Development of sorghum arabinoxylan-soy protein isolate composite nanoparticles for delivery of curcumin: Effect of polysaccharide content on stability and in vitro digestibility.

The purpose of this study was to fabricate composite nanoparticles using soy protein isolate (SPI) and sorghum bran arabinoxylan (AX) for the delivery of curcumin (Cur). The influences of AX concentrations on the physicochemical characteristic, stability and bioaccessibility of curcumin were investigated. The findings showed that the encapsulation efficiency of curcumin obviously increased upon incorporating AX in comparison to SPI-Cur particles. Hydrogen bonds and hydrophobic interactions were the primary driving forces for the formation of SPI-Cur-AX nanoparticles (SCA). SCA nanoparticles with 1.00 % AX exhibited a uniform size with orderly distribution, suggesting its remarkable physical stability due to the strengthened electrostatic repulsion. However, excessive AX led to aggregation of particles, a noticeable increase in size, and subsequently, a reduction in stability. Due to the heightened free radical scavenging capacity of sorghum AX, SCA nanoparticles exhibited superior antioxidant capabilities. Compared to free curcumin, encapsulation within composite particles significantly enhanced the retention rate and bioaccessibility of curcumin. This improvement was attributed to the potent emulsification ability of AX, which coordinated with bile salt to promote the transfer of curcumin into micelles. The research provides an effective strategy for developing food-grade delivery carriers aimed at enhancing dispersibility, stability and bioaccessibility of the fat-soluble bioactives.

Cancer-cell-derived fumarate suppresses the anti-tumor capacity of CD8+ T cells in the tumor microenvironment.

Metabolic alterations in the microenvironment significantly modulate tumor immunosensitivity, but the underlying mechanisms remain obscure. Here, we report that tumors depleted of fumarate hydratase (FH) exhibit inhibition of functional CD8+ T cell activation, expansion, and efficacy, with enhanced malignant proliferative capacity. Mechanistically, FH depletion in tumor cells accumulates fumarate in the tumor interstitial fluid, and increased fumarate can directly succinate ZAP70 at C96 and C102 and abrogate its activity in infiltrating CD8+ T cells, resulting in suppressed CD8+ T cell activation and anti-tumor immune responses in vitro and in vivo. Additionally, fumarate depletion by increasing FH expression strongly enhances the anti-tumor efficacy of anti-CD19 CAR T cells. Thus, these findings demonstrate a role for fumarate in controlling TCR signaling and suggest that fumarate accumulation in the tumor microenvironment (TME) is a metabolic barrier to CD8+ T cell anti-tumor function. And potentially, fumarate depletion could be an important strategy for tumor immunotherapy.

Ultrasensitive HPLC-MS Quantification of S-(2-Succino) Cysteine Based on Ethanol/Acetyl Chloride Derivatization in Fumarate Accumulation Cells.

Succination is a nonenzymatic and irreversible post-translational modification (PTM) with important biological significance, yielding S-(2-succino) cysteine (2SC) residue. This PTM is low in abundance and often requires a large amount of protein samples for 2SC quantification. In this work, an efficient quantification method based on ethanol/acetyl chloride chemical derivatization was developed. The three carboxyl groups of 2SC were all esterified to increase hydrophobicity, greatly improving its ionization efficiency. The sensitivity was increased by 112 times; the limit of detection was reduced to 0.885 fmol, and the protein usage was reduced by at least 10 times. The established method was used to detect the overall concentration of 2SC in fumarate accumulation cells quantitatively.

Fumarate suppresses B-cell activation and function through direct inactivation of LYN.

Activated B cells increase central carbon metabolism to fulfill their bioenergetic demands, yet the mechanistic basis for this, as well as metabolic regulation in B cells, remains largely unknown. Here, we demonstrate that B-cell activation reprograms the tricarboxylic acid cycle and boosts the expression of fumarate hydratase (FH), leading to decreased cellular fumarate abundance. Fumarate accumulation by FH inhibition or dimethyl-fumarate treatment suppresses B-cell activation, proliferation and antibody production. Mechanistically, fumarate is a covalent inhibitor of tyrosine kinase LYN, a key component of the BCR signaling pathway. Fumarate can directly succinate LYN at C381 and abrogate LYN activity, resulting in a block to B-cell activation and function in vitro and in vivo. Therefore, our findings uncover a previously unappreciated metabolic regulation of B cells, and reveal LYN is a natural sensor of fumarate, connecting cellular metabolism to B-cell antigen receptor signaling.

Preparation and Characterization of Polysaccharide-Based Hydrogels for Cutaneous Wound Healing.

Natural hydrogels are growing in interest as a priority for wound healing. Plant polysaccharides have a variety of biological pharmacological activities, and chitosan hydrogels have proven strong antimicrobial effects, but hydrogels prepared with polysaccharides alone have certain deficiencies. Polysaccharides from flowers of Lonicera japonica Thunb. (LP) and the aerial parts of Mentha canadensis L. (MP) were extracted and oxidized by sodium periodate (NaIO4) and then cross-linked with oxidized-carboxymethylated chitosan (O-CCS) to develop oxidized plant- polysaccharides-chitosan hydrogels (OPHs). SEM observation showed that OPHs had porous interior structures with interconnecting pores. The OPHs showed good swelling, water-retention ability, blood coagulation, cytocompatibility properties, and low cytotoxicity (classed as grade 1 according to United States Pharmacopoeia), which met the requirements for wound dressings. Then the cutaneous wound-healing effect was evaluated in BALB/C mice model, after 7 days treatment, the wound-closure rate of OPHs groups were all greater than 50%, and after 14 days, all were greater than 90%, while the value of the control group was only 72.6%. Of them, OPH-2 and OPH-3 were more favorable to the wound-healing process, as the promotion was more significant. The plant polysaccharides and CS-based hydrogel should be a candidate for cutaneous wound dressings.

Fabrication and Characterization of Ultra-High-Pressure (UHP)-Induced Whey Protein Isolate/κ-Carrageenan Composite Emulsion Gels for the Delivery of Curcumin.

The emulsion gels have attracted extensive interests due to their unique physical characters, remarkable stability, and control release properties of flavor and functional components compared to emulsions in liquid. In the current work, whey protein isolate (WPI)/κ-carrageenan (κ-CG) composite emulsion gels were fabricated based on the ultra-high-pressure (UHP) technology, in replacement of the traditional thermal, acid, or enzyme processing. Uniform composite emulsion gels could be fabricated by UHP above 400 MPa with minimum WPI and κ-CG concentrations of 8.0 and 1.0 wt%, respectively. The formation of UHP-induced emulsion gels is mostly attributed to the hydrophobic interaction and hydrogen bonding. The emulsion gels with different textures, rheology properties, and microstructures could be fabricated through adjusting the formulations (WPI concentration, κ-CG concentration, and oil phase fraction) as well as processing under different conditions (pressure and time). Afterward, curcumin-loaded emulsion gels were fabricated and subjected to an in vitro simulated gastrointestinal digestion in order to investigate the gastrointestinal fate of curcumin. In vitro simulated digestion results demonstrated that the UHP treatment significantly retarded the release of curcumin but had little impact on the bioaccessibility of curcumin. The results in this work provide useful information for the construction of emulsion gels through a non-thermal process, which showed great potential for the delivery of heat-sensitive bioactive components.

Double-Network Hydrogels of Corn Fiber Gum and Soy Protein Isolate: Effect of Biopolymer Constituents and pH Values on Textural Properties and Microstructures.

Corn fiber gum (CFG) -soy protein isolate (SPI) double-network (DN) hydrogels were fabricated using laccase and a heat treatment process, in which CFG solution formed the first gel network via laccase oxidation, while SPI formed the second network through heating, as described in our previous research. The aim of this study was to investigate the influences of CFG/SPI constituents (CFG concentration 0-3%, w/v; SPI concentration 8-10%, w/v) and pH values (5.0-7.5) on the textural properties, microstructures and water-holding capacities (WHC) of the CFG-SPI DN hydrogels. Confocal Laser Scanning Microscopy (CLSM) results showed an apparent phase separation when the CFG concentration was above 1% (w/v). The textural characteristics and WHC of most DN hydrogels were enhanced with increasing concentrations of CFG and SPI. Scanning Electron Microscopy (SEM) observations revealed that the microstructures of DN hydrogels were converted from coarse and irregular to smooth and ordered as pH values increased from 5.0 to 7.5. Excellent textural properties and WHC were observed at pH 7.0. This study developed various CFG-SPI DN hydrogels with diverse textures and structures, governed by the concentrations of protein/polysaccharide and pH values, and also contributes to the understanding of gum-protein interactions in DN hydrogels obtained under different conditions.

Snakegourd root/Astragalus polysaccharide hydrogel preparation and application in 3D printing.

Hydrogels have good water retention, biocompatibility and biodegradability, so they are well used in the medical industry. Here, we have active polysaccharide exacted from Chinese traditional medicine and carboxymethyl chitosan cross-linked to form hydrogel and characterized them by Scanning electron microscopy, FTIR analysis, swelling, degradation, release and cytotoxicity tests. We printed the composite hydrogel into patches with three different shapes by Hot-Melt extruded 3D printer and studied the effects of different shapes on the release of drug. The results show, under acidic or alkaline conditions, the BSA cumulative release rate of the three hydrogel patches with different shape range from 49% to 89%. Therefore, there is a significant difference in the release between circular, cube and rectangular shape. Through the study, we found that the hydrogels we prepared have excellent potential for future applications in drug delivery system.

Engineering of glycerol utilization in Gluconobacter oxydans 621H for biocatalyst preparation in a low-cost way.

BACKGROUND: Whole cells of Gluconobacter oxydans are widely used in various biocatalytic processes. Sorbitol at high concentrations is commonly used in complex media to prepare biocatalysts. Exploiting an alternative process for preparation of biocatalysts with low cost substrates is of importance for industrial applications. RESULTS: G. oxydans 621H was confirmed to have the ability to grow in mineral salts medium with glycerol, an inevitable waste generated from industry of biofuels, as the sole carbon source. Based on the glycerol utilization mechanism elucidated in this study, the major polyol dehydrogenase (GOX0854) and the membrane-bound alcohol dehydrogenase (GOX1068) can competitively utilize glycerol but play no obvious roles in the biocatalyst preparation. Thus, the genes related to these two enzymes were deleted. Whole cells of G. oxydans ∆GOX1068∆GOX0854 can be prepared from glycerol with a 2.4-fold higher biomass yield than that of G. oxydans 621H. Using whole cells of G. oxydans ∆GOX1068∆GOX0854 as the biocatalyst, 61.6 g L-1 xylonate was produced from 58.4 g L-1 xylose at a yield of 1.05 g g-1. CONCLUSION: This process is an example of efficient preparation of whole cells of G. oxydans with reduced cost. Besides xylonate production from xylose, other biocatalytic processes might also be developed using whole cells of metabolic engineered G. oxydans prepared from glycerol.

Increased glutarate production by blocking the glutaryl-CoA dehydrogenation pathway and a catabolic pathway involving L-2-hydroxyglutarate.

Glutarate is a five carbon platform chemical produced during the catabolism of L-lysine. It is known that it can be catabolized through the glutaryl-CoA dehydrogenation pathway. Here, we discover that Pseudomonas putida KT2440 has an additional glutarate catabolic pathway involving L-2-hydroxyglutarate (L-2-HG), an abnormal metabolite produced from 2-ketoglutarate (2-KG). In this pathway, CsiD, a Fe2+/2-KG-dependent glutarate hydroxylase, is capable of converting glutarate into L-2-HG, and LhgO, an L-2-HG oxidase, can catalyze L-2-HG into 2-KG. We construct a recombinant strain that lacks both glutarate catabolic pathways. It can produce glutarate from L-lysine with a yield of 0.85 mol glutarate/mol L-lysine. Thus, L-2-HG anabolism and catabolism is a metabolic alternative to the glutaryl-CoA dehydrogenation pathway in P. putida KT2440; L-lysine can be both ketogenic and glucogenic.

A Bacterial Multidomain NAD-Independent d-Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d-Lactate Oxidization.

Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from Pseudomonas putida KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome c as the electron acceptor, but it completely lost the ability to use coenzyme Q10 Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the in vivo d-lactate utilization function of Fe-S d-iLDH.IMPORTANCE Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been continually reported, including the unique NAD-independent d-lactate dehydrogenase that contains an Fe-S oxidoreductase domain besides the typical flavin-containing domain (Fe-S d-iLDH). Although Fe-S d-iLDH is widely distributed among bacterial species, the investigation of it is insufficient. Fe-S d-iLDH from Pseudomonas putida KT2440, which is the major d-lactate-oxidizing enzyme for the strain, might be a representative of this type of enzyme. A study of it will be helpful in understanding the detailed mechanisms underlying the lactate utilization processes.