scSPLAT, a scalable plate-based protocol for single cell WGBS library preparation

DNA methylation is a central epigenetic mark that has diverse roles in gene regulation, development, and maintenance of genome integrity. 5 methyl cytosine (5mC) can be interrogated at base resolution in single cells by using bisulfite sequencing (scWGBS). Several different scWGBS strategies have been described in recent years to study DNA methylation in single cells. However, there remain limitations with respect to cost-efficiency and yield. Herein, we present a new development in the field of scWGBS library preparation; single cell Splinted Ligation Adapter Tagging (scSPLAT). scSPLAT employs a pooling strategy to facilitate sample preparation at a higher scale and throughput than previously possible. We demonstrate the accuracy and robustness of the method by generating data from 225 single K562 cells and from 309 single liver nuclei and compare scSPLAT against other scWGBS methods.

DNA accessibility is not the primary determinant of chromatin-mediated gene regulation

ABSTRACTDNA accessibility is thought to be of major importance in regulating gene expression. We test this hypothesis using a restriction enzyme as a probe of chromatin structure and as a proxy for transcription factors. We measured the digestion rate and the fraction of accessible DNA at all genomicAluI sites in budding yeast and mouse liver nuclei. Hepatocyte DNA is more accessible than yeast DNA, consistent with longer linkers between nucleosomes, and indicating that nucleosome spacing is a major determinant of accessibility. DNA accessibility varies from cell to cell, such that essentially no sites are accessible or inaccessible in every cell.AluI sites in inactive mouse promoters are accessible in some cells, implying that transcription factors could bind without activating the gene. Euchromatin and heterochromatin have very similar accessibilities, suggesting that transcription factors can penetrate heterochromatin. Thus, DNA accessibility is not likely to be the primary determinant of gene regulation.

BIOCHEMICAL AND MORPHOMETRIC PARAMETERS OF SOME ORGANS AND TISSUES OF HYBRID TILAPIA (OREOCHROMIS SPP.) FOR GROWING WITH USE OF PREPARATION ES-2

The article studies the effect of addition into the feed of Sapropel extract (ES-2 preparation) on the intensity of lipid peroxidation in the liver and gills of hybrid tilapia ( Oreochromis spp. ), as well as on the morphofunctional state of its liver. Sapropel extract caused a decrease in the content of TBA-reactants in the tissues of tilapia liver by 17% compared to the control group. In gills the bioadditive resulted in the increased content of peroxide products by 24%. The introduction of ES-2 in fish feed resulted in reduction of spontaneous and ascorbate-dependent lipid peroxidation rate in the liver by 18%. In the gills of fish, under the influence of Sapropel, the rate of spontaneous lipid peroxidation increased by 27%, the rate of ascorbate-dependent lipid peroxidation - by 23%. The change in the intensity of peroxide processes under the influence of the fodder additive in fish organs is tissue-specific: antioxidant effect was recorded in the liver, prooxidant effect was observed in the gills. The introduction of the Sapropel extract does not lead to a change in the volume of liver nuclei in the test groups of tilapia, while the average cell volume in the experimental group was 37% lower than in the control group. The decrease in cell volume led to the increase in the nuclear-cytoplasmic ratio by 1.9 times in the experimental group compared to the control group. Hepatocyte cytoplasm volume decrease and nuclear-cytoplasmic ratio increase due to addition of ES-2 preparation into productive feed of hybrid tilapia would indicate a rise of functional activity of liver cells.

Insulin induction of SREBP-1c in rodent liver requires LXRα-C/EBPβ complex

Insulin increases lipid synthesis in liver by activating transcription of the gene encoding sterol regulatory element-binding protein-1c (SREBP-1c). SREBP-1c activates the transcription of all genes necessary for fatty acid synthesis. Insulin induction of SREBP-1c requires LXRα, a nuclear receptor. Transcription of SREBP-1c also requires transcription factor C/EBPβ, but a connection between LXRα and C/EBPβ has not been made. Here we show that LXRα and C/EBPβ form a complex that can be immunoprecipitated from rat liver nuclei. Chromatin immunoprecipitation assays showed that the LXRα-C/EBPβ complex binds to the SREBP-1c promoter in a region that contains two binding sites for LXRα and is known to be required for insulin induction. Knockdown of C/EBPβ in fresh rat hepatocytes or mouse livers in vivo reduces the ability of insulin to increase SREBP-1c mRNA. The LXRα-C/EBPβ complex is bound to the SREBP-1c promoter in the absence or presence of insulin, indicating that insulin acts not by increasing the formation of this complex, but rather by activating it.