Genetic and functional characterization of the hyaluronate lyase HylB and the beta-N-acetylglucosaminidase HylZ in Streptococcus zooepidemicus.

Hyaluronic acid (hyaluronan) is a high molecular weight glycosaminoglycan composed of repeating disaccharides of glucuronic acid and N-acetylglucosamine. The molecular weight of hyaluronan is important for its rheological property, biological function, and application. However, genes important for regulation of hyaluronan production or molecular weight remain poorly understood. Here, we address the roles of two predicated hyaluronate lyase-encoding genes, hylB and hylZ in Streptococcus zooepidemicus. Semi-quantitative RT-PCR assays showed that hylZ was constitutively expressed while the expression level of hylB was growth-phase dependent. Using recombinantly expressed 6His-HylB and -HylZ protein, enzyme assays revealed that HylB was a hyaluronate lyase, and its K(m) and V(max) were 0.57 µM and 1.43 mM min(-1), respectively. 6His-HylZ showed no activity against hyaluronan while it hydrolyzed pNp-ß-GlcNAc suggesting that HylZ was a beta-N-acetylglucosaminidase. Under the optimal conditions (pH 6.0 and 50 °C), the K(m) and V(max) for 6His-HylZ to degrade pNp-ß-GlcNA were 1.16 mM and 26.18 µmol min(-1) mg(-1), respectively. Characterization of ΔhylB and ΔhylZ demonstrated that loss of hylB or/and hylZ had no significant effects on cell growth, lactic acid, and hyaluronan yields. Significantly, as compared to the wild type, ΔhylB produced hyaluronan with an 18 % increase in molecular weight. Our results strongly suggest that hylB encodes a hyaluronate lyase while hylZ encodes a ß-N-acetylglucosaminidase. hylB-deficient strain has the potential to produce high molecular weight hyaluronan.

Exopolysaccharide riclin and anthocyanin-based composite colorimetric indicator film for food freshness monitoring.

Food freshness monitoring is vital to ensure food safety. Recently, packaging materials incorporating pH-sensitive films have been employed to monitor the freshness of food products in real time. The film-forming matrix of the pH-sensitive film is essential to maintain the desired physicochemical functions of the packaging. Conventional film-forming matrices, such as polyvinyl alcohol (PVA), have drawbacks of low water resistance, poor mechanical properties, and weak antioxidant ability. In this study, we successfully synthesise PVA/riclin (P/R) biodegradable polymer films to overcome these limitations. The films feature riclin, an agrobacterium-derived exopolysaccharide. The uniformly dispersed riclin conferred outstanding antioxidant activity to the PVA film and significantly improved its tensile strength and barrier properties by forming hydrogen bonds. Purple sweet potato anthocyanin (PSPA) was used as a pH indicator. The intelligent film with added PSPA provided robust surveillance of volatile ammonia and changed its color within 30 s in the pH range of 2-12. This multifunctional colorimetric film also engendered discernible color changes when the quality of shrimp deteriorated, demonstrating its great potential as an intelligent packaging material to monitor food freshness.

Rapid Feedback Stabilization of Quantum Systems With Application to Preparation of Multiqubit Entangled States.

For stochastic quantum systems with measurement feedback, this article proposes a rapid switching control scheme based on state space partition and realizes the rapid stabilization of an eigenstate of an observable operator. Meanwhile, we apply the proposed scheme to the preparation of typical entangled states in multiqubit systems. In view of the convergence obstacle caused by the symmetric structure of the state space, especially in the case with degenerate observable operators, we first partition the state space into a subset containing the target state and its complement to distinguish the target state from its antipodal points, and then design the corresponding control laws in these two subsets, respectively, by using different Lyapunov functions. The interaction Hamiltonians are also constructed to drive the system state to the desired subset first, and further to the target state. In particular, the control law designed in the undesired subset guarantees the strictly monotonic descent of the corresponding Lyapunov function, which makes the system trajectory switch between the two subsets at most twice and has the potential to speed up the convergence process. We also prove the stability of the closed-loop system with the proposed switching control law based on the stochastic Lyapunov stability theory. By applying the proposed switching control scheme to a three-qubit system, we achieve the preparation of a GHZ state and a W state.

Safety assessment of functional oligooctasaccharide riclinoctaose: A pilot study of genotoxicity, acute toxicity, and subchronic toxicity.

Riclinoctaose was produced by enzymatic hydrolysis of a succinoglycan-type exopolysaccharide riclin. It can be used as a prebiotic to regulate the composition of gut microbiota. Therefore, a safety evaluation is needed. Here, we reported the safety data generated on riclinoctaose. Standard in vitro genotoxicity tests such as the bacterial reverse mutation assay and in vivo micronucleus assay were performed and no mutagenic or clastogenic potential was found. In the acute toxicity study, ICR mice were administered with riclinoctaose via gavage in 14-day studies at the level corresponding to 3000 mg/kg BW/day. In the subchronic study, the diets containing 0%, 1.0%, 2.5%, and 5.0% of riclinoctaose (weight/weight) were prepared for ICR mice for 13 weeks. No test item-related adverse effects were observed in the acute and subchronic studies. No riclinoctaose-induced differences in the overall health, body weight gain, food and water consumption, hematology, blood chemistry, gross pathology, histopathology, or animal death were observed. A no-observed-adverse-effect level of 8842 mg/kg BW/day for male and 9230 mg/kg BW/day for female mice was identified for riclinoctaose when administered for 13 weeks. In conclusion, these findings demonstrated the safety of riclinoctaose and indicated the possibility that riclinoctaose may be used as a functional food. PRACTICAL APPLICATION: Functional oligosaccharide is a low-calorie sweetener, which is beneficial to human health. Dietary riclinoctaose can improve intestinal health and understanding the safety of riclinoctaose is the first step to evaluate its potential use in functional food. Therefore, genotoxicity, acute toxicity, and subchronic toxicity of riclinoctaose were studied.

Engineering CpG-ASO-Pt-Loaded Macrophages (CAP@M) for Synergistic Chemo-/Gene-/Immuno-Therapy.

Adoptive cell therapy by natural cells for drug delivery has achieved encouraging progress in cancer treatment over small-molecule drugs. Macrophages have a great potential in antitumor drug delivery due to their innate capability of sensing chemotactic cues and homing toward tumors. However, major challenge in current macrophage-based cell therapy is loading macrophages with adequate amounts of therapeutic, while allowing them to play a role in immunity without compromising cell functions. Herein, a potent strategy to construct a macrophage-mediated drug delivery platform loaded with a nanosphere (CpG-ASO-Pt) (CAP) composed of functional nucleic acid therapeutic (CpG-ASO) and chemotherapeutic drug cisplatin (Pt) is demonstrated. These CAP nanosphere loaded macrophages (CAP@M) are employed not only as carriers to deliver this nanosphere toward the tumor sites, but also simultaneously to guide the differentiation and maintain immunostimulatory effects. Both in vitro and in vivo experiments indicate that CAP@M is a promising nanomedicine by macrophage-mediated nanospheres delivery and synergistically immunostimulatory activities. Taken together, this study provides a new strategy to construct a macrophage-based drug delivery system for synergistic chemo-/gene-/immuno-therapy.

Bacterial exopolysaccharides: Chemical structures, gene clusters and genetic engineering.

In recent decades, the composition, structure, biosynthesis, and function of bacterial extracellular polysaccharides (EPS) have been extensively studied. EPS are synthesized through different biosynthetic pathways. The genes responsible for EPS synthesis are usually clustered on the genome or large plasmids of bacteria. Generally, different EPS synthesis gene clusters direct the synthesis of EPS with different chemical structures and biological activities. A better understanding of the gene functions involved in EPS biosynthesis is critical for the production of EPS with special biological activities. Genetic engineering methods are usually used to study EPS synthesis related genes. This review organizes the available information on EPS, including their structures, synthesis of related genes, and highlights the research progress of modifying EPS gene clusters through gene-editing methods.

The succinoglycan riclin restores beta cell function through the regulation of macrophages on Th1 and Th2 differentiation in type 1 diabetic mice.

Bacterial succinoglycan is found suitable as a viscosifying and emulsifying agent in the food industry. Riclin is a de-succinyl succinoglycan from an Agrobacterium isolate. Our previous study has revealed that riclin exerts special anti-inflammatory effects in vitro and in vivo. This study aims to determine the effects of riclin on preventing against immunological injury of beta cells in a type 1 diabetic model. We found that orally riclin effectively restores beta-cell function and improves the complications of streptozotocin (STZ)-induced diabetes. Riclin also reduces STZ-induced liver and kidney damage, and balances the inappropriate ratio of T helper type 1 cell (Th1)/type 2 cell (Th2) in the spleen and pancreatic draining lymph nodes of the STZ-induced diabetic mice. In a co-culture system with the islet ß cell MIN6 and macrophage RAW 264.7, riclin reduces the levels of IFN-γ and IL-1ß, protecting against STZ-caused MIN6 cell injury. We identified that riclin specifically binds to the membrane of macrophages and regulates the ratio of IL-10 and IL-12, thereby inhibiting the macrophage-mediated polarization of Th1 cells and promoting the differentiation of Th2 cells, which depends on the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) receptor. Moreover, orally riclin significantly decreases the incidence of STZ-induced hyperglycemia (7.1% in riclin vs. 92.9% in STZ), and prevents autoimmune diabetes in non-obese diabetic (NOD) mice, with 87.5% of mice free of diabetes compared to 46.6% of the control mice. These results suggest that riclin has potential to be a functional food to prevent and improve autoimmune diabetes and related diseases.

Anti-tumor activity and immunogenicity of a succinoglycan riclin.

Natural polysaccharides have attracted considerable interests due to diverse biological activities. Succinoglycan is an extracellular polysaccharide produced by most Agrobacterium strains. Here, we confirmed riclin was a typical succinoglycan by NMR and methylation analysis, and investigated the antitumor effects of riclin in sarcoma 180 tumor-bearing mice. The results showed that riclin inhibited the tumor growth significantly as well as cyclophosphamide (CTX). While CTX caused serious damage to spleen structure, riclin increased the spleen index and promoted lymphocytes proliferation in peripheral blood, spleen and lymph nodes. Riclin decreased splenocytes apoptosis as evidenced by alterations of B-cell lymphoma-2 family proteins and Cleaved Caspase-3 protein. Moreover, 1H nuclear magnetic resonance (NMR)-based metabolomics analysis revealed that riclin partially altered the metabolic profiles of splenocytes. In conclusion, riclin is a succinoglycan that performed strong immunogenicity and suppressed sarcoma growth in mice. Succinoglycan riclin could be a potential antitumor agent for functional food and pharmaceutical purpose.

Preparation and Gut Microbiota Modulatory Property of the Oligosaccharide Riclinoctaose.

In relation to available polysaccharides, oligosaccharides have a low molecular weight, less viscosity, and complete water solubility. These properties endow oligosaccharides with significant biological properties including the microbiota regulation ability. In this study, a homogeneous oligooctasaccharide, riclinoctaose, was biosynthesized from succinylglycan riclin by enzymatic degradation. Monosaccharide composition, Fourier-transform infrared, electrospray ionization mass spectrometry, and nuclear magnetic resonance spectrometry analysis indicated that riclinoctaose is an oligooctasaccharide consisting of one galactose and seven glucose residues, with a pyruvate group linked to the terminal glucose residue. The effects of dietary riclinoctaose on the gut microbiota of mice were evaluated. We found that the dietary riclinoctaose significantly altered intestinal microbiota with the increased growth of beneficial intestinal bacteria including Bifidobacteria and Lactobacillus and decreased the abundance of pernicious bacteria such as Gammaproteobacteria. The level of short-chain fatty acids (SCFAs) was significantly elevated in the riclinoctaose cecum. Our results suggested that riclinoctaose as a prebiotic may have a great potential application in functional foods.

The chemical properties and hygroscopic activity of the exopolysaccharide lubcan from Paenibacillus sp. ZX1905.

This study aims to explore the production and physicochemical properties of an exopolysaccharide (EPS) produced from soil isolate, Paenibacillus sp. ZX1905 in submerged culture. The highest EPS production of 15.67 g/L was achieved in a medium containing soluble starch, peptone and inorganic salts. The purified EPS exhibited excellent skin lubricating properties and was named lubcan. The chemical analysis reviewed that lubcan was an acidic heteropolysaccharide consisted of glucuronic acid, glucose, mannose, galactose, and rhamnose in a molar ratio of 2:3:1:2:2, and the average molecular mass was about 3.27 × 106 Da. NMR and methylation analysis revealed that lubcan backbone was composed of 1,4-α-Man, 1,4,6-α-Glc, 1,3-α-Gal, 1,3-ß-Rha, and 1,3-ß-Gal, together with the branches of 1,3-α-Glc, 1,3-α-Rha, two 1,4-α-GlcA, and terminal-α-Glc(4,6-pyr). The lubcan solution exhibited stability at pH ranging from 5.0 to 7.0, temperature between 5 and 50 °C, and monovalent salts (0.2 M) and divalent salts (0.05 M) addition. The moisture absorption rates of lubcan were 16.98% and 40.41% under the conditions of 43% and 81% relative humidity, which were close to that of hyaluronic acid (17.28% and 41.20%, respectively). These properties make lubcan a good alternative to more expensive hyaluronic acid in the cosmetic industry.

In vitro and in vivo anti-Listeria effect of Succinoglycan Riclin through regulating MAPK/IL-6 axis and metabolic profiling.

Infectious diseases such as Listeria monocytogenes infection pose a great threat to the health of human beings and the development of livestock and poultry farming. Currently the treatment of Listeria infection mainly relies on antibiotics, which may result in excessive antibiotic residues in livestock and poultry products, as well as causing an increase in the occurrence of zoonotic diseases. Here, we demonstrate that Succinoglycan Riclin promoted the clearance of Listeria in the in vitro and in vivo infection model. The expression and secretion of inflammatory cytokines including IL-6 and IL-1ß were significantly increased after Riclin treatment upon infection. The protective effect of Riclin was mainly through activating MAPK/IL-6 axis. HO-1/IL-1ß signaling pathway was less important in this process. Moreover, Riclin caused significant metabolic changes including pathways involved in glycolysis, protein synthesis and oxidative stress during Listeria infection. These results suggest a potential use of Succinoglycan Riclin as non-antibiotic preventive and therapeutic anti-microbial agent in livestock and poultry farming and human diseases.

Soluble beta-glucan salecan improves vaginal infection of Candida albicans in mice.

Vulvovaginal candidiasis (VVC) is one of the most common infections in women. The purpose of this study was to evaluate the ability of a water-soluble ß-glucan salecan to protect against Candida albicans (C. albicans) vaginal infection. The model was reproduced with intravaginal inoculation of yeast blastoconidia in pseudoestrus mice. We found that mice that received salecan (0.5 mg per mouse) after infection had 85% fewer CFU than infected mice given saline. Compared with the C.albicans group, salecan reduced the migration of polymorphonuclear neutrophils (PMNs) in the vagina, decreased mRNA levels of cytokines IL23, IL22, IL17a, and IL17f, anti-candidal genes S100a8 and S100a9 and C.albicans pattern recognition receptor Dectin1. The analysis for vaginal microbial community composition at different taxa levels revealed that the bacterial flora composition in the vagina of the salecan-treated mice was similar to that of the uninfected mice, and distinguished from the infected mice. The vaginal lavages from the salecan treated group had more Enterococcus and its metabolite lactate. Our results suggest salecan might be a potential therapeutic agent for vaginal infection of C.albicans.

Characterization of an alkali-stable xyloglucanase/mixed-linkage ß-glucanase Pgl5A from Paenibacillus sp. S09.

Xyloglucans and mixed-linkage ß-glucans are the major components of hemicelluloses in lignocellulosic biomass. In this study, a novel ß-1,4-glucanase Pgl5A belonging to the glycoside hydrolase family 5 subfamily 4 (GH5_4), was identified from Paenibacillus sp. S09. Pgl5A is a 70.9-kDa protein containing an N-terminal GH5_4 module, a carbohydrate-binding module (CBM)_X2 and a CBM3. Full-length Pgl5A and its CBM deletion mutants Pgl5A∆C and Pgl5A-CD were expressed in E. coli. All three enzymes showed maximal activity at 55 °C and pH 4.5-5.0, and possessed similar activity toward xyloglucan, barley ß-glucan, and lichenan. Deletion of the CBM modules can improve thermostability and acid-tolerant properties of Pgl5A. Circular dichroism (CD) and intrinsic fluorescence spectroscopy analysis verified that C-terminus truncation improves the enzyme acid-tolerant properties. Homology modeling and CD spectra indicated that Pgl5A has an architectural (ß/α)8 fold of GH5_4 enzymes. The catalytic efficiency (kcat/Km) of Pgl5A toward xyloglucan, but not mixed-linkage ß-glucan, was reduced due to C-terminus truncation. TLC and LC-MS analysis showed that Pgl5A cleaves xyloglucan and mixed-linkage ß-glucan into a series of xyloglucan oligosaccharides and gluco-oligosaccharides, respectively. The favorable enzymatic characteristics and high catalytic activities toward both xyloglucan and mixed-linkage ß-glucan make Pgl5A a promising candidate for biotechnological industrial applications.

Thermally controllable Mie resonances in a water-based metamaterial.

Active control of metamaterial properties is of great significance for designing miniaturized and versatile devices in practical engineering applications. Taking advantage of the highly temperature-dependent permittivity of water, we demonstrate a water-based metamaterial comprising water cubes with thermally tunable Mie resonances. The dynamic tunability of the water-based metamaterial was investigated via numerical simulations and experiments. A water cube exhibits both magnetic and electric response in the frequency range of interest. The magnetic response is primarily magnetic dipole resonance, while the electric response is a superposition of electric dipole resonance and a smooth Fabry-Pérot background. Using temporal coupled-mode theory (TCMT), the role of direct scattering is evaluated and the Mie resonance modes are analyzed. As the temperature of water cube varies from 20 °C to 80 °C, the magnetic and electric resonance frequencies exhibit obvious blue shifts of 0.10 and 0.14 GHz, respectively.

Oral Administration of Succinoglycan Riclin Improves Diet-Induced Hypercholesterolemia in Mice.

Epidemiological studies have demonstrated that hypercholesterolemia is associated with an elevated risk of atherosclerosis and cardiovascular diseases. In addition to the available cholesterol-lowering drugs, nutritionally balanced diets containing functional foods have attracted much interest as potential candidates to improve hypercholesterolemia. In the study, we demonstrated that dietary succinoglycan riclin effectively alleviated diet-induced hypercholesterolemia. Compared with the high-cholesterol-diet (HCD) group, the high-riclin group significantly decreased levels of the serum total cholesterol, low-density lipoprotein cholesterol (LDL-C), and hepatic cholesterol (34, 40, and 51%, respectively), consequently improving hepatic steatosis and reducing proinflammatory cytokine expressions. 1H nuclear magnetic resonance (NMR)-based lipidomics and metabolomics analyses revealed that the riclin group partially reversed metabolic profile changes induced by the HCD, approaching that of the normal diet (ND) group. Riclin has no direct effects on cholesterol metabolism-related gene expression among the three HCD model groups. Basically, riclin increased the solution viscosity and interfered in the process of bile acid-cholesterol emulsification, decreasing cholesterol digestion and promoting cholesterol and bile acid excretion in the feces. These results suggested potential therapeutic utility of succinoglycan riclin as a food additive for people suffering from hypercholesterolemia and related diseases.