In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes

DNA methylation is one of the most important epigenetic alterations involved in the control of gene expression. Bisulfite sequencing of genomic DNA is currently the only method to study DNA methylation patterns at single-nucleotide resolution. Hence, next-generation sequencing of bisulfite-converted DNA is the method of choice to investigate DNA methylation profiles at the genome-wide scale. Nevertheless, whole genome sequencing for analysis of human methylomes is expensive, and a method for targeted gene analysis would provide a good alternative in many cases where the primary interest is restricted to a set of genes.Here, we report the successful use of a custom Agilent SureSelect Target Enrichment system for the hybrid capture of bisulfite-converted DNA. We prepared bisulfite-converted next-generation sequencing libraries, which are enriched for the coding and regulatory regions of 174 ADME genes (i.e. genes involved in the metabolism and distribution of drugs). Sequencing of these libraries on Illumina’s HiSeq2000 revealed that the method allows a reliable quantification of methylation levels of CpG sites in the selected genes, and validation of the method using pyrosequencing and the Illumina 450K methylation BeadChips revealed good concordance.     

Exome sequencing reveals CCDC111 mutation associated with high myopia

Myopia is a refractive error of the eye that is prevalent worldwide. The most extreme form, high myopia, is usually associated with other ocular disorders such as retinal detachment, macular degeneration, cataract, and glaucoma, and is one of leading causes of blindness. The etiology is complex and has not been fully elucidated. In this study, we identified a novel missense variant of the CCDC111 gene (NM_152683.2: c.265T > G; p.Y89D) in a high myopia family by exome sequencing. The variant was identified in 4 patients from an additional 270 sporadic high myopia patients, but not found in 270 controls. The amino acid is highly conserved across species, and variants giving rise to amino acid substitutions are predicted to be functionally damaging. The CCDC111 gene was ubiquitously expressed in primary cell cultures from human eye tissue, including corneal epithelial cells, choroidal melanoma cells, scleral fibroblasts, retinal epithelial cells, retinal Müller cells, and lens capsule epithelial cells. In summary, our results suggested that the CCDC111 may be a susceptibility gene for high myopia.     

Synergistical effect of 20-HETE and high salt on NKCC2 protein and blood pressure via ubiquitin–proteasome pathway

We previously generated a cytochrome P450 4F2 (CYP4F2) transgenic mouse model and demonstrated that overexpressed CYP4F2 and overproduced 20-HETE in the kidneys contribute to the increase of blood pressure in the CYP4F2 transgenic mice with normal salt intake. We currently expect to elucidate a potential mechanism of salt-related hypertension whereby diverse levels of 20-HETE interact with dietary salt on Na⁺-K⁺-2Cl^? cotransporter, isoform 2 (NKCC2) in the kidneys of the transgenic and wild-type mice with high salt intake. High salt intake reduced about 85 % abundance of renal NKCC2 protein in the transgenic mice and about 24 % in the wild-type mice by Western blot. Furthermore, we first found that NKCC2 was ubiquitinated and interacted with Nedd4-2 by immunoprecipitation in the transgenic mice with high salt intake. In addition, inhibition of 20-HETE synthesis or proteasome activity reversed the reduction of NKCC2 expression induced by 20-HETE and high salt intake. These results suggest that 20-HETE and high salt intake synergistically decrease the expression of NKCC2 protein via Nedd4-2-mediated ubiquitin–proteasome pathway, and thereby modulate natriuresis and blood pressure. We propose that diverse levels of 20-HETE have diverse effects on blood pressure in different salt concentrations.     

Store-Operated Ca2+ Channels Blockers Inhibit Lipopolysaccharide Induced Astrocyte Activation

The destruction of calcium homeostasis is an important factor leading to neurological diseases. Store-operated Ca²⁺ (SOC) channels are essential for Ca²⁺ homeostasis in many cell types. However, whether SOC channels are involved in astrocyte activation induced by lipopolysaccharide (LPS) still remains unknown. In this study, we used LPS as an exogenous stimulation to investigate the role of SOC channels in astrocyte activation. Using calcium imaging technology, we first found that SOC channels blockers, 1-[h-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365) and 2-aminoethyldiphenyl borate (2-APB), inhibited LPS induced [Ca²⁺]_i increase, which prompted us to speculate that SOC channels may be involved in LPS induced astrocyte activation. Further experiments confirmed our speculation shown as SOC channels blockers inhibited LPS induced astrocyte activation characterized as cell proliferation by MTS and BrdU assay, raise in glial fibrillary acidic protein expression by immunofluorescence and Western Blot and secretion of interleukin 6 (IL-6) and interleukin 1β (IL-1β) by ELISA. So, our studies showed that SOC channels are involved in LPS-induced astrocyte activation.     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

Cloning and truncation modification of trehalose-6-phosphate synthase gene from Selaginella pulvinata

A homologous sequence was amplified from resurrection plant Selaginella pulvinta by RACE technique, proved to be the full-length cDNA of trehalose-6-phosphate synthase gene by homologous alignment and yeast complementation assay, and nominated as SpTPS1 gene. The open reading frame of this gene was truncated 225 bp at the 5′-end, resulting the N-terminal truncation modification of 75 amino acids for its encoding protein. The TPS1 deletion mutant strain YSH290 of the brewer's yeast transformed by the truncated gene SpTPS1Δ and its original full-length version restored growth on the medium with glucose as a sole carbon source and displayed growth curves with no significant difference, indicating their encoding proteins functioning as TPS enzyme. The TPS activity of the mutant strain transformed by the truncated gene SpTPS1Δ was about six fold higher than that transformed by its original version, reasoning that the extra N-terminal extension of the full-length amino acid sequence acts as an inhibitory domain to trehalose synthesis. However, the trehalose accumulation of the mutant strain transformed by the truncated gene SpTPS1Δ was only 8% higher than that transformed by its original version. This result is explained by the feedback balance of trehalose content coordinated by the comparative activities between trehalose synthase and trehalase. The truncated gene SpTPS1Δ is suggested to be used in transgenic operation, together with the inhibition of trehalase activity by the application of validamycin A or genetic deficiency of the endogenous trehalase gene, for the enhancement of trehalose accumulation and improvement of abiotic tolerance in transgenic plants.