A polysaccharide utilization locus from the gut bacterium Dysgonomonas mossii encodes functionally distinct carbohydrate esterases.

The gut microbiota plays a central role in human health by enzymatically degrading dietary fiber and concomitantly excreting short chain fatty acids that are associated with manifold health benefits. The polysaccharide xylan is abundant in dietary fiber but noncarbohydrate decorations hinder efficient cleavage by glycoside hydrolases (GHs) and need to be addressed by carbohydrate esterases (CEs). Enzymes from carbohydrate esterase families 1 and 6 (CE1 and 6) perform key roles in xylan degradation by removing feruloyl and acetate decorations, yet little is known about these enzyme families especially with regard to their diversity in activity. Bacteroidetes bacteria are dominant members of the microbiota and often encode their carbohydrate-active enzymes in multigene polysaccharide utilization loci (PULs). Here we present the characterization of three CEs found in a PUL encoded by the gut Bacteroidete Dysgonomonas mossii. We demonstrate that the CEs are functionally distinct, with one highly efficient CE6 acetyl esterase and two CE1 enzymes with feruloyl esterase activities. One multidomain CE1 enzyme contains two CE1 domains: an N-terminal domain feruloyl esterase, and a C-terminal domain with minimal activity on model substrates. We present the structure of the C-terminal CE1 domain with the carbohydrate-binding module that bridges the two CE1 domains, as well as a complex of the same protein fragment with methyl ferulate. The investment of D. mossii in producing multiple CEs suggests that improved accessibility of xylan for GHs and cleavage of covalent polysaccharide-polysaccharide and lignin-polysaccharide bonds are important enzyme activities in the gut environment.

Untargeted Metabolomics Based on Liquid Chromatography-Mass Spectrometry for the Analysis of Plasma and Erythrocyte Samples in Childhood Obesity.

The circulating metabolome of human peripheral blood provides valuable information to investigate the molecular mechanisms underlying the development of diseases and to discover candidate biomarkers. In particular, erythrocytes have been proposed as potential systemic indicators of the metabolic and redox status of the organism. To accomplish wide-coverage metabolomics analysis, the combination of complementary analytical techniques is necessary to manage the physicochemical complexity of the human metabolome. Herein, we describe an untargeted metabolomics method to capture the plasmatic and erythroid metabolomes based on ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry, combining reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography. The method provides comprehensive metabolomics fingerprinting of plasma and erythrocyte samples, thereby enabling the elucidation of the distinctive metabolic disturbances behind childhood obesity and associated comorbidities, such as insulin resistance.

Screening of stationary phase selectivities for global lipid profiling by ultrahigh performance supercritical fluid chromatography.

The performance of seven sub-2-µm particle packed columns (2-picolylamine, 2-PIC; charged surface hybrid fluoro-phenyl, CSH-FP; high strength silica C18 SB, HSS-C18; diethylamine, DEA; 1-aminoanthracene, 1-AA; high density diol and ethylene bridged hybrid; BEH) was examined for lipid separation in ultra-high performance supercritical fluid chromatography (UHPSFC) coupled to quadrupole time-of-flight mass spectrometry. Based on the results of the column screening a method for profiling of multiple lipid species from the major lipid classes was developed. Stationary phases containing ß-hydroxy amines, i.e. 1-AA, DEA and 2-PIC, yielded strong retention and poor peak shapes of zwitterionic lipids with primary amine groups, such as phosphatidylserines, phosphatidylethanolamines and its lyso forms. The BEH and HSS-C18 columns showed strong retention of polar and nonpolar lipids, respectively. The Diol column retained the majority of major lipid classes and also produced symmetric peaks. In addition, this column also produced the highest resolution within and between major lipid classes. An injection solvent composed of methanol:chloroform (1:2, v:v) and the addition of 20 mM ammonium formate in the mobile phase improved chromatographic separation and mass spectrometry detection in comparison to ammonium acetate or absence of additive. Finally, chromatographic and mass spectrometric parameters were optimized for the Diol column using a design of experiments approach. The separation mechanism on the Diol column depended on the lipid functionality and the length and degree of unsaturation of the acyl groups. The developed method could resolve 18 lipid classes and multiple lipids within each class, from blood serum and brain tissue in 11 min.

Ultrasound-assisted dispersive extraction for the high pressure liquid chromatographic determination of tetracyclines residues in milk with diode array detection.

Ultrasound assisted matrix solid phase dispersive extraction was applied for the selective isolation and clean-up of tetracyclines (oxytetracycline, tetracycline, epi-chlorotetracycline, chlorotetracycline and doxycycline) from milk. Target analytes were determined by an accurate and sensitive chromatographic analytical method, which was validated to meet the European Legislation criteria. The separation was performed on a LiChroCART-LiChrospher® 100 RP-18 (5µm, 250×4mm) analytical column, operated at ambient temperature, followed by diode array detection. Validation included investigation of linearity, selectivity, stability, limits of detection and quantitation, decision limit, detection capability, trueness, precision and ruggedness according to the Youden's approach. Limits of quantitation of examined tetracyclines were from 14.5 to 56.6µg/kg significantly lower than respective Maximum Residue Limits, whereas recoveries ranged from 82.0% to 108%. The applicability of the method was evaluated using milk samples purchased from local market. Accuracy of the method was additionally proved by analysis of bovine milk certified reference material (BCR®-492).