Design, Synthesis, and Anti-Osteoporotic Characterization of Arginine N-Glycosylated Teriparatide Analogs via the Silver-catalyzed Solid-Phase Glycosylation Strategy.

In spite of effective antiosteoporosis potency, teriparatide, a bone-building agent approved by the FDA (Food and Drug Administration), was proven to exhibit various side effects. In our previous work, we developed a universal strategy for synthesizing arginine N-glycosylated peptides termed silver-promoted solid-phase glycosylation (SSG) strategy. However, it is unknown whether the SSG strategy can be applied in the peptide drug design. Herein, we first reported the optimization of teriparatide via SSG strategy. Using Arg20 and/or Arg25 as the modifying positions, three series of arginine N-glycosylated teriparatide analogs were successfully synthesized, of which the introduced sugar groups included glucose, galactose, mannose, rhamnose, ribose, 2-acetamino-2-deoxy-glucose, xylose, lactose, and maltose. Among the 27 arginine N-glycosylated derivatives, Arg20-xylose and Arg25-maltose teriparatide analogs, termed PTH-1g and PTH-2i, respectively, indicated enhanced serum stability and significantly improved antiosteoporotic activities in vitro and in vivo compared with the native counterpart. They may serve as effective therapeutic candidates for treating osteoporosis.

Evaluation of multienzyme supplementation and fiber levels on nutrient and energy digestibility of diets fed to gestating sows and growing pigs.

The objective was to investigate the effect of a multienzyme blend (MEblend) and inclusion level on apparent total tract digestibility (ATTD) of energy and nutrients, as well as ileal digestibility of crude protein (CP) and amino acids (AA) in gestation diets with low (LF) or high-dietary fiber (HF) fed to gestation sows. For comparison, growing pigs were fed the same HF diets to directly compare ATTD values with the gestating sows. In experiment 1, 45 gestating sows (parity 0 to 5; 187 ±â€…28 kg bodyweight; BW) were blocked by parity in a 2 × 3 factorial arrangement and fed 2.2 kg/d of the HF (17.5% neutral detergent fiber; NDF) or LF (13% NDF) diet and one of three levels of MEblend (0.0%, 0.08%, and 0.1%) to determine impacts of MEblend on ATTD. Twenty-seven growing pigs (initial 35.7 ±â€…3.32 kg BW) were fed the same HF diet (5% of BW) and one of three MEblend inclusions. The MEblend at both 0.08% and 0.1% increased ATTD of energy, NDF, and acid detergent fiber (ADF) (P < 0.05) in gestating sows but ATTD of total non-starch polysaccharides (NSP) and its residues were not affected. Sows fed HF, regardless of MEblend, had greater ATTD of NDF, xylose, and total NSP (P < 0.05) in comparison to grower pigs. In experiment 2, ileal cannulas were placed in 12 gestating sows (parity 0 to 2; BW 159 ±â€…12 kg) to determine apparent and standardized ileal digestibility (AID and SID) of AA and NSP. In a crossover design, sows were fed the same six diets, as in experiment 1, and a nitrogen-free diet during five periods of seven days each to achieve eight replicates per diet. There was no interaction between diet fiber level and MEblend inclusion. Supplementation of MEblend to gestating sow diets did not impact SID of CP and AA regardless of dietary fiber level. The SID of His, Ile, Lys, Phe, Thr, Trp, and Val were 3% to 6% lower (P < 0.09) in HF than LF independent of MEblend. Supplementation of MEblend did not impact AID of NSP components, but sows fed HF had higher AID of arabinose (LF: 26.5% vs. HF: 40.6%), xylose (LF: 3.5% vs. HF: 40.9%), and total NSP (LF: 25.9% vs. HF: 40.0%) compared to sows fed LF (P < 0.05). Dietary supplementation of MEblend increased ATTD of nutrients, NSP, and energy in diets fed to gestating sows regardless of inclusion level, with MEblend having a greater incremental increase in diets with lower NDF levels. Inclusion of MEblend impacted neither SID of AA nor AID of NSP in low- or high-fiber gestation diets, but high-fiber diet, negatively affected SID of AA.

Fiber-degrading enzymes have been extensively studied in growing pigs with minimal studies focusing on gestating sows; however, commercial gestating sow diets often contain more fiber than grower pig diets to stimulate the sensation of satiety without influencing weight gain. A challenge with dietary fiber is its hindrance on digestibility of nutrients. Supplementation of multienzyme blends increases nutrient digestibility of fibrous diets in grower pigs, but there is little data characterizing the effects of fiber-degrading enzymes in gestation diets for pregnant sows. In this study, inclusion of a multienzyme comprised of various carbohydrases and a protease in gestation diets increased apparent total tract digestibility of nutrients and energy for both gestating sows and growing pigs; however, digestibility of non-starch polysaccharides was only improved in growing pigs. Enzyme supplementation to gestating sow diets had limited impact on the ileal digestibility of nutrients, but ileal digestibility of amino acids and crude protein was reduced when gestating sows were fed diets higher in neutral detergent fiber. When formulating high-fiber diets for gestating sows and growing pigs using similar ingredients, it is critical to consider the differences in digestibility of fibrous components, particularly regarding ileal digestibility of amino acids.

Antibacterial Effect of Melanoidins Derived From Xylose and Phenylalanine Against Bacillus cereus and Clostridium perfringens.

Melanoidins produced from the combination of D-xylose and L-phenylalanine have been reported to exhibit strong antibacterial effects. This study investigated the influence of environmental factors, such as temperatures (10, 15, 20, 25, 30, 35, 40, and 45°C), pH (5.5, 6.0, 6.5, 7.0, 7.5, and 8.0), and water activity (aw: 0.99, 0.96, and 0.93), on the antibacterial effect of the melanoidins produced from the combination of D-xylose with L-phenylalanine against Bacillus cereus and Clostridium perfringens in culture media. Furthermore, freeze-dried powdered melanoidin was used to determine the minimum concentration for growth inhibition, to compare the antibacterial effect of the melanoidin with conventional food preservatives. The liquid melanoidins significantly inhibited the growth of B. cereus (up to 4 log CFU/mL at the maximum) and C. perfringens (up to 6.5 log CFU/mL at the maximum) regardless of the incubation temperatures. However, the remarkable difference between the presence and absence of the melanoidins was demonstrated in the range of 20-35°C as 4 log-cycle lower in B. cereus and 2 log-cycle lower in C. perfringens than those without the melanoidins. The antibacterial effect of the melanoidin on B. cereus was not influenced by pH from 5.5 to 7.0, which exhibited 2-3 log-cycle lower viable counts than those without the melanoidin. Only one log-cycle difference between with and without the melanoidin was shown in C. perfringens growth under the pH range of 5.5-7.0. Although there was no significant difference in the growth of B. cereus between three aw conditions, the melanoidin exhibited a significant antibacterial effect at aw 0.99 demonstrating 4 log-cycle lower viable numbers than those without the melanoidin. Minimum inhibitory concentration of the melanoidin powder for B. cereus and C. perfringens was 7 mg/mL and 15 mg/mL, respectively, regardless of the kind of foods. Furthermore, the melanoidin exhibited comparable antibacterial effect on B. cereus and C. perfringens to potassium sorbate and sodium benzoate under the same concentration as the minimum inhibitory concentration of the melanoidin, demonstrating 2 log-cycle reduction during 3 days of incubation period at 25°C. The results presented here suggest that the xylose- and phenylalanine-based melanoidin demonstrates the possibility to be an alternative food preservative.

Kinetic modeling of xylonic acid production by Gluconobacter oxydans: effects of hydrodynamic conditions.

In this study, the synthesis of xylonic acid from xylose by Gluconobacter oxydans NL71 has been investigated. According to the relationship between oxygen transfer rate and oxygen uptake rate, three different kinetic models of product formation were established and the nonlinear fitting was carried out. The results showed that G. oxydans has critical dissolved oxygen under different strain concentrations, and the relationship between respiration intensity and dissolved oxygen conformed to the Monod equation [Formula: see text]. The maximum reaction rate per unit cell mass and the theoretical maximum specific productivity of G. oxydans obtained by the kinetic model are 0.042 mol/L/h and 6.97 g/gx/h, respectively. These results will assist in determining the best balance between the airflow rate and cell concentration in the reaction and improve the production efficiency of xylonic acid.

A novel cytoskeletal action of xylosides.

Proteoglycan glycosaminoglycan (GAG) chains are attached to a serine residue in the protein through a linkage series of sugars, the first of which is xylose. Xylosides are chemicals which compete with the xylose at the enzyme xylosyl transferase to prevent the attachment of GAG chains to proteins. These compounds have been employed at concentrations in the millimolar range as tools to study the role of GAG chains in proteoglycan function. In the course of our studies with xylosides, we conducted a dose-response curve for xyloside actions on neural cells. To our surprise, we found that concentrations of xylosides in the nanomolar to micromolar range had major effects on cell morphology of hippocampal neurons as well as of Neuro2a cells, affecting both actin and tubulin cytoskeletal dynamics. Such effects/morphological changes were not observed with higher xyloside concentrations. We found a dose-dependent alteration of GAG secretion by Neuro2a cells; however, concentrations of xylosides which were effective in altering neuronal morphology did not cause a large change in the rate of GAG chain secretion. In contrast, both low and high concentrations of xylosides altered HS and CS composition. RNAseq of treated cells demonstrated alterations in gene expression only after treatment with millimolar concentration of xylosides that had no effect on cell morphology. These observations support a novel action of xylosides on neuronal cells.

Comparative study on the gastrointestinal- and immune- regulation functions of Hedysari Radix Paeparata Cum Melle and Astragali Radix Praeparata cum Melle in rats with spleen-qi deficiency, based on fuzzy matter-element analysis.

CONTEXT: Hedysari Radix Praeparata Cum Melle (HRPCM) and Astragali Radix Praeparata Cum Melle (ARPCM) are used interchangeably in clinics to treat spleen-qi deficiency (SQD) symptom mainly including gastrointestinal dysfunction and decreased immunity, which has unknown differences in efficacy. OBJECTIVE: To investigate the differences between HRPCM and ARPCM on intervening gastrointestinal- and immune-function with SQD syndrome. MATERIALS AND METHODS: After the SQD model was established, the Sprague-Dawley (SD) rats were randomly divided into nine groups (n = 10): normal; model; Bu-Zhong-Yi-Qi Pills; 18.9, 12.6 and 6.3 g/kg dose groups of HRPCM and ARPCM. Gastrointestinal function including d-xylose, gastrin, amylase vasoactive intestinal peptide, motilin, pepsin, H+/K+-ATPase, Na+/K+-ATPase, sodium-glucose cotransporter 1 (SGLT1), glucose transporter 2 (GLUT2) and immune function including spleen and thymus index, blood routine, interleukin (IL)-2, IL-6, interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α), immunoglobulin (Ig) M, IgA, IgG and delayed-type hypersensitivity (DTH) were detected. Finally, the efficacy differences were analysed comprehensively by the fuzzy matter-element method. RESULTS: In regulating immune, the doses differences in efficacy between HRPCM and ARPCM showed in the high-dose (18.9 g/kg), but there were no differences in the middle- and low- dose (12.6 and 6.37 g/kg); the efficacy differences were primarily reflected in levels of IL-6, IFN-γ, TNF-α and IgM in serum, and the mRNA expression of IL-6 and IFN-γ in the spleen. In regulating gastrointestinal, the efficacy differences were primarily reflected in the levels of D-xylose, MTL, and GAS in serum, and the mRNA and protein expression of SGLT1 and GLUT2 in jejunum and ileum. DISCUSSION AND CONCLUSIONS: HRPCM is more effective than ARPCM on regulating gastrointestinal function and immune function with SQD syndrome. Therefore, we propose that HRPCM should be mainly used to treat SQD syndrome in the future.

Enhancing Hydrogen Productivity of Photosynthetic Bacteria from the Formulated Carbon Source by Mixing Xylose with Glucose.

To develop an efficient photofermentative process capable of higher rate biohydrogen production using carbon components of lignocellulosic hydrolysate, a desired carbon substrate by mixing xylose with glucose was formulated. Effects of crucial process parameters affecting cellular biochemical reaction of hydrogen by photosynthetic bacteria (PSB), i.e., variation in initial concentration of total carbon, glucose content in initial carbon substrate, and light intensity, were experimentally investigated using response surface methodology (RSM) with a Box-Behnken design (BBD). Hydrogen production rate (HPR) in the maximum value of 30.6 mL h-1 L-1 was attained under conditions of 39 mM initial concentration of total carbon, 59% (mol/mol) glucose content in initial carbon substrate, and 12.6 W m-2 light intensity at light wavelength of 590 nm. Synergic effects of metabolizing such a well-formulated carbon substrate for sustaining the active microbial synthesis to sufficiently accumulate biomass in bioreactor, as well as stimulating enzyme activity of nitrogenase for the higher rate biohydrogen production, were attributed to this carbon substrate that can enable PSB to maintain the relatively consistent microenvironment in suitable culture pH condition during the optimized photofermentative process.

Xylo-oligosaccharide alleviates Salmonella induced inflammation by stimulating Bifidobacterium animalis and inhibiting Salmonella colonization.

Xylo-oligosaccharide (XOS), which is considered as a potential prebiotic, exhibits multiple beneficial effects on modulation of gut microbiota, strength of intestinal barrier, and inhibition of intestinal inflammation. The objective of this study is to investigate whether XOS protects against Salmonella infection by modulating gut microbiota, enhancing the intestinal barrier, and resisting colonization. C57BL/6 male mice received water supplementation with 5% XOS for 14 days before Salmonella Typhimurium infection. The results showed that XOS suppressed the Salmonella-induced inflammation, but had limited effects on tight junction molecules and mRNA expression of mucus proteins, except for claudin-1 in the colon. Data of 16S rDNA sequencing indicated that XOS modulated gut microbiota composition by significantly stimulating Bifidobacterium animalis (B. animalis), and reducing Salmonella counts. Therefore, the potential protective effects of B. animalis against Salmonella challenge were investigated as well. Bifidobacterium animalis subsp lactis BB-12 (BB12), which could markedly increase in XOS, was selected to treat mice. Similarly, Salmonella-induced inflammatory reactions were alleviated by BB12 but tight junction molecules and mucin proteins in the colonic tissues were not affected. Administration of BB12 remarkably decreased the copies of Salmonella in cecal digesta post Salmonella infection. Additionally, the decrease concentrations of cecal propionate and total short-chain fatty acids (SCFAs) in Salmonella-infected mice were reversed by BB12 treatment, and propionate performed a strong inhibitory effect on Salmonella growth in vitro. Besides that, BB12 could directly restrict Salmonella proliferation in vitro. Moreover, BB12 reduced the adhesion ability of Salmonella on the Caco-2 cells model. Our results suggest that XOS could be considered as a candidate of functional food to protect against Salmonella infection by stimulating Bifidobacterium, which then resists Salmonella colonization by maintaining the intestinal SCFAs levels and suppressing adhesibility.

Xylose-Configured Cyclophellitols as Selective Inhibitors for Glucocerebrosidase.

Glucocerebrosidase (GBA), a lysosomal retaining ß-d-glucosidase, has recently been shown to hydrolyze ß-d-xylosides and to transxylosylate cholesterol. Genetic defects in GBA cause the lysosomal storage disorder Gaucher disease (GD), and also constitute a risk factor for developing Parkinson's disease. GBA and other retaining glycosidases can be selectively visualized by activity-based protein profiling (ABPP) using fluorescent probes composed of a cyclophellitol scaffold having a configuration tailored to the targeted glycosidase family. GBA processes ß-d-xylosides in addition to ß-d-glucosides, this in contrast to the other two mammalian cellular retaining ß-d-glucosidases, GBA2 and GBA3. Here we show that the xylopyranose preference also holds up for covalent inhibitors: xylose-configured cyclophellitol and cyclophellitol aziridines selectively react with GBA over GBA2 and GBA3 in vitro and in vivo, and that the xylose-configured cyclophellitol is more potent and more selective for GBA than the classical GBA inhibitor, conduritol B-epoxide (CBE). Both xylose-configured cyclophellitol and cyclophellitol aziridine cause accumulation of glucosylsphingosine in zebrafish embryo, a characteristic hallmark of GD, and we conclude that these compounds are well suited for creating such chemically induced GD models.

Antibacterial activity of xylose-derived LpxC inhibitors - Synthesis, biological evaluation and molecular docking studies.

LpxC inhibitors represent a promising class of novel antibiotics selectively combating Gram-negative bacteria. In chiral pool syntheses starting from D- and L-xylose, a series of four 2r,3c,4t-configured C-furanosidic LpxC inhibitors was obtained. The synthesized hydroxamic acids were tested for antibacterial and LpxC inhibitory activity, the acquired biological data were compared with those of previously synthesized C-furanosides, and molecular docking studies were performed to rationalize the observed structure-activity relationships. Additionally, bacterial uptake and susceptibility to efflux pump systems were investigated for the most promising stereoisomers.

Identification of a xylose-inducible promoter and its application for improving vitamin B12 production in Sinorhizobium meliloti.

Sinorhizobium meliloti 320 is a vitamin B12 (VB12 ) high-producing strain that has been isolated and identified in our previous study. Because the regulatory toolbox for S. meliloti is limited, we searched for new genetic components and identified the two xylose-inducible promoters PA and PB based on a promoter-probe vector with a green fluorescent protein (GFP) as reporter. Compared with the ParaA promoter from S. meliloti, both promoters exhibited higher induced expression and lower basal expression. Subsequently, the influence of glucose or sucrose on the expression of GFP driven by these three promoters was assayed. Glucose repressed all three promoters, and the expression of ParaA was the lowest in the presence of glucose. Although sucrose repressed the expression of PA by 35% and improved the expression of ParaA by 16%, the expression level of PA was the highest and was 13% higher than that of ParaA . Lastly, we overexpressed the hemA gene in the C4 pathway using the PA promoter in S. meliloti 320, and the VB12 production of the engineered strain increased by 11%. The VB12 production was further increased by 11% by adding 0.1% sodium succinate to the culture medium.

Fed-batch polyhydroxybutyrate production by Paraburkholderia sacchari from a ternary mixture of glucose, xylose and arabinose.

Polyhydroxybutyrate (PHB) is a biodegradable bioplastic that is comparable with many petroleum-based plastics in terms of mechanical properties and is highly biocompatible. Lignocellulosic biomass conversion into PHB can increase profit and add sustainability. Glucose, xylose and arabinose are the main monomer sugars derived from upstream lignocellulosic biomass processing. The sugar mixture ratios may vary greatly depending on the pretreatment and enzymatic hydrolysis conditions. Paraburkholderia sacchari DSM 17165 is a bacterium strain that can convert all three sugars into PHB. In this study, fed-batch mode was applied to produce PHB on three sugar mixtures (glucose:xylose:arabinose = 4:2:1, 2:2:1, 1:2:1). The highest PHB concentration produced was 67 g/L for 4:2:1 mixture at 41 h corresponding to an accumulation of 77% of cell dry weight as PHB. Corresponding sugar conversion efficiency and productivity were 0.33 g PHB/g sugar consumed and 1.6 g/L/h, respectively. The results provide references for process control to maximize PHB production from real sugar streams derived from corn fibre.

Structural characterization of a novel polysaccharide from Pleurotus citrinopileatus and its antitumor activity on H22 tumor-bearing mice.

In this research, a novel polysaccharide (PCP) was extracted from Pleurotus citrinopileatus and purified by Sephadex G-150 gel column, and its antitumor activity was investigated using the model H22 tumor-bearing mice. PCP was found to be composed of arabinose, galactose, glucose, xylose, mannose and glucuronic acid in a proportion of 0.66: 14.59: 10.77: 1: 0.69: 0.23 with average molecular weight of 7.30 × 105 Da. Further analysis suggested that PCP was a pyranose with α-type and ß-type glycosidic residues. The antitumor assays in vivo indicated that PCP could effectively suppress H22 solid tumor growth, protect immune organs and improve inflammation and anemia. Besides, Annexin V-FITC/PI double staining and JC-1 staining demonstrated that PCP could induce apoptosis of H22 hepatoma cells. The PI staining assay revealed that PCP induced H22 hepatoma cells apoptosis by arresting cell cycle in S phase. These results suggest that the polysaccharide from Pleurotus citrinopileatus possesses potential value in the treatment of liver cancer.

High throughput, small scale methods to characterise the growth of marine fungi.

Various marine fungi have been shown to produce interesting, bioactive compounds, but scaling up the production of these compounds can be challenging, particularly because little is generally known about how the producing organisms grow. Here we assessed the suitability of using 100-well BioScreen plates or 96-well plates incubated in a robot hotel to cultivate eight filamentous marine fungi, six sporulating and two non-sporulating, to obtain data on growth and substrate (glucose, xylose, galactose or glycerol) utilisation in a high throughput manner. All eight fungi grew in both cultivation systems, but growth was more variable and with more noise in the data in the Cytomat plate hotel than in the BioScreen. Specific growth rates between 0.01 (no added substrate) and 0.07 h-1 were measured for strains growing in the BioScreen and between 0.01 and 0.27 h-1 for strains in the plate hotel. Three strains, Dendryphiella salina LF304, Penicillium chrysogenum KF657 and Penicillium pinophilum LF458, consistently had higher specific growth rates on glucose and xylose in the plate hotel than in the BioScreen, but otherwise results were similar in the two systems. However, because of the noise in data from the plate hotel, the data obtained from it could only be used to distinguish between substrates which did or did not support growth, whereas data from BioScreen also provided information on substrate preference. Glucose was the preferred substrate for all strains, followed by xylose and galactose. Five strains also grew on glycerol. Therefore it was important to minimise the amount of glycerol introduced with the inoculum to avoid misinterpreting the results for growth on poor substrates. We concluded that both systems could provide physiological data with filamentous fungi, provided sufficient replicates are included in the measurements.

Engineering Prokaryotic Transcriptional Activator XylR as a Xylose-Inducible Biosensor for Transcription Activation in Yeast.

Biosensors regulated by specific substrates are needed to develop genetic tools to meet the needs of engineering microbial cell factories. Here, a xylose-inducible biosensor (xylbiosensor), comprising the Escherichia coli activation factor XylR, fusion activation domain (AD) VPRH, and a hybrid promoter with operator xylO, was established in Yarrowia lipolytica. The addition of xylose to an engineered Y. lipolytica strain harboring the xylbiosensor could trigger significant transcriptional activation of target genes, such as mcherry and the xylose utilization gene. Furthermore, a novel promoter Pleu-Pxo-Ptef was developed to construct a bidirectional expression system. The xylbiosensor showed good portability in Saccharomyces cerevisiae, suggesting its potential value in other eukaryotic cells. This study is the first to construct a "turn-on" xylbiosensor induced by xylose addition based on a prokaryotic activator XylR and eukaryotic universal AD. The xylbiosensor exhibits potential in pathway engineering for xylose utilization and xylose-derived product biosynthesis in yeast.

Optimization of polyhydroxybutyrate production by experimental design of combined ternary mixture (glucose, xylose and arabinose) and process variables (sugar concentration, molar C:N ratio).

Conversion of lignocellulosic feedstocks to polyhydroxybutyrate (PHB) could make lignocellulosic biorefineries more profitable and sustainable. Glucose, xylose and arabinose are the main sugars derived from pretreatment and hydrolysis of herbaceous feedstocks. Burkholderia sacchari DSM 17165 is a bacterium that can convert these sugars into PHB. However, the effects of sugar ratio, sugar concentration, and molar C:N ratio on PHB production have not been studied. In this study, a seven-run mixture design for sugar ratio combined with a 32 full factorial design for process variables was performed to optimize PHB production. A polynomial model was built based on experimental data, and optimum conditions for different sugar streams were derived and validated. The highest PHB production (3.81 g/L) was achieved with arabinose at a concentration of 25.54 g/L and molar C:N ratio of 74.35. Results provide references for manipulation of sugar mixture and process control to maximize PHB production.

Steam-exploded sugarcane bagasse as a potential beef cattle feedstock: effects of different pretreatment conditions1.

Sugarcane bagasse, a lignocellulosic biomass produced by sugar production, is rarely utilized directly due to economic concerns. However, pretreatment of this biomass could make it suitable as a feedstock for the beef cattle industry. Accordingly, this study investigated the effects of different steam explosion conditions on bagasse digestibility using in vitro fermentation techniques, scanning electron microscopy, and detailed chemical analyses. In vitro incubation of an untreated sample and samples pretreated at 6 different pressures ranging from 0.6 to 1.6 MPa at intervals of 0.2 MPa for 5 min was conducted out for 96 h. Results showed that increasing the pressure in the steam explosion pretreatment induced degradation of the hemicellulose and increased soluble sugar content, especially for arabinose (L; P < 0.01) and xylose contents (Q; P < 0.01). In vitro incubation showed that compared with untreated bagasse, gas production and degradation rate of the bagasse improved linearly (L; P < 0.01) after all treatments. The lag time disappeared with steam pressure above 1.0 MPa and the maximum gas production was obtained under pretreatment at 1.4 MPa for 5 min. Furthermore, pretreatment of bagasse by steam explosion enhanced (Q: P < 0.01) quadratically estimated energy values and OM digestibility. Thus, the current results demonstrate that sugarcane bagasse may be effectively used as a potential beef cattle feedstock after steam explosion pretreatment.

Xylose rich heteroglycan from flaxseed gum mediates the immunostimulatory effects on macrophages via TLR2 activation.

Immunostimulatory activity of the flaxseed gum neutral fraction (NFG) was investigated. NFG was characterized as a xylose rich heteroglycan through monosaccharide composition analysis, FT-IR, methylation/GC-MS, and 1D/2D-NMR. NFG stimulated NO production and phagocytic activity of macrophages. Secretion of interleukin-6, interleukin-1ß, and tumor necrosis factor-α (254.7 pg/mL, 2.5 ng/mL, and 42.9 pg/mL, respectively) was significantly induced by NFG. Mitogen-activated protein kinases of JNK and P38 were activated by NFG with increased phosphorylation of JNK and P38, while NO production was reduced to 6.05 and 4.42 µM by JNK and P38 inhibitor, respectively. Nuclear factor-κB signaling pathway was also activated by NFG with the suppression of IκBα and up-regulation of phosphorylation of IκBα and nuclear factor-κB P65. Toll like receptor-2 was the molecular target of NFG and responsible for the activation of down-stream signaling pathways. Thus, NFG from flaxseed gum may potentially be used as a natural immunomodulator in functional foods.

Efficacy of a novel sequential enzymatic hydrolysis of lignocellulosic biomass and inhibition characteristics of monosugars.

Efficient production of sugar monomers from lignocellulose is often hampered by serious bottle-necks in biomass hydrolysis. The present study reveals that ultra-sonication assisted pretreatment following autoclaving, termed as combined pretreatment, can lead to more efficient delignification of lignocellulosic biomass and an open, deformed polysaccharide matrix, found favorable for subsequent enzymatic hydrolysis, is formed. The pattern of inhibition for the enzymatic hydrolysis reaction on combined-pretreated saw dust is identified. Two main inhibition models (competitive and noncompetitive) are proposed and a better fit of experimental values with the theoretical values for the competitive inhibition model validates the proposition that in the present experiment, glucose inhibits the enzymes competitively. Additionally, accuracy of the inhibitory kinetics based models is estimated over a series of enzyme and substrate concentrations.

A Xylose-Inducible Expression System and a CRISPR Interference Plasmid for Targeted Knockdown of Gene Expression in Clostridioides difficile.

Here we introduce plasmids for xylose-regulated expression and repression of genes in Clostridioides difficile The xylose-inducible expression vector allows for ∼100-fold induction of an mCherryOpt reporter gene. Induction is titratable and uniform from cell to cell. The gene repression plasmid is a CRISPR interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of the Streptococcus pyogenes Cas9 protein (dCas9) that is targeted to a gene of interest by a constitutively expressed single guide RNA (sgRNA). Expression of dCas9 is induced by xylose, allowing investigators to control the timing and extent of gene silencing, as demonstrated here by dose-dependent repression of a chromosomal gene for a red fluorescent protein (maximum repression, ∼100-fold). To validate the utility of CRISPRi for deciphering gene function in C. difficile, we knocked down the expression of three genes involved in the biogenesis of the cell envelope: the cell division gene ftsZ, the S-layer protein gene slpA, and the peptidoglycan synthase gene pbp-0712 CRISPRi confirmed known or expected phenotypes associated with the loss of FtsZ and SlpA and revealed that the previously uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell division, and protection against lysis.IMPORTANCEClostridioides difficile has become the leading cause of hospital-acquired diarrhea in developed countries. A better understanding of the basic biology of this devastating pathogen might lead to novel approaches for preventing or treating C. difficile infections. Here we introduce new plasmid vectors that allow for titratable induction (P xyl ) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins in C. difficile Besides bypassing the lengthy process of mutant construction, CRISPRi can be used to study the function of essential genes, which are particularly important targets for antibiotic development.