Anti-proliferation and radiosensitization effects of chitooligosaccharides on human lung cancer line Hep G2

Objective: To observe the anti-proliferation and radiosensitization effect of chitooligosaccharides(COS) on human lung cancer cell line Hep G2. Methods: CCK-8 assay was employed to obtain the inhibition ratio of COS on Hep G2 cells at 24 h after treatment. The clonogenic assay was used to analyze the cell viability of RAY group and RAY+COS group with X-ray of 0, 1, 2, 4, 6 and 8 Gy, and the cell survival curve was used to analyze the sensitization ratio of COS. Flow cytometry was employed to detect cell cycle and apoptosis rate in control group, RAY group and RAY+COS group after 24 h treatment. Results: COS inhibited the proliferation of Hep G2 cells, and the inhibition rate positively correlated with the concentration of COS. The cell viability decreased with increasing exposure dose in RAY group and RAY+COS group. The cell viabilities of RAY+COS group were lower than those of RAY group at the dose of 4, 6 and 8 Gy(P<0.05), and the sensitization ratio of COS was 1.19. There were higher percentage at G2/M phase and apoptosis rate, and lower percentage at S phase in RAY+COS group versus the other two groups(P<0.01). Conclusions: COS can inhibit the proliferation of Hep G2 cells, and enhance the radiosensitization of Hep G2 cells, induce apoptosis and G2/M phase arrest.

Comparative study of the chitooligosaccharides effect on the proliferation inhibition and radiosensitization of three types of human gastric cancer cell line

Objective:To observe the chitooligosaccharides(COS) effect on the proliferation inhibition and radiosensitivity of three types of human gastric cancer cell line.Mothods:CCK-8 assay was employed to obtain the inhibition ratio of COS on BGC823 cells,MKN45 cells and SGC7901 cells at 48 h after treatment and the proliferation-inhibition curve was drawn with the inhibition ratio of COS on three types of cells.The clonogenic assay was used to detect the cell viability of 0,1,2,4,6 and 8 Gy(6 dose grades) in RAY group and RAY+COS group after X-ray,and the cell survival curve was used to analyze the sensitization enhancement ratio of COS.Flow cytometry was employed to detect cell cycle and apoptosis rate in control group,RAY group and RAY+COS group after 48 h treatment.Results:COS inhibited the proliferation of three types of cells.The inhibition rate was positively correlated with the concentration of COS,and the susceptibility of MKN45 cells,SGC7901 cells and BGC823 cells to COS decreased in turn.The cell viability decreased gradually with the increasing radiation dose in RAY group and RAY+COS group(P<0.01).The cell viabilities of RAY+COS group were lower than those of RAY group at all the dose grades under X-ray exposure(P<0.01),and the sensitization enhancement ratios of COS on BGC823 cells,MKN45 cells and SGC7901 cells were 1.06,1.28 and 1.15 respectively.In controlled trials,apoptosis rate and percentage in the G_2/M phase of three types of cells in RAY+COS group were higher than those in control group and RAY group,and percentage in the S phase and the G_0/G_1 phase in RAY+COS group were lower than those in the other two groups(P<0.01).Conclusions:COS can inhibit the proliferation of three types of human gastric cancer cells and enhance the radiosensitivity by inducing apoptosis and G_2/M phase arrest.

Studies on the Antimicrobial Activity of Chitooligosaccharides from Housefly Larvae, Musca domestica vicina Macquart(Diptera: Muscidae)

The inhibition effect of chitooligosaccharides from housefly larvae on pathogens ofcrops seeds, fruits or vegetables was studied and the main factors influencing theantimicrobial activity of chitooligosaccharides were also investigated. It was foundthat chitooligosaccharides from housefly larvae had wide spectrum fungistasis. It canstrongly inhibit the development of 31 kinds of plant pathogenic fungi such as corticiumrolfsii Saccardo. The results indicated that the degree of deacelylation (DD) or theaverage molecular weight(MW)of chitooligosaccharides were related to the antimicrobialactivity. The antimicrobial activity increased with the rising of DD or the declining ofMW. Pot culture results showed that chitooligosaccharide could enhance the rate ofgermination and emergence of the seeds of maize, wheat and cotton. Chitooligosaccharidehad certain effect on corn southern leaf blight caused by Helminthosporium maydis.

Preparation,characterization,and antifungal evaluation of a new type of aminourea chitooligosaccharide derivatives

Phytopathogenic fungi cause heavy negative impact on the agricultural economy,but most existing fungicides are toxic and pose a threat to both human health and environments.A green and efficient fungicide is urgently needed.Chitooligosaccharides(COSs),the degradation products of natural polysaccharide chitosan,are nontoxic and biodegradable antifungal substances.In this study,a novel type of aminourea chitooligosaccharide derivatives(AUCOS)was synthesized by successively grafting a hydrazine group and an amine-carbonyl group onto a chitooligosaccharide backbone to enhance the antifungal capability of COSs.The structures of the target compounds were identified by FTIR,1H NMR,and 13C NMR,and the degree of substitution of each product was calculated from the results of the elemental analysis.The antifungal activities of the prepared chitooligosaccharide derivatives against Fusarium solani,Verticillium albo-atrum and Phytophthora capsici were tested in vitro.The AUCOSs had better inhibitory efficiencies against the three plant pathogen fungi than that of chitooligosaccharide,of which aminourea chitooligosaccharide 2(AUCOS2)was the most promising antifungal compound,whose highest inhibition rates were 60.12%,82.95%,and 85.23%against F.solani,V.albo-atrum and P.capsici,respectively.The synthesized derivatives have good application prospects in crop protection and deserve further research.

Adsorption characteristics of chitooligosaccharides onto activated charcoal in aqueous solutions

To investigate the adsorption characteristics of chitooligosaccharides in solution onto activated charcoal,we studied the optimal adsorption conditions and the adsorption mechanisms of the chitooligosaccharides onto activated charcoal,which will greatly promote the application of activated charcoal in the chitooligosaccharides separation and purification.We studied the effects of particle size of activated charcoal,pH of solution,contact time,temperature,and initial concentration of chitooligosaccharides on the adsorption behavior in batch mode experiments.Activated charcoal in fine particle size showed a high uptake of chitooligosaccharides.Weak alkaline solution(pH 8–9)was the most favorable to the adsorption.The adsorption equilibrium after 60 min was established,which followed a pseudo-second-order kinetic model.The adsorption capacity(Q_(max))reached 0.195 g/g(chitooligosaccharides/activated charcoal)at 298 K.The adsorption was temperature-insensitive,and the adsorption isotherms could be best described by the Langmuir equation.Chitooligosaccharides adsorbed on activated charcoal could be desorbed in 50%ethanol solution in combination with an acidic condition(pH 2),reaching desorption efficiency of 96.0%.These findings are of great significance for the production and purification of amino oligosaccharides including chitooligosaccharides using activated charcoal.

Improving the protective effects of a FGF for peripheral nerve injury repair using sulfated chitooligosaccharides

Injury to the peripheral nerves can result in temporary or life-long neuronal dysfunction and subsequent economic or social disability. Acidic fibroblast growth factor(a FGF) promotes the growth and survival of neurons and is a possible treatment for peripheral nerve injury. Yet,the actual therapeutic utility of a FGF is limited by its short half-life and instability in vivo. In the present study,we prepared sulfated chitooligosaccharides(SCOS),which have heparinlike properties,to improve the bioactivity of a FGF. We investigated the protective effects of SCOS with or without a FGF on RSC96 cells exposed to Na2 S2 O4 hypoxia/reoxygenation injury. Cell viability was measured by MTT assay and cytotoxicity induced by Na2 S2 O4 was assessed by lactate dehydrogenase(LDH) release into the culture medium. Pretreatment with a FGF and SCOS dramatically decreased LDH release after injury compared to pretreatment with a FGF or SCOS alone. We subsequently prepared an a FGF/SCOS thermo-sensitive hydrogel with poloxamer and examined its effects in vivo. Paw withdrawal thresholds and thermal withdrawal latencies were measured in rats with sciatic nerve injury. Local injection of the a FGF/SCOS hydrogels(a FGF: 40,80 μg/kg) increased the efficiency of sciatic nerve repair compared to a FGF(80 μg/kg) hydrogel alone. Especially a FGF/SCOS thermo-sensitive hydrogel decreased paw withdrawal thresholds from 117.75 ± 8.38(g,4 d) to 65.74 ± 3.39(g,10 d),but a FGF alone group were 140.58 ± 27.54(g,4 d) to 89.12 ± 5.60(g,10 d)(a FGF dose was 80 μg/kg,P < 0.05,n = 8). The thermal withdrawal latencies decreased from 11.61 ± 2.26(s,4 d) to 2.37 ±0.67(s,10 d). However,a FGF alone group were from 17.69 ± 1.47(s,4 d) to4.65 ± 1.73(s,10 d)(P < 0.05,n = 8). Furthermore,the a FGF/SCOS hydrogels also exhibited good biocompatibility in mice. In summary,SCOS improved the protective effects of a FGF in RSC96 cells injured with Na2S2O4 and increased the efficiency of nerve repair and recovery of function in rats with sciatic nerve injury. These findings pave an avenue for the development of novel prophylactic and therapeutic strategies for peripheral nerve injury.

Control Efficacy of Streptomyces sp. SY-L12 and Chitooligosaccharide on Kiwifruit Bacterial Canker, Pseudomonas syringae pv. actinidiae

[ Objective] The paper was to develop a priority scheme for prevention and control of kiwifruit bacterial canker with biological agents. [ Method ] The inhibitory effect of Streptomyces sp. SY-L12 on the growth of Pseudomonas syringae pv. actinidiae were assessed using liquid co-culture and inhibition zone meth- ods. The control efficiency of Streptomyces sp. fermentation broth against kiwifruit bacterial canker was studied via potted trial. [ Result ] As Streptomyces sp. SY- L12 was incubated for 24 h, the growth inhibition rate of its fermentation broth on P. syr/ngae pv. actinidiae was 94.0%. The control efficiency of fermentation broth against P. syringae pv. actinidiae was the best of 85.4% in potted trial when Streptomyces sp. SY-L12 grew at 28~C, 150 rpm for 8 -9 d, better than that of control agent copper hydroxide. The control efficiency was improved as the agent was mixed with 1 mg/L chitooligosaccharide. [ Conclusion] Streptomyces sp. SY-L12 could effectively inhibit the growth of P. syr/ngae pv. actinidiae, and can be developed as biological agent against kiwifruit bacterial canker.

Enzymatic Synthesis of Sialyl Lactosamine Grafted Chitooligosaccharides

An efficient enzymatic assembly strategy was developed for the concise synthesis of structurally well-defined sialylated chitooligosaccharides.Two enzyme modules for theβ-1,3-N-acetyl-glucosaminylation andβ-1,4-galactosylation were applied for the grafting N-acetyl lactosamine(LacNAc)unit(s)onto the chitooligosaccharides.The LacNAc grafted chitooligosaccharides were further modified withα-2,3-orα-2,6-sialylation by two enzymatic sialylation modules to generate a total of 20 sialylated chitooligosaccharides.The inhibition study of influenza virus-induced cytopathy with synthetic sialylated chitooligosaccharides indicated that the sialic acid linkage and chain length both contribute to the binding preference and inhibition potency.

Proteomic Analysis of Rice Plasma Membrane-associated Proteins in Response to Chitooligosaccharide Elicitors

Chitooligomers or chitooligosaccharides （COS） are elicitors that bind to the plasma membrane （PM） and elicit various defense responses. However, the PM-bound proteins involved in elicitor-mediated plant defense responses still remain widely unknown. In order to get more information about PM proteins involved in rice defense responses, we conducted PM proteomic analysis of the rice suspension cells elicited by COS. A total of 14 up- or downregulated protein spots were observed on 2-D gels of PM fractions at 12 h and 24 h after COS incubation. Of them, eight protein spots were successfully identified by MS （mass spectrography） and predicted to be associated to the PM and function in plant defense, including a putative PKN/PRK1 protein kinase, a putative pyruvate kinase isozyme G, a putative zinc finger protein, a putative MAR-binding protein MFP1, and a putative calcium-dependent protein kinase. Interestingly, a COS-induced pM5-like protein was identified for the first time in plants, which is a transmembrane nodal modulator in transforming growth factor-β（TGFβ） signaling in vertebrates. We also identified two members of a rice polyprotein family, which were up-regulated by COS. Our study would provide a starting point for functionality of PM proteins in the rice basal defense.

Eco-friendly preparation of chitooligosaccharides with different degrees of deacetylation from shrimp shell waste and their effects on the germination of wheat seeds

Production of chitosan and its derivatives by traditional methods involves the excessive use of a reaction solution comprisedof sodium hydroxide and hydrochloric acid. Waste water resulting from this process has lim让ed the application of chitosanas a fertilizer as the process causes serious environmental pollution. Specifically, the resulting waste water contains highlevels of dissolved nitrogen and minerals from shrimp shells. In this study, an eco-friendly method was established to produce chitooligosaccharides (COS) with different degrees of deacetylation (DDAs) from shrimp shell waste. At a solid-tosolventratio of 1:6, the degree of demineralization was above 90% with the treatment of 30 g-L_1 H3PO4, and the degree ofdeproteinization was above 80% when treated with 30 g-L_1 KOH at 70 °C. Chitosans with different DDAs were obtainedby microwave-assisted KOH metathesis and the COS with Mw approximately 1500 Da were then prepared by oxidativedegradation. In summary, 33.73 kg H3PO4,12.77 kg, and 241.31 kg KOH were supplied during the processes of demineralization,deproteinization, and deacetylation of 100 kg shrimp shell waste, respectively. The process water was totally recycled,demonstrating that the shrimp shell could be wholly transformed into fertilize The entire process created a product withthe fractions of N:P2O5:K2O:COS = 7.94:24.44:10.72:1 &27. The test on the germination promotion of wheat seeds revealedthat the COS with 72」2% DDA significantly promoted germination. This work demonstrated the use of an eco-friendlypreparation method of COS with a specific degree of deacetylation that can be applied as a fertilizer.

Chitooligosaccharides from squid pen prepared using different enzymes:characteristics and the effect on quality of surimi gel during refrigerated storage

Chitooligosaccharides(COS)from squid pen produced using amylase,lipase and pepsin were characterized.COS produced by 8%(w/w)lipase(COS-L)showed the maximum FRAP and ABTS radical scavenging activity than those prepared using other two enzymes.COS-L had the average molecular weight(MW)of 79 kDa,intrinsic viscosity of 0.41 dL/g and water solubility of 49%.DPPH,ABTS radical scavenging activities,FRAP and ORAC of COS-L were 5.68,322.68,5.66 and 42.20μmol TE/g sample,respectively.Metal chelating activity was 2.58μmol EE/g sample.For antibacterial activity,minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of COS-L against the targeted bacteria were in the range of 0.31–4.91 mg/mL and 0.62–4.91 mg/mL,respectively.Sardine surimi gel added with 1%(w/w)COS-L showed the lower PV,TBARS and microbial growth during 10 days of storage at 4°C.COS-L from squid pen could inhibit lipid oxidation and extend the shelf-life of refrigerated sardine surimi gel.

Immobilization of Chitosanase on Magnetic Nanoparticles: Preparation, Characterization and Properties

Chitosanase could cleaveβ-1,4-linkage of chitosan to produce chitooligosaccharides(COS)with diverse biological activities.However,there are many limitations on the use of free chitosanase in industrial production.Enzyme immobilization is generally considered a valuable strategy in industrial-scale applications.In this study,the chitosanase Csn-BAC from Bacillus sp.MD-5 was immobilized on Fe_(3)O_(4)-SiO_(2) magnetic nanoparticles(MNPs)to enhance its properties,which could be recovered easily from reaction media using magnetic separation.The activities of Csn-BAC immobilized with MNPs(MNPs@Csn-BAC)were de-termined with temperature and pH,and the thermal-and pH-stabilities,respectively.The reusability of the MNPs@Csn-BAC was determined in repeated reaction cycles.Immobilization enhanced the thermal and pH stability of Csn-BAC compared with the free enzyme.After eight reaction cycles using MNPs@Csn-BAC,the residual enzyme activity was 72.15%.Finally,the amount of COS released by MNPs@Csn-BAC was 1.86 times higher than that of the free Csn-BAC in the catalytic performance experiment.The immobilized Csn-BAC exhibits broad application prospects in the production of COS.

“海之星”免疫增强型壳寡糖生物制剂对红花檵木冻害恢复的研究

“海之星”免疫增强型壳寡糖生物制剂是中国科学院”糖链植物疫苗”技术成果转化研究中以壳寡糖为主要原料经过多种对低温抗性有效的成份复配成的对低温冷害有很好效果的水溶性制剂。该研究通过现场实地操作,得出试验数据证明,规律地喷施750倍的”海之星”免疫增强型壳寡糖制剂能够有效地恢复低温冷害对红花檵木造成的损伤,并能提高红花檵木的免疫机能,促进恢复后期叶片增大,叶片颜色变深,促进花芽分花量,可为壳寡糖在红花檵木冻害后的恢复防治应用中提供依据。同时也为将“海之星”免疫增强型壳寡糖生物制剂推广至园林植物冻害后的修复与救治中提供可能。

Chitin oligosaccharides for the food industry:production and applications

Chitin oligosaccharides(CHOS),high-value-added oligomers linked by N-acetyl-d-glucosamine(GlcNAc,NAG),and a small amount of d-glucosamine(GlcN,GA),have aroused increasing interest due to their excellent biological properties,including antibacterial,anti-inflammatory,and immunoprotective activities,and intestinal regulation.The efficient production and utilization of CHOS with high performance can solve problems from chitin as biowaste.However,the large-scale production of well-defined CHOS has not been fully accomplished due to the limited biotechnology and separation methods,thus impeding the research on their biological functions as well as their accurate applications.In this review,we comprehensively summarize the current preparation methods of CHOS,including the chemical,physical,enzymatic and biosynthetic methods.The advantages and disadvantages of the methods are discussed in terms of efficiency,economy,and environmental effects.Furthermore,the applications of CHOS in the food industry and their contributions to human health based on their excellent bioactivities are expounded.It is hoped that this review will help in providing new insights into the production of CHOS with high precision,and support the application of CHOS in serving the food industry as nutritional supplements or foods for special medical purposes.

Signaling events during initiation of arbuscular mycorrhizal symbiosis

Under nutrient-limiting conditions, plants will enter into symbiosis with arbuscular mycorrhizal （AM） fungi for the enhancement of mineral nutrient acquisition from the surrounding soil. AM fungi live in close, intracellular association with plant roots where they transfer phosphate and nitrogen to the plant in exchange for carbon. They are obligate fungi, relying on their host as their only carbon source. Much has been discovered in the last decade concerning the signaling events during initiation of the AM symbiosis, including the identification of signaling molecules generated by both partners. This signaling occurs through symbiosis-specific gene products in the host plant, which are indispensable for normal AM development. At the same time, plants have adapted complex mechanisms for avoiding infection by pathogenic fungi, including an innate immune response to general microbial molecules, such as chitin present in fungal cell walls. How it is that AM fungal colonization is maintained without eliciting a defensive response from the host is still uncertain. In this review, we present a summary of the molecular signals and their elicited responses during initiation of the AM symbiosis, including plant immune responses and their suppression.

Chitin deacetylase:from molecular structure to practical applications

Chitosan and chitooligosaccharides(COS),as derivatives of chitin through deacetylation reaction,have broad applications due to their good biodegradability,biocompatibility,and solubility.In addition,chitosan and COS are involved in cell wall morphogenesis and host–pathogen interactions in vivo.Chitin deacetylases(CDAs)are enzymes that can catalyze the deN-acetylation of chitin.They are widely distributed in protozoa,algae,bacteria,fungi,and insects with important physiological functions.Compared with the traditional chemical method,enzymatic catalysis by CDAs provides an enzymatic catalysis method to produce chitosan and COS with controllable deacetylation site and environmental friendliness.These characteristics attract researchers to produce CDAs by fungicides or pesticides.However,researches on heterologous expression and directed evolution of CDAs are still lacking.In this review,we summarize the latest knowledge of CDAs,especially for heterologous expression systems and directed evolution strategies,which may contribute to the industrial production and future application of CDAs.