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The present study reports the structural characteristics of 3 polysaccharide fractions (SPS-F1, SPS-F2 and SPS-F3) isolated and purified from squash. SPS-F1 (molecular weight (Mw) = 12.30 kDa) and SPS-F2 (Mw= 19.40 kDa) were likely to contain HG and RG-I domain of pectic polysaccharide, respectively. SPS-F2 (Mw= 270.4 kDa) was mainly composed of rhamnose, galactose and arabinose. The treatment with SPS decreased body weight gain, glucose and TG levels in type 2 diabetes rats. Besides, 25 differential metabolites were identified based on urinary metabolomics analysis, which are crucial to the anti-diabetic effect of SPS. The regulation of nicotinamide N-oxide, histamine, cis-aconitate, citrate, L-malic acid, 3-(3-hydroxyphenyl) propanoic acid and N-acetyl-L-aspartic acid were mainly associated with energy metabolism, gut microbiota and inflammation. Study of surface plasmon resonance revealed the binding kinetics with galectin-3 (Gal-3) and fibroblast growth factor 2 (FGF2). The KDvalues of SPS-F2 and SPS-F3 to Gal-3 were 4.97 × 10-3and 1.48 × 10-3mol/L, indicating a weak binding affinity. All 3 fractions showed moderate binding to FGF2 and the affinity was SPS-F3 > SPS-F2 > SPS-F1. Thus, the metabolomics and SPR approach were proved to be a promising tool in exploring the anti-diabetes effects of SPS and provided a deep understanding of the mechanisms.

Hericium erinaceusis a nutritious edible and medicinal fungi, rich in a variety of functional active ingredients, with various physiological functions such as antioxidation, anticancer, and enhancing immunity. It is also effective in protecting the digestive system and preventing neurodegenerative diseases. In this review paper, we summarize the sources, structures and efficacies of the main active components in H. erinaceusfruiting body, mycelium, and culture media, and update the latest research progress on their biological activities and the related molecular mechanisms. Based on this information, we provide detailed challenges in current research, industrialization and information on the active ingredients of H. erinaceus. Perspectives for future studies and new applications of H. erinaceusare proposed.

Heparin is a highly sulfated linear glycosaminoglycan that is used as an anticoagulant to prevent and treat thrombotic diseases. Herein, we find that heparin specifically inhibits the activation of the Cas12 protein through the competitive binding of heparin and crRNA to Cas12. Studies illustrate that heparin’s high molecular weight and strong negative charge are critical parameters for its inhibitory effect. This unexpected finding was engineered for the detection of heparin, affording a low detection limit of 0.36 ng/mL for fluorometric quantification. We further developed a rapid lateral flow-based system named HepaStrip (heparin strip), allowing heparin monitoring in clinical samples within 20 min. Finally, in vivoinvestigations revealed that heparin can regulate gene editing with the clusters of the regularly spaced short palindromic repeat (CRISPR)/Cas12 system in Escherichia coli. Heparin blocks the formation of Cas12-crRNA ribonucleoprotein, allowing the application of CRISPR for rapid and field-deployable detection of heparin and unleashing the potential use of heparin in future anti-CRISPR applications.

The multienzyme cascade has received growing attention to obtain structurally defined glycans in vitro. However, due to poor enzyme stability and low compatibility between glycoenzymes, artificially designed multienzyme pathways to access glycans are often inefficient. Herein, based on the strategy “Modular-Enzymes Assembly by Spatial Segregation” (MASS), we developed a universal immobilization platform to assemble multiple glycoenzymes in compartmentalized MOF particles, inside and outside, significantly reducing the undesired interference and cross-inhibitions. By changing the enzyme modules, a series of glycosyl donor, disaccharides, oligosaccharides, and polysaccharides bearing cofactor regeneration were efficiently prepared. This bioreactor was further successfully applied to the reaction system with high substrate concentration to demonstrate its industrial potential. This robust multienzyme immobilization platform should serve to promote the enzymatic synthesis of more complex glycans.

Glycosaminoglycans (GAGs) have high negative charge and are biologically and pharmaceutically important because their high charge promotes a strong interaction with many proteins. Due to the inherent heterogeneity of GAGs, multiple oligosaccharides, containing certain common domains, often can interact with clusters of basic amino acid residues on a target protein. The specificity of many GAG–protein interactions remains undiscovered since there is insufficient structural information on the interacting GAGs. Herein, we establish a cluster sequencing strategy to simultaneously deduce all major sequences of the affinity GAG oligosaccharides, leading to a definition of the consensus sequence they share that corresponds to the specific binding domain for the target protein. As a proof of concept, antithrombin III-binding oligosaccharides were examined, resulting in a heptasaccharide domain containing the well-established anticoagulant pentasaccharide sequence. Repeating this approach, a new pentasaccharide domain was discovered corresponding to the heparin motif responsible for binding interferon-γ (IFNγ). Our strategy is fundamentally important for the discovery of saccharide sequences needed in the development of novel GAG-based therapeutics.

In this work, the first version of “Glycomapping” software is developed for the analysis of the most common low-molecular-weight heparin (LMWH), enoxaparin. Using ultrahigh-performance liquid chromatography-mass spectrometry, size exclusion chromatography is applied, and a virtual database of glycans in enoxaparin is established for the initial searching. With “Glycomapping”, a complex chromatogram can be fitted, significantly improving resolution and confirming an accurate distribution range for each size of glycan within enoxaparin. In addition, randomly matched MS data can be corrected, with the constraint of the corresponding chromatographic retention time range, to remove most false positive data. The analytical stability of “Glycomapping” software was confirmed. Enoxaparin, prepared by different manufacturers and from different animal sources, was analyzed using “Glycomapping.” Compared to raw data, data processed with “Glycomapping” are more robust and accurate. Another two LMWHs, nadroparin and dalteparin could also be analyzed with this software. This work lays a solid foundation for the automated analysis of heterogeneous mixtures of natural glycans, such as LMWHs and other complex oligosaccharides and polysaccharides.

Fibroblast growth factor (FGF) 23 is a phosphate-regulating hormone that is elevated in patients with chronic kidney disease and associated with cardiovascular mortality. Experimental studies showed that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4). A recent structural analysis revealed that the complex of FGF23 and FGFR1, the physiologic FGF23 receptor in the kidney, includes soluble α-klotho (klotho) and heparin, which both act as co-factors for FGF23/FGFR1 signaling. Here, we investigated whether soluble klotho, a circulating protein with cardio-protective properties, and heparin, a factor that is routinely infused into patients with kidney failure during the hemodialysis procedure, regulate FGF23/FGFR4 signaling and effects in cardiac myocytes. We developed a plate-based binding assay to quantify affinities of specific FGF23/FGFR interactions and found that soluble klotho and heparin mediate FGF23 binding to distinct FGFR isoforms. Heparin specifically mediated FGF23 binding to FGFR4 and increased FGF23 stimulatory effects on hypertrophic growth and contractility in isolated cardiac myocytes. When repetitively injected into two different mouse models with elevated serum FGF23 levels, heparin aggravated cardiac hypertrophy. We also developed a novel procedure for the synthesis and purification of recombinant soluble klotho, which showed anti-hypertrophic effects in FGF23-treated cardiac myocytes. Thus, soluble klotho and heparin act as independent FGF23 co-receptors with opposite effects on the pathologic actions of FGF23, with soluble klotho reducing and heparin increasing FGF23-induced cardiac hypertrophy. Hence, whether heparin injections during hemodialysis in patients with extremely high serum FGF23 levels contribute to their high rates of cardiovascular events and mortality remains to be studied.

Metal–organic frameworks (MOFs) are rarely applied as solid supports in the enzymatic synthesis of oligosaccharides and polysaccharides, as glycosyltransferases are readily inactivated by traditional MOFs due to the poor compatibility and the limited mass transfer for complex carbohydrates in MOFs. Here, on the basis of the synthetic methods of zeolitic imidazolate framework-90 (ZIF-90), we prepared bimetal organic material (BMOM) microreactors that successfully encapsulated Pasteurella multocidaheparosan synthase 2 (PmHS2), a critical glycosyltransferase in the enzymatic synthesis of heparin and heparan sulfate. The second metal ion introduced can increase the mesopores in the BMOM, stabilize the active pocket of glycosyltransferase, and facilitate the deprotonation of critical amino acid residues, Asp and Glu of PmHS2, to initiate the catalyzation. On the basis of this bimetallic microreactor, heparosan disaccharide, oligosaccharide, and polysaccharide are successfully prepared in quantitative yield, providing a viable BMOM-based immobilization strategy to simulate the physiological microenvironment for glycosyltransferase.

The objective of this present study is to evaluate the effect of different drying methods (spray drying, freeze drying and hot-air drying) on physicochemical properties and antioxidant ability of G. lucidumextracts (GLEs) using various techniques. Meanwhile, combined with free amino acids (FAA) analysis, taste activity values (TAV) analysis and electronic tongue to investigate the relationship between the drying method and the sensory quality of GLEs. The study demonstrates that spray drying GLEs (SDE) has a high water solubility of 96.04% ± 0.10% due to its low crystallinity and hollow spherical microstructure. SDE and freeze drying GLEs (FDE) contain high levels of polysaccharides and proteins, which facilitates the formation of polysaccharide-protein chelates, thereby improving antioxidant capacity. However, SDE has higher antioxidant activity when its polysaccharide content is the same. SDE has higher thermal stability to maintain the molecular structure of GLEs. Meanwhile, FDE has a bitter FAAs content up to 65.84 ± 1.12 mg/g and TAV of Histidine is 178.70. Overall, the results suggest that spray drying is an appropriate and preferred drying method to maintain the bioactivity and bioavailability of GLEs. These findings may enhance the understanding of the GLEs flavor property and bioavailability associated with drying methods and provide a theoretical basis for G. lucidumprocessing.

This study examined poly(glycerol-1,8-octanediol-sebacate) (PGOS) copolymers with low-level substitution of O (1,8-octanediol) for G (glycerol) units (G/O ratios 0.5:0.5, 0.66:0.33, 0.75:0.25, 0.8:0.2, and 0.91:0.09) prepared in bulk by immobilized Candida antarcticaLipase B (N435) catalysis. The central question explored was the extent that exchanging less than half of poly(glycerol sebacate) (PGS) glycerol units with 1,8-octanediol can be used as a strategy to fine-tune biomaterial properties. Synthesized copolymers having G/O ratios of 0.66:0.33, 0.75:0.25, 0.8:0.2, and 0.91:0.09 have similar molecular weights, where Mwvaried from 52,800 to 63,800 g/mol, Mnvaried from 5100 to 6450 g/mol, and ĐM(molecular mass dispersity, Mw/Mn) values were also similar (8.4–11.4). All of the copolymers were branched, and dendritic glycerol units reached 11% for PGOS-0.91:0.09:1.0. Analysis of DSC second heating scans revealed that copolymers with higher 1,8-octanediol contents have relatively higher Tmand ΔHfvalues. Over the copolymer compositional range studied herein, Tmand ΔHfvalues varied from 5.3 to 21.1 °C and 8.0 to 23.1 J/g, respectively. Stress–strain curves of PGOS copolymers cured at 140 °C for 48 h exhibited either a unimodal, bimodal, or trimodal response to tensile loading. Varying G/O from 10:1 to 2:1 resulted in significant increases in the peak stress (0.26–4.01 MPa), preyield modulus (0.65–62.59 MPa), failure to strain (64–110%), and failure toughness (0.1–0.56 MPa). This demonstrates that altering the G/O ratio over a narrow compositional range provides biomaterials with widely different yet tunable mechanical properties. Further investigation of PGOS-0.75:0.25:1.0 films revealed that varying the cure conditions from 120 to 160 °C for periods of 24–72 h provides access to biomaterials with a failure strain range from 15 to 224% and Young’s modulus from 1.17 to 10.85 MPa. Hence, using the dual variables of compositional variation and changes in cure conditions provides an exciting platform for PGS analogues to optimize material–tissue interactions. Increased contents of 1,8-octanediol slowed in vitrodegradation. Slowed degradation of PGOS relative to PGS will be valuable for use in slower healing wounds.

Heparin products are widely used clinical anticoagulants essential in the practice of modern medicine. Low-molecular-weight heparins (LMWHs) are currently prepared by the controlled chemical or enzymatic depolymerization of unfractionated heparins (UFHs) that are extracted from animal tissues. In many clinical applications, LMWHs have displaced UFHs and currently comprise over 60% of the heparin market. In the past, our laboratory has made extensive efforts to prepare bioengineered UFHs relying on a chemoenzymatic process to address concerns about animal-sourced UFHs. The current study describes the use of a novel chemoenzymatic process to prepare a chemobiosynthetic LMWH from a low-molecular-weight heparosan. The resulting chemobiocatalytic LMWH matches most of the United States pharmacopeial specifications for enoxaparin, a LMWH prepared through the base-catalyzed depolymerization of animal-derived UFH.

Heparin is a highly charged, polysulfated polysaccharide and serves as an anticoagulant. Heparin binds to multiple proteins throughout the body, suggesting a large range of potential therapeutic applications. Although its function has been characterized in multiple physiological contexts, heparin’s solution conformational dynamics and structure–function relationships are not fully understood. Molecular dynamics (MD) simulations facilitate the analysis of a molecule’s underlying conformational ensemble, which then provides important information necessary for understanding structure–function relationships. However, for MD simulations to afford meaningful results, they must both provide adequate sampling and accurately represent the energy properties of a molecule. The aim of this study is to compare heparin’s conformational ensemble using two well-developed force fields for carbohydrates, known as GLYCAM06 and CHARMM36, using replica exchange molecular dynamics (REMD) simulations, and to validate these results with NMR experiments. The anticoagulant sequence, an ultra-low-molecular-weight heparin, known as Arixtra (fondaparinux, sodium), was simulated with both parameter sets. The results suggest that GLYCAM06 matches experimental nuclear magnetic resonance three-bond J-coupling values measured for Arixtra better than CHARMM36. In addition, NOESY and ROESY experiments suggest that Arixtra is very flexible in the sub-millisecond time scale and does not adopt a unique structure at 25 C. Moreover, GLYCAM06 affords a much more dynamic conformational ensemble for Arixtra than CHARMM36.

Emerging viral diseases can substantially threaten national and global public health. Central to our ability to successfully tackle these diseases is the need to quickly detect the causative virus and neutralize it efficiently. Here we present the rational design of DNA nanostructures to inhibit dengue virus infection. The designer DNA nanostructure (DDN) can bind to complementary epitopes on antigens dispersed across the surface of a viral particle. Since these antigens are arranged in a defined geometric pattern that is unique to each virus, the structure of the DDN is designed to mirror the spatial arrangement of antigens on the viral particle, providing very high viral binding avidity. We describe how available structural data can be used to identify unique spatial patterns of antigens on the surface of a viral particle. We then present a procedure for synthesizing DDNs using a combination of in silico design principles, self-assembly, and characterization using gel electrophoresis, atomic force microscopy and surface plasmon resonance spectroscopy. Finally, we evaluate the efficacy of a DDN in inhibiting dengue virus infection via plaque-forming assays. We expect this protocol to take 2–3 d to complete virus antigen pattern identification from existing cryogenic electron microscopy data, ~2 weeks for DDN design, synthesis, and virus binding characterization, and ~2 weeks for DDN cytotoxicity and antiviral efficacy assays.

Bovine intestinal heparins are structurally distinct from porcine intestinal heparins and exhibit lower specific anticoagulant activity (units/mg). The reduced content of N-sulfo, 3-O-sulfo glucosamine, the central and critical residue in heparin’s antithrombin III binding site, is responsible for bovine intestinal heparin’s reduced activity. Previous studies demonstrate that treatment of bovine intestinal heparin with 3-O-sulfotransferase in the presence of 3′-phosphoadenosine-5′-phosphosulfate afforded remodeled bovine heparin with an enhanced activity reaching the United States Pharmacopeia’s requirements. Starting from this remodeled bovine intestinal heparin, we report the preparation of a bovine intestinal low molecular weight heparin having the same structural properties and anti-factor IIa and anti-factor Xa activities of Enoxaparin. Moreover, this bovine intestinal heparin-derived “Enoxaparin” showed comparable platelet factor-4 binding affinity, suggesting that it should exhibit similarly low levels of heparin induced thrombocytopeneia, HIT.

The biological functions of natural polyelectrolytes are strongly influenced by the presence of ions, which bind to the polymer chains and thereby modify their properties. Although the biological impact of such modifications is well recognized, a detailed molecular picture of the binding process and of the mechanisms that drive the subsequent structural changes in the polymer is lacking. Here, we study the molecular mechanism of the condensation of calcium, a divalent cation, on hyaluronan, a ubiquitous polymer in human tissues. By combining two-dimensional infrared spectroscopy experiments with molecular dynamics simulations, we find that calcium specifically binds to hyaluronan at millimolar concentrations. Because of its large size and charge, the calcium cation can bind simultaneously to the negatively charged carboxylate group and the amide group of adjacent saccharide units. Molecular dynamics simulations and single-chain force spectroscopy measurements provide evidence that the binding of the calcium ions weakens the intramolecular hydrogen-bond network of hyaluronan, increasing the flexibility of the polymer chain. We also observe that the binding of calcium to hyaluronan saturates at a maximum binding fraction of ∼10–15 mol %. This saturation indicates that the binding of Ca2+strongly reduces the probability of subsequent binding of Ca2+at neighboring binding sites, possibly as a result of enhanced conformational fluctuations and/or electrostatic repulsion effects. Our findings provide a detailed molecular picture of ion condensation and reveal the severe effect of a few, selective and localized electrostatic interactions on the rigidity of a polyelectrolyte chain.

Glycosaminoglycans (GAGs) are heterogeneous, negatively charged, macromolecules that are found in animal tissues. Based on the form of component sugar, GAGs have been categorized into four different families: heparin/heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate, and hyaluronan. GAGs engage in biological pathway regulation through their interaction with protein ligands. Detailed structural information on GAG chains is required to further understanding of GAG–ligand interactions. However, polysaccharide sequencing has lagged behind protein and DNA sequencing due to the non-template-driven biosynthesis of glycans. In this review, we summarize recent progress in the analysis of GAG chains, specifically focusing on techniques related to mass spectroscopy (MS), including separation techniques coupled to MS, tandem MS, and bioinformatics software for MS spectrum interpretation. Progress in the use of other structural analysis tools, such as nuclear magnetic resonance (NMR) and hyphenated techniques, is included to provide a comprehensive perspective.

Glycosaminoglycans (GAGs) contribute to the treatment of many human diseases, especially in the field of thrombosis, because of their anticoagulant activity. GAGs interrupt the coagulation process by interacting with multiple coagulation factors through defined sequences within their linear and negatively charged chains, which are not fully elucidated. Numerous methods have been developed to characterize the structure of pharmaceutical GAGs, including intravenouslyor subcutaneouslyadministered heparin and orally administered sulodexide. However, most currently available methods only focus on the oligosaccharide portion or analyze the whole mixture because longer-chain polysaccharides are extremely difficult to resolve by chromatographic separation. We have established two novel electrophoresis–mass spectrometry methods to provide a panoramic view of the structures of pharmaceutical GAGs. In the first method, an in-gel digestion procedure was developed to recover GAGs from the polyacrylamide gels, while in the second method, a strong anion exchange ultrafiltration procedure was developed to extract multiple GAG species from the agarose gels. Both procedures are compatible with liquid chromatography–tandem mass spectrometry, and structural information, such as disaccharide composition and chain length, can be revealed for each GAG fraction. The applications of these two methods on analysis of two different GAG drugs, heparin and sulodexide, were demonstrated. The current study offers the first robust tool to directly elucidate the structure of larger GAG chains with more biological importance rather than obtaining a vague picture of all chains as a mixture, which is fundamental for better understanding the structure–activity relationship and quality control of the GAG drugs.

Neural tissue engineering strategies are paramount to create fully mature neurons, necessary for new therapeutic strategies for neurological diseases or the creation of reliable in vitromodels. Scaffolds can provide physical support for these neurons and enable cues for enhancing neural cell differentiation, such as electrical current. Coaxial electrospinning fibers, designed to fulfill neural cell needs, bring together an electroconductive shell layer (PCL-PANI), able to mediate electrical stimulation of cells cultivated on fibers mesh surface, and a soft core layer (PGS), used to finetune fiber diameter (951 ± 465 nm) and mechanical properties (1.3 ± 0.2 MPa). Those dual functional coaxial fibers are electroconductive (0.063 ± 0.029 S cm−1, stable over 21 days) and biodegradable (72% weigh loss in 12 hours upon human lipase accelerated assay). For the first time, the long-term effects of electrical stimulation on induced neural progenitor cells were studied using such fibers. The results show increase in neural maturation (upregulation of MAP2, NEF-H and SYP), up-regulation of glutamatergic marker genes (VGLUT1 – 15-fold) and voltage-sensitive channels (SCN1α – 12-fold, CACNA1C – 32-fold), and a down-regulation of GABAergic marker (GAD67 – 0.09-fold), as detected by qRT-PCR. Therefore, this study suggest a shift from an inhibitory to an excitatory neural cell profile. This work shows that the PGS/PCL-PANI coaxial fibers here developed have potential applications in neural tissue engineering.

Granulocyte macrophage colony stimulating factor (GMCSF) is an immunomodulatory cytokine that is harnessed as a therapeutic. GMCSF is known to interact with other clinically important molecules, such as heparin, suggesting that endogenous and administered GMCSF has the potential to modulate orthogonal treatment outcomes. Thus, molecular level characterization of GMCSF and its interactions with biologically active compounds is critical to understanding these mechanisms and predicting clinical consequences. Here, we dissect the biophysical factors that facilitate the GMCSF–heparin interaction, previously shown to be pH-dependent, using nuclear magnetic resonance spectroscopy, surface plasmon resonance, and molecular dynamics simulations. We find that the affinity of GMCSF for heparin increases not only with a transition to acidic pH but also with an increase in heparin chain length. Changes in local flexibility, including a disruption of the N-terminal helix at acidic pH, also accompany the binding of heparin to GMCSF. We use molecular dynamics simulations to propose a mechanism in which a positive binding pocket that is not fully solvent accessible at neutral pH becomes more accessible at acidic pH, facilitating the binding of heparin to the protein.

Fucosylated chondroitin sulfate (FCS) oligosaccharides extracted from sea cucumber and depolymerized exhibit potent anticoagulant activity. Knowledge of the antithrombotic activity of different size oligosaccharides and their fucose (Fuc) branch sulfation pattern should promote their development for clinical applications. We prepared highly purified FCS trisaccharide repeating units from hexasaccharide (6-mer) to octadecasaccharide (18-mer), including those with 2,4-disulfated and 3,4-disulfated Fuc branches. All 10 oligosaccharides were identified by their nuclear magnetic resonance structures and ESI-FTMS spectroscopy. In vitroanticoagulant activities and surface plasmon resonance binding tests indicated those of larger molecular sizes and 2,4-disulfated Fuc branches showed stronger anticoagulant effects with respect to anti-FXase activity, as well as stronger binding to FIXa among various clotting proteins. However, both types of FCS 9-mer to 18-mer exhibited molecular size-independent potent antithrombotic activity in vivoat the same dose. In addition, both types of the FCS 6-mer exhibited favorable antithrombotic activity in vivo, although they showed weak anticoagulant activity in vitro. Combining absorption and metabolism studies, we conclude that FCS 9–18 oligomers could remain in the circulation to interact with various clotting proteins to prevent thrombus formation, and appreciable quantities of these oligomers could be excreted through the kidneys. All FCS 9–18 oligomers also resulted in no bleeding, hypotension, or platelet aggregation risk during blood circulation. Thus, FCS 9–18 oligomers with 2,4-disulfated or 3,4-disulfated Fuc branches exhibit potent and safe antithrombotic activity needed for clinical applications.

Obesity is associated with gut microbiome dysbiosis. Our previous research has shown that highly branched rhamnogalacturonan type I (RG-I)-enriched pectin (WRP, 531.5 kDa, 70.44% RG-I, Rha/(Gal + Ara) = 20) and its oligosaccharide with less branched RG-I [DWRP, 12.1 kDa, 50.29% RG-I, Rha/(Gal + Ara) = 6] are potential prebiotics. The present study is conducted to uncover the impact of the content, molecular size, and branch degrees of RG-I on the inhibiting effect of high-fat diet (HFD)-induced obesity. The commercial pectin (CP, 496.2 kDa, 35.77% RG-I, Rha/(Gal + Ara) = 6), WRP, and DWRP were orally administered to HFD-fed C57BL/6J mice (100 mg kg–1d–1) to determine their individual effects on obesity. WRP significantly prevented bodyweight gain, insulin resistance, and inflammatory responses in HFD-fed mice. No obvious anti-obesity effect was observed in either CP or DWRP supplementation. A mechanistic study revealed that CP and DWRP could not enhance the diversity of gut microbiota, while WRP treatment positively modulated the gut microbiota of obese mice by increasing the abundance of Butyrivibrio, Roseburia, Barnesiella, Flavonifractor, Acetivibrio, and Clostridiumcluster IV. Furthermore, WRP significantly promoted browning of white adipose tissues in HFD-fed mice, while CP and DWRP did not. WRP can attenuate the HFD-induced obesity by modulation of gut microbiota and lipid metabolism. Highly branched RG-I domain enrichment is essential for pectin mitigating against the HFD-induced obesity.

Chondroitin sulfate E (CS-E) is a sulfated polysaccharide that contains repeating disaccharides of 4,6-disulfated N-acetylgalactosamine and glucuronic acid residues. Here, we report the enzymatic synthesis of three homogeneous CS-E oligosaccharides, including CS-E heptasaccharide (CS-E 7-mer), CS-E tridecasaccharide (CS-E13-mer), and CS-E nonadecasaccharide (CS-E 19-mer). The anti-inflammatory effect of CS-E 19-merwas investigated in this study. CS-E 19-merneutralizes the cytotoxic effect of histones in a cell-based assay and in mice. We also demonstrate that CS-E 19-mertreatment improves survival and protects against organ damage in a mouse model of endotoxemia induced by bacterial lipopolysaccharide (LPS). CS-E19-merdirectly interacts with circulating histones in the plasma from LPS-challenged mice. CS-E 19-merdoes not display anticoagulant activity nor react with heparin-induced thrombocytopenia antibodies isolated from patients. The successful synthesis of CS-E oligosaccharides provides structurally defined carbohydrates for advancing CS-E research and offers a potential therapeutic agent to treat life-threatening systemic inflammation.

Heparin is a widely used biotherapeutic produced from animal tissues. However, it might be possible to produce a bioengineered version using a multienzyme process, relying on the isolation of the E. coliK5 capsule heparosan and its chemical conversion to N-sulfoheparosan, NSH. Glucuronyl C5-epimerase, the first enzyme that acts on NSH, catalyzes the reversible conversion of glucuronic acid (GlcA) to iduronic acid (IdoA). Using full-length NSH, containing different amounts of N-acetylglucosamine (GlcNAc) residues, we demonstrate that C5-epimerase specificity relates to polysaccharide sequence, particularly the location of GlcNAc residues within the chain. We leveraged the deuterium exchange and the novel β-glucuronidase heparanase BP, which cleaves at the GlcA residue. Liquid chromatography–mass spectrometry and gel permeation chromatography of partial/complete heparanase BP digestion products from various NSH substrates treated with C5-epimerase provide information on C5-epimerase activity and action pattern. This study provides insight into optimizing the large-scale production of bioengineered heparin.

Electrospinning is a simple method for producing nanoscale or microscale fibers from a wide variety of materials. Intrinsically conductive polymers (ICPs), such as polyaniline (PANI), show higher conductivities with the use of secondary dopants like m-cresol. However, due to the low volatility of most secondary dopants, it has not been possible to electrospin secondary doped ICP fibers. In this work, the concept of secondary doping has been applied for the first time to electrospun fibers. Using a novel design for rotating drum electrospinning, fibers were efficiently and reliably produced from a mixture of low- and high-volatility solvents. The conductivity of electrospun PANI–poly(ethylene oxide) (PEO) fibers prepared was 1.73 S/cm, two orders of magnitude higher than the average value reported in the literature. These conductive fibers were tested as electrodes for supercapacitors and were shown to have a specific capacitance as high as 3121 F/g at 0.1 A/g, the highest value reported, thus far, for PANI–PEO electrospun fibers.