Smart-RRBS for single-cell methylome and transcriptome analysis

The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study. Here, we present details of the protocol and provide guidance on its utility. Our Smart-RRBS (reduced representation bisulfite sequencing) protocol combines Smart-seq2 and RRBS and entails physically separating mRNA from the genomic DNA. It generates paired epigenetic promoter and RNA-expression measurements for ~24% of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes ~3 d to process up to 192 samples manually. It requires basic molecular biology expertise and laboratory equipment, including a PCR workstation with UV sterilization, a DNA fluorometer and a microfluidic electrophoresis system

Loss of DNA methyltransferase activity in primed human ES cells triggers increased cell-cell variability and transcriptional repression

Maintenance of pluripotency and specification towards a new cell fate are both dependent on precise interactions between extrinsic signals and transcriptional and epigenetic regulators. Directed methylation of cytosines by the de novo methyltransferases DNMT3A and DNMT3B plays an important role in facilitating proper differentiation, whereas DNMT1 is essential for maintaining global methylation levels in all cell types. Here, we generated single-cell mRNA expression data from wild-type, DNMT3A, DNMT3A/3B and DNMT1 knockout human embryonic stem cells and observed a widespread increase in cellular and transcriptional variability, even with limited changes in global methylation levels in the de novo knockouts. Furthermore, we found unexpected transcriptional repression upon either loss of the de novo methyltransferase DNMT3A or the double knockout of DNMT3A/3B that is further propagated upon differentiation to mesoderm and ectoderm. Taken together, our single-cell RNA-sequencing data provide a high-resolution view into the consequences of depleting the three catalytically active DNMTs in human pluripotent stem cells

Egg performance, egg quality, and nutrient utilization in laying hens fed diets with different levels of rapeseed expeller cake

The aim of this study was to evaluate the effect of different levels of rapeseed expeller cake (RC) in the diet of laying hens on egg performance, egg quality, retention and excretion of nitrogen, calcium and phosphorus, and metabolizability of energy. The experiment was carried out with 72 Bovans Brown hens, from 28 to 53 weeks of age. Experimental treatment consisted of four isonitrogenous and isocaloric corn-soybean diets (as calculated: 17% crude protein, 11.6 MJ/kg metabolizable energy, 0.81% lysine, 0.36% methionine, 3.60% calcium and 0.37% available phosphorus), containing 0, 4, 6 or 8% RC. The RC used was produced from double zero rapeseed and contained 29.3% crude protein, 17.4% crude fat, 10.8% crude fibre, 0.63% calcium, 0.97% phosphorus, 1.91% lysine and 0.84% methionine. During the experimental period, the dietary level of RC had no significant effects on egg performance and egg quality parameters. Boiled eggs from hens fed a diet with 8% RC were characterized by an inferior flavour to those from other groups. There were no treatment effects on nitrogen balance or metabolizability of energy, though the highest dietary level of RC negatively affected retention and excretion of calcium and phosphorus.

Effects on performance and eggshell quality of particle size of calcium sources in laying hens' diets with different Ca concentrations

The objective of this study was to evaluate the effect of particle size of a dietary Ca source on egg production and eggshell quality when added to hens' diets that have different levels of calcium. The experiment was carried out on 216 ISA Brown hens (25 to 70 weeks of age), allocated to 9 groups of 12 replicates (cages), with two birds in each cage. A 3 × 3 factorial arrangement was used, with three dietary levels of calcium (3.20, 3.70 and 4.20 %) and three levels of dietary substitutions (0, 25 and 50 %) of fine particles of limestone (FPL, diameter 0.2–0.6 mm) with large particles of limestone (LPL, diameter 1.0–1.4 mm) as a Ca source. The level of Ca in the diet had no effect on egg production, mean egg weight, feed intake, feed conversion ratio or eggshell quality parameters (P > 0.05). Substitution of FPL with LPL did not affect laying performance indices or eggshell quality at 30, 43 and 53 weeks of age (P > 0.05); however, it increased (P < 0.05) eggshell percentage, thickness, density and breaking strength in older hens (69 weeks of age). In conclusion, the results of this study demonstrated that a level of 3.20 % Ca in a layer's diet is sufficient through the entire laying cycle to maintain good egg production and eggshell quality and that partial (25 or 50 %) substitution of fine- with large-particle limestone can, irrespective of the level of Ca in the diet, improve eggshell quality in aged laying hens

Antimicrobial efficacy of mixtures of silver nanoparticles and polyhydric alcohols against health-promoting bacteria

In the present study, the effectiveness of a mixture of silver nanoparticles with polyhydric alcohols (glycerol, erythritol, mannitol and xylitol) against six species of healthpromoting bacteria have been examined. Synthesis of silver nanoparticles was carried out using trisodium citrate as the reducing and stabilizing agent. The nanoparticles were characterized by electronic absorption, scanning electron microscopy and powder X-ray diffraction measurements. Electronic absorption spectrum revealed high uniform of synthesized nanoparticles. Practically no aggregation was observed when nanoparticles were mixed with polyhydric alcohols, suggesting weak interaction between ingredients of the mixture. Spherical silver nanoparticles, as depicted by scanning electron microscopy, were found to have diameters in the range of 10 to 30 nm; mean diameter was 18 ± 4 nm. The X-ray diffraction pattern of the prepared samples indicated the face-centred cubic crystalline structure of the metallic silver nanoparticles. In biological study, quite interesting protective effect of polyalcohols on the growth inhibition of health-promoting bacteria by silver nanoparticles was observed. The most substantial protective effect of the tested silver nanoparticles-polyalcohol mixtures was estimated for B. bifidum, L. paraplantarum, and L. phamnosus species

Assessing the possibility of genetically modified DNA transfer from GM feed to broiler, laying hen, pig and calf tissues

The aim of this study was to assess the possibility of genetically modified DNA transfer from feed containing RR soybean or/and MON810 maize to animal tissues, gut bacterial flora, food of animal origin, and the fate of GM DNA in the animal digestive tract. The experiment was carried out on broilers, laying hens, pigs and calves. All animals were divided into four groups: I – control group (non-modified feed), II – GM soybean group (non-modified maize, RR soybean), III – GM maize group (MON810 maize, non-modified soybean), and IV – GM maize and soybean group (MON810 maize, RR soybean). Samples of blood, organs, tissues, digesta from the gastrointestinal tract, and eggs were analysed for the presence of plant species specific genes, and transgenic sequences of CaMV 35S promoter and NOS terminator. PCR amplifications of these GM sequences were conducted to investigate the GM DNA transfer from feed to animal tissues and bacterial gut flora. In none of the analysed samples of blood, organs, tissues, eggs, excreta and bacterial DNA were plant reference genes or GM DNA found. A GM crop diet did not affect bacterial gut flora as regards diversity of bacteria species, quantity of particular bacteria species in the animal gut, or incorporation of transgenic DNA to the bacteria genome. It can be concluded that MON810 maize and RR soybean used for animal feeding are substantially equivalent to their conventional counterparts. Genetically modified DNA from MON810 maize and RR soybean is digested in the same way as plant DNA, with no probability of its transfer to animal tissues or gut bacterial flora

APOE4 disrupts intracellular lipid homeostasis in human iPSC-derived glia

The E4 allele of the apolipoprotein E gene (APOE) has been established as a genetic risk factor for many diseases including cardiovascular diseases and Alzheimer’s disease (AD), yet its mechanism of action remains poorly understood. APOE is a lipid transport protein, and the dysregulation of lipids has recently emerged as a key feature of several neurodegenerative diseases including AD. However, it is unclear how APOE4 perturbs the intracellular lipid state. Here, we report that APOE4, but not APOE3, disrupted the cellular lipidomes of human induced pluripotent stem cell (iPSC)–derived astrocytes generated from fibroblasts of APOE4 or APOE3 carriers, and of yeast expressing human APOE isoforms. We combined lipidomics and unbiased genome-wide screens in yeast with functional and genetic characterization to demonstrate that human APOE4 induced altered lipid homeostasis. These changes resulted in increased unsaturation of fatty acids and accumulation of intracellular lipid droplets both in yeast and in APOE4-expressing human iPSC-derived astrocytes. We then identified genetic and chemical modulators of this lipid disruption. We showed that supplementation of the culture medium with choline (a soluble phospholipid precursor) restored the cellular lipidome to its basal state in APOE4-expressing human iPSC-derived astrocytes and in yeast expressing human APOE4. Our study illuminates key molecular disruptions in lipid metabolism that may contribute to the disease risk linked to the APOE4 genotype. Our study suggests that manipulating lipid metabolism could be a therapeutic approach to help alleviate the consequences of carrying the APOE4 allele