9 research outputs found
Three-Component Functionalized Dihydropyridine Synthesis via a Formal Inverse Electron-Demand Hetero-Diels–Alder Reaction
A mild
three-component synthetic approach to versatile 2-amino-1,4-dihydropyridines
from terminal alkynes, sulfonyl azides, and <i>N</i>-sulfonyl-1-aza-1,3-butadienes
was successfully developed and relied on the in situ generation of
metalated ynamide intermediates <b>Ib</b> to achieve a formal
inverse electron-demand hetero-Diels–Alder reaction. Experimental
results suggest that alkali metal cations (Li<sup>+</sup> and Cs<sup>+</sup> ions) might play a critical role to achieve the cycloaddition
process
Variation of DNA Methylome of Zebrafish Cells under Cold Pressure
<div><p>DNA methylation is an essential epigenetic mechanism involved in multiple biological processes. However, the relationship between DNA methylation and cold acclimation remains poorly understood. In this study, Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) was performed to reveal a genome-wide methylation profile of zebrafish (Danio rerio) embryonic fibroblast cells (ZF4) and its variation under cold pressure. MeDIP-seq assay was conducted with ZF4 cells cultured at appropriate temperature of 28°C and at low temperature of 18°C for 5 (short-term) and 30 (long-term) days, respectively. Our data showed that DNA methylation level of whole genome increased after a short-term cold exposure and decreased after a long-term cold exposure. It is interesting that metabolism of folate pathway is significantly hypomethylated after short-term cold exposure, which is consistent with the increased DNA methylation level. 21% of methylation peaks were significantly altered after cold treatment. About 8% of altered DNA methylation peaks are located in promoter regions, while the majority of them are located in non-coding regions. Methylation of genes involved in multiple cold responsive biological processes were significantly affected, such as anti-oxidant system, apoptosis, development, chromatin modifying and immune system suggesting that those processes are responsive to cold stress through regulation of DNA methylation. Our data indicate the involvement of DNA methylation in cellular response to cold pressure, and put a new insight into the genome-wide epigenetic regulation under cold pressure.</p></div
Enriched GO categories under cold pressure.
<p>GO enrichment analysis was performed in both 5-day and 30-day cold treated cells (cultured at 18°C) compared with control cells (cultured at 28°C). Genes related with DMRs were subjected to the analysis. GOs with ratio > = 1.5 and P value < = 0.05 are shown in the figure. P values for enrichment of GO categories are shown in the figure.</p
Variation of DNA methylation pattern of ZF4 cells under cold pressure.
<p>Genomic DNAs isolated from ZF4 cells cultured at 28°C, 18°C for 5 days and 30 days were subjected to MeDIP-seq analysis. <b>A</b>. Methylation peaks were distributed in promoter, exon, intron, downstream, intergenic regions as shown in the figure. <b>B</b>. Heat map of hierarchical clustering performed for all detected methylation peaks in all three samples shows variation of DNA methylation patterns under cold pressure. Each row represents a detected methylation peak.</p
FDRs of the methylation level comparisons from three different samples (28°C, 18°C /5d and 18°C /30d).
<p>FDRs of the methylation level comparisons from three different samples (28°C, 18°C /5d and 18°C /30d).</p
DNA methylation levels of different genomic regions under cold pressure.
<p>DNA methylation level of each genomic region from three samples (28°C, 18°C /5d, 18°C /30d) was shown as log2(RPKM+1). The RPKM value of each genomic region was compared as 18°C /5d vs 28°C, 18°C /30d vs 28°C and 18°C /30d vs 18°C /5d using T test. The corrected P values of FDR are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160358#pone.0160358.t001" target="_blank">Table 1</a>. CpG: CpG island; codexon: coding regions of exon; UP3k: regions within 3 kbs upstream gene; Down5k: regions within 5 kbs downstream gene; TSS 1k: regions within ±1 kb around transcription start site; LINE: long interspersed nuclear element; SINE: short interspersed nuclear element; Low complexity: low complexity DNA; Satellite: Satellite DNA; LTR: long terminal repeat.</p
Folate biosynthesis pathway (ko00790) was differentially distributed in 5- day cold treated cells.
<p>A. Differentially distributed KEGG pathways in cold treated cells compared with control cells. Wilcoxon signed-rank test was applied to identify KEGG pathways of which related peak-RPM values are differentially distributed in any of the two sample pairs (5d/0d: 18°C /5d vs 28°C, 30d/0d: 18°C /30d vs 28°C). We identified 5 KEGG pathways as differentially distributed that meet the criteria of P value < = 0.05 and Median RPM Ratio > = 1.1 or < = 1/1.1 (marked by blue solid triangles). Red dots present non-significantly changed KEGG. Folate biosynthesis pathway (ko00790) is marked by red circle. B. RPM heatmap of methylation peaks of folate biosynthesis pathway. Peak-RPM of folate biosynthesis pathway in three different samples (28°C, 18°C /5d, 18°C /30d) is shown by the density of the red color. C. RT-qPCR analysis of <i>gch1</i> and <i>gch2</i> genes. Relative expression was carried out by the comparative threshold cycle (CT) method. Statistical analysis was performed using GraphPad Prism 5 software. The Student <i>t</i> test was used on measurements from18°C /5d, 18°C /30d and 28°C samples from 3 experimental replicates.</p
Anti-ROS genes were hypomethylated under cold pressure.
<p>A. Cold stress induced ROS production in ZF4 cells. ZF4 cells were treated with cold stress at 18°C for 3 h, 6 h, 12 h, 1 d, 3 d and 5 d. The DCF positive cells were measured by flow cytometric analysis. Error bars represent standard deviations (SD) (n = 3). An asterisk represents significant difference of ROS level compared to 28°C sample (P<0.05). **: P<0.01, ***: P<0.001. B. Heatmap of RPM values of DNA methylation at promoter regions of anti-ROS genes. C. IGV image created from MeDIP-seq data for <i>selenbp1</i> gene. Black arrow shows the transcriptional direction of the gene. Red box shows differentially methylated peaks upstream of the gene. D. MeDIP-qPCR analysis of <i>selenbp1</i> gene. Frequency of DNA methylation was calculated by the comparative threshold cycle (CT) method. E. RT-qPCR analysis of <i>selenbp1</i> gene. Relative quantitation was carried out by the comparative threshold cycle (CT) method. Error bars represent standard deviations (SD) (n = 3). Data are normalized to β-actin. An asterisk represents significant difference compared to 28°C sample (P<0.05). **: P<0.01, ***: P<0.001.</p
Enrichment of H3K4me3 and H3K27me3 at promoter regions of p53 and selenbp1 gene.
<p>ChIP assays were performed in 28°C, 18°C /5d and 18°C /30d samples with H3K4me3, H3K27me3 and IgG antibodies. Primers specific for the promoters of <i>p53</i> (A) and <i>selenbp1</i> (B) were used for quantitative PCR of ChIP DNAs. Relative quantitation was carried out by the comparative threshold cycle (CT) method. Statistical analysis was performed using GraphPad Prism 5 software. The Student <i>t</i> test was used on measurements of enrichment from 18°C /5d, 18°C /30d and 28°C samples from 3 replicates. Error bars represent standard deviations (SD) (n = 3). An asterisk represents significant difference of enrichment compared to 28°C sample (P<0.05). **: P<0.01, ***: P<0.001.</p