Comparison study between single enzyme and multienzyme complex in distiller's dred grains with soluble supplemented diet in broiler chicken.

Upregulation of the nutritional value of feed is the major target of various studies in the livestock industry, and dietary enzyme supplementation could aid in digesting the nondegrading nutrients of grains in feed ingredients. Dried distillers' grains with solubles (DDGS) is a byproduct of the fermentation process in the beverage industry and can be used as a large supply source of fiber in feed. Therefore, we conducted an experiment with male broiler chickens to investigate the effect of various types of enzymes on DDGS and compare the efficacy of single enzyme and multienzyme complexes on growth performance and gut environments in broiler chickens. We used 420 1-day-old broiler chickens (Ross 308), and they were allotted into 4 dietary treatments with seven replications (CON, corn-soybean meal [SBM] diet; NC, DDGS supplemented diet; SE, 0.05 % of mannanase supplemented DDGS-based diet; MC, 0.10% of multienzyme complex (mannanase and xylanase, glucanase) supplemented DDGS-based diet. The dietary exogenous enzyme in the DDGS-supplemented diet could improve growth performance as much as the growth of the control group, and digestibility of dry matter, crude protein, and gross energy were significantly increased by enzyme addition in groups of chicks fed DDGS-supplementation diet. Moreover, the populations of pathogenic bacteria, coliforms, and Bacteroidetes were significantly decreased by enzyme supplementation, which might lead to improved gut mucus-secreting cells and inflammatory cytokines in the jejunum. Collectively, dietary single enzyme and multienzyme complexes could improve gut environments, including intestinal immune responses and gut microbial population, and lead to improvement of growth performance in broiler chickens.

Protective and restorative effects of sophorolipid on intestinal dystrophy in dextran sulfate sodium-induced colitis mouse model.

The public has gradually begun to regard inflammatory bowel disease (IBD) as a crucial health issue; however, its mode of action has not been fully elucidated. Sophorolipid (SPL), a glycolipid-type biosurfactant, could be used as a potential treatment in physical intestinal dystrophy. We conducted a 2 × 2 factorial experiment to investigate the protective effect of SPL in a dextran sulfate sodium (DSS)-induced colitis mouse model (first factor, presence of SPL in feed; second factor, presence of DSS in water). Forty C57BL/6 mice (8-week-old) were used, and they were allocated to treatments according to their initial body weight. After a 7 d adjustment period, the DSS treatment was initiated in specific groups. At day 14, DSS was withdrawn from mice, and half of the mice were randomly selected and euthanized to collect colon and colon content samples. Three days after the end of DSS treatment, the rest of the mice were euthanized to investigate the therapeutic effect of SPL. Dietary SPL improved the growth performance in 3 d after DSS treatment, and the histopathological score was lower in the DSS-treated SPL group than in the DSS-treated control group. Mucosal thickness and goblet cell numbers significantly increased in the SPL-supplemented groups compared to in the control group. Similarly, SPL supplementation upregulated the gene expression levels of mucin-2, interleukin-10, and transforming growth factor-ß, and increased the concentration of short chain fatty acid compared to the control groups. In conclusion, dietary supplementation with SPL attenuated the pathological response against acute and chronic inflammation by the maintenance of the mucosal barrier and wound healing capacity.

Dietary Whey Protein Supplementation Increases Immunoglobulin G Production by Affecting Helper T Cell Populations after Antigen Exposure.

Whey protein is a by-product of cheese and casein manufacturing processes. It contains highly bioactive molecules, such as epidermal growth factor, colony-stimulating factor, transforming growth factor-α and -ß, insulin-like growth factor, and fibroblast growth factor. Effects of whey protein on immune responses after antigen (hemagglutinin peptide) injection were evaluated in rats. Experimental diets were formulated based on NIH-31M and supplemented with 1% amino acids mixture (CON) or 1% whey protein concentrate (WPC) to generate isocaloric and isonitrogenous diets. Rats were fed the experimental diets for two weeks and then exposed to antigen two times (Days 0 and 14). Blood was collected on Days 0, 7, 14, and 21 for hematological analysis. The WPC group showed decreased IgA and cytotoxic T cells before the antigen injection (Day 0) but increased IgG, IL-2, and IL-4 after antigen injection due to increased B cells and T cells. Helper T cells were increased at Days 14 and 21, but cytotoxic T cells were not affected by WPC. WPC may activate adaptive immunity (IgG) against antigen by modulating helper T cells. Bioactive molecules might contribute to the immune-enhancing effects of whey protein concentrate.

Simultaneous quantification of methylene blue and its major metabolite, azure B, in plasma by LC-MS/MS and its application for a pharmacokinetic study.

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of methylene blue (MB) and its major metabolite, azure B (AZB), in rat plasma. A simple protein precipitation using acetonitrile was followed by injection of the supernatant on to a Zorbax HILIC Plus column (3.5 µm, 2.1 × 100 mm) with isocratic mobile phase consisting of 5 mM ammonium acetate in 10:90 (v/v) water:methanol at a flow rate of 0.3 mL/min and detection in positive ionization mode. The standard curve was linear over the concentration range from 1 to 1000 ng/mL for MB and AZB with coefficient of determination above 0.9930. The lower limit of quantification was 1 ng/mL using 20 µL of rat plasma sample. The intra- and inter-assay precision and accuracy were <12%. The developed analytical method was successfully applied to the pharmacokinetic study of MB and AZB in rats.

Liquid chromatography-tandem mass spectrometry for quantification of lacosamide, an antiepileptic drug, in rat plasma and its application to pharmacokinetic study.

A rapid, simple and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) was developed for the determination of an antiepileptic drug, lacosamide, in rat plasma. The method involves the addition of acetonitrile and internal standard solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on column packed with octadecylsilica (5 µm, 2.0 × 50 mm) with 0.1% formic acid and acetonitrile as mobile phase, and the detection was performed on tandem mass spectrometry by the multiple-reaction monitoring via an electrospray ionization source. The standard curve was linear over the concentration range from 0.3 to 1000 ng/mL. The lower limit of quantification was 0.3 ng/mL using 50 µL of rat plasma sample. The intra- and inter-assay precision and accuracy were found to be less than 11.7 and 8.8%, respectively. The developed analytical method was successfully applied to the pharmacokinetic study of lacosamide in rats.

Quantification of lurasidone, an atypical antipsychotic drug, in rat plasma with high-performance liquid chromatography with tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometric (LC/MS/MS) method was developed for the determination of an atypical antipsychotic drug, lurasidone, in rat plasma. The method involves the addition of acetonitrile and ziprasidone (internal standard) solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on a column packed with octadecylsilica (5 µm, 2.0 × 50 mm) with 0.1% formic acid and 0.1% formic acid in acetonitrile as mobile phase and the detection was performed using tandem mass spectrometry by multiple-reaction monitoring via an electrospray ionization source. The standard curve was linear (r = 0.9982) over the concentration range 0.002-1 µg/mL. The intra- and inter-assay precisions were 1.7 and 8.6%, respectively. The accuracy range was from 90.3 to 101.8%. The lower limit of quantification was 2.0 ng/mL using 50 µL of rat plasma sample. The developed analytical method was successfully applied to the pharmacokinetic study of lurasidone in rats.

Pharmacokinetics, brain distribution, and plasma protein binding of the antiepileptic drug lacosamide in rats.

The study aimed to characterize the pharmacokinetics of lacosamide, a new antiepileptic drug, in rats after intravenous and oral administration at doses of 1, 3, 10, and 30 mg/kg. Moreover, brain distribution and plasma protein binding were estimated. After intravenous injection, terminal half-life, systemic clearance, and steady state volumes of distribution remained unaltered as a function of dose with values in the range 3.01-3.53 h, 221-241 mL/h/kg and 702-732 mL/kg, respectively. Following oral administration, absolute oral bioavailability was not dose dependent and was at 93.3-106%. However, the time to peak concentration and the dose-normalized peak concentration for 30 mg/kg were significantly different with those for other doses. The extent of urinary excretion of lacosamide was 17.1% and 16.5% for intravenous and oral doses, respectively, whereas fecal excretion was negligible. The brain to plasma ratio of lacosamide was consistent regardless of post-dosing time and the brain to plasma partition coefficient was 0.553. Further, the plasma protein binding of lacosamide was concentration independent with free fraction at 95.9%. Lacosamide showed linear pharmacokinetics at an intravenous dose of 1-30 mg/kg and an oral dose of 1-10 mg/kg but non-linear pharmacokinetics at a 30 mg/kg oral dose.

Proteome analysis of Halobacterium salinarum and characterization of proteins related to the degradation of isopropyl alcohol.

We reported in a previous study that proteomic approach, coupled with genomic techniques, could be used to screen and develop multiple candidates for halophilic enzymes from Halobacterium salinarum. In order to evaluate the biodegradation of isopropyl alcohol (IPA) by H. salinarum, the amounts of residual IPA and acetone generated in the growth media were determined using a gas chromatography-flame ionization detector (GC-FID). The protein expression profiles of cells which had been cultured with IPA were obtained with the two-dimensional gel electrophoresis. Proteins evidencing different expression levels in the presence of 0.5% IPA were identified by electrospray ionization-quadruple-time of flight (ESI-Q-TOF) mass spectrometry. We found 12 proteins which were down-regulated, and another 12 proteins which were up-regulated, in the presence of 0.5% IPA and we further identified 17 proteins among them using ESI-TOF MS/MS. Among these identified proteins, we selected glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for further characterization as a halophilic enzyme. We have demonstrated for the first time that H. salinarum possesses the ability to degrade IPA and GAPDH was both stable and active at high salt concentrations, with maximum activity occurring at 1 M NaCl, although the optimal salt concentration with regard to the growth of H. salinarum is 4.3 M.

Improvement of the two-dimensional gel electrophoresis analysis for the proteome study of Halobacterium salinarum.

Inherent problems exist in the use of two-dimensional gel electrophoresis (2-DE) for sample preparation and separation of proteins from Halobacterium salinarum. In particular, proteins from cells grown in 25% NaCl are difficult to resolve by 2-DE due to the abundance of salt. To remove salts, a 3 kDa molecular weight cut-off column was used. When soluble proteins were separated by 2-DE, most of the proteins were concentrated in the acidic range. For separation of proteins in the pH 3-6 range, ultrazoom immobilized pH gradient strips were used. In addition, sample separation using a IPGphor/Multiphor combined system was a more effective method for the proteome analysis of acidic proteins than using IPGphor for the isoelectric focusing step.