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Lipase activity from Aspergillus fumigatus (AFL) were modulated by an effector (HMD) that was discovered for an allosteric site on the bioactive macromolecule. Experimental evaluation and computational modeling of allosteric effects revealed alterations in the structure of AFL. It was found that AFL's activity in HMD solution increased by approximately 46 % due to mainly enhanced lid mobility. HMD-AFL interaction was driven by enthalpy and entropy. However, when AFL was coupled to HMD-modified microspheres (PS-HMD-p), its hydrolysis activity decreased by ~14.3 % due to reduced lid mobility. After immobilization, AFL's ester-synthesis activity also decreased, due to changes in the conformational dynamics and the geometric characteristics of active site. Investigating the structural dynamics of allosteric regulation of the lipase not only reveals its structural changes underlying the functional variation but also enhances the understanding of the allosteric property that is underappreciated in exoenzymes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Candidalysin, a cytolytic peptide produced by the fungal pathogen Candida albicans, is a key virulence factor. However, its host cell targets remain elusive. Here we performed a genome-wide loss-of-function CRISPR screen in the TR146 human oral epithelial cell line and identified that disruption of genes (XYLT2, B3GALT6 and B3GAT3) in glycosaminoglycan (GAG) biosynthesis conferred resistance to damage induced by candidalysin and live C. albicans. Surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin binds to sulfated GAGs, facilitating its enrichment on the host cell surface. Adding exogenous sulfated GAGs or the analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate also inhibited C. albicans invasion and fungal-induced epithelial cell cytokine production. In mice with vulvovaginal candidiasis, topical dextran sulfate administration reduced intravaginal tissue damage and inflammation. Collectively, sulfated GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The biological activities of heparan sulfate (HS) are intimately related to their molecular weights, degree and pattern of sulfation and homogeneity. The existing methods for synthesizing homogeneous sugar chains of low dispersity involve multiple steps and require stepwise isolation and purification processes. Here, we designed a mesoporous metal-organic capsule for the encapsulation of glycosyltransferase and obtained a microreactor capable of enzymatically catalyzing polymerization reactions to prepare homogeneous heparosan of low dispersity, the precursor of HS and heparin. Since the sugar chain extension occurs in the pores of the microreactor, low molecular weight heparosan can be synthesized through space-restricted catalysis. Moreover, the glycosylation co-product, uridine diphosphate (UDP), can be chelated with the exposed metal sites of the metal-organic capsule, which inhibits trans-cleavage to reduce the molecular weight dispersity. This microreactor offers the advantages of efficiency, reusability, and obviates the need for stepwise isolation and purification processes. Using the synthesized heparosan, we further successfully prepared homogeneous 6-O-sulfated HS of low dispersity with a molecular weight of approximately 6 kDa and a polydispersity index (PDI) of 1.032. Notably, the HS generated exhibited minimal anticoagulant activity, and its binding affinity to fibroblast growth factor 1 was comparable to that of low molecular weight heparins., Competing Interests: Declaration of competing interest We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Low Molecular Weight Heparins (LMWHs) are well-established for use in the prevention and treatment of thrombotic diseases, and as a substitute for unfractionated heparin (UFH) due to their predictable pharmacokinetics and subcutaneous bioavailability. LMWHs are produced by various depolymerization methods from UFH, resulting in heterogeneous compounds with similar biochemical and pharmacological properties. However, the delicate supply chain of UFH and potential contamination from animal sources require new manufacturing approaches for LMWHs. Various LMWH preparation methods are emerging, such as chemical synthesis, enzymatic or chemical depolymerization and chemoenzymatic synthesis. To establish the sameness of active ingredients in both innovator and generic LMWH products, the Food and Drug Administration has implemented a stringent scientific method of equivalence based on physicochemical properties, heparin source material and depolymerization techniques, disaccharide composition and oligosaccharide mapping, biological and biochemical properties, and in vivo pharmacodynamic profiles. In this review, we discuss currently available LMWHs, potential manufacturing methods, and recent progress for manufacturing quality control of these LMWHs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hepatitis B virus (HBV) infection remains a major health threat with limited treatment options. One of various new antiviral strategies is based on a fusion of Staphylococcus aureus nuclease (SN) with the capsid-forming HBV core protein (HBc), termed coreSN. Through co-assembly with wild-type HBc-subunits, the fusion protein is incorporated into HBV nucleocapsids, targeting the nuclease to the encapsidated viral genome. However, coreSN expression was based on transfection of a plasmid vector. Here, we explored whether introducing protein transduction domains (PTDs) into a fluorescent coreSN model could confer cell-penetrating properties for direct protein delivery into cells. Four PTDs were inserted into two different positions of the HBc sequence, comprising the amphiphilic translocation motif (TLM) derived from the HBV surface protein PreS2 domain and three basic PTDs derived from the Tat protein of human immunodeficiency virus-1 (HIV-1), namely Tat4, NP, and NS. To directly monitor the interaction with cells, the SN in coreSN was replaced with the green fluorescent protein (GFP). The fusion proteins were expressed in E. coli , and binding to and potential uptake by human cells was examined through flow cytometry and fluorescence microscopy. The data indicate PTD-dependent interactions with the cells, with evidence of uptake in particular for the basic PTDs. Uptake was enhanced by a triplicated Simian virus 40 (SV40) large T antigen nuclear localization signal (NLS). Interestingly, the basic C terminal domain of the HBV core protein was found to function as a novel PTD. Hence, further developing cell-permeable viral capsid protein fusions appears worthwhile.

Major developmental events occurring in the hippocampus during the third trimester of human gestation and neonatally in altricial rodents include rapid and synchronized dendritic arborization and astrocyte proliferation and maturation. We tested the hypothesis that signals sent by developing astrocytes to developing neurons modulate dendritic development in vivo . We altered neuronal development by neonatal (third trimester-equivalent) ethanol exposure in mice; this treatment increased dendritic arborization in hippocampal pyramidal neurons. We next assessed concurrent changes in the mouse astrocyte translatome by translating ribosomal affinity purification (TRAP)-seq. We followed up on ethanol-inhibition of astrocyte Chpf2 and Chsy1 gene translation because these genes encode for biosynthetic enzymes of chondroitin sulfate glycosaminoglycan (CS-GAG) chains (extracellular matrix components that inhibit neuronal development and plasticity) and have not been explored before for their roles in dendritic arborization. We report that Chpf2 and Chsy1 are enriched in astrocytes and their translation is inhibited by ethanol, which also reduces the levels of CS-GAGs measured by Liquid Chromatography/Mass Spectrometry. Finally, astrocyte-conditioned medium derived from Chfp2 -silenced astrocytes increased neurite branching of hippocampal neurons in vitro . These results demonstrate that CS-GAG biosynthetic enzymes in astrocytes regulates dendritic arborization in developing neurons., Competing Interests: Disclosure All the authors reported no biomedical financial interests or potential conflicts of interest.

Seaweeds represent a rich source of sulfated polysaccharides with similarity to heparan sulfate, a facilitator of myriad virus host cell attachment. For this reason, attention has been drawn to their antiviral activity, including the potential for anti-SARS-CoV-2 activity. We have identified and structurally characterized several fucoidan extracts, including those from different species of brown macroalga, and a rhamnan sulfate from a green macroalga species. A high molecular weight fucoidan extracted from Saccharina japonica (FSjRPI-27), and a rhamnan sulfate extracted from Monostroma nitidum (RSMn), showed potent competitive inhibition of spike glycoprotein receptor binding to a heparin-coated SPR chip. This inhibition was also observed in cell-based assays using hACE2 HEK-293 T cells infected by pseudotyped SARS-CoV-2 virus with IC 50 values <1 μg/mL. Effectiveness was demonstrated in vivo using hACE2-transgenic mice. Intranasal administration of FSjRPI-27 showed protection when dosed 6 h prior to and at infection, and then every 2 days post-infection, with 100 % survival and no toxicity at 10 4 plaque-forming units per mouse vs. buffer control. At 5-fold higher virus dose, FSjRPI-27 reduced mortality and yielded reduced viral titers in bronchioalveolar fluid and lung homogenates vs. buffer control. These findings suggest the potential application of seaweed-based sulfated polysaccharides as promising anti-SARS-CoV-2 prophylactics., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jonathan S. Dordick reports financial support was provided by Rensselaer Polytechnic Institute. Robert Linhardt reports financial support was provided by Rensselaer Polytechnic Institute. Amit Singh reports financial support was provided by Albany Medical College. Dennis Metzger reports financial support was provided by Albany Medical College. Jonathan S. Dordick reports a relationship with Lavaage, Inc. that includes: equity or stocks. Jonathan S. Dordick has patent #Compositions incorporating sulfated polysaccharides for inhibiting SARS-CoV-2 pending to Rensselaer Polytechnic Institute. Robert J. Linhardt has patent #Compositions incorporating sulfated polysaccharides for inhibiting SARS-CoV-2 pending to Rensselaer Polytechnic Institute. Weihua Jin has patent #Compositions incorporating sulfated polysaccharides for inhibiting SARS-CoV-2 pending to Rensselaer Polytechnic Institute. Fuming Zhang has patent #Compositions incorporating sulfated polysaccharides for inhibiting SARS-CoV-2 pending to Rensselaer Polytechnic Institute. Jonathan S. Dordick has patent #SYSTEMS AND METHODS UTILIZING IN VIVO EFFECTIVE SULFATED GLYCANS FOR COVID-19 PREVENTION AND TREATMENT pending to Rensselaer Polytechnic Institute. Amit Singh has patent #SYSTEMS AND METHODS UTILIZING IN VIVO EFFECTIVE SULFATED GLYCANS FOR COVID-19 PREVENTION AND TREATMENT pending to Albany Medical College. Dennis Metzger has patent #SYSTEMS AND METHODS UTILIZING IN VIVO EFFECTIVE SULFATED GLYCANS FOR COVID-19 PREVENTION AND TREATMENT pending to Albany Medical College. Jonathan S. Dordick is cofounder of Lavaage, Inc., which may license the disclosed patents from Rensselaer Polytechnic Institute. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Candidalysin is a cytolytic peptide produced by the opportunistic fungal pathogen Candida albicans. This peptide is a key virulence factor in mouse models of mucosal and hematogenously disseminated candidiasis. Despite intense interest in the role of candidalysin in C. albicans pathogenicity, its host cell targets have remained elusive. To fill this knowledge gap, we performed a genome-wide loss-of-function CRISPR screen in a human oral epithelial cell line to identify specific host factors required for susceptibility to candidalysin-induced cellular damage. Among the top hits were XYLT2 , B3GALT6 and B3GAT3 , genes that function in glycosaminoglycan (GAG) biosynthesis. Deletion of these genes led to the absence of GAGs such as heparan sulfate on the epithelial cell surface and increased resistance to damage induced by both candidalysin and live C. albicans. Biophysical analyses including surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin physically binds to sulfated GAGs, facilitating its oligomerization or enrichment on the host cell surface. The addition of exogenous sulfated GAGs or the GAG analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate, but not non-sulfated dextran, also inhibited epithelial cell endocytosis of C. albicans and fungal-induced epithelial cell cytokine and chemokine production. In a murine model of vulvovaginal candidiasis, topical dextran sulfate administration reduced host tissue damage and decreased intravaginal IL-1β and neutrophil levels. Collectively, these data indicate that GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage.

Mycoplasma pneumoniae , a notable pathogen behind respiratory infections, employs specialized proteins to adhere to the respiratory epithelium, an essential process for initiating infection. The role of glycosaminoglycans, especially heparan sulfate, is critical in facilitating pathogen-host interactions, presenting a strategic target for therapeutic intervention. In this study, we assembled a glycan library comprising heparin, its oligosaccharide derivatives, and a variety of marine-derived sulfated glycans to screen the potential inhibitors for the pathogen-host interactions. By using Surface Plasmon Resonance spectroscopy, we evaluated the library's efficacy in inhibiting the interaction between M. pneumoniae adhesion proteins and heparin. Our findings offer a promising avenue for developing novel therapeutic strategies against M. pneumoniae infections.

Endothelial dysfunction induced by oxidative stress is an early predictor of atherosclerosis, which can cause various cardiovascular diseases. The glycocalyx layer on the endothelial cell surface acts as a barrier to maintain endothelial biological function, and it can be impaired by oxidative stress. However, the mechanism of glycocalyx damage during the development of atherosclerosis remains largely unclear. Herein, we established a novel strategy to address these issues from the glycomic perspective that has long been neglected. Using countercharged fluorescence protein staining and quantitative mass spectrometry, we found that heparan sulfate, a major component of the glycocalyx, was structurally altered by oxidative stress. Comparative proteomics and protein microarray analysis revealed several new heparan sulfate-binding proteins, among which alpha-2-Heremans-Schmid glycoprotein (AHSG) was identified as a critical protein. The molecular mechanism of AHSG with heparin was characterized through several methods. A heparan analog could relieve atherosclerosis by protecting heparan sulfate from degradation during oxidative stress and by reducing the accumulation of AHSG at lesion sites. In the present study, the molecular mechanism of anti-atherosclerotic effect of heparin through interaction with AHSG was revealed. These findings provide new insights into understanding of glycocalyx damage in atherosclerosis and lead to the development of corresponding therapeutics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Heparins have been invaluable therapeutic anticoagulant polysaccharides for over a century, whether used as unfractionated heparin or as low molecular weight heparin (LMWH) derivatives. However, heparin production by extraction from animal tissues presents multiple challenges, including the risk of adulteration, contamination, prion and viral impurities, limited supply, insecure supply chain, and significant batch-to-batch variability. The use of animal-derived heparin also raises ethical and religious concerns, as well as carries the risk of transmitting zoonotic diseases. Chemoenzymatic synthesis of animal-free heparin products would offer several advantages, including reliable and scalable production processes, improved purity and consistency, and the ability to produce heparin polysaccharides with molecular weight, structural, and functional properties equivalent to those of the United States Pharmacopeia (USP) heparin, currently only sourced from porcine intestinal mucosa. We report a scalable process for the production of bioengineered heparin that is biologically and compositionally similar to USP heparin. This process relies on enzymes from the heparin biosynthetic pathway, immobilized on an inert support and requires a tailored N -sulfoheparosan with N -sulfo levels similar to those of porcine heparins. We also report the conversion of our bioengineered heparin into a LMWH that is biologically and compositionally similar to USP enoxaparin. Ultimately, we demonstrate major advances to a process to provide a potential clinical and sustainable alternative to porcine-derived heparin products., Competing Interests: Competing interests statement:The following authors are inventors on U.S. Patent 11,591,628, which has been licensed commercially and may benefit from royalties: M.D., E.E.P., L.F., N.G., P.R.B., T.J.S., P.D., A.O., M.H., D.M., R.J.L., and J.S.D.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide COVID-19 pandemic, leading to 6.8 million deaths. Numerous variants have emerged since its outbreak, resulting in its significantly enhanced ability to spread among humans. As with many other viruses, SARS‑CoV‑2 utilizes heparan sulfate (HS) glycosaminoglycan (GAG) on the surface of host cells to facilitate viral attachment and initiate cellular entry through the ACE2 receptor. Therefore, interfering with virion-HS interactions represents a promising target to develop broad-spectrum antiviral therapeutics. Sulfated glycans derived from marine organisms have been proven to be exceptional reservoirs of naturally existing HS mimetics, which exhibit remarkable therapeutic properties encompassing antiviral/microbial, antitumor, anticoagulant, and anti-inflammatory activities. In the current study, the interactions between the receptor-binding domain (RBD) of S-protein of SARS-CoV-2 (both WT and XBB.1.5 variants) and heparin were applied to assess the inhibitory activity of 10 marine-sourced glycans including three sulfated fucans, three fucosylated chondroitin sulfates and two fucoidans derived from sea cucumbers, sea urchin and seaweed Saccharina japonica, respectively. The inhibitory activity of these marine derived sulfated glycans on the interactions between RBD of S-protein and heparin was evaluated using Surface Plasmon Resonance (SPR). The RBDs of S-proteins from both Omicrion XBB.1.5 and wild-type (WT) were found to bind to heparin, which is a highly sulfated form of HS. All the tested marine-sourced sulfated glycans exhibited strong inhibition of WT and XBB.1.5 S-protein binding to heparin. We believe the study on the molecular interactions between S-proteins and host cell glycosaminoglycans provides valuable insight for the development of marine-sourced, glycan-based inhibitors as potential anti-SARS-CoV-2 agents., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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 ( hepa rin strip ), allowing heparin monitoring in clinical samples within 20 min. Finally, in vivo investigations 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.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus with high contagion and mortality rates. Heparan sulfate proteoglycans (HSPGs) are ubiquitously expressed on the surface of mammalian cells. Owing to its high negatively charged property, heparan sulfate (HS) on the surface of host cells is used by many viruses as cofactor to facilitate viral attachment and initiate cellular entry. Therefore, inhibition of the interaction between viruses and HS could be a promising target to inhibit viral infection. In the current study, the interaction between the receptor-binding domain (RBD) of MERS-CoV and heparin was exploited to assess the inhibitory activity of various sulfated glycans such as glycosaminoglycans, marine-sourced glycans (sulfated fucans, fucosylated chondroitin sulfates, fucoidans, and rhamnan sulfate), pentosan polysulfate, and mucopolysaccharide using Surface Plasmon Resonance. We believe this study provides valuable insights for the development of sulfated glycan-based inhibitors as potential antiviral agents.

Traumatic brain injury (TBI) is the leading cause of death and disability due to injury worldwide. Extracellular matrix (ECM) remodeling is known to significantly contribute to TBI pathophysiology. Glycosaminoglycans, which are long-chain, variably sulfated polysaccharides abundant within the ECM, have previously been shown to be substantially altered after TBI. In this study, we sought to delineate the dynamics of glycosaminoglycan alterations after TBI and discover the precise biologic processes responsible for observed glycosaminoglycan changes after injury. We performed state-of-the art mass spectrometry on brain tissues isolated from mice after TBI or craniotomy-alone. We observed dynamic changes in glycosaminoglycans at Day 1 and 7 post-TBI, with heparan sulfate, chondroitin sulfate, and hyaluronan remaining significantly increased after a week vis-à-vis craniotomy-alone tissues. We did not observe appreciable changes in circulating glycosaminoglycans in mice after experimental TBI compared to craniotomy-alone nor in patients with TBI and severe polytrauma compared to control patients with mild injuries, suggesting increases in injury site glycosaminoglycans are driven by local synthesis. We subsequently performed an unbiased whole genome transcriptomics analysis on mouse brain tissues 7 days post-TBI and discovered a significant induction of hyaluronan synthase 2, glypican-3, and decorin. The functional role of decorin after injury was further examined through multimodal behavioral testing comparing wild-type and Dcn -/- mice. We discovered that genetic ablation of Dcn led to an overall negative effect of TBI on function, exacerbating motor impairments after TBI. Collectively, our results provide a spatiotemporal characterization of post-TBI glycosaminoglycan alterations in the brain ECM and support an important adaptive role for decorin upregulation after TBI., Competing Interests: Declarations of Competing Interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

β-Glucosidase is a biological macromolecule that catalyzes the hydrolysis of various glycosides and oligosaccharides. It may also be used to catalyze the synthesis of glycosides under suitable conditions. Carrier-bound β-glucosidase can enhance the enzymatic activity in the synthesis of glycosides in organic solvent solutions, although the molecular mechanism regulating activity is yet unknown. This study investigated the impact of utilizing montmorillonite (Mmt), attapulgite (Attp), and kaolinite (Kao) as carriers on the activity of β-glucosidase from Prunus dulcis (PdBg). When Attp was used as carriers, the molecular dynamic (MD) simulations found the distance between pNPG and the active site residues E183 and E387 was minimally impacted by the adsorptions, hence PdBg maintained about 81.3 ± 0.89 % of its native activity. Out of the three clay minerals, the relative activity of PdBg loaded on Mmt was the lowest because of the highest electrostatic energy. The substrate channel of PdBg on Kao is directed towards the surface, limiting the accessibility of substrates. Secondary structure and conformation studies revealed that the conformational stability of PdBg in solvent solutions was enhanced by coupling to Attp. Unlike dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and 1,2-dimethoxyethane (DME), tert-butanol (t-BA) did not penetrate into the active site of PdBg interfering with its binding to the substrate. The maximum yield of n-octyl-β-glucoside (OGP) synthesis catalyzed by Attp-immobilized PdBg reached 48.3 %., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Heparin-induced thrombocytopenia (HIT) is a serious complication caused by heparin drugs. The ultralarge complexes formed by platelet factor 4 (PF4) with heparin or low molecular weight heparins (LMWHs) are important participants in inducing the immune response and HIT., Objectives: We aim at characterizing the interaction between PF4 and long-chain heparin oligosaccharides and providing robust analytical methods for the analysis of PF4-heparin complexes., Methods: In this work, the characteristics of PF4-enoxaparin complexes after incubation in different molar ratios and concentrations were analyzed by multiple analytical methods, especially liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry with multiple reaction monitoring were developed to qualitatively and quantitatively monitor heparin oligosaccharides and PF4 in HIT-inducing complexes., Results: The results showed that the largest proportion of ultralarge complexes formed by PF4 and enoxaparin was at a specific molar ratio, ie, a PF4/enoxaparin ratio of 2:1, while the ultralarge complexes contained PF4 tetramer and enoxaparin at a molar ratio of approximately 2:1., Conclusion: A binding model of PF4 and enoxaparin in ultralarge complexes is proposed with one heparin oligosaccharide chain (∼ dp18) bound to 2 PF4 tetramers in different morphologies to form ultralarge complexes, while PF4 tetramer is surrounded by multiple heparin chains in smaller complexes. Our study provides new insights into the structural mechanism of PF4-LMWH interaction, which help to further understand the mechanism of LMWH immunogenicity and develop safer heparin products., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Decellularized lung scaffolds and hydrogels are increasingly being utilized in ex vivo lung bioengineering. However, the lung is a regionally heterogenous organ with proximal and distal airway and vascular compartments of different structures and functions that may be altered as part of disease pathogenesis. We previously described decellularized normal whole human lung extracellular matrix (ECM) glycosaminoglycan (GAG) composition and functional ability to bind matrix-associated growth factors. We now determine differential GAG composition and function in airway, vascular, and alveolar-enriched regions of decellularized lungs obtained from normal, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) patients. Significant differences were observed in heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA) content and CS/HS compositions between both different lung regions and between normal and diseased lungs. Surface plasmon resonance demonstrated that HS and CS from decellularized normal and COPD lungs similarly bound fibroblast growth factor 2, but that binding was decreased in decellularized IPF lungs. Binding of transforming growth factor β to CS was similar in all three groups but binding to HS was decreased in IPF compared to normal and COPD lungs. In addition, cytokines dissociate faster from the IPF GAGs than their counterparts. The differences in cytokine binding features of IPF GAGs may result from different disaccharide compositions. The purified HS from IPF lung is less sulfated than that from other lungs, and the CS from IPF contains more 6-O-sulfated disaccharide. These observations provide further information for understanding functional roles of ECM GAGs in lung function and disease. STATEMENT OF SIGNIFICANCE: Lung transplantation remains limited due to donor organ availability and need for life-long immunosuppressive medication. One solution, the ex vivo bioengineering of lungs via de- and recellularization has not yet led to a fully functional organ. Notably, the role of glycosaminoglycans (GAGs) remaining in decellularized lung scaffolds is poorly understood despite their important effects on cell behaviors. We have previously investigated residual GAG content of native and decellularized lungs and their respective functionality, and role during scaffold recellularization. We now present a detailed characterization of GAG and GAG chain content and function in different anatomical regions of normal diseased human lungs. These are novel and important observations that further expand knowledge about functional GAG roles in lung biology and disease., Competing Interests: Declaration of Competing Interest This manuscript has not been published and is not under consideration for publication elsewhere. We have no conflicts of interest to disclose., (Copyright © 2023. Published by Elsevier Ltd.)

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) and influenza viruses have spread around the world at an unprecedented rate. Despite multiple vaccines, new variants of SARS-CoV-2 and influenza have caused a remarkable level of pathogenesis. The development of effective antiviral drugs to treat SARS-CoV-2 and influenza remains a high priority. Inhibiting viral cell surface attachment represents an early and efficient means to block virus infection. Sialyl glycoconjugates, on the surface of human cell membranes, play an important role as host cell receptors for influenza A virus and 9-O-acetyl-sialylated glycoconjugates are receptors for MERS, HKU1 and bovine coronaviruses. We designed and synthesized multivalent 6'-sialyllactose-counjugated polyamidoamine dendrimers through click chemistry at room temperature concisely. These dendrimer derivatives have good solubility and stability in aqueous solutions. SPR, a real-time analysis quantitative method for of biomolecular interactions, was used to study the binding affinities of our dendrimer derivatives by utilizing only 200 micrograms of each dendrimer. Three SARS-CoV-2 S-protein receptor binding domain (wild type and two Omicron mutants) bound to multivalent 9-O-acetyl-6'-sialyllactose-counjugated and 6'-sialyllactose-counjugated dendrimers bound to a single H3N2 influenza A virus's HA protein (A/Hong Kong/1/1968), the SPR study results suggest their potential anti-viral activities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The structural and functional relationships of glycosaminoglycans (GAGs) derived from marine organisms have been investigated, suggesting that marine invertebrates, particularly Bivalvia, are abundant sources of highly sulfated or branched GAGs. In this study, we identified a novel fucosylated heparan sulfate (Fuc-HS) from the midgut gland of the Japanese scallop, Patinopecten yessoensis. Scallop HS showed resistance to GAG-degrading enzymes, including chondroitinases and heparinases, and susceptibility to heparinases increased when scallop HS was treated with mild acid hydrolysis, which removes the fucosyl group. Moreover, 1 H NMR detected significant signals near 1.2-1.3 ppm corresponding to the H-6 methyl proton of fucose residues and small H-3 (3.59 ppm) or H-2 (3.39 ppm) signals of glucuronate (GlcA) were detected, suggesting that the fucose moiety is attached to the C-3 position of GlcA in scallop HS. GC-MS detected peaks corresponding to 1, 3, 5-tri-O-acetyl-2, 4-di-O-methyl- L -fucitol and 1, 4, 5-tri-O-acetyl-2, 3-di-O-methyl- L -fucitol, suggesting that the fucose moiety is 3-O- or 4-O-sulfated. Furthermore, scallop HS showed anti-coagulant and neurite outgrowth-promoting (NOP) activities. These results suggest that the midgut gland of scallops is a valuable source of Fuc-HS with biological activities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Ganoderenic acid D (GAD), extracted from the Chinese herb Ganoderma lucidum, was loaded onto a graphene oxide-polyethylene glycol-anti-epidermal growth factor receptor (GO-PEG-EGFR) carrier to develop a targeting antitumor nanocomposite (GO-PEG@GAD). The carrier was fabricated from PEG and anti-EGFR aptamer modified GO. Targeting was mediated by the grafted anti-EGFR aptamer, which targets the membrane of HeLa cells. Physicochemical properties were characterized by transmission electron microscopy, dynamic light scattering, X-ray powder diffraction, and Fourier transform infrared spectroscopy. High loading content (77.3 % ± 1.08 %) and encapsulation efficiency (89.1 % ± 2.11 %) were achieved. Drug release continued for approximately 100 h. The targeting effect both in vitro and in vivo was confirmed by confocal laser scanning microscopy (CLSM) and imaging analysis system. The mass of the subcutaneous implanted tumor was significantly decreased by 27.27 ± 1.23 % after treatment with GO-PEG@GAD compared with the negative control group. Moreover, the in vivo anti-cervical carcinoma activity of this medicine was due to activation of the intrinsic mitochondrial pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Heparin is a commonly used anticoagulant drug, derived from the tissues of animals including pigs, cows, and sheep. Measuring heparin concentration in plasma is challenging due to its complex molecular structure. Existing methods rely on measuring heparin's anticoagulant activity, which provides pharmacodynamic (PD) data but not pharmacokinetic (PK) data, measuring concentration over time. To overcome this limitation, we used liquid chromatography-mass spectrometry (LC-MS) and the multiple reaction monitoring (MRM) method to directly measure heparin's concentration in non-human primates after administering porcine, bovine, and ovine heparin. A protocol was developed to enable an MRM method for application to small plasma volumes without purification. The PK data obtained from LC-MS are then compared with the data obtained using the Heparin Red assay and the PD data determined using biochemical clinical assays. Results showed that LC-MS and Heparin Red assay measurements closely correlated with unfractionated heparin's biological activities, supporting the use of mass spectra and dye-binding assays to determine heparin levels in plasma. This study builds a way for the measurement of heparin concentration in plasma, which could lead to an improved understanding of heparin's metabolism and dosing safety., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

In this work, we isolated two new sulfated glycans from the body wall of the sea cucumber Thyonella gemmata : one fucosylated chondroitin sulfate (TgFucCS) (17.5 ± 3.5% kDa) and one sulfated fucan (TgSF) (383.3 ± 2.1% kDa). NMR results showed the TgFucCS backbone composed of [→3)-β- N -acetylgalactosamine-(1→4)-β-glucuronic acid-(1→] with 70% 4-sulfated and 30% 4,6-disulfated GalNAc units and one-third of the GlcA units decorated at the C3 position with branching α-fucose (Fuc) units either 4-sulfated (65%) or 2,4-disulfated (35%) and the TgSF structure composed of a tetrasaccharide repeating unit of [→3)-α-Fuc2,4S-(1→2)-α-Fuc4S-(1→3)-α-Fuc2S-(1→3)-α-Fuc2S-(1→] n . Inhibitory properties of TgFucCS and TgSF were investigated using SARS-CoV-2 pseudovirus coated with S-proteins of the wild-type (Wuhan-Hu-1) or the delta (B.1.617.2) strains and in four different anticoagulant assays, comparatively with unfractionated heparin. Molecular binding to coagulation (co)-factors and S-proteins was investigated by competitive surface plasmon resonance spectroscopy. Among the two sulfated glycans tested, TgSF showed significant anti-SARS-CoV-2 activity against both strains together with low anticoagulant properties, indicating a good candidate for future studies in drug development.

Protein synthesis in Corynebacterium glutamicum is critical for applications in biotechnology and medicine. However, the use of C. glutamicum for protein production is limited by its low expression and aggregation. To overcome these limitations, a molecular chaperone plasmid system was developed in this study to improve the efficiency of recombinant protein synthesis in C. glutamicum. The effect of molecular chaperones on target protein synthesis (Single-chain variable fragment, Scfv) under three different promoter strengths was tested. In addition, the plasmid containing the molecular chaperone and target protein was verified for growth stability and plasmid stability. This expression model was further validated using two recombinant proteins, human interferon-beta (Hifn) and hirudin variant III (Rhv3). Finally, the Rhv3 protein was purified, and analysis of Rhv3 activity confirmed that the use of a molecular chaperone led to an improvement in test protein synthesis. Thus, the use of molecular chaperones is believed to will improve recombinant proteins synthesis in C. glutamicum.

Electrical stimulation is a powerful strategy to improve the differentiation of neural stem cells into neurons. Such an approach can be implemented, in association with biomaterials and nanotechnology, for the development of new therapies for neurological diseases, including direct cell transplantation and the development of platforms for drug screening and disease progression evaluation. Poly(aniline):camphorsulfonic acid (PANI:CSA) is one of the most well-studied electroconductive polymers, capable of directing an externally applied electrical field to neural cells in culture. There are several examples in the literature on the development of PANI:CSA-based scaffolds and platforms for electrical stimulation, but no review has examined the fundamentals and physico-chemical determinants of PANI:CSA for the design of platforms for electrical stimulation. This review evaluates the current literature regarding the application of electrical stimulation to neural cells, specifically reviewing: (1) the fundamentals of bioelectricity and electrical stimulation; (2) the use of PANI:CSA-based systems for electrical stimulation of cell cultures; and (3) the development of scaffolds and setups to support the electrical stimulation of cells. Throughout this work, we critically evaluate the revised literature and provide a steppingstone for the clinical application of the electrical stimulation of cells using electroconductive PANI:CSA platforms/scaffolds.

Apolipoprotein E (ApoE)'s ϵ4 alle is the most important genetic risk factor for late onset Alzheimer's Disease (AD). Cell-surface heparan sulfate (HS) is a cofactor for ApoE/LRP1 interaction and the prion-like spread of tau pathology between cells. 3-O-sulfo (3-O-S) modification of HS has been linked to AD through its interaction with tau, and enhanced levels of 3-O-sulfated HS and 3-O-sulfotransferases in the AD brain. In this study, we characterized ApoE/HS interactions in wildtype ApoE3, AD-linked ApoE4, and AD-protective ApoE2 and ApoE3-Christchurch. Glycan microarray and SPR assays revealed that all ApoE isoforms recognized 3-O-S. NMR titration localized ApoE/3-O-S binding to the vicinity of the canonical HS binding motif. In cells, the knockout of HS3ST1-a major 3-O sulfotransferase-reduced cell surface binding and uptake of ApoE. 3-O-S is thus recognized by both tau and ApoE, suggesting that the interplay between 3-O-sulfated HS, tau and ApoE isoforms may modulate AD risk., (© 2023 Wiley-VCH GmbH.)

The structure of the sulfated galactan from the red alga Botryocladia occidentalis (BoSG) was originally proposed as a simple repeating disaccharide of alternating 4-linked α-galactopyranose (Galp) and 3-linked β-Galp units with variable sulfation pattern. Abundance was estimated only for the α-Galp units: one-third of 2,3-disulfation and one-third of 2-monosulfation. Here, we isolated again the same BoSG fractions from the anion-exchange chromatography, obtaining the same NMR profile of the first report. More careful NMR analysis led us to revise the structure. A more complex sulfation pattern was noted along with the occurrence of 4-linked α-3,6-anhydro-Galp (AnGalp) units. Interestingly, the more sulfated BoSG fraction showed slightly reduced in vitro anti-SARS-CoV-2 activities against both wild-type and delta variants, and significantly reduced anticoagulant activity. The BoSG fractions showed no cytotoxic effects. The reduction in both bioactivities is attributed to the presence of the AnGalp unit. Docking scores from computational simulations using BoSG disaccharide constructs on wild-type and delta S-proteins, and binding analysis through competitive SPR assays using blood (co)-factors (antithrombin, heparin cofactor II and thrombin) and four S-proteins (wild-type, delta, gamma, and omicron) strongly support the conclusion about the deleterious impact of the AnGalp unit., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design, writing, or decision of this manuscript., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Appearance of senescent beta cells in the pancreas leads to the onset of type 2 diabetes (T2D). The structural analysis of a sulfated fuco-manno-glucuronogalactan (SFGG) indicated SFGG had the backbones of interspersing 1, 3-linked β-D-GlcpA residues, 1, 4-linked α-D-Galp residues, and alternating 1, 2-linked α-D-Manp residues and 1, 4-linked β-D-GlcpA residues, sulfated at C6 of Man residues, C2/C3/C4 of Fuc residues and C3/C6 of Gal residues, and branched at C3 of Man residues. SFGG effectively alleviated senescence-related phenotypes in vitro and in vivo, including cell cycle, senescence-associated β-galactosidase, DNA damage and senescence-associated secretory phenotype (SASP) -associated cytokines and hall markers of senescence. SFGG also alleviated beta cell dysfunction in insulin synthesis and glucose-stimulated insulin secretion. Mechanistically, SFGG attenuated senescence and improved beta cell function via PI3K/AKT/FoxO1 signaling pathway. Therefore, SFGG could be used for beta cell senescence treatment and alleviation of the progression of T2D., Competing Interests: Declaration of competing interest We have no financial interests/personal relationships with other people or organizations that can inappropriately influence our work, and no other conflict of interest exists., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

Heparan sulfate (HS) is a heterogeneous, cell-surface polysaccharide critical for transducing signals essential for mammalian development. Imaging of signaling proteins has revealed how their localization influences their information transfer. In contrast, the contribution of the spatial distribution and nanostructure of information-rich, signaling polysaccharides like HS is not known. Using expansion microscopy (ExM), we found striking changes in HS nanostructure occur as human pluripotent stem (hPS) cells differentiate, and these changes correlate with growth factor signaling. Our imaging studies show that undifferentiated hPS cells are densely coated with HS displayed as hair-like protrusions. This ultrastructure can recruit fibroblast growth factor for signaling. When the hPS cells differentiate into the ectoderm lineage, HS is localized into dispersed puncta. This striking change in HS distribution coincides with a decrease in fibroblast growth factor binding to neural cells. While developmental variations in HS sequence were thought to be the primary driver of alterations in HS-mediated growth factor signaling, our high-resolution images indicate a role for the HS nanostructure. Our study highlights the utility of high-resolution glycan imaging using ExM. In the case of HS, we found that changes in how the polysaccharide is displayed link to profound differences in growth factor binding.

Sulfated glycans from marine organisms are excellent sources of naturally occurring glycosaminoglycan (GAG) mimetics that demonstrate therapeutic activities, such as antiviral/microbial infection, anticoagulant, anticancer, and anti-inflammation activities. Many viruses use the heparan sulfate (HS) GAG on the surface of host cells as co-receptors for attachment and initiating cell entry. Therefore, virion-HS interactions have been targeted to develop broad-spectrum antiviral therapeutics. Here we report the potential anti-monkeypox virus (MPXV) activities of eight defined marine sulfated glycans, three fucosylated chondroitin sulfates, and three sulfated fucans extracted from the sea cucumber species Isostichopus badionotus , Holothuria floridana , and Pentacta pygmaea , and the sea urchin Lytechinus variegatus , as well as two chemically desulfated derivatives. The inhibitions of these marine sulfated glycans on MPXV A29 and A35 protein-heparin interactions were evaluated using surface plasmon resonance (SPR). These results demonstrated that the viral surface proteins of MPXV A29 and A35 bound to heparin, which is a highly sulfated HS, and sulfated glycans from sea cucumbers showed strong inhibition of MPXV A29 and A35 interactions. The study of molecular interactions between viral proteins and host cell GAGs is important in developing therapeutics for the prevention and treatment of MPXV.

Introduction: The unexpected surge of respiratory syncytial virus (RSV) cases following pandemic phase of COVID-19 has drawn much public attention. Drawing on the latest antiviral research, revisiting this heightened annual outbreak of respiratory disease could lead to new treatments. The ability of sulfated polysaccharides to compete for a variety of viruses binding to cell surface heparan sulfate, suggests several drugs that might have therapeutic potential for targeting RSV-glycosaminoglycan interactions. Methods: In the current study, the binding affinity and kinetics of two RSV glycoproteins (RSV-G protein and RSV-F protein) to heparin were investigated by surface plasmon resonance. Furthermore, solution competition studies using heparin oligosaccharides of different lengths indicated that the binding of RSV-G protein to heparin is size-dependent, whereas RSV-F protein did not show any chain length preference. Results and discussion: The two RSV glycoproteins have slightly different preferences for heparin sulfation patterns, but the N -sulfo group in heparin was most critical for the binding of heparin to both RSV-G protein and RSV-F protein. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for their inhibition of the RSV-G protein and RSV-F protein-heparin interaction, and both highly negative compounds showed strong inhibition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shi, He, Song, Linhardt, Dordick, Chi and Zhang.)

Nanotechniques for curcumin (Cur) encapsulation provided a potential capability to avoid limitations and improve biological activities in food and pharmaceutics. Different from multi-step encapsulation systems, in this study, zein-curcumin (Z-Cur) core-shell nanoparticles could be self-assembled within Eudragit S100 (ES100) fibers through one-pot coaxial electrospinning with Cur at an encapsulation efficiency (EE) of 96% for ES100-zein-Cur (ES100-Z-Cur) and EE of 67% for self-assembled Z-Cur. The resulting structure realized the double protection of Cur by ES100 and zein, which provided both pH responsiveness and sustained release performances. The self-assembled Z-Cur nanoparticles released from fibermats were spherical (diameter 328 nm) and had a relatively uniform distribution (polydispersity index 0.62). The spherical structures of Z-Cur nanoparticles and Z-Cur nanoparticles loaded in ES100 fibermats could be observed by transmission electron microscopy (TEM). Fourier transform infrared spectra (FTIR) and X-ray diffractometer (XRD) revealed that hydrophobic interactions occurred between the encapsulated Cur and zein, while Cur was amorphous (rather than in crystalline form). Loading in the fibermat could significantly enhance the photothermal stability of Cur. This novel one-pot system much more easily and efficiently combined nanoparticles and fibers together, offering inherent advantages such as step economy, operational simplicity, and synthetic efficiency. These core-shell biopolymer fibermats which incorporate Cur can be applied in pharmaceutical products toward the goals of sustainable and controllable intestine-targeted drug delivery.

SARS-CoV-2 receptor binding domains (RBDs) interact with both the ACE2 receptor and heparan sulfate on the surface of host cells to enhance SARS-CoV-2 infection. We show that suramin, a polysulfated synthetic drug, binds to the ACE2 receptor and heparan sulfate binding sites on the RBDs of wild-type, Delta, and Omicron variants. Specifically, heparan sulfate and suramin had enhanced preferential binding for Omicron RBD, and suramin is most potent against the live SARS-CoV-2 Omicron variant (B.1.1.529) when compared to wild type and Delta (B.1.617.2) variants in vitro. These results suggest that inhibition of live virus infection occurs through dual SARS-CoV-2 targets of S-protein binding and previously reported RNA-dependent RNA polymerase inhibition and offers the possibility for this and other polysulfated molecules to be used as potential therapeutic and prophylactic options against COVID-19., (© 2023. The Author(s).)

In this study we investigated the effects of a neonatal handling protocol that mimics the handling of sham control pups in protocols of neonatal exposure to brain insults on dendritic arborization and glycosaminoglycan (GAG) levels in the developing brain. GAGs are long, unbranched polysaccharides, consisting of repeating disaccharide units that can be modified by sulfation at specific sites and are involved in modulating neuronal plasticity during brain development. In this study, male and female Sprague-Dawley rats underwent neonatal handling daily between post-natal day (PD)4 and PD9, with brains analyzed on PD9. Neuronal morphology and morphometric analysis of the apical and basal dendritic trees of CA1 hippocampal pyramidal neurons were carried out by Golgi-Cox staining followed by neuron tracing and analysis with the software Neurolucida. Chondroitin sulfate (CS)-, Hyaluronic Acid (HA)-, and Heparan Sulfate (HS)-GAG disaccharide levels were quantified in the hippocampus by Liquid Chromatography/Mass Spectrometry analyses. We found sex by neonatal handling interactions on several parameters of CA1 pyramidal neuron morphology and in the levels of HS-GAGs, with females, but not males, showing an increase in both dendritic arborization and HS-GAG levels. We also observed increased expression of glucocorticoid receptor gene Nr3c1 in the hippocampus of both males and females following neonatal handling suggesting that both sexes experienced a similar stress during the handling procedure. This is the first study to show sex differences in two parameters of brain plasticity, CA1 neuron morphology and HS-GAG levels, following handling stress in neonatal rats., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Sepsis is a lethal syndrome manifested by an unregulated, overwhelming inflammation from the host in response to infection. Here, we exploit the use of a synthetic heparan sulfate octadecasaccharide (18-mer) to protect against sepsis. The 18-mer not only inhibits the pro-inflammatory activity of extracellular histone H3 and high mobility group box 1 (HMGB1), but also elicits the anti-inflammatory effect from apolipoprotein A-I (ApoA-I). We demonstrate that the 18-mer protects against sepsis-related injury and improves survival in cecal ligation and puncture mice and reduces inflammation in an endotoxemia mouse model. The 18-mer neutralizes the cytotoxic histone-3 (H3) through direct interaction with the protein. Furthermore, the 18-mer enlists the actions of ApoA-I to dissociate the complex of HMGB1 and lipopolysaccharide, a toxic complex contributing to cell death and tissue damage in sepsis. Our study provides strong evidence that the 18-mer mitigates inflammatory damage in sepsis by targeting numerous mediators, setting it apart from other potential therapies with a single target.

Fucosylated chondroitin sulfate (FucCS) is a unique marine glycosaminoglycan that exhibits diverse biological functions, including antiviral and anticoagulant activity. In previous work, the FucCS derived from Pentacta pygmaea (PpFucCS) showed moderate anticoagulant effect but high inhibitory activity against the Wuhan strain of severe acute respiratory syndrome coronavirus (SARS-CoV-2). In this study, we perform free-radical depolymerization of PpFucCS by the copper-based Fenton method to generate low molecular weight (MW) oligosaccharides. PpFucCS oligosaccharides were structurally analyzed by 1H nuclear magnetic resonance spectroscopy and were used to conduct structure-activity relationship studies regarding their effects against SARS-CoV-2 and clotting. Anticoagulant properties were measured by activated partial thromboplastin time, protease (factors Xa and IIa) inhibition by serine protease inhibitors (antithrombin [AT] and heparin cofactor II [HCII]), and competitive surface plasmon resonance (SPR) assay using AT, HCII, and IIa. Anti-SARS-CoV-2 properties were measured by the concentration-response inhibitory curves of HEK-293T-human angiotensin-converting enzyme-2 cells infected with a baculovirus pseudotyped SARS-CoV-2 Delta variant spike (S)-protein and competitive SPR assays using multiple S-proteins (Wuhan, N501Y [Alpha], K417T/E484K/N501Y [Gamma], L542R [Delta], and Omicron [BA.2 subvariant]). Cytotoxicity of native PpFucCS and oligosaccharides was also assessed. The PpFucCS-derived oligosaccharide fraction of the highest MW showed great anti-SARS-CoV-2 Delta activity and reduced anticoagulant properties. Results have indicated no cytotoxicity and MW dependency on both anti-SARS-CoV-2 and anticoagulant effects of PpFucCS, as both actions were reduced accordingly to the MW decrease of PpFucCS. Our results demonstrate that the high-MW structures of PpFucCS is a key structural element to achieve the maximal anti-SARS-CoV-2 and anticoagulant effects., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tau spreading in Alzheimer's disease is mediated by cell surface heparan sulfate (HS). As a class of sulfated polysaccharides, fucoidans might compete with HS to bind tau, resulting in the cessation of tau spreading. The structural determinants of fucoidans for competition with HS binding to tau are not well understood. Sixty previously prepared fucoidans/glycans with different structural determinants were used to determine their binding abilities to tau using SPR and AlphaLISA. Finally, it was found that fucoidans had two fractions (sulfated galactofucan (SJ-I) and sulfated heteropolysaccharide (SJ-GX-3)), which exhibited strong binding abilities than heparin. Tau cellular uptake assays using wild type mouse lung endothelial cell lines were performed. It was shown SJ-I and SJ-GX-3 inhibited tau-cell interaction and tau cellular uptake, suggesting that fucoidans might be good candidates for inhibiting tau spreading. NMR titration mapped fucoidans binding sites, which could provide the theoretical basis for the design of tau spreading inhibitors., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2022. Published by Elsevier Ltd.)

In December 2019, the global coronavirus disease 2019 (COVID-19) pandemic began in Wuhan, China. COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which infects host cells primarily through the angiotensin-converting enzyme 2 (ACE2) receptor. In addition to ACE2, several studies have shown the importance of heparan sulfate (HS) on the host cell surface as a co-receptor for SARS-CoV-2-binding. This insight has driven research into antiviral therapies, aimed at inhibiting the HS co-receptor-binding, e.g., by glycosaminoglycans (GAGs), a family of sulfated polysaccharides that includes HS. Several GAGs, such as heparin (a highly sulfated analog of HS), are used to treat various health indications, including COVID-19. This review is focused on current research on the involvement of HS in SARS-CoV-2 infection, implications of viral mutations, as well as the use of GAGs and other sulfated polysaccharides as antiviral agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Complex carbohydrates (glycans) are major players in all organisms due to their structural, energy, and communication roles. This last essential role involves interacting and/or signaling through a plethora of glycan-binding proteins. The design and synthesis of glycans as potential drug candidates that selectively alter or perturb metabolic processes is challenging. Here we describe the first reported sulfur-linked polysaccharides with potentially altered conformational state(s) that are recalcitrant to digestion by heparanase, an enzyme important in human health and disease. An artificial sugar donor with a sulfhydryl functionality is synthesized and enzymatically incorporated into polysaccharide chains utilizing heparosan synthase. Used alone, this donor adds a single thio-sugar onto the termini of nascent chains. Surprisingly, in chain co-polymerization reactions with a second donor, this thiol-terminated heparosan also serves as an acceptor to form an unnatural thio-glycosidic bond ('S-link') between sugar residues in place of a natural 'O-linked' bond. S-linked heparan sulfate analogs are not cleaved by human heparanase. Furthermore, the analogs act as competitive inhibitors with > ~200-fold higher potency than expected; as a rationale, molecular dynamic simulations suggest that the S-link polymer conformations mimic aspects of the transition state. Our analogs form the basis for future cancer therapeutics and modulators of protein/sugar interactions., (© 2022. The Author(s).)

Objective: While lipid-lowering drugs have become a mainstay of clinical therapy these treatments only slow the progression of the disease and can have side effects. Thus, new treatment options are needed to supplement the effects of lipid lowering therapy for treating atherosclerosis. We examined the use of an inexpensive and widely available marine polysaccharide rhamnan sulfate as an oral therapeutic for limiting vascular inflammation and atherosclerosis., Methods and Results: We found rhamnan sulfate enhanced the barrier function of endothelial cells, preventing the deposition of LDL and maintaining barrier function even in the presence of glycocalyx-degrading enzymes. Rhamnan sulfate was also found to bind directly to FGF-2, PDGF-BB and NF-κB subunits with high affinity. In addition, rhamnan sulfate was a potent inhibitor of NF-κB pathway activation in endothelial cells by TNF-α. We treated ApoE -/- mice with a high fat diet for 4 weeks and then an addition 9 weeks of high fat diet with or without rhamnan sulfate. Rhamnan sulfate reduced vascular inflammation and atherosclerosis in both sexes of ApoE -/- mice but had a stronger therapeutic effect in female mice. Oral consumption of rhamnan sulfate induced a significant decrease in cholesterol plasma levels in female mice but not in male mice. In addition, there was a marked reduction in inflammation for female mice in the liver and aortic root in comparison to male mice., Conclusions: Rhamnan sulfate has beneficial effects in reducing inflammation, binding growth factors and NF-κB, enhancing endothelial barrier function and reducing atherosclerotic plaque formation in ApoE -/- mice., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Aaron Baker reports financial support was provided by National Institutes of Health. Aaron Baker reports financial support was provided by US Office of Congressionally Directed Medical Research Programs. Aaron Baker reports financial support was provided by American Heart Association. Robert Linhardt reports financial support was provided by National Institutes of Health., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The now prevalent Omicron variant and its subvariants/sub-lineages have led to a significant increase in COVID-19 cases and raised serious concerns about increased risk of infectivity, immune evasion, and reinfection. Heparan sulfate (HS), located on the surface of host cells, plays an important role as a co-receptor for virus-host cell interaction. The ability of heparin and HS to compete for binding of the SARS-CoV-2 spike (S) protein to cell surface HS illustrates the therapeutic potential of agents targeting protein-glycan interactions. In the current study, phylogenetic tree of variants and mutations in S protein receptor-binding domain (RBD) of Omicron BA.2.12.1, BA.4 and BA.5 were described. The binding affinity of Omicron S protein RBD to heparin was further investigated by surface plasmon resonance (SPR). Solution competition studies on the inhibitory activity of heparin oligosaccharides and desulfated heparins at different sites on S protein RBD-heparin interactions revealed that different sub-lineages tend to bind heparin with different chain lengths and sulfation patterns. Furthermore, blind docking experiments showed the contribution of basic amino acid residues in RBD and sulfo groups and carboxyl groups on heparin to the interaction. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for inhibition on the interaction of heparin and S protein RBD of Omicron BA.2.12.1, BA.4/BA.5, and both showed much stronger inhibition than heparin.