Comparison of Methyl-capture Sequencing vs. Infinium 450K methylation array for methylome analysis in clinical samples

Interindividual variability in the epigenome has gained tremendous attention for its potential in pathophysiological investigation, disease diagnosis, and evaluation of clinical intervention. DNA methylation is the most studied epigenetic mark in epigenome-wide association studies (EWAS) as it can be detected from limited starting material. Infinium 450K methylation array is the most popular platform for high-throughput profiling of this mark in clinical samples, as it is cost-effective and requires small amounts of DNA. However, this method suffers from low genome coverage and errors introduced by probe cross-hybridization. Whole-genome bisulfite sequencing can overcome these limitations but elevates the costs tremendously. Methyl-Capture Sequencing (MC Seq) is an attractive intermediate solution to increase the methylome coverage in large sample sets. Here we first demonstrate that MC Seq can be employed using DNA amounts comparable to the amounts used for Infinium 450K. Second, to provide guidance when choosing between the 2 platforms for EWAS, we evaluate and compare MC Seq and Infinium 450K in terms of coverage, technical variation, and concordance of methylation calls in clinical samples. Last, since the focus in EWAS is to study interindividual variation, we demonstrate the utility of MC Seq in studying interindividual variation in subjects from different ethnicities.     

Efficacy of acidic pretreatment for the saccharification and fermentation of alginate from brown macroalgae

This study was performed to evaluate the effectiveness of acidic pretreatment in increasing the enzymatic digestibility of alginate from brown macroalgae. Pretreatment with 1 % (w/v) sulfuric acid at 120 °C for 30 min produced oligosaccharides, mannuronic acid, and guluronic acid. Enzymatic saccharification of pretreated alginate by alginate lyases produced 52.2 % of the theoretical maximal sugar yield, which was only 7.5 % higher than the sugar yield obtained with unpretreated alginate. Mass spectrometric analyses of products of the two reactions revealed that acidic pretreatment and enzymatic saccharification produced saturated monomers (i.e., mannuronic and guluronic acid) with saturated oligosaccharides and unsaturated monomers (i.e., 4-deoxy-l-erythro-5-hexoseulose uronic acid; DEH), respectively. While DEH is further metabolized by microorganisms, mannuronic acid and guluronic acid are not metabolizable. Because of the poor efficacy in increasing enzymatic digestibility and owing to the formation of non-fermentable saturated monomers, acidic pretreatment cannot be recommended for enzymatic saccharification and fermentation of alginate.     

The role of ATP and free ADP in metabolic coupling during fuel-stimulated insulin release from islet beta-cells in the isolated perfused rat pancreas.

The isolated perfused rat pancreas was used to test the hypothesis that total cellular ATP or the ratio of ATP/free ADP plays the primary role in coupling intermediary metabolism to the biophysical events that are the basis of glucose-stimulated insulin release. The pancreas was preperfused for 20 min with 4.0 mM of a physiological mixture of 20 amino acids plus 4.2 mM glucose, and insulin release was then stimulated for 150 s by suddenly increasing the glucose to 8.3 mM. The pancreas was sampled at 24, 48, 72, and 150 s after the switch. The content of total ATP, ADP, AMP, Pi, phosphocreatine, and creatine were measured in beta-cell enriched cores of pancreatic islets microdissected from freeze-dried pancreas cryostat sections. Metabolites were measured by quantitative histochemical enzymatic cycling techniques. Modeling studies were carried out to assess the impact of biochemical analytical results on the membrane potential of the beta-cells. The level of free ADP was calculated using the creatine kinase equilibrium reaction and an intracellular pH of 7.2. First phase insulin release was stimulated at least 10-fold with the maximum reached 45 s after adding high glucose. The biochemical analytical data demonstrate that the total cellular level of the putative coupling factor ATP and of the ratios ATP/free ADP and ATP/free ADP x Pi are not significantly influenced by a glucose level change that causes a more than 10-fold surge of insulin release. The strength and limitations of the present experimental strategy and the implications of the results for our understanding of metabolic coupling in glucose-stimulated insulin release are discussed.     

Thermodynamic studies of base pairing involving 2,6-diaminopurine.

The thermal stabilities of oligodeoxyribonucleotide duplexes containing 2,6-diaminopurine (D) matched with each of the four normal DNA bases were determined by optical melting techniques. Comparison of optical melting curves yielded relative stabilities for the D-containing standard base pairs in an otherwise identical base-pair sequence. The D:T pair was found to be more stable than the A:T pair in dC3DG3:dC3TG3, as stable as the A:T in dCT3DT3G:dCA3TA3G, and less stable than the A:T in dCA3DA3G:dCT7G. The order of stabilities for X:Y in the DNA duplex dCA3XA3G:dCT3YT3G is: (A:T) greater than (T:D) congruent to (D:T) greater than or equal to (T:A) greater than (C:D) congruent to (D:A) congruent to (D:G) greater than or equal to (D:C) congruent to (G:D) congruent to (D:D) greater than or equal to (A:D). Implications of these results for design of DNA oligonucleotide probes are discussed.     

Evidence for separate networks of classical and novel basement membrane collagen. Characterization of alpha 3(IV)-alport antigen heterodimer.

The COOH-terminal non-collagenous domains (NC1) of type IV collagen from glomerular basement membranes (GBM), lens capsule basement membranes, and Descemet's membrane varied in the distribution of their NC1 subunits. All of these basement membranes (BMs) contained both classical (alpha 1(IV) and alpha 2(IV)) and novel collagen chains (alpha 3(IV), alpha 4(IV) and the Alport antigen). Whereas GBM had a predominance of disulfide-bonded subunits, the lens capsule and Descemet's membrane were primarily monomeric, differences that are likely related to the functional and structural diversity of collagen in various tissues. A heterodimer formed from monomeric subunits of alpha 3(IV) and the Alport antigen exists in human and bovine GBM. This dimer represents an important cross-link of the NC1 domain of novel collagen. Additionally, immunoaffinity methodology showed that the novel BM collagen hexamers segregate into populations containing only novel BM subunits without the participation of the classical subunits (alpha 1(IV) and alpha 2(IV)). These data provided evidence for the presence of two separate networks of BM collagen: one containing alpha 1(IV) and alpha 2(IV), and the other consisting of the novel collagen chains.     

Dry Etching Characteristics of 16-nm Amorphous Carbon Layer in a Dual-Frequency Plasma Etcher

Plasma Physics Reports - The etching characteristics of a patterned amorphous carbon layer (ACL) in a magnetized inductively coupled plasma (MICP) etcher were investigated. The etch rate and etched...