintenance. In this work, we combine statistical analysis and stochastic mathematical modeling to analyze Repli-BS data from human embryonic stem cells. We estimate site-specific kinetic rate constants for the restoration of methyl marks on >10 million uniquely mapped cytosines within the CpG (cytosine-phosphate-guanine) dinucleotide context across the genome using Maximum Likelihood Estimation. We find that post-replication remethylation rate constants span approximately two orders of magnitude, with half-lives of per-site recovery of steady-state methylation levels ranging from shorter than ten minutes to five hours and longer. Furthermore, we find that kinetic constants of maintenance methylation are correlated among neighboring CpG sites. Stochastic mathematical modeling provides insight to the biological mechanisms underlying the inference results, suggesting that enzyme processivity and/or collaboration can produce the observed kinetic correlations. Our combined statistical/mathematical modeling approach expands the utility of genomic datasets and disentangles heterogeneity in methylation patterns arising from replication-associated temporal dynamics versus stable cell-to-cell differences