Extraction, characterization, antioxidation and anti-inflammatory activity of polysaccharides from Bupleurum chinense based on different molecular weights.

Bupleurum chinense polysaccharide has a wide range of biological activities. In this study, Bupleurum chinense polysaccharides (BPs), BPs-1 (30 kDa) and BPs-2 (2000 kDa) with different molecular weights were isolated and prepared by ultrafiltration interception method. The structures of BPs, BPs-1 and BPs-2 were characterised by monosaccharide composition, GC-MS, Fourier transform infra-red spectroscopy and nuclear magnetic resonance. The results showed that the monosaccharide composition of BPs with different molecular weights was the same, but the proportion was different. BPs, BPs-1 and BPs-2 were mainly connected by Glup-(1→,→2,4)-Araf-(1→,→6)-Glup-(1→). The anti-inflammatory activity screening experiment in vitro showed that BPs-1 had stronger anti-inflammatory effect. Antioxidant experiments showed that BPs-2 had high free radical scavenging activity. This study laid a foundation for elucidating the fine structure and structure-activity relationship of Bupleurum chinense polysaccharides and will promote the product development of Bupleurum chinense polysaccharides.

Automated chemoenzymatic modular synthesis of human milk oligosaccharides on a digital microfluidic platform.

Glycans, along with proteins, nucleic acids, and lipids, constitute the four fundamental classes of biomacromolecules found in living organisms. Generally, glycans are attached to proteins or lipids to form glycoconjugates that perform critical roles in various biological processes. Automatic synthesis of glycans is essential for investigation into structure-function relationships of glycans. In this study, we presented a method that integrated magnetic bead-based manipulation and modular chemoenzymatic synthesis of human milk oligosaccharides (HMOs), on a DMF (Digital Microfluidics) platform. On the DMF platform, enzymatic modular reactions were conducted in solution, and purification of products or intermediates was achieved by using DEAE magnetic beads, circumventing the intricate steps required for traditional solid-phase synthesis. With this approach, we have successfully synthesized eleven HMOs with highest yields of up to >90% on the DMF platform. This study would not only lay the foundation for OPME synthesis of glycans on the DMF platform, but also set the stage for developing automated enzymatic glycan synthesizers based on the DMF platform.

Synthesis of acylated derivatives of alginate oligosaccharides and evaluation of their potential antibacterial activities against Staphylococcus aureus.

Alginate oligosaccharides (AOS) are crucial carbohydrate-based biomaterial used in the synthesis of potential drugs and biological agents, but their antibacterial activities are not significant. In this study, AOS acylated derivatives were synthesized by grafting maleic anhydride (MA) onto AOS at varying ratios. Additionally, their inhibitory effects against Staphylococcus aureus were thoroughly investigated. Characterization of the AOS acylated derivatives (AOS-MA-x, where x = 1, 5, 10, and 20) was conducted using Fourier-transformed infrared spectroscopy, 1H nuclear magnetic resonance spectroscopy, and X-ray diffraction, which confirmed the successful synthesis of these derivatives. The bacteriostatic activity of the AOS-MA derivatives was assessed using growth curves and plate coating method, demonstrating significant antibacterial effects against S. aureus, as compared with AOS. Among these derivatives, AOS-MA-20 exhibited the most potent bacteriostatic activity and was selected for further investigation of its inhibitory mechanism. Scanning electron microscopy analysis revealed that treatment with AOS-MA-20 led to the lysis and rupture of S. aureus cells, expelling their intracellular contents. Moreover, AOS-MA-20 disrupted the integrity of cell wall and cell membrane, impacted ATPase activity, and inhibited the formation of biofilm to some extent, ultimately resulting in bacterial death. These findings lay a foundational framework for the development of environmentally friendly antimicrobial agents.

Structural Characterization and Screening for Anti-inflammatory Activity of Polysaccharides with Different Molecular Weights from Astragali Radix.

Astragali Radix polysaccharides (APSs) exhibit a broad spectrum of biological activity, which is mainly related to immune regulation. At present, most available studies focus on total APSs or a certain component of APSs. However, systematic structural study and screening for the anti-inflammatory activity of polysaccharides with different molecular weights (MW) have yet to be conducted. In this study, lipopolysaccharide (LPS)-induced RAW264.7 macrophages were used as a model to investigate the anti-inflammatory activity of APSs and its fractions. The results revealed that fraction APS-I had better anti-inflammatory effects than APS-II. After APS-I was hydrolyzed by trifluoroacetic acid (TFA), the resulting degradation products oligosaccharides were fully methylated. These derivatized oligosaccharides were further analyzed by MALDI-TOF-MS and UPLC-Q-Exactive-MS/MS. The results showed that APS-I was a hetero-polysaccharide with a molecular weight of about 2.0×106 Da, mainly consisting of glucose (46.8 %) and galactose (34.4 %). The degree of polymerization of Astragali Radix oligosaccharides (APOS) was 2-16. APOS were identified as 1,4-glucooligosaccharides and 1,4-galactooligosaccharides. The findings of this study lay the foundation for further elucidation of structure-function relationships of APSs and provide guidance for the development of anti-inflammatory drugs.

Ultra-sensitive fluorescent biosensor for multiple bacteria detection based on CDs/QDs@ZIF-8 and microfluidic fluidized bed.

An ultra-sensitive fluorescent biosensor based on CDs/QDs@ZIF-8 and microfluidic fluidized bed was developed for rapid and ultra-sensitive detection of multiple target bacteria. The zeolitic imidazolate frameworks (ZIF-8) act as the carrier to encapsulate three kinds of fluorescence signal molecules from the CDs/QDs@ZIF-8 signal amplification system. Besides, three kinds of target pathogenic bacteria were automatically, continuously, and circularly captured by the magnetic nanoparticles (MNPs) in the microfluidic fluidized bed. The neutral Na2EDTA solution was the first time reported to not only dissolve the ZIF-8 frameworks from the MNPs-bacteria-CDs/QDs@ZIF-8 sandwich complexes, but also release the CDs/QDs from sandwich complexes with no loss of fluorescence signal. Due to the advantages of signal amplification and automated sample pretreatment, the proposed fluorescent biosensor can simultaneously detect Escherichia coli O157:H7, Salmonella paratyphi A, and Salmonella paratyphi B as low as 101 CFU/mL within 1.5 h, respectively. The mean recovery in spiked milk samples can reach 99.18%, verifying the applicability of this biosensor in detecting multiple bacteria in real samples.

In Silico Study on a Binding Mechanism of ssDNA Aptamers Targeting Glycosidic Bond-Containing Small Molecules.

Aptamer-based detection targeting glycoconjugates has attracted significant attention for its remarkable potential in identifying structural changes in saccharides in different stages of various diseases. However, the challenges in screening aptamers for small carbohydrates or glycoconjugates, which contain highly flexible and diverse glycosidic bonds, have hindered their application and commercialization. In this study, we investigated the binding conformations between three glycosidic bond-containing small molecules (GlySMs; glucose, N-acetylneuraminic acid, and neomycin) and their corresponding aptamers in silico, and analyzed factors contributing to their binding affinities. Based on the findings, a novel binding mechanism was proposed, highlighting the central role of the stem structure of the aptamer in binding and recognizing GlySMs and the auxiliary role of the mismatched bases in the adjacent loop. Guided by this binding mechanism, an aptamer with a higher 6'-sialyllactose binding affinity was designed, achieving a KD value of 4.54 ± 0.64 µM in vitro through a single shear and one mutation. The binding mechanism offers crucial guidance for designing high-affinity aptamers, enhancing the virtual screening efficiency for GlySMs. This streamlined workflow filters out ineffective binding sites, accelerating aptamer development and providing novel insights into glycan-nucleic acid interactions.

Unveiling the inverse antimicrobial impact of a hetero-chitooligosaccharide on Candida tropicalis growth and biofilm formation.

Chitosan and chitooligosaccharide (COS) are renowned for their potent antimicrobial prowess, yet the precise antimicrobial efficacy of COS remains elusive due to scant structural information about the utilized saccharides. This study delves into the antimicrobial potential of COS, spotlighting a distinct hetero-chitooligosaccharide dubbed DACOS. In contrast to other COS, DACOS remarkably fosters the growth of Candida tropicalis planktonic cells and fungal biofilms. Employing gradient alcohol precipitation, DACOS was fractionated, unveiling diverse structural characteristics and differential impacts on C. tropicalis. Notably, in a murine model of systemic candidiasis, DACOS, particularly its 70 % alcohol precipitates, manifests a promotive effect on Candida infection. This research unveils a new pathway for exploring the intricate nexus between the structural attributes of chitosan oligosaccharides and their physiological repercussions, underscoring the imperative of crafting chitosan and COS with meticulously defined structural configurations.

Characterization and application of active human α2,6-sialyltransferases ST6GalNAc V and ST6GalNAc VI recombined in Escherichia coli.

Eukaryotic sialyltransferases play key roles in many physiological and pathological events. The expression of active human recombinant sialyltransferases in bacteria is still challenging. In the current study, the genes encoding human N-acetylgalactosaminide α2,6-sialyltransferase V (hST6GalNAc V) and N-acetylgalactosaminide α2,6-sialyltransferase VI (hST6GalNAc VI) lacking the N-terminal transmembrane domains were cloned into the expression vectors, pET-32a and pET-22b, respectively. Soluble and active forms of recombinant hST6GalNAc V and hST6GalNAc VI when coexpressed with the chaperone plasmid pGro7 were successfully achieved in Escherichia coli. Further, lactose (Lac), Lacto-N-triose II (LNT II), lacto-N-tetraose (LNT), and sialyllacto-N-tetraose a (LSTa) were used as acceptor substrates to investigate their activities and substrate specificities. Unexpectedly, both can transfer sialic acid onto all those substrates. Compared with hST6GalNAc V expressed in the mammalian cells, the recombinant two α2,6-sialyltransferases in bacteria displayed flexible substrate specificities and lower enzymatic efficiency. In addition, an important human milk oligosaccharide disialyllacto-N-tetraose (DSLNT) can be synthesized by both human α2,6-sialyltransferases expressed in E. coli using LSTa as an acceptor substrate. To the best of our knowledge, these two active human α2,6-sialyltransferases enzymes were expressed in bacteria for the first time. They showed a high potential to be applied in biotechnology and investigating the molecular mechanisms of biological and pathological interactions related to sialylated glycoconjugates.

Advances in the preparation, characterization, and biological functions of chitosan oligosaccharide derivatives: A review.

Chitosan oligosaccharide (COS), which represent the positively charged basic amino oligosaccharide in nature, is the deacetylated and degraded products of chitin. COS has become the focus of intensive scientific investigation, with a growing body of practical and clinical studies highlighting its remarkable health-enhancing benefits. These effects encompass a wide range of properties, including antibacterial, antioxidant, anti-inflammatory, and anti-tumor activities. With the rapid advancements in chemical modification technology for oligosaccharides, many COS derivatives have been synthesized and investigated. These newly developed derivatives possess more stable chemical structures, improved biological activities, and find applications across a broader spectrum of fields. Given the recent interest in the chemical modification of COS, this comprehensive review seeks to consolidate knowledge regarding the preparation methods for COS derivatives, alongside discussions on their structural characterization. Additionally, various biological activities of COS derivatives have been discussed in detail. Lastly, the potential applications of COS derivatives in biomedicine have been reviewed and presented.

[Preparation, characterization and activity evaluation of Spirulina-chitooligosaccharides capable of inhibiting biofilms].

The biofilms formed by pathogenic microorganisms seriously threaten human health and significantly enhance drug resistance, which urgently call for developing drugs specifically targeting on biofilms. Chitooligosaccharides extracted from shrimp and crab shells are natural alkaline oligosaccharides with excellent antibacterial effects. Nevertheless, their inhibition efficacy on biofilms still needs to be improved. Spirulina (SP) is a microalga with negatively charged surface, and its spiral structure facilitates colonization in the depth of the biofilm. Therefore, the complex of Spirulina and chitooligosaccharides may play a synergistic role in killing pathogens in the depth of biofilm. This research first screened chitooligosaccharides with significant bactericidal effects. Subsequently, Spirulina@Chitooligosaccharides (SP@COS complex was prepared by combining chitooligosaccharides with Spirulina through electrostatic adsorption. The binding of the complex was characterized by zeta potential, z-average size, and fluorescence labeling. Ultraviolet-visible spectroscopy (UV-Vis) showed the encapsulation efficiency and the drug loading efficiency reached up to 90% and 16%, respectively. The prepared SP@COS2 exhibited a profound synergistic inhibition effect on bacterial and fungal biofilms, which was mainly achieved by destroying the cell structure of the biofilm. These results demonstrate the potential of Spirulina-chitooligosaccharides complex as a biofilm inhibitor and provide a new idea for addressing the harm of pathogenic microorganisms.

Evaluation and Mechanistic Investigation of Human Milk Oligosaccharide against SARS-CoV-2.

Four human milk oligosaccharides (HMOs), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 2'-fucosyllactose (2'-FL), and 3-fucosyllactose (3-FL), were assessed for their possible antiviral activity against the SARS-CoV-2 spike receptor binding domain (RBD) in vitro. Among them, only 2'-FL/3-FL exhibited obvious antibinding activity against direct binding and trans-binding in competitive immunocytochemistry and enzyme-linked immunosorbent assays. The antiviral effects of 2'-FL/3-FL were further confirmed by pseudoviral assays with three SARS-Cov-2 mutants, with a stronger inhibition effect of 2'-FL than 3-FL. Then, 2'-FL/3-FL were studied with molecular docking and microscale thermophoresis analysis, showing that the binding sites of 2'-FL on RBD were involved in receptor binding, in addition to a tighter bond between them, thus enabling 2'-FL to be more effective than 3-FL. Moreover, the immunomodulation effect of 2'-FL was preliminary evaluated and confirmed in a human alveolus chip. These results would open up possible applications of 2'-FL for the prevention of SARS-CoV-2 infections by competitive binding inhibition.

Variations in metabolic enzymes cause differential changes of heparan sulfate and hyaluronan in high glucose treated cells on chip.

Glycocalyx dysfunction is believed as the first step in diabetic vascular disease. However, few studies have systematically investigated the influence of HG on the glycocalyx as a whole and its major constituent glycans towards one type of cell. Furthermore, most studies utilized traditional two-dimensional (2D) cultures in vitro, which can't provide the necessary fluid environment for glycocalyx. Here, we utilized vascular glycocalyx on chips to evaluate the changes of glycocalyx and its constituent glycans in HG induced HUVECs. Fluorescence microscopy showed up-regulation of hyaluronan (HA) but down-regulation of heparan sulfate (HS). By analyzing the metabolic enzymes of both glycans, a decrease in the ratio of synthetic/degradative enzymes for HA and an increase in that for HS were demonstrated. Two substrates (UDP-GlcNAc, UDP-GlcA) for the synthesis of both glycans were increased according to omics analysis. Since they were firstly pumped into Golgi apparatus to synthesize HS, less substrates may be left for HA synthesis. Furthermore, the differential changes of HA and HS were confirmed in vessel slides from db/db mice. This study would deepen our understanding of impact of HG on glycocalyx formation and diabetic vascular disease.

Digital microfluidics-engaged automated enzymatic degradation and synthesis of oligosaccharides.

Glycans are an important group of natural biopolymers, which not only play the role of a major biological energy resource but also as signaling molecules. As a result, structural characterization or sequencing of glycans, as well as targeted synthesis of glycans, is of great interest for understanding their structure-function relationship. However, this generally involves tedious manual operations and high reagent consumptions, which are the main technical bottlenecks retarding the advances of both automatic glycan sequencing and synthesis. Until now, automated enzymatic glycan sequencers or synthesizers are still not available on the market. In this study, to promote the development of automation in glycan sequencing or synthesis, first, programmed degradation and synthesis of glycans catalyzed by enzymes were successfully conducted on a digital microfluidic (DMF) device by using microdroplets as microreactors. In order to develop automatic glycan synthesizers and sequencers, a strategy integrating enzymatic oligosaccharide degradation or synthesis and magnetic manipulation to realize the separation and purification process after enzymatic reactions was designed and performed on DMF. An automatic process for enzymatic degradation of tetra-N-acetyl chitotetraose was achieved. Furthermore, the two-step enzymatic synthesis of lacto-N-tetraose was successfully and efficiently completed on the DMF platform. This work demonstrated here would open the door to further develop automatic enzymatic glycan synthesizers or sequencers based on DMF.

In Silico Selection and Validation of High-Affinity ssDNA Aptamers Targeting Paromomycin.

Glycans are promising for disease diagnosis since glycan biosynthesis is significantly affected by disease states, and glycosylation changes are probably more pronounced than protein expression during the transformation to the diseased condition. Glycan-specific aptamers can be developed for challenging applications such as cancer targeting; however, the high flexibility of glycosidic bonds and scarcity of studies on glycan-aptamer binding mechanisms increased the difficulty of screening. In this work, the model of interactions between glycans and ssDNA aptamers synthesized based on the sequence of rRNA genes was developed. Our simulation-based approach revealed that paromomycin as a representative example of glycans is preferred to bind base-restricted stem structures of aptamers because they are more critical in stabilizing the flexible structures of glycans. Combined experiments and simulations have identified two optimal mutant aptamers. Our work would provide a potential strategy that the glycan-binding rRNA genes could act as the initial aptamer pools to accelerate aptamer screening. In addition, this in silico workflow would be potentially applied in the more extensive in vitro development and application of RNA-templated ssDNA aptamers targeting glycans.

Synthesis of acylated derivatives of chitosan oligosaccharide and evaluation of their potential antifungal agents on Fusarium oxysporum.

Chitosan oligosaccharide (COS) is an important carbohydrate-based biomaterial for synthesizing candidate drugs and biological agents. This study synthesized COS derivatives by grafting acyl chlorides of different alkyl chain lengths (C8, C10, and C12) onto COS molecules and further investigated their physicochemical properties and antimicrobial activity. The COS acylated derivatives were characterized using Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance spectroscopy, X-ray diffraction, and thermogravimetric analysis. COS acylated derivatives were successfully synthesized and possessed high solubility and thermal stability. As for the evaluation of antibacterial activity, COS acylated derivatives did not significantly inhibit Escherichia coli and Staphylococcus aureus, but they significantly inhibited Fusarium oxysporum, which was superior to that of COS. Transcriptomic analysis revealed that COS acylated derivatives exerted antifungal activity mainly by downregulating the expression of efflux pumps, disrupting cell wall integrity, and impeding normal cell metabolism. Our findings provided a fundamental theory for the development of environmentally friendly antifungal agents.

Ulvan and Ulva oligosaccharides: a systematic review of structure, preparation, biological activities and applications.

Ulva is one of the main green algae causing green tide disasters. Ulvan is the primarily component polysaccharide of the cell wall of Ulva and its complex structure and monosaccharide composition resulted in various biological activities. However, the high-value and effective utilization of extracted ulvan have been obstructed by limitations ranging from large molecular weight and low solubility to poor bioavailability. Ulva oligosaccharide obtained by degrading ulvan can not only ideally retain the various biological activities of ulvan very well but also effectively solve the problems of low solubility and poor bioavailability. The preparation and biological activity studies of ulvan and Ulva oligosaccharides have become a hot spot in the field of marine biological resources development research. At present, the comprehensive reviews of ulvan and Ulva oligosaccharides are still scarce. What are overviewed in this paper are the chemical composition, structure, extraction, and purification of ulvan and Ulva oligosaccharides, where research progress on the biological activities of ulvan and Ulva oligosaccharides is summarized and prospected. A theoretical and practical basis has been provided for further research on ulvan and Ulva oligosaccharides, as well as the high-value development and effective utilization of marine algae resources.

Recent Research and Application Prospect of Functional Oligosaccharides on Intestinal Disease Treatment.

The intestinal tract is an essential digestive organ of the human body, and damage to the intestinal barrier will lead to various diseases. Functional oligosaccharides are carbohydrates with a low degree of polymerization and exhibit beneficial effects on human intestinal health. Laboratory experiments and clinical studies indicate that functional oligosaccharides repair the damaged intestinal tract and maintain intestinal homeostasis by regulating intestinal barrier function, immune response, and intestinal microbial composition. Functional oligosaccharides treat intestinal disease such as inflammatory bowel disease (IBD) and colorectal cancer (CRC) and have excellent prospects for therapeutic application. Here, we present an overview of the recent research into the effects of functional oligosaccharides on intestinal health.

Chitosan Oligosaccharide Supplementation Affects Immunity Markers in Ewes and Lambs during Gestation and Lactation.

Chitosan oligosaccharide (COS) is derived through deacetylation of chitin from crustacean shells. Previous studies reported the benefits of COS to gut microbiota, immunity and health of host species. In this study, 120 pregnant composite ewes were subdivided into treatment and control groups in duplicate. COS was supplemented via a loose lick to provide an estimated intake of COS @100−600 mg/d/ewe for five weeks pre-lambing until lamb marking. Body weight was recorded pre-treatment for ewes, and at lamb marking and weaning for both ewes and lambs. Serum immunity markers immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), secretory immunoglobulin A (sIgA), interleukin (IL)-2, IL10 and faecal sIgA were determined for ewes and lambs at lamb marking and weaning by enzyme-linked immunosorbent assay (ELISA). We found that COS can be incorporated in sheep feed without compromising palatability. Maternal COS supplementation did not influence the body weight of ewes or lambs. It did, however, significantly increase the concentrations of serum IL2 in ewes at marking and weaning (p < 0.001). In lambs, COS also significantly increased the IL2 concentration at making (p = 0.018) and weaning (p = 0.029) and serum IgM at marking (p < 0.001). No significant effect was observed in the concentration of any other immune marker or cytokine in either ewes or lambs. In conclusion, maternal COS supplementation significantly modulated some immunity markers in both ewes and lambs. The short duration of maternal COS supplementation and optimal seasonal conditions during the trial may explain the lack of significant body weight in ewes and lambs from the COS supplementation.

[Glycosylation, glycan receptors recognition of SARS-CoV-2 and discoveries of glycan inhibitors against SARS-CoV-2].

COVID-19 represents the most serious public health event in the past few decades of the 21st century. The development of vaccines, neutralizing antibodies, and small molecule chemical agents have effectively prevented the rapid spread of COVID-19. However, the continued emergence of SARS-CoV-2 variants have weakened the efficiency of these vaccines and antibodies, which brought new challenges for searching novel anti-SARS-CoV-2 drugs and methods. In the process of SARS-CoV-2 infection, the virus firstly attaches to heparan sulphate on the cell surface of respiratory tract, then specifically binds to hACE2. The S protein of SARS-CoV-2 is a highly glycosylated protein, and glycosylation is also important for the binding of hACE2 to S protein. Furthermore, the S protein is recognized by a series of lectin receptors in host cells. These finding implies that glycosylation plays important roles in the invasion and infection of SARS-CoV-2. Based on the glycosylation pattern and glycan recognition mechanisms of SARS-CoV-2, it is possible to develop glycan inhibitors against COVID-19. Recent studies have shown that sulfated polysaccharides originated from marine sources, heparin and some other glycans display anti-SARS-CoV-2 activity. This review summarized the function of glycosylation of SARS-CoV-2, discoveries of glycan inhibitors and the underpinning molecular mechanisms, which will provide guidelines to develop glycan-based new drugs against SARS-CoV-2.

Structural and Biochemical Analysis Reveals Catalytic Mechanism of Fucoidan Lyase from Flavobacterium sp. SA-0082.

Fucoidans represent a type of polyanionic fucose-containing sulfated polysaccharides (FCSPs) that are cleaved by fucoidan-degrading enzymes, producing low-molecular-weight fucoidans with multiple biological activities suitable for pharmacological use. Most of the reported fucoidan-degrading enzymes are glycoside hydrolases, which have been well studied for their structures and catalytic mechanisms. Little is known, however, about the rarer fucoidan lyases, primarily due to the lack of structural information. FdlA from Flavobacterium sp. SA-0082 is an endo-type fucoidan-degrading enzyme that cleaves the sulfated fuco-glucuronomannan (SFGM) through a lytic mechanism. Here, we report nine crystal structures of the catalytic N-terminal domain of FdlA (FdlA-NTD), in both its wild type (WT) and mutant forms, at resolutions ranging from 1.30 to 2.25 Å. We show that the FdlA-NTD adopts a right-handed parallel ß-helix fold, and possesses a substrate binding site composed of a long groove and a unique alkaline pocket. Our structural, biochemical, and enzymological analyses strongly suggest that FdlA-NTD utilizes catalytic residues different from other ß-helix polysaccharide lyases, potentially representing a novel polysaccharide lyase family.