Distinct growth factor-induced dynamic mass redistribution (DMR) profiles for monitoring oncogenic signaling pathways in various cancer cells

Targeting dysregulated signaling pathways in tumors has led to the development of a novel class of signal transduction inhibitors, including inhibitors of the epidermal growth factor (EGF) receptor (EGFR). To dissect oncogenic pathways, identify key pathway determinants, and evaluate the efficacy of targeted agents, it is vital to develop technologies that allow the detection of temporal signaling events under physiological conditions. Here we report the application of a label-free optical biosensor to reveal the rapid response of cancer cells to EGF, expressed as a dynamic mass redistribution (DMR) signal. In response to EGF, squamous cell carcinoma of the head and neck cells exhibited a rapid rise in DMR signal, whereas lung adenocarcinoma cells showed a biphasic DMR profile, suggesting a cell type-dependent DMR response. Pharmacological studies suggested the importance of EGFR and the phosphatidylinositol-3 kinase pathway in mediating the EGF-induced DMR response. The defined DMR signatures offer a simple yet sensitive tool for evaluating EGFR-targeted agents, as shown with gefitinib and erlotinib. The assay can also be used for cell-based high-throughput screening of EGF pathway inhibitors, as demonstrated by its robust performance in a 384-well plate format (Z′?>?0.5). This technology is applicable to other oncogenic pathways for the discovery of novel therapeutic agents for the treatment of various cancers.     

Label-free optical biosensor for ligand-directed functional selectivity acting on beta(2) adrenoceptor in living cells

Recent realization of ligand-directed functional selectivity demands high-resolution tools for studying receptor biology and ligand pharmacology. Here we use label-free optical biosensor to examine the dynamic mass redistribution of human epidermoid A431 cells in response to diverse beta(2)-adrenoceptor ligands. Multi-parameter analysis reveals distinct patterns in activation and signaling of the receptor induced by different agonists. Sequential and co-stimulation assays categorize various ligands for their ability to modulate signaling induced by catechol, a structural component of catecholamines. This study documents multiple ligand-specific states of the beta(2)-adrenoceptor and highlights the power of the biosensor assays for screening pathway-biased ligands.     

Sequences in the H19 ICR that are transcribed as small RNA in oocytes are dispensable for methylation imprinting in YAC transgenic mice

Allele-specific methylation of the endogenous H19 imprinting control region (ICR) is established in sperm. We previously showed that the paternal H19 ICR in yeast artificial chromosome (YAC) transgenic mice (TgM) was preferentially methylated in somatic cells, but not in germ cells, suggesting that differential methylation could be established after fertilization. In this report, we discovered small RNA molecules in growing oocytes, the nucleotide sequences of which mapped to the H19 ICR. To test if these small RNA sequences play a role in the establishment of differential methylation, we deleted the sequences from the H19 ICR DNA and generated YAC TgM. In somatic cells of these mice, methylation imprinting of the transgene was normally established. In addition, the mutant fragment was not methylated in sperm and eggs. These data demonstrate that sequences in the H19 ICR that correspond to the small RNA sequences are dispensable for methylation imprinting in YAC TgM.     

DNA methylation-mediated silencing of neuronatin (NNAT) in pig parthenogenetic fetuses

It is generally believed that aberrant expression of imprinted genes participates in growth retardation of mammalian parthenogenesis. Neuronatin (NNAT), a paternally expressed gene, plays important roles in neuronal growth and metabolic regulation. Here we have compared the gene expression and promoter methylation pattern of NNAT between pig normally fertilized (Con) and parthenogenetic (PA) embryos. The results showed loss of NNAT expression (p < 0.001) and hypermethylation of NNAT promoter in PA samples. Additionally, partial methylation was observed in Con fetuses, while almost full methylation and unmethylation of NNAT promoter were apparent in Metaphase II (MII) oocytes and mature sperms, respectively, which identified the CpG promoter region as a putative differentially methylated region (DMR) of NNAT. The data demonstrate that promoter hypermethylation is associated with the silencing of NNAT in pig PA fetuses, which may be related to developmental failure of pig parthenogenesis at early stages.     

Duodenal Mucosa: A New Target for the Treatment of Type 2 Diabetes

ObjectiveAfter a high-fat and high-sugar diet, the duodenal mucosa of rodents proliferate and trigger the signal of insulin resistance, which may be the cause of type 2 diabetes (T2D). In response to this phenomenon, researchers have designed the duodenal mucosal resurfacing (DMR) procedure, mainly through the hydrothermal ablation procedure, to restore the normal mucosal surface, thereby correcting this abnormal metabolic signal. This article aims to understand the changes in duodenum before and after the onset or treatment of T2D, and the potential mechanisms of DMR procedure.MethodsA literature search of PubMed and Web of Science was conducted using appropriate keywords.ResultsBoth animal and clinical studies have shown that the villus thickness, intestinal cells, glucose transporters, enteric nerves, and gut microbiota and their metabolites in the duodenum undergo corresponding changes before and after the onset or treatment of T2D. These changes may be related to the pathogenesis of T2D. DMR procedure may produce beneficial glycemic and hepatic metabolic effects by regulating these changes.ConclusionThe duodenum is an important metabolic signaling center, and limiting nutrient exposure to this critical region will have powerful metabolic benefits. The DMR procedure may regulate glycemic and hepatic parameters through various mechanisms, which needs to be further confirmed by a large number of animal and clinical studies.     

Inheritance Patterns and Stability of DNA Methylation Variation in Maize Near-Isogenic Lines

DNA methylation is a chromatin modification that contributes to epigenetic regulation of gene expression. The inheritance patterns and trans-generational stability of 962 differentially methylated regions (DMRs) were assessed in a panel of 71 near-isogenic lines (NILs) derived from maize (Zea mays) inbred lines B73 and Mo17. The majority of DMRs exhibit inheritance patterns that would be expected for local (cis) inheritance of DNA methylation variation such that DNA methylation level was coupled to local genotype. There are few examples of DNA methylation that exhibit trans-acting control or paramutation-like patterns. The cis-inherited DMRs provide an opportunity to study the stability of inheritance for DNA methylation variation. There was very little evidence for alterations of DNA methylation levels at these DMRs during the generations of the NIL population development. DNA methylation level was associated with local genotypes in nearly all of the >30,000 potential cases of inheritance. The majority of the DMRs were not associated with small RNAs. Together, our results suggest that a significant portion of DNA methylation variation in maize exhibits locally (cis) inherited patterns, is highly stable, and does not require active programming by small RNAs for maintenance.DNA methylation may contribute to heritable epigenetic information in many eukaryotic genomes. In this study, we have documented the inheritance patterns and trans-generational stability for nearly 1000 DNA methylation variants in a segregating maize population. At most loci studied, the DNA methylation differences are locally inherited and are not influenced by the other allele or other genomic regions. The inheritance of DNA methylation levels across generations is quite robust with almost no examples of unstable inheritance, suggesting that DNA methylation differences can be quite stably inherited, even in segregating populations.