Hyperglycemia is a principal driver of β cell failure and multiple-organ complications in diabetes. Chronic exposure to hyperglycemia overstimulates mTORC1, disrupting glucose metabolism and promoting ER stress, oxidative stress, and inflammation; however, the upstream metabolic signal(s) linking glucose to mTORC1 activation remains unclear. Here, we identified glucosamine as a key metabolite connecting elevated glucose to mTORC1 signaling in pancreatic islets and kidney, both major targets of hyperglycemic damage. Using 13C6-glucose metabolic labeling in diabetic rodents treated with or without the SGLT2 inhibitor dapagliflozin or insulin, combined with targeted metabolomics and metabolic flux analysis, we found that tissue glucose concentrations strongly correlated with glucosamine. A similar correlation with plasma glucose was conserved in humans with or without type 2 diabetes, and inversely associated with β cell function. In vitro, low-dose glucosamine stimulated mTORC1 in islets and kidney proximal tubule cells in an O-GlcNAcylation-dependent manner. Broad phosphoproteomics and transcriptomics analyses in β cells showed that glucosamine activated mTORC1-regulating pathways, induced oxidative stress, ER stress, and dedifferentiation. Genetic inhibition of β cell mTORC1 via heterozygous Raptor knockout, as well as pharmacologic inhibition of the glucosamine/mTORC1 axis through SGLT2 inhibition, alleviated β cell stress, improved glycemic control, and restored β cell function. These findings identified the glucosamine/mTORC1 pathway as an important mediator of β cell and kidney dysfunction in diabetes.
Glucosamine (GlcN) is an essential amino monosaccharide widely used in pharmaceuticals, nutraceuticals, and cosmetics. Microbial fermentation presents a sustainable alternative to its traditional chemical production. However, in Saccharomyces cerevisiae, competitive carbon flux towards ethanol significantly limits GlcN yields. In this study, an S. cerevisiae strain for GlcN biosynthesis was engineered by integrating heterologous GlmD (glucosamine-6-phosphate deaminase) and GlmP (glucosamine-6-phosphate phosphatase) genes. To redirect carbon flux, the pyruvate decarboxylase genes pdc1, pdc5, and pdc6 were sequentially knocked out using the Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) approach, generating strains S. cerevisiaeGlmDP/pdc1Δ, GlmDP/pdc1Δpdc5Δ, and GlmDP/pdc1Δpdc5Δpdc6Δ. S. cerevisiae GlmDP/pdc1Δpdc5Δpdc6Δ achieved a GlcN titer of 2.20 ± 0.11 g/L, a 1.54-fold increase over the parental S. cerevisia GlmDP strain, while its ethanol yield decreased by 26%. This enhancement was achieved without significantly affecting cell growth or glucose consumption. Comparative transcriptomics between the triple-knockout and parental yeasts revealed 892 differentially expressed genes. Pathways related to glycolysis and ethanol formation were predominantly downregulated, whereas pathways potentially supporting GlcN synthesis were upregulated. The engineered strain demonstrated high genetic stability over 50 generations. Our findings demonstrate that disrupting ethanol formation is an effective strategy to enhance GlcN production in S. cerevisiae, providing valuable insights for carbon flux redistribution.
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.
A body of evidence suggests that upregulating O-GlcNAcylation, a reversible post-translational modification of serine and threonine residues on target proteins, is beneficial in neurological diseases. However, this phenomenon is currently underexplored in the pharmacotherapy of epilepsy. Therefore, we aimed to explore the potential effects of combining N-acetylglucosamine (GlcNAc), a precursor for O-GlcNAcylation, and a centrally acting benzodiazepine (diazepam) on oxidative stress, a known driver of epilepsy, and some epileptogenesis-associated genes. Mice (n = 10) were randomly assigned to treatment groups and treated with varied oral doses (100, 200, and 400 mg/kg) of GlcNAc in combination with diazepam (1 mg/kg) for 14 days. Following this, seizure was chemically induced with 70 mg/kg pentylenetetrazol intraperitoneally. Brains of treated mice were excised for antioxidant assays and to determine the expression of genes associated with epileptogenesis: potassium chloride co-transporter (KCC4), interleukin (IL-6), tumour necrosis factor-α (TNF-α), and brain-derived neurotrophic factor (BDNF). Our findings suggest that GlcNAc, when concurrently administered with diazepam, prevents oxidative stress and reduces the gene expression of IL-6, a cytokine associated with neuroinflammation and seizures, whilst increasing the gene expression of KCC4, an ion co-transporter that promotes antiepileptogenesis.
Sanfilippo syndrome type B, also known as mucopolysaccharidosis type IIIB (MPS IIIB), is a rare autosomal recessive lysosomal storage disorder caused by mutations in the N-acetyl-α-D-glucosaminidase (NAGLU) gene, which encodes the enzyme α-N-acetylglucosaminidase. This enzyme is essential for degrading heparan sulfate. The deficiency leads to toxic accumulation within cells. To investigate the impact of NAGLU mutations, mutational data were retrieved from public databases including NCBI, UniProt, and HGMD. A total of 162 variants were evaluated using sequence-based prediction tools to identify deleterious mutations, followed by structure-based in silico analyses to assess changes in protein stability, biophysical properties, and ligand-binding potential. Among the analyzed mutations, the I154R variant was identified as the most deleterious, showing disease-associated characteristics, structural instability, and impaired functional properties. Molecular docking with N-acetylglucosamine (NAG) revealed binding affinities of -4.17 kcal/mol for the native protein and -3.97 kcal/mol for the I154R mutant, suggesting a retained yet slightly reduced binding potential. Molecular dynamics simulations supported these findings, indicating stable trajectories, favorable interaction profiles, and moderate flexibility for both complexes. These results enhance our understanding of NAGLU-related pathogenicity in MPS IIIB, contributing to improved health care strategies and offering a valuable foundation for future therapeutic developments targeting enzyme dysfunction in Sanfilippo syndrome type B.
Chondroitin sulfate, alone or associated with glucosamine (CS/CS+GLU), is an effective knee osteoarthritis (KOA) treatment, with fewer adverse effects (AEs) than non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase 2 inhibitors (COXIBs). To estimate the CS/CS+GLU cost-effectiveness versus NSAIDs/COXIBs, due to the avoidance of mild-moderate or severe gastrointestinal AEs (GIAE), ischemic heart disease (IHD), acute kidney insufficiency (AKI), chronic kidney failure (CKF) and ischemic stroke (IS) from the Spanish National Health System (NHS) perspective. Two analyses were considered: 1) savings to the NHS from current CS/CS+GLU treatment; and 2) maximum savings that could be achieved by the NHS if all patients with KOA currently treated with NSAIDs/COXIB would be switched to CS/CS+GLU. AEs frequency, associated utilities loss and managing cost were obtained from medical literature and Spanish sources. A probabilistic model (second-order Monte Carlo simulation) was carried out for a 3-year time horizon. Treatment duration: 180 days (base case); 90 and 240 days (sensitivity analysis). First analysis: 45,087 mild-moderate GIAE, 3,217 severe GIAE, 211 IHD, 1,087 AKI, 746 CKF and 3,359 IS, would be avoided with CS/CS+GLU. Discounting drugs cost, the three-year savings would be 57.1 million euros (savings probability: 80.7%). The savings per patient treated with CS/CS+GLU would amount to €38.02 (95% CI 14.06; €75.69), with a mean gain of 0.0023 (95% CI 0.0018-0.0027) QALY. The probability that CS/CS+GLU treatment was dominant (lower costs and QALY gain) or cost-effective (cost per QALY gained less than €25,000) was 80.7% and 98.1%, respectively. In the second analysis, savings for the NHS would amount to 387 million euros, with the CS/CS+GLU-only option being dominant and cost-effective in 100% of the analyses. The improved tolerability of CS/CS+GLU versus NSAIDs/COXIBs is expected to prevent thousands of AEs and generate considerable savings for the NHS, making it cost-effective treatment.
Two series of compounds were designed and synthesized based on a glycine methyl ester-quinoline core scaffold combined with a fragment containing nitrogen using either an isoxazole or a piperidine carboxamide fragment as a linker as dual inhibitors targeting both chitin synthase (CHS) and glucosamine-6-phosphate synthase (GlmS). The inhibition percentage (IP) values of these compounds against CHS ranged from 29.9% to 84.5%, while their inhibition percentages against GlmS spanned 38.1% to 86.9% at the concentration of 300 μg mL-1. The IC50(CHS)/IC50(GlmS) ratios of compounds 8c, 9, 14c, 14g, and 14i, which were approximately equal to 1 (between 0.97 and 1.15), demonstrated that these compounds were dual inhibitors targeting both CHS and GlmS. Kinetic analysis revealed that compounds 8c (K i = 0.109 mM) and 14e (K i = 0.125 mM) acted as noncompetitive inhibitors of CHS, exhibiting stronger binding affinities for CHS than that of UDP-GlcNAc (K m = 4.11 mM). Similarly, 8c (K i = 0.227 mM) and 14e (K i = 0.248 mM) displayed higher binding affinities for GlmS than that of UDP-GlcNAc (K m = 6.28 mM). Antifungal activity assays in vitro demonstrated that compounds 8c, 8i, 9, 14d, 14e, and 14g exhibited potent antifungal effects comparable to those of fluconazole or polyoxin B. The results of antifungal activity experiments against drug-resistant fungal strains and sorbitol protection assays, coupled with the results of drug combination studies, further indicated that the target of these compounds is the chitin in the cell wall. Molecular docking showed that compounds had good affinities with both CHS and GlmS. Furthermore, ADME (absorption, distribution, metabolism, excretion) profiling revealed that these compounds possess favorable pharmacokinetic properties and drug-likeness, representing viable lead compounds for the development of novel antifungal agents.
The overexpression of glucose transporters (GLUTs) in cancer cells represents a promising diagnostic target. While 2-NBDG, a fluorescent glucose analog, enables GLUT-mediated imaging, it suffers from low brightness and requires high concentrations and washing. Herein, we report the development of turn-on fluorescent probes for GLUTs based on the viscosity-sensitive fluorophore in GFP for a wash-free imaging of prostate cancer cell, PC-3. A series of fluorescent molecular rotors (FMRs) were synthesized by modifying the 2-methyl-4-(p-dimethylaminobenzylidene)-5-imidazolinone (DMAB) core. Structural modifications, such as cyclic amine substitution and julolidine incorporation, enhanced fluorescence quantum yields (ΦF up to 0.17 in glycerol) and viscosity sensitivity (χ up to 0.87). Among them, the julolidine-containing fluorophore Julo-Ph showed the best performance. The DMAB-based fluorophores were conjugated to glucose or glucosamine to create GLUT-targeted probes. A glucosamine-bound conjugate, GlcN-Julo-Ph, was found to enable a wash-free bright fluorescence imaging of PC-3 cancer cells. Inhibition studies and docking simulations strongly suggested a GLUT-mediated uptake of the fluorophore. GlcN-Julo-Ph outperformed 2-NBDG in emitting a brighter intracellular fluorescence at a lower concentration, notably, under wash-free conditions. Our findings shed light on the utility of viscosity-sensitive FMRs for the design of turn-on imaging probes and offer a promising platform for a rapid, low-background cancer cell detection.
Whitening is a core function of cosmetics， and ingredients including acetyl glucosamine， tranexamic acid， nicotinamide， phenethyl resorcinol， glabridin， and ascorbyl tetraisopalmitate are widely used due to their clearly established whitening mechanisms. However， existing analytical methods face notable limitations： photodiode array detectors （PDA） cannot reliably detect weakly or non-ultraviolet （UV）-absorbing components； evaporative light scattering detectors （ELSD） suffer from inadequate sensitivity for low-concentration analytes； mass spectrometry （MS） is costly； and single-column cannot achieve the separation of ingredients with significantly different polarities. To address these challenges， this study established an analytical method for the determination of the six whitening ingredients in cosmetics using ultra performance liquid chromatography coupled with a photodiode array detector and a corona charged aerosol detector （UPLC-PDA-CAD）. For sample pretreatment： 0.2 g of sample was weighed， and 10 mL dichloromethane and 10 mL deionized water were added for vortex extraction of whitening ingredients. The mixture was then centrifuged for phase separation. The aqueous layer and dichloromethane layer were collected separately. The aqueous layer was washed twice with dichloromethane； all dichloromethane fractions were combined， concentrated to less than 1 mL under a nitrogen stream， and finally made up to volume with isopropanol. Both the aqueous layer and the reconstituted isopropanol solution were filtered through 0.22 μm hydrophilic polytetrafluoroethylene （PTFE） membranes before UPLC injection. For chromatographic separation， a Waters HSS T3 column （150 mm×2.1 mm， 1.7 μm） was selected. The column temperature was set at 40 ℃， the injection volume was 1 μL， and the flow rate was 0.3 mL/min. The mobile phase consisted of isopropanol， acetonitrile， and 20 mmol/L ammonium acetate solution （pH adjusted to 4.5 using formic acid）， using a gradient elution program. For detection， a tandem PDA-CAD system was used： PDA （scan range 200-400 nm） was employed for components with UV absorption， while CAD （nebulization temperature 35 °C， acquisition frequency 5 Hz） was used for components with weak UV absorption. Quantification was performed using the external standard method.Method validation results showed good linear relationships for all six whitening ingredients within their respective concentration ranges. The correlation coefficients （r） were all greater than 0.999. The limits of detection （LODs， S/N=3） were 5.0-50.0 µg/g， and the limits of quantification （LOQs， S/N=10） were 12.0-120.0 µg/g. Spiked recovery tests were conducted on negative cosmetic matrices （emulsion， cream， oil） at low， medium， and high levels. The recoveries ranged from 92.8% to 110.1%， and the relative standard deviations （RSDs， n=6） were 0.12%-5.45%， indicating excellent precision and accuracy. This method was applied to seven commercially cosmetics. The results revealed that all target whitening ingredients declared on the product labels were detected， with significant differences in their content. Nicotinamide was the most frequently detected compound， found in five products， and also exhibited the highest concentrations， ranging from 0.19% to 2.29%. Phenethyl resorcinol was detected in three products， with contents ranging from 0.02% to 0.52%. Ascorbyl tetraisopalmitate was detected in two products， at 0.09% and 3.08%， respectively. Acetyl glucosamine， tranexamic acid， and glabridin were each detected in only one product. In conclusion， this established UPLC-PDA-CAD method is simple， efficient， sensitive and accurate. It effectively overcomes the technical challenge of detecting whitening ingredients with widely varying polarities， offering reliable technical support for cosmetic quality control， regulatory supervision， and evaluation of product whitening efficacy and potential sensitization risks. 建立了一种超高效液相色谱-二极管阵列检测器串联电雾式检测器（UPLC-PDA-CAD）测定化妆品中乙酰壳糖胺、氨甲环酸、烟酰胺、苯乙基间苯二酚、光甘草定及抗坏血酸四异棕榈酸酯6种美白成分的分析方法。样品用二氯甲烷和水涡旋提取，收集二氯甲烷层和水层；水层经二氯甲烷洗涤后，合并二氯甲烷相，氮吹浓缩至<1 mL并以异丙醇定容。水层和异丙醇定容液分别过滤，注入液相色谱仪，通过Waters HSS T3柱（150 mm×2.1 mm，1.7 μm）分离。流动相为异丙醇-乙腈-20 mmol/L乙酸铵缓冲液（甲酸调节pH至4.0），采用梯度洗脱方式，以二极管阵列检测器串联电雾式检测器进行检测，外标法定量。分别对样品前处理方法和色谱条件进行了优化。在优化的实验条件下，6种美白功效成分在一定范围内线性关系良好，相关系数（r）均>0.999，在乳、霜、油3种化妆品基质中的加标回收率为92.8%~110.1%，相对标准偏差（RSD，n=6）为0.12%~5.54%。对市售的7款化妆品进行检测，检出的美白成分与产品包装标示成分一致，但各成分含量差异较大。烟酰胺的使用频率和测得含量均较高，5款产品的测试结果为0.19%~2.29%。该方法操作简便，稳定可靠，重复性好，适用于化妆品中这几种极性差异显著的美白成分的检测。
Chitosan is a linear binary copolymer composed of glucosamine and N-acetylglucosamine units, exhibiting excellent bioactivities and broad application potential. The structural architecture of chitosan is defined by three fundamental parameters: degree of polymerization (DP), fraction of acetylation (FA), and pattern of acetylation (PA). However, due to long-standing constraints in preparation and analytical methodologies, current research predominantly concentrates on DP and FA, while largely overlooking the structural parameter of PA. This review systematically summarizes the production and characterization methods of chitosan with well-defined PA, and highlights that PA is an important factor regulating the structure-function relationship of chitosan. Importantly, PA can significantly modify its physicochemical properties and bioactivities independently of DP and FA. These advances have primarily relied on the increasing availability of well-characterized recombinant chitin deacetylases (CDAs), as well as continuously optimized techniques such as chromatographic separation and mass spectrometry. Together, these developments have enabled systematic research on chitosan with well-defined sequences and predictable properties.
Sialic acids are important for cellular communication, with N-acetylneuraminic acid (Neu5Ac) being the canonical form of sialic acid in humans. Presence of non-canonical sialic acids, derived from dietary intake or as metabolic side product, has been linked to immune disorders and cancer. As homeostasis of different sialic acids remains poorly understood in humans, we studied the role of N-acetylneuraminate lyase (NPL) in their catabolism. In vitro expression of NPL in different biological systems revealed broad substrate specificity towards sialic acids and related 2-keto-3-deoxy metabolites. In agreement with the broad substrate specificity, NPL-deficient red blood cells accumulated Neu5Ac and 3-deoxy-d-glycero-d-galacto-nonulosonic acid (KDN). Interestingly, endogenous levels of non-canonical sialic acids, including N-glycolylneuraminic acid (Neu5Gc) and KDN, were depleted in HEK293T cells upon NPL overexpression, while Neu5Ac and CMP-Neu5Ac levels remained stable. This was further confirmed by supplementation with different sialic acids. Detailed analysis of sugar phosphate intermediates of the hexosamine and sialic acid biosynthesis pathways showed strongly elevated ManNAc-6P (N-acetyl-d-mannosamine 6-phosphate) and Neu5Ac-9P, indicating efficient recycling of ManNAc to increase de novo Neu5Ac biosynthesis. However, this recycling was not efficient for Neu5Gc and KDN. While GlcNAc-6P (N-acetyl-d-glucosamine 6-phosphate) levels were slightly elevated, no evidence was found for further metabolism towards GlcN-6P (glucosamine 6-phosphate) and energy production via glycolysis as shown for bacterial neuraminate lyases. In conclusion, human NPL catabolizes a broad range of sialic acids. However, depending on the cellular context, NPL contributes to a net cellular reduction in non-canonical sialic acids, such as KDN, due to a lack of efficient recycling.
Background/Objective(s)/Introduction: Prebiotics are substances that metabolically favor certain microorganisms of a microbiome, promoting homeostasis. Dental biofilm microorganisms are enmeshed in a matrix of extracellular polymeric substances that they produce. A diet rich in sucrose can lead to a dysbiotic biofilm associated with microbial acid production and a change in the matrix's composition (mostly water-insoluble glucans), which allows acids to accumulate within biofilms and contribute to teeth demineralization. Thus, the effects of putative prebiotics were evaluated to verify their impact on exopolysaccharides, the microbial population, and biofilm formation. Materials and methods: Five potential prebiotics (N-acetyl-d-glucosamine, arginine, proline, sodium nitrate, and urea) were evaluated compared with a substance-free control. A Streptococcus mutans biofilm model on polystyrene plates was used to determine the concentrations of substances that would inhibit sucrose-derived biofilm formation. Selected concentrations were then used to verify the production of insoluble glucans by glucosyltransferase B. Afterward, S. mutans and mixed-species (S. mutans, Actinomyces naeslundii, and Streptococcus gordonii) biofilms were grown on saliva-coated hydroxyapatite discs with sucrose to evaluate the microbial population and 3D biofilm structure (exopolysaccharides and bacterial biovolume). Lastly, a microcosm biofilm formed on polystyrene plates was used to assess the effects of the substances on biomass and the proportion of distinct viable microbial populations. Results: Only arginine inhibited insoluble glucan production and S. mutans biofilm accretion (≅ 90%). Arginine and proline inhibited a biofilm build-up in mixed-species and microcosm models and modulated microbial counts of species associated with cariogenic biofilms. In the microcosm biofilm, urea hindered biomass accretion in initial biofilms and the counts of aciduric microbiota and fungi, but N-acetyl-d-glucosamine stimulated microbial growth. Sodium nitrate affected the size and shape of microcolonies in S. mutans and mixed-species biofilms. Conclusion(s): Among the substances tested, arginine and proline modulated the microbial population and hindered biofilm accretion, especially arginine, which hampered glucan production. However, urea is the only substance able to impede fungal growth.
Chitooligosaccharides (COS) are short-chain chitosan derivatives with a wide range of biomedical, agricultural, and environmental applications, including antimicrobial therapy, wound healing, and pollutant removal. Reliable quantification of COS is essential but currently relies on high-performance liquid chromatography, mass spectrometry, or capillary electrophoresis, which require costly equipment, complex sample preparation, and are unsuitable for routine or on-site applications. This study reports a rapid, solvent-free, colorimetric assay for COS based on the reaction of 5% aqueous ninhydrin with free amino groups in McIlvaine buffer. The assay was optimized using glucosamine as a model analyte, yielding maximal sensitivity at pH 7.0. The chromophore generated (Ruhemann's purple) remained stable for over 120 min after reaction, allowing measurements to be taken without strict time constraints. Calibration was linear from 0.4 to 2.2 mM (R2 = 0.9926), with low limits of detection (0.006 mM) and quantification (0.018 mM). Increasing absorbance with COS polymerization degree (DP1-DP6) demonstrates specificity for free amino groups, while N-acetyl glucosamine showed a negligible response. Furthermore, the assay was successfully adapted for solid-phase detection on ninhydrin-pretreated filter paper and nitrocellulose, with enhanced sensitivity. This simple, efficient, and low-cost method provides an accessible alternative to instrumental techniques, supporting COS monitoring in laboratory workflows and enabling portable applications in biomedicine, agriculture, and environmental diagnostics.
Chitosan is a natural biopolymer derived from the deacetylation of chitin, a structural polysaccharide abundantly found in the shells and exoskeletons of crustaceans, insects, and other arthropods. Its unique physicochemical properties have led to widespread applications in biomedical, pharmaceutical, agricultural, and industrial sectors. In Nigeria, abundant freshwater crustaceans and insects remain underutilized as sources of chitosan. This study evaluated the potential of West African river prawns (Macrobrachium vollenhovenii) and American cockroaches (Periplaneta americana) as local chitosan sources using a modified chemical extraction process. Exoskeletal materials were pretreated, demineralized, deproteinized, and subjected to autoclave assisted alkaline deacetylation (50% NaOH, 121 °C, 15 psi, 30 min). Chitin yields were 29.53% for prawns and 17.78% for cockroaches, while chitosan yields were 28.13% and 11.56%, respectively. Fourier Transform Infrared Spectroscopy confirmed characteristic functional groups of chitosan in both sources. The degree of deacetylation (DD) was 68.79% for prawn-derived chitosan and 81.21% for cockroach-derived chitosan, indicating effective conversion of N-acetyl. D-glucosamine to D-glucosamine units. These findings demonstrate that both species are viable alternative sources for chitosan production, with pressurized deacetylation enhancing yield and polymer quality. This approach provides a scalable, reproducible strategy for sustainable chitosan extraction in Nigeria, supporting potential applications in biotechnology, medicine, and industry.
Effective treatment of central nervous system diseases is limited by the blood-brain barrier, which restricts the entry of most therapeutics, especially large molecules such as biologics. Here, we demonstrate that coating non-ionic surfactant vesicles with selected ligands markedly enhances their transport across brain endothelium. A focused screen of ligands targeting endothelial receptors and transporters identified glucosamine, transferrin, Angiopep-2, and L-carnitine as promising candidates with enhanced blood-brain barrier transport properties. Mannosamine, which lacks a known endothelial target, served as a non-targeted control. Mechanistic studies indicated dynamin- and clathrin-dependent pathways drove uptake of all functionalised nanoparticles, while caveolae-mediated endocytosis enhanced transport of glucosamine- and L-carnitine-coated vesicles. L-carnitine was of interest because it engages the organic cation transporter OCTN2 that is the primary transporter facilitating carnitine influx at the blood-brain interface; despite reports of its overexpression on brain endothelium, this transporter remains underexplored for targeting drugs. Vesicles coated with L-carnitine enhanced delivery of an antibody across an in vitro barrier model by an order of magnitude compared to free antibody. In a murine Venezuelan equine encephalitis virus infection model, L-carnitine-functionalised vesicles carrying a therapeutic antibody produced substantial reductions in viral burden within both brain and peripheral tissues. These vesicles performed comparably to systems directed at glucose transport pathways and outperformed those targeting transferrin or low-density lipoprotein-related receptors. Overall, the data highlights the flexibility of our vesicle platform for targeted drug delivery and specifically the potential of carnitine functionalisation for effective transport across the blood-brain barrier.
Chitin, one of the most abundant marine polysaccharides, plays a pivotal role in global carbon‑nitrogen cycles, and its degradation is mediated by marine chitinolytic microorganisms. The widely distributed genus Tenacibaculum is well known for its fish pathogenicity and its capacity to degrade diverse polysaccharides; however, its potential for chitin degradation remains poorly characterized. Here, we isolated a novel bacterium, Tenacibaculum sp. IMCC1, from the Philippine Sea, sharing 98% 16S rRNA gene similarity with Tenacibaculum mesophilum. Genome sequencing yielded a 3,357,366 bp circular chromosome (G + C content 31.73%) containing 3124 protein-coding genes, 55 tRNAs, and 5 rRNA operons (no plasmids). CAZy annotation identified 126 carbohydrate-active enzymes (CAZymes). Genome analysis further revealed a putative chitin degradation pathway, encompassing extracellular depolymerization of chitin into chitooligosaccharides, their subsequent hydrolysis into N-acetylglucosamine (GlcNAc) and glucosamine (GlcN), transport into the cytoplasm, and downstream metabolism to UDP-GlcNAc, a key precursor for peptidoglycan biosynthesis. Together, these results predict the chitin-degrading potential of Tenacibaculum sp. IMCC1, expanding the functional repertoire of marine chitinolytic bacteria and providing genetic resources for ecological studies and chitin biotechnological valorization.
Multi-functionality in extant enzymes, including the ability to transform multiple substrates, is thought to arise, in part, from conformational flexibility. The hexokinase protein family represents a classic model system for investigating the relationship between substrate specificity and conformational change. Within this family, human glucokinase (hGCK) displays notable degrees of conformational heterogeneity, including an intrinsically disordered loop. The extent to which these structural features contribute to the breadth of hGCK's substrate scope is unknown. Here, we investigate the substrate specificities of extant and ancestral glucokinases that span the evolutionary emergence of conformational heterogeneity in this family. We show that extant hGCK catalyzes the ATP-dependent phosphorylation of glucose, 2-deoxyglucose, mannose, glucosamine, fructose, allose and galactose with catalytic efficiencies ranging from 6.3 x 10 3 M -1 sec -1 to 0.33 M -1 sec -1 . A glucokinase ancestor from early vertebrate evolution (vGCK), which also displays conformational heterogeneity and disorder, phosphorylates these same seven substrates with similar k cat / K m values. An antecedent, chordate glucokinase (cGCK), which displays reduced conformational heterogeneity and lacks intrinsic disorder, also transforms these same substrates, but with higher overall catalytic efficiencies and markedly lower K m values. Notably, however, the ratios of k cat / K m values for individual substrate pairs, which define specificity, are unchanged for all three enzymes. Our results demonstrate that substrate specificity is not correlated with conformational diversity in GCKs and support a model in which the differences in catalytic efficiencies of various substrates arise from differences in the ability to form the ground state enzyme-carbohydrate binary complex.
Fully functional neural competence and integrity require 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 axons and EVs, the molecular mechanisms of those miRNAs present in EVs and their functional role in nervous system development have 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 compartmentalized 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.
We have developed a compact tape drive (CoT) with on-demand sample delivery for time-resolved serial femtosecond crystallography (SFX) experiments, which can deliver sample droplets and/or initiate reactions with a drop-on-drop strategy. Two disposable piezoelectric injectors are positioned in tandem along the tape to produce a queue of nanolitre-scale droplets. X-ray free-electron laser pulses arrive perpendicular to and pass through the broad face of the tape. The pulse is synchronized and aligned to the droplets, thereby enabling highly efficient SFX data collection. The tape transport speed and the delivery distance can be varied to control the mixing time from approximately 130 ms to tens of seconds. We conducted time-resolved SFX experiments utilizing a basic enzymatic reaction model of hen egg white lysozyme (HEWL) and N-acetyl-D-glucosamine (GlcNAc) to demonstrate the drop-on-drop capabilities of the CoT, and the full binding process of GlcNAc to HEWL was observed at 1.3-9.7 s.
The Endoglycosidase CC N180H mutant (Endo-CC N180H) is extensively utilized in the synthesis of homogeneous glycopeptides and glycoconjugates due to its high transglycosylation activity. However, its potential in analytical glycomics remains underexplored. In this study, various structural acceptors were synthesized using N-acetylglucosamine (GlcNAc) as a substrate, and their transglycosylation reactions catalyzed by Endo-CC N180H were investigated. Modifications at the C6 position of GlcNAc completely abolished transglycosylation activity, while alterations at the C1 position were well tolerated. Among these acceptors, d0-Bn-β-GlcNAc exhibited the highest transglycosylation efficiency, with optimal reaction conditions identified as 150 mU/mL Endo-CC N180H at 45 °C for 36 h. Leveraging these conditions, d5-Bn-β-GlcNAc was further synthesized, enabling the development of a stable isotope-labeled chemoenzymatic strategy for N-glycan relative quantification. Using sialylated glycopeptides as a model, the method achieved excellent linearity over a 1:10-10:1 molar ratio range (R2 = 0.9991), high precision (CV = 0.22-6.24%), and a limit of detection of 50 fmol. Moreover, the strategy was successfully applied to the profiling and relative quantification of high-mannose N-glycans in ribonuclease B. Collectively, this work expands the analytical utility of Endo-CC N180H and establishes a robust and sensitive platform for quantitative glycomics for relative quantification of selected N-glycan species in glycomics research.