Vascular injury and coagulopathy are key drivers of mortality in bacterial sepsis. In Neisseria meningitidis infection, endothelial adhesion and thrombosis cause the characteristic petechial rash and, in the most severe cases, purpura fulminans. Although antibiotics rapidly kill bacteria, inflammation and vascular injury often persist or worsen after bacterial clearance, suggesting ongoing toxicity from released bacterial components. Here we identify bacterial histone-like proteins (HLPs), small positively charged DNA-binding proteins conserved across bacterial species, as previously unrecognized mediators of vascular damage. In vitro HLPs are released following antibiotic exposure, disrupting endothelial integrity. In patients with severe sepsis, they are detectable in plasma and tissue, colocalising with areas of vascular leak and coagulopathy. Non-anticoagulant heparins and anti-HLP antibodies neutralize HLP-induced endothelial disruption and toxicity in vitro and in vivo. These findings reveal HLPs as antibiotic-released bacterial toxins and suggest new therapeutic strategies to prevent vascular injury in sepsis.

Excessive foraging of colonic mucin glycans by gut bacteria is associated with diseases such as inflammatory bowel disease. Although Akkermansia muciniphila is an important mucin degrader, the role of carbohydrate sulfatases that facilitate digestion of these heavily sulfated glycans remains unclear. Combining in vitro digestion assays, proteomics and structural biology, we show that A. muciniphila sulfatases, such as Amuc1755 and Amuc0953, have rare adaptations targeted towards known sulfated mucin structures. They show larger degrees of modularity, including a previously unknown mucin-binding domain. When grown on colonic mucin substrates, glycoproteins of reduced size were important for the growth of A. muciniphila. Further mutational analysis and localization studies revealed that desulfation of N-acetyl-D-glucosamine was periplasmic, while desulfation of D-galactose occurred extracellularly and in the periplasm. These data improve our understanding of contexts for the positive health correlations of A. muciniphila while metabolizing colonic mucin as its sole carbon source.

Acidic glycans are essential for the biology of multicellular eukaryotes. To utilize them, microbial life including symbionts and pathogens has evolved polysaccharide lyases (PL) that cleave their 1,4 glycosidic linkages via a β-elimination mechanism. PL family 33 (PL33) enzymes have the unusual ability to target a diverse range of glycosaminoglycans (GAGs), as well as the bacterial polymer, gellan gum. In order to gain more detailed insight into PL33 activities we recombinantly expressed 10 PL33 members derived from all major environments and further elucidated the detailed biochemical and biophysical properties of five, showing that their substrate specificity is conferred by variations in tunnel length and topography. The key amino acids involved in catalysis and substrate interactions were identified, and employing a combination of complementary biochemical, structural, and modeling approaches, we show that the tunnel topography is induced by substrate binding to the glycan. Structural and bioinformatic analyses revealed that these features are conserved across several lyase families as well as in mammalian GAG epimerases.

The windowpane oyster (Placuna placenta) is common in coastal areas of the Philippines, thriving in brackish waters. Its shells underpin the local craft industries. While its meat is edible, only small amounts are consumed locally, most going to waste. Utilization of this potential nutrient source is hindered by the lack of information concerning its organic and mineral content, the possible presence of heavy metal ions, and the risk of microbial pathogens. We report extensive analysis of the meat from Placuna placenta, harvested during three different seasons to account for potential variations. This comprises proximate analysis, mineral, antioxidant, and microbial analyses. While considerable seasonal variation was observed, the windowpane oyster was found to be a rich source of protein, fats, minerals, and carbohydrates, comparing well with the meats of other shellfish and land animals. Following pre-cooking (~90 °C, 25–30 min), the standard local method for food preparation, no viable E. coli or Salmonella sp. were detected. Mineral content was broadly similar to that reported in fish, although iron, zinc, and copper were more highly represented, nevertheless, heavy metals were below internationally acceptable levels, with the exception of one of three samples, which was slightly above the only current standard, FSANZ. Whether the arsenic was in the safer organic form, which is commonly the case for shellfish, or the more toxic inorganic form remains to be established. This and the variation of arsenic over time will need to be considered when developing food products. Overall, the meat of the windowpane oyster is a valuable food resource and its current (albeit low-level) use should lower any barriers to its acceptance, making it suitable for commercialization. The present data support its development for high-value food products in urban markets.

Common methods for establishing the presence of enteric bacteria polluting water supplies, or in other samples, rely on detecting the hydrolysis of model glucuronide substrates by glucuronidases to release a phenolic product quantifiable by absorbance or fluorescence. Substrates include the β-D-glucuronides of p-nitrophenol, and umbelliferyl or quercetin derivatives. One limitation is that it may be difficult or impossible to quantify the released phenolic moiety in samples that are strongly coloured or, that contain fluorescent compounds. Exploiting the sensitivity available from the 19F nucleus to changes in chemical environment which can be detected by 19F NMR spectroscopy, and the almost complete absence of 19F from naturally-occurring samples containing organic matter, which provides background-free signals, we propose a model substrate; 4-fluorophenyl β-D-glucuronide (4FP-glucuronide). The 19F NMR chemical shift position of 4FP-glucuronide changes from -121.0 ppm upon hydrolysis to release 4-fluorophenol, at -124.9 ppm (at pH 6.8), enabling detection of β-glucuronidase activity. We illustrate the use of this substrate with environmental samples from forest soil, standing water, and mud from cattle pasture. Each of these would challenge conventional methods, owing to their opacity or the presence of coloured organic material. The technique enables detection of glucuronidases, a widely-used proxy for enteric bacteria, extending the scope of testing beyond water to include environmental and other challenging samples.

Gradients of FGFs are an essential feature of many developmental processes. Thus, FGF2 stimulates different responses at high and low concentrations, while FGF4 gradients are critical, e.g., in limb development., but it is not known if responses to FGF4 differ in a concentration-dependent manner. Employing rat mammary (Rama) 27 fibroblasts, we therefore measured the FGF4 concentration- and time-dependence of the simulation of phosphorylation of FRS2α, MAPK1 and MAPK3 which are downstream of the FGF receptor (FGFR1c). At 10 pg/mL FGF4 caused a very weak phosphorylation of FRS2α at Y435 and Y196 and a stronger one of MAPK1 and MAPK3 that oscillated with maximum levels after 30 min and 240 min stimulation. The phosphorylation of these proteins at 1 ng/mL FGF4 was considerably stronger and showed a similar oscillation. At 10 ng/mL FGF4, the phosphorylation of FRS2α reached an early peak after 5 min, declined at 15 min and then rose again at 30 min before declining to the end of the time-course at 240 min, whereas the phosphorylation of MAPK1 and MAPK3 was strong after 5 minutes, reached a maximum after 30 minutes and then declined gradually. At 1 µg/mL and 3 µg/mL FGF4 after 15 min and 60 min the phosphorylation of FRS2α, MAPK1 and MAPK3 was much lower at 60 min compared to that observed at lower concentrations of FGF4. The data indicate that at low concentrations FGF4 elicits an oscillatory response at the level of phosphorylation of FRS2α and of MAPK1 and MAPK3, whereas a bell-shaped dose response may occur at the highest concentrations of FGF4. The addition of exogenous heparin, an effective mimic of endogenous heparan sulfate, suggests that there may be an influence of the interaction of FGF4 with pericellular matrix and the dose-and time-dependence of the phosphorylation of FRS2α and MAPK1and MAPK3. ### Competing Interest Statement The authors have declared no competing interest. University of Tabuk, https://ror.org/04yej8x59 European Commission, https://ror.org/00k4n6c32, FET-OPEN ArrestAD 737390

Akkermansia muciniphila , an obligate mucin degrader, is a major member of the human colonic microbiota and has been associated positive health outcomes. Mucins are complex glycoproteins that contain heavily sulfated O -glycans and form the protective colonic mucus layer. Bacterial carbohydrate sulfatases are required to metabolise these heavily sulfated mucin glycans and excessive bacterial foraging has been associated with several diseases. Sulfatases have been linked with inflammatory bowel disease, making these microbiota enzymes potential drug targets. A. muciniphila expresses carbohydrate sulfatases that can act on colonic mucins yet their roles in its metabolism remain opaque. Our data reveal that A. muciniphila requires glycopeptides/protein forms of colonic mucin for metabolism and its sulfatases have unique adaptations compared to Bacteroides species. Localisation studies reveal that desulfation of N -acetyl-D-glucosamine, but not D-galactose, is exclusively periplasmic. A cell surface sulfatase has a novel carbohydrate binding module that binds to colonic mucin. This paints a contrasting picture of sulfated mucin metabolism by Akkermansia muciniphila versus Bacteroides species. These data will be important for understanding the contexts for Akkermansia muciniphila ’s positive health correlations.

Vesicle trafficking is pivotal in heparan sulfate (HS) biosynthesis, influencing its spatial and temporal regulation within distinct Golgi compartments. This regulation modulates the sulfation pattern of HS, which is crucial for governing various biological processes. Here, we investigate the effects of silencing Rab1A and Rab2A expression on the localisation of 3-O-sulfotransferase-5 (3OST5) within Golgi compartments and subsequent alterations in HS structure and levels. Interestingly, silencing Rab1A led to a shift in 3OST5 localization towards the trans-Golgi, resulting in increased HS levels within 24 and 48 h, while silencing Rab2A caused 3OST5 accumulation in the cis-Golgi, with a delayed rise in HS content observed after 48 h. Furthermore, a compensatory mechanism was evident in Rab2A-silenced cells, where increased Rab1A protein expression was detected. This suggests a dynamic interplay between Rab1A and Rab2A in maintaining the fine balance of vesicle trafficking processes involved in HS biosynthesis. Additionally, we demonstrate that the trafficking of 3OST5 in COPI vesicles is facilitated by GOLPH3 protein. These findings identify novel vesicular transport mechanisms regulating HS biosynthesis and reveal a compensatory relationship between Rab1A and Rab2A in maintaining baseline HS production.

Covering up to 2025.Plant-derived polyphenols of various chemical classes are widely distributed in dietary substances, e.g. fruits, nuts, vegetables and teas. Such phenolic derivatives are natural antioxidants and have been linked with numerous health benefits, notably anti-cancer and anti-inflammatory properties. Additionally, they may behave as mild estrogens, as in the case of genistein. However, there has often been no clear correlation between in vitro properties, as measured in cell lines for instance, and in vivo performance. Moreover, it is not always clear what the true active species might be, as most phenols are readily subject to phase II metabolism, generating predominantly glucuronides and sulfates. In this highlight, we seek to address the question of whether dietary substance metabolites, especially glucuronides, which have been more widely studied, do indeed possess distinct activities in their own right compared to their parent substances. In most cases this will refer to enzyme inhibition and/or interaction with cell lines. General observations concerning glucuronidation are provided, accompanied by practical comments concerning the synthesis of glucuronides, which are not always available or marketed in useful quantities. The main structural classes of natural polyphenols are introduced, with comments including synthetic details and biological properties for important members of each class.

Excessive degradation of the colonic mucin layer by Bacteroides within the human gut microbiota drives inflammatory bowel disease (IBD) in mice. Bacterial carbohydrate sulfatases are key enzymes in gut colonization, and they are elevated in human IBD and correlate with disease severity. Selective inhibitors of carbohydrate sulfatases could function as sulfatase-selective drugs, allowing precise control of sulfatase activity while preserving these otherwise beneficial bacteria. Arylsulfamates are covalent inhibitors that target a catalytic formylglycine residue of steroid sulfatases, a residue that is also conserved in carbohydrate sulfatases. Here, we find that a library of aryl- and carbohydrate sulfamates is ineffective against carbohydrate sulfatases, yet can inhibit human gut microbiota (HGM) species grown on sulfated glycans. Leveraging thermal proteome profiling (TPP), we identify a lipid kinase as the target responsible for these effects. This work highlights the imperative for developing specific inhibitors targeting carbohydrate sulfatases and reveals the adverse effects that arylsulfamates have on Bacteroides species of the HGM.

Heparin manufacturing generates significant volumes of by-products, which are typically treated as waste, and whose potential as a source of alternative bioactive materials is yet to be fully developed. Here, we employed a circular economy approach to support the repurposing of this waste stream for potential anti-inflammatory applications. Glycosaminoglycan fractions were extracted and purified from porcine-derived heparin by-products and structurally characterised using nuclear magnetic resonance and high performance size exclusion chromatography. The fractions exhibited distinct saccharide compositions, sulfation patterns, and molecular weight profiles. Bioactivity assays demonstrated that selected fractions attenuated LPS-induced NF-κB activation in RAW-Blue™ cells and enhanced IL-10 production ex vivo, indicating immunomodulatory potential. Heparin by-products have negligible anticoagulant activity, which supports their safe use in non-anticoagulant biomedical applications. These findings illustrate the potential of heparin by-products as therapeutic agents, while contributing to sustainable pharmaceutical manufacturing.

The heparan sulfate (HS)-rich extracellular matrix (ECM) serves as an initial interaction site for the homotrimeric spike (S) protein of SARS-CoV-2 to facilitate subsequent docking to angiotensin-converting enzyme 2 (ACE2) receptors and cellular infection. More recent variants, notably Omicron, have evolved by swapping several amino acids to positively charged residues to enhance the interaction of the S-protein trimer with the negatively charged HS. However, these enhanced interactions may reduce Omicron’s ability to move through the HS-rich ECM to effectively find ACE2 receptors and infect cells, raising the question of how to mechanistically explain HS-associated viral movement. In this work, we show that Omicron S proteins have evolved to balance HS interaction stability and dynamics, resulting in enhanced mobility on an HS-functionalized artificial matrix. This property is achieved by the ability of Omicron S-proteins to cross-link at least two HS chains, allowing direct S-protein switching between chains as a prerequisite for cell surface mobility. Optimized HS interactions can be targeted pharmaceutically, as an HS mimetic significantly suppressed surface binding and cellular infection specifically of the Omicron variant. These findings suggest a robust way to interfere with SARS-CoV-2 Omicron infection and potentially future variants.

Heparan sulfate (HS) is a glycosaminoglycan, polysaccharides that are considered to have arisen in the last common unicellular ancestor of multicellular animals. In this light, the large interactome of HS and its myriad functions in relation to the regulation of cell communication are not surprising. The binding of proteins to HS determines their localisation and diffusion, essential for embryonic development and homeostasis. Following the biosynthesis of the initial heparosan polymer, the subsequent modifications comprise an established canonical pathway and a minor pathway. The more frequent former starts with N-deacetylation and N-sulfation of GlcNAc residues, the latter with C-5 epimerisation of a GlcA residue adjacent to a GlcNAc. The binding of proteins to HS is driven by ionic interactions. The multivalent effect arising from the many individual ionic bonds between a single protein and a polysaccharide chain results in a far stronger interaction than would be expected from an ion-exchange process. In many instances, upon binding, both parties undergo substantial conformational change, the resulting hydrogen and van der Waal bonds contributing significant free energy to the binding reaction. Nevertheless, ionic bonds dominate the protein-polysaccharide interaction kinetically. Together with the multivalent effect, this provides an explanation for the observed trapping of HS-binding proteins in extracellular matrix. Importantly, individual ionic bonds have been observed to be dynamic; breaking and reforming, while the protein remains bound to the polysaccharide. These considerations lead to a model for 1D diffusion of proteins in extracellular matrix on HS, involving mechanisms such as sliding, chain switching and rolling.

IgA nephropathy (IgAN) is the most common type of glomerulonephritis that frequently progresses to kidney failure. However, the molecular pathogenesis underlying IgAN remains largely unknown. Here, we investigated the role of galectin-3 (Gal-3), a galactoside-binding protein in IgAN pathogenesis, and showed that Gal-3 expression by the kidney was significantly enhanced in patients with IgAN. In both TEPC-15 hybridoma-derived IgA-induced, passive, and spontaneous “grouped” ddY IgAN models, Gal-3 expression was clearly increased with disease severity in the glomeruli, peri-glomerular regions, and some kidney tubules. Gal-3 knockout (KO) in the passive IgAN model had significantly improved proteinuria, kidney function and reduced severity of kidney pathology, including neutrophil infiltration and decreased differentiation of Th17 cells from kidney-draining lymph nodes, despite increased percentages of regulatory T cells. Gal-3 KO also inhibited the NLRP3 inflammasome, yet it enhanced autophagy and improved kidney inflammation and fibrosis. Moreover, administration of 6-de-O-sulfated, N-acetylated low-molecular-weight heparin, a competitive Gal-3 binding inhibitor, restored kidney function and improved kidney lesions in passive IgAN mice. Thus, our results suggest that Gal-3 is critically involved in IgAN pathogenesis by activating the NLRP3 inflammasome and promoting Th17 cell differentiation. Hence, targeting Gal-3 action may represent a new therapeutic strategy for treatment of this kidney disease.

The monoglucuronides of resveratrol were prepared from resveratrol triacetate. They showed contrasting properties in aqueous solution: facile E / Z isomerisation for the 3- O -isomer and ready H/D exchange, without isomerisation, for the 4′- O -isomer.

The vast structural diversity of sulfated polysaccharides demands an equally diverse array of enzymes known as polysaccharide sulfotransferases (PSTs). PSTs are present across all kingdoms of life, including algae, fungi and archaea, and their sulfation pathways are relatively unexplored. Sulfated polysaccharides possess anti-inflammatory, anticoagulant and anti-cancer properties and have great therapeutic potential. Current identification of PSTs using Pfam has been predominantly focused on the identification of glycosaminoglycan (GAG) sulfotransferases because of their pivotal roles in cell communication, extracellular matrix formation and coagulation. As a result, our knowledge of non -GAG PSTs structure and function remains limited. The major sulfotransferase families, Sulfotransfer 1 and Sulfotransfer 2 , display broad homology and should enable the capture of a wide assortment of sulfotransferases but are limited in non -GAG PST sequence annotation. In addition, sequence annotation is further restricted by the paucity of biochemical analyses of PSTs. There are now high-throughput and robust assays for sulfotransferases such as colorimetric PAPS (3 ' -phosphoadenosine 5 ' -phosphosulfate) coupled assays, Europium-based fluorescent probes for ratiometric PAP (3 ' -phosphoadenosine-5 ' -phosphate) detection, and NMR methods for activity and product analysis. These techniques provide real-time and direct measurements to enhance the functional annotation and subsequent analysis of sulfated polysaccharides across the tree of life to improve putative PST identification and characterisation of function. Improved annotation and biochemical analysis of PST sequences will enhance the utility of PSTs across biomedical and biotechnological sectors.

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

The S1 family of sulfatases is the sole family of enzymes that desulfate carbohydrates, linking them to inflammatory bowel diseases and various cancers. There is an unmet need for therapeutics against carbohydrate sulfatases, and while effective, orally available inhibitors of S1 steroid sulfatases exist, their efficacy against S1 carbohydrate sulfatases remains largely unexplored. In this study we assess a library of aryl- and carbohydrate sulfamates/sulfonates for their ability to inhibit carbohydrate sulfatases from human colonic bacteria. Surprisingly, these compounds prove ineffective. We show that arylsulfamate inhibitors inhibit the growth of anti-cancer human gut microbiota species independent of sulfatase activity. Leveraging thermal proteome profiling, we identify the non-sulfatase targets responsible for these effects. This work highlights the imperative for developing specific inhibitors targeting carbohydrate sulfatases and unveils the adverse effects of arylsulfamates, a class of drugs designed for hormone cancer treatment, on the human gut microbiota, potentially influencing their therapeutic efficacy. ### Competing Interest Statement The authors have declared no competing interest.

Hyaluronic acid (HA) is a natural and biocompatible polysaccharide that is able to interact with CD44 receptors to regulate inflammation, fibrosis, and tissue reconstruction. It is a suitable chemical scaffold for drug delivery that can be functionalized with pharmacophores and/or vectorizable groups. The derivatization of HA is achieved to varying extents by reacting 1-amino-1-deoxy-lactitol via the carboxyl group to form amide linkages, giving rise to the grafted polymer, HYLACH. This retains the broad properties of HA, even though, as in most HA-grafted polymers, the detailed conformational effects of such substitutions, while crucial in the design or optimization of drug delivery systems, remain unknown. Here, the conformation, size, secondary structure, hydrogen bond network, and hydration features of lactosylated HA derivatives were evaluated by using multiple independent molecular dynamics simulations. This revealed subtle but nevertheless significant changes in the HA scaffold, establishing the density of grafting as the key parameter determining its properties.