Oral joint supplements (OJSs), specifically those containing glucosamine and chondroitin sulfate, are some of the most popular feed additives fed by horse owners. However, evidence of the efficacy of these nutraceuticals in vivo is lacking. To investigate the impacts of an OJS on equine lameness, 40 geldings (18 ± 7 yr) with chronic lameness and AAEP lameness grades between 2 and 4 (as scored by a veterinary sports medicine and rehabilitation specialist) were enrolled in the study. Horses were stratified by lameness grade, BCS, and weight, and then randomly assigned to receive a placebo powder or an OJS daily for 6 wks. Stride length measurements and lameness evaluations were performed every 2 wk. There was no effect of treatment or treatment-by-day interaction for stride length, though day affected both walk (p = 0.04) and trot (p < 0.01). Only day affected lameness grade, with the lowest average score on d28 (p = 0.04). There was no evidence of supplementation improving gait symmetry over time. As such, there is no evidence to support efficacy of the tested OJS when fed for 6 wk. These results emphasize the importance of well-controlled studies and support continued development of evidence-based management strategies for equine joint health.

This study evaluated the effects of in ovo feeding with glycosaminoglycans and vitamins, combined with amphotericin and gentamicin, on incubation traits, intestinal morphometry, organ development, and cadherin-1 gene relative abundance in broilers. Fertile Ross 308 breeder eggs were randomly assigned to the following five treatments: uninjected eggs; eggs injected with a solution containing amphotericin, gentamicin, ascorbic acid, and pyridoxine; the same solution supplemented with chondroitin sulfate; glucosamine sulfate; or both glycosaminoglycans. Hatching rate, embryo mortality, and total mortality were not affected by treatments (p > 0.05). Broilers from the solution-based treatment showed higher relative heart weight at 1 day of age (p < 0.05). Intestinal morphometric parameters increased with age in both the jejunum and ileum. In ovo feeding modulated villus height, crypt depth, and villus height-crypt depth ratio, particularly in the ileum during early posthatch development. The relative abundance of the cadherin-1 (CDH1) gene was influenced by the interaction between in ovo feeding and age, with higher values observed in broilers receiving glycosaminoglycan-supplemented solutions at specific posthatch periods. In conclusion, in ovo feeding with glycosaminoglycans and vitamins modulated early intestinal development and relative heart weight without impairing hatchability in broilers.

Fully functional neural competence and integrity requires a complex array of communication means among neurons, with extracellular vesicles (EVs) emerging as a relevant mechanism for cell-cell interaction in the CNS. Despite the growing number of studies demonstrating the presence of microRNAs (miRNAs) in axon and EVs, the molecular mechanisms of those miRNAs present in EVs and their functional role in nervous system development has not been fully explored. In this study, we investigated whether neuronal EVs can have a role in neuron-to-neuron communication during the development of neuron connectivity in mouse primary cortical neuron cultures. Our results demonstrate how miR-99a can regulate axonal growth via its EV-mediated delivery and through the targeting of HS3ST2, a heparan sulphate glucosamine 3-O-sulphotransferase, which is predominantly expressed in the brain and generates rare 3-O-sulphated domains in heparan sulphate proteoglycans, with growing importance in development and neurodegenerative mechanisms. Importantly, we show how in compartmentalised microfluidic cultures, where axons are isolated from neuronal somas, the growth-promoting effects of neuron-derived EVs are local to the axon. These findings establish that neuronal EVs can deliver miRNAs to discrete subcellular domains to acutely modulate local gene expression, thereby driving axonal growth and shaping neurodevelopment.

Graphene quantum dots (GQDs) belong to the carbon quantum dot family, with low toxicity, high biocompatibility, and excellent colloidal stability. Mesoporous silica nanoparticles (MSNs) are recognized as effective drug delivery systems due to their high drug-loading capacity, while glucosamine sulfate (GS) exhibits immunemodulatory properties. In this study, to maximize the immune effect of GS, a graphene quantum dot-coated mesoporous silica loaded with glucosamine sulfate (MSN-G-Q) is synthesized, and its immunoactivation effects are evaluated in vitro and in vivo. In vitro, MSN-G-Q enhances macrophage uptake activity, stimulates nitric oxide (NO) production, and upregulates CD80[+] and CD86[+] expression. In vivo, MSN-G-Q/OVA increases immune organ index, facilitates dendritic cell (DC) maturation, promotes T lymphocyte proliferation and differentiation, elevates antigen-specific IgG antibodies and cytokines, including IFN-γ, IL-4, and IL-1β, and prolongs ovalbumin (OVA) residence in immune organs and lymph nodes. The results show that MSN-G-Q/OVA is a promising immunostimulatory antigen delivery system.

Osteoarthritis (OA) is a progressive degenerative joint disease characterized by cartilage deterioration, leading to chronic pain and impaired immobility particularly in older dogs.

A series of sulfated oligosaccharides (one tetra- and ten disaccharides) have been synthesized to study their interactions with midkine, a heparin-binding growth factor involved in cancer and inflammation. These compounds were prepared as glycosaminoglycan (GAG) mimetics and displayed hydrophobic groups at specific positions to enhance midkine binding. For the synthesis of the tetrasaccharide, a fluoro-assisted strategy was adopted. The use of an N-phenyltrifluoroacetimidate donor, instead of the analogous trichloroacetimidate, proved to be crucial in obtaining the desired tetramer with good yield. On the other hand, the synthesized disaccharides differed in the number of sulfates present and in the substituent at position 2 of the glucosamine unit. Fluorescence polarization competition experiments provided relative binding affinities for each glycomimetic expressed as IC50 values. Our results indicated that all the mimetics interacted with midkine in the micromolar range, highlighting the affinity of the 4,6-di-O-sulfated disaccharides with aromatic rings on the 2-amide group (IC50's from 3.4 ± 0.8 to 4.3 ± 1.1 μM). Overall, this study offers valuable data for the design and synthesis of high-affinity midkine ligands with potential biological applications.

Glycosylation changes are pivotal in colorectal cancer (CRC) development. The role of bisecting GlcNAc, a specific N-glycosylation type catalyzed by glycosyltransferase MGAT3, in CRC progression remains elusive. Previous studies indicated that dietary interventions can be beneficial for patients with certain congenital disorders of glycosylation. However, the impact of dietary fatty acids, such as palmitic acid (PA), on glycosylation regulation remains largely unclear. Here, we observed markedly decreased levels of bisecting GlcNAc and MGAT3 in colonic tissues of CRC patients. Downregulation of bisecting GlcNAc in CRC cells increased cell proliferation, migration, and invasion, while decreasing apoptosis. Moreover, a PA-rich diet inhibited CRC carcinogenesis in azoxymethane/dextran sodium sulfate-induced CRC mice by elevating bisecting GlcNAc levels. However, in Mgat3fl/fl Villin-Cre mice the inhibitory effects of the PA-rich diet were abolished. Intact glycopeptide analysis revealed that PA enhanced the bisecting GlcNAc modification on desmoglein 2 (DSG2). Additionally, DSG2 was identified to inhibit CRC carcinogenesis through the EGFR/AKT signaling pathway. In conclusion, dietary PA suppresses CRC carcinogenesis by regulating bisecting GlcNAc modification on DSG2, providing a direct mechanistic link between dietary fatty acids and CRC.

Polyethylene terephthalate microplastics (PET-MPs), a major microplastics component identified in human vasculature, pose emerging environmental health risks. This study systemically profiled MPs in human aortic tissues and investigated the mechanisms underlying PET-MPs-induced aortic injury in vivo and in vitro. Chronic oral exposure of Sprague-Dawley rats to PET-MPs resulted in endothelial glycocalyx loss and structural impairment of aortic elastic fibers. Transcriptomic and proteomic analyses elucidated that PET-MPs triggered endoplasmic reticulum stress and reactive oxygen species generation, initiating glycocalyx loss and inflammatory activation. This response further pinpointed interleukin-1β (IL-1β) as a pivotal mediator inducing smooth muscle cell phenotypic switching. Crucially, restoration of the glycocalyx using sulodexide mitigated endothelial dysfunction and downstream smooth muscle cells phenotypic switching. These findings establish endothelial glycocalyx degradation via endoplasmic reticulum stress-reactive oxygen species as a novel mechanism for PET-MPs-induced vascular injury and highlight glycocalyx protection as a potential strategy against environmental microplastic hazards.

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of Acetyl Glucosamine, Glucosamine, Glucosamine HCl, and Glucosamine Sulfate. Two of the ingredients, Acetyl Glucosamine and Glucosamine Sulfate, are reported to function in cosmetics as skin-conditioning agents, Glucosamine HCl is reported to function as a pH adjustor, and the function of Glucosamine in cosmetics is not reported. The Panel reviewed the available data and concluded that these glucosamine ingredients are safe in cosmetics in the present practices of use and concentration described in this safety assessment when formulated to be non-irritating.

Safe, potent, and low-bleeding anticoagulants remain an unmet medical need. Here we report a homogeneous glycosaminoglycan (GAG), designated TPG, isolated from the brittlestar Trichaster palmiferus. TPG (30.3 kDa, 32% sulfate) is composed of an almost equimolar ratio of L-Iduronic acid (IdoA) and N-acetyl-D-galactosamine (GalNAc) together with minor D-glucosamine (GlcN), D-galacturonic acid (GalA), and D-galactose (Gal). These physicochemical properties are distinct from heparin, dermatan sulfate, and chondroitin sulfate. Using one- and two-dimensional NMR of its desulfated derivative, free-radical depolymerized fragments, and oligosaccharide fractions generated by partial N-deacetylation-deamination cleavage, we established that the polymer backbone is built from four repeating disaccharides: L-IdoA2S3S-α-1,3-D-GalNAc4S/6S-β-1,4-, L-IdoA2S-α-1,3-D-GalNAc4S/6S-β-1,4-, D-GalA-α-1,3-D-GalNAc4S-β-1,4-, and D-GalNAc4S-β-1,4-GlcN0S/6S-α-1,3-. The IdoA2S3S and GalA residues and the D-GalNAc4S-β-1,4-GlcN0S/6S-α-1,3- unit are rare in natural GAGs, rendering TPG resistant to chondroitinase ABC. NMR detected terminal Gal in desulfated TPG and large oligosaccharide fragments, but its connection pattern remains unclear. TPG prolongs clotting times by simultaneously blocking intrinsic factor Xase and potentiating HCII-mediated thrombin inhibition, and it achieves potent antithrombotic efficacy in vivo with virtually no bleeding, an advantage over enoxaparin. These findings establish a brittlestar GAG whose unusual sulfation pattern defines a new class of high-efficacy, low-bleeding anticoagulants.

Deciphering rare 3-O-sulfated motifs in heparan sulfate (HS) is the key to understanding critical biological processes, such as viral infection and tumor progression. However, the structural analysis of these elusive structures remains highly challenging due to their low abundance and the limitations of conventional analytical approaches. In this study, we report the application of human lysosomal arylsulfatase G (ARSG), a sulfatase specifically targeting 3-O-sulfated glucosamine residues, as a novel enzymatic tool, which, in combination with heparinase and heparanase digestion, facilitates the identification of 3-O-sulfated glucosamine moieties within HS. Coupling ARSG-mediated regioselective desulfation with hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) enables the efficient detection and quantification of 3-O-sulfated oligosaccharides in HS and heparin derivatives. This approach overcomes a major analytical barrier in HS characterization and provides new insights into the distribution and functional roles of 3-O-sulfation. The integration of ARSG expands the enzymatic toolkit available for HS structural characterization and represents a significant advance toward elucidating the structure-function relationships of HS in health and disease.

ConspectusHeparan sulfate (HS), a highly sulfated glycosaminoglycan, varies in its disaccharide units, chain length, and sulfation patterns. HS structural diversity and its localization at cell surfaces and in the extracellular matrix enable HS interaction with a breadth of HS-binding proteins (HSBPs), HS thus being a co-receptor for other proteins and initiating various biological responses. Several designed and studied HS mimetics modulate HSBP activity implicated in various diseases. A key HSBP is heparanase (HPSE), which can cleave HS into smaller fragments, facilitating release of angiogenic growth factors, activating biological signals that may contribute to pathological conditions (promoting tumor development and metastasis), and enabling autoreactive immune cells to target insulin-producing β-cells. Thus, HPSE serves as a crucial target for disease therapy strategies. Several saccharide-based HS mimetics, designed as HSPE inhibitors, have advanced to clinical trials, but these sugar molecules were discontinued or suspended due to adverse effects from off-target HSBP interactions. Glycopolymers engineered to incorporate functionalized glycan residues into their polymeric backbones are a promising approach to retain endogenous HS' native biological activity, thereby enhancing therapeutic efficacy. Stereoselective formation of α-1,2-cis-glycosidic linkages that connect the glucosamine unit to the uronic acid disaccharide core is challenging during development of HS mimetics as HPSE inhibitors. Computational modeling and a stereoselective catalytic glycosylation method were used to design and synthesize glycopolymer-based HS mimetics with repeating units of the glucosamine-glucuronic acid disaccharide core and a controlled degree of polymerization and incorporate glycan residues with precisely tailored sulfation patterns. This strategy ensures targeted biological activity and maintains structural specificity toward its intended HSBP. Glycopolymers were synthesized using ring-opening metathesis polymerization with the third-generation Grubbs catalyst, enabling precise control over both the degree of polymerization and molecular weight by tuning the catalyst loading. The most potent glycopolymer displayed superior potency and selectivity compared to previously reported monovalent and polymeric HPSE inhibitors and demonstrated remarkable antimetastatic activity in models of mammary carcinoma and myeloma cancer. It also protected pancreatic β-cells and human islets from HPSE-induced damage, suggesting a possible diabetes therapeutic agent. Prioritizing multivalency and precise structural control in polymeric HS mimetics facilitates targeted interactions with specific HSBPs and enhances their potential for precision therapeutic applications.