Due to its high specificity and peak expression in hepatocellular carcinoma (HCC), Glypican-3 (GPC3) is a heavily researched target. While current GPC3-targeted therapies demonstrate remarkable efficacy, traditional GPC3 assessment methods are limited by time-dependent and spatial heterogeneity, invasiveness, and prolonged waiting periods, highlighting an urgent need for new GPC3 evaluation strategies. In this study, first, we selected MHCC97H as a GPC3-high expressing HCC cell line (named GPC3high MHCC97H) and constructed a GPC3-low expressing MHCC97H cell line (named GPC3low MHCC97H) using lentiviral transfection technology. Second, a novel radiopharmaceutical precursor, CS03, was synthesized. Molecular docking analysis revealed that CS03 exhibits the potential to bind to multiple amino acid residues of GPC3, indicating its promise as a targeting molecule for GPC3. Therefore, we labeled it with Ga-68 to synthesize [68Ga]Ga-CS03. The radiolabeling yield of [68Ga]Ga-CS03 exceeded 80%. After purification, the radiochemical purity of [68Ga]Ga-CS03 was greater than 98%, with a specific activity of (3.00 ± 0.22) × 106 MBq/mmol. [68Ga]Ga-CS03 exhibited excellent in vitro stability, rapid blood clearance (T 1/2α = 0.93 ± 0.49 min and T 1/2β = 17.22 ± 1.42 min), and good in vitro affinity (36.30 ± 5.17 nM). Micro-PET/CT imaging of HCC models revealed significant [68Ga]Ga-CS03 radiotracer accumulation in GPC3high MHCC97H tumors 0.5 h postinjection. In contrast, minimal or no significant accumulation was detected in GPC3low MHCC97H tumors and blocking group tumors, indicating the excellent in vivo affinity and specificity of [68Ga]Ga-CS03. [68Ga]Ga-CS03 showed rapid tumor clearance, with a significant decrease in tumor radiotracer distribution by 1 h, a finding corroborated by subsequent biodistribution studies. Furthermore, micro-PET/CT images and biodistribution results indicated that [68Ga]Ga-CS03 was cleared by the kidneys, which explains its rapid blood clearance. Finally, we collected tumor tissues from the animal models after PET imaging and performed hematoxylin and eosin (H&E) staining and GPC3 immunofluorescence (IF) staining. We found that the fluorescence intensity of IF staining was positively correlated with the radiotracer distribution in the tumor sites on PET images, which further validated the specificity of [68Ga]Ga-CS03 for GPC3 binding. In conclusion, we developed [68Ga]Ga-CS03, a novel GPC3-targeted PET radiotracer. Extensive in vitro and in vivo evaluations demonstrated its favorable stability, high affinity, and specificity. These promising preclinical findings highlight its potential for the noninvasive assessment of GPC3 expression in HCC.

Milk oligosaccharides (MOs) are essential for the development of mammalian offspring, yet their fine-scale structural evolutionary divergence remains unelucidated, largely due to isomeric complexity and the limitations of analytical methods. Here, we present GlycoBond X, an online platform that couples high-resolution separation with parallel structural characterization of glycan isomers through multi-stage chemical derivatization and RP-HPLC-MS/MS. By applying this strategy to four evolutionarily distinct mammals, we uncovered a conserved transition from acidic MO dominance (>75% in mouse and tree shrew) to predominantly fucosylated neutral MOs (>62% in macaque and human). GlycoBond X unveiled unprecedented structural diversity, including 22 unique fucosylation motifs and 66 previously undescribed human MOs. Notably, tree shrew MOs exhibited human-like structures and shared over 69% FUT2 sequence homology with primates. This study established a high-throughput, high-sensitivity platform, elucidated the adaptive structural evolution of oligosaccharides via evolutionary glycomics, and provided a foundation for exploring their biosynthetic pathways.

Influenza viruses significantly threaten human health, making it crucial to develop effective anti-influenza drugs. Viral hemagglutinin (HA) was found to bind specifically to glycan receptors. Here, we aim to investigate how the structure of different fucoidan oligosaccharide monomers affects binding to the influenza A virus. First, we employed a "layer by layer" degradation strategy to prepare structurally diverse oligosaccharides from Sargassum fusiforme. Using two-dimensional ion-pair reversed-phase high-performance liquid chromatography (2D-IPRP-HPLC), 46 fucoidan oligosaccharides were successfully isolated and purified. Subsequently, we investigated the interactions of these monomers with HA from four subtypes of influenza A viruses (H1N1, H3N2, H5N1, and H7N9) by "shotgun" glycan microarray approach. The structure of fucoidan oligosaccharides that exhibit specific binding was characterized through multistep chemical derivatization combined with electrospray ionization-tandem mass spectrometry (ESI-MSn). The results revealed that the monomers bound well to H1N1 and H3N2 HA proteins, but weakly to H5N1 and H7N9 HA proteins. The binding affinity was significantly influenced by the degree of polymerization, the level of sulfation, the position of sulfate groups, and the linkage patterns of the oligosaccharides. This study is essential for elucidating the active structural domains responsible for anti-influenza activity within fucoidan and will contribute to the development of fucoidan-based inhibitors against influenza A viruses.

Drugs that inhibit tumor angiogenesis, promote vascular normalization and improve the tumor microenvironment. However, their application is limited by adaptive or compensatory resistance. Chondroitin sulfate (CS) regulates numerous proteins including pro-angiogenic growth factors, for whom binding affinity depends on sulfation of CS. In this study, we aimed to determine how sulfation of natural tetrasaccharides and hexasaccharides of CS affected binding to the vascular endothelial growth factor (VEGF-A) and fibroblast growth factor 2 (FGF-2). Twenty-eight CS oligosaccharide isomers were obtained by preparative HPLC, tagged with the AEAB fluorescent linker, and identified using an improved chemical derivatization strategy combined with tandem mass spectrometry. CS oligosaccharide microarrays revealed that VEGF-A and FGF-2 bound preferentially to highly sulfated CS, and the GalNAc(4S)GlcA(2S)GalNAc(6S) sequence was found to be indispensable for binding to these proteins. By integrating glycan microarrays with computational modeling, this study revealed the relationship between the structure of CS and its interactions with pro-angiogenic factors. The degree and the specific sulfation patterns on CS should be taken into account when designing anti-angiogenic drugs.

Konjac glucomannan (KGM) is a polysaccharide with potential medical and functional properties. Here, the antioxidant and cytoprotective effects of sulfated and differently sized KGM fractions were investigated using various in vitro assays. The sulfated KGMs (SKGMs) were prepared via a relay strategy. First, Vitamin C (Vc)-H2O2 degradation was employed to obtain three soluble KGM fractions with different molecular weights. Second, nine KGM derivatives with varying sulfate content were obtained by the sulfur trioxide-pyridine method. The scavenging of DPPH, superoxide, and hydroxyl radicals was measured in vitro. The antioxidant activity of SKGM correlated positively with sulfate content. SKGM-I-2 displayed the most potent radical scavenging activity. Its purification by cellulose DEAE-52 column chromatography yielded four homogeneous fractions (SKGM-I-2a, SKGM-I-2b, SKGM-I-2c, and SKGM-I-2d). Pretreatment with SKGM-I-2d increased the viability of RAW264.7 cells exposed to H2O2. Moreover, SKGM-I-2d significantly increased the activity of superoxide dismutase and catalase, as well as the levels of glutathione, while regulating the expression of Keap1, Nrf2, and HO-1 in RAW264.7 cells. The present study suggests that SKGM-I-2d protects RAW264.7 cells against H2O2-induced oxidative injury through the activation of the Nrf2/Keap1 signaling pathway. These results provide a scientific basis for future studies linking the structural and functional features of KGM.

As the main active glycoprotein of egg white, the biological functions of chicken ovomucin α- and β-subunit are closely related to the structure of glycans. However, the exact composition and structure of the subunit glycans are still unknown. We obtained highly pure chicken ovomucin α-subunit and β-subunit protein bands by the strategy combined with two-step isoelectric precipitation and SDS-PAGE gel electrophoresis. The ammonia-catalyzed one-pot procedure was then used to release and capture α-and β-subunit protein glycans with 1-phenyl- 3-Methyl-5-pyrazolone (PMP). The N/O-glycans of bis-PMP derivatives were purified and analyzed by LC-MS. More importantly, an effective dual modification was performed to accurately quantify neutral and sialylated O-glycans through methylamidation of sialic acid residues and simultaneously through carbonyl condensation reactions of reducing ends with PMP. We first showed that the α-subunit protein has only N-glycosylation modification, and the β-subunit only O-glycosylation, a total of 22 N-glycans and 20 O-glycans were identified in the α- and β-subunit, respectively. In addition, the complex N-glycan (47%) and the sialylated O-glycan (77%) are each major types of the above subunits. Such findings in this study provide a basis for studying the functional and biological activities of chicken ovomucin glycans.

In this study, crude polysaccharide (LAG -C) and homogeneous arabinogalactan (LAG -W) were isolated from Qinling Larix kaempferi of Shaanxi Province. Bioactivity assays showed that LAG -W and LAG -C enhanced the phagocytic ability, NO secretion, acid phosphatase activity, and cytokine production (IL -6, IL-1 beta, and TNF- alpha) of RAW264.7 macrophages. Notably, LAG -W exhibited a significantly stronger immunomodulatory effect than LAGC. The primary structure of LAG -W was characterised by chemical methods (monosaccharide composition, methylation analysis, and alkali treatment) and spectroscopic techniques (gas chromatography -mass spectrometry, high-performance liquid chromatography -mass spectrometry, and 1D/2D nuclear magnetic resonance). LAG -W was identified as a 22.08 kilodaltons (kDa) neutral polysaccharide composed of arabinose and galactose at a 1:7.5 molar ratio. Its backbone consisted of repeated -> 3)- beta-Gal p -(1 -> residues. Side chains, connected at the O-6 position, were mainly composed of T- beta-Gal p -(1 -> and T- beta-Gal p -(1 -> 6)- beta-Gal p -(1 -> residues. And it also contained small amounts of T- beta-Ara p -(1 ->, T- alpha-Ara f -(1 -> 6)- beta-Gal p -(1 -> 6)- beta-Gal p -(1 ->, and T- alpha-Ara f - (1 -> 3)- alpha-Ara f -(1 -> 6)- beta-Gal p -(1 -> residues. By structurally and functionally characterising L. kaempferi polysaccharides, this study opens the way for the valorisation of this species.

Glycans mediate various biological processes through carbohydrate-protein interactions, and glycan microarrays have become indispensable tools for understanding these mechanisms. However, advances in functional glycomics are hindered by the absence of convenient and universal methods for obtaining natural glycan libraries with diverse structures from glycoconjugates. To address this challenge, we have developed an integrative approach that enables one-pot release and simultaneously capture, separation, structural characterization, and functional analysis of N/O-glycans. Using this approach, glycoconjugates are incubated with a pyrazolone-type heterobifunctional tag-ANPMP to obtain glycan-2ANPMP conjugates, which are then converted to glycan-AEPMP conjugates. We prepared a tagged glycan library from porcine gastric mucin, soy protein, human milk oligosaccharides, etc. Following derivatization by N-acetylation and permethylation, glycans were subjected to detailed structural characterization by ESI-MSn analysis, which revealed >83 highly pure glycan-AEPMPs containing various natural glycan epitopes. A shotgun microarray is constructed to study the fine details of glycan-bindings by proteins and antisera.

The nutritious goat milk has low allergenicity. Oligosaccharides represent one of the crucial functional constituents in goat milk, which are structurally similar to human milk oligosaccharides (HMOs). Currently, the anti-allergic activity of GMOs has not been reported. In this study, GMOs were efficiently separated into neutral (NGMOs) and sialylated (SGMOs) fractions, following by qualitative and quantitative analysis at the isomer level using online LC-MS/MS. Fifteen NGMOs and 28 SGMOs were detected in goat milk, with 10 SGMOs reported for the first time. Distinctly, α2,6-linked SGMOs were 3.9 times more abundant in goat milk than in bovine milk, with the total relative content of 6'SL, 3'SLN and 6'NGL in SGMOs approach to 60%, which is more similar to HMOs. Orally administering GMOs, especially α2,6-linked sialylated oligosaccharides, significantly alleviated food allergy in ovalbumin-induced BALB/c mice. SGMOs restored the balance of Lachnospiraceae, Erysipelotrichaceae, and Bacteroidaceae, reconstructed the intestinal mucosal barrier, especially restored the levels of fucosylation, sialylation, and sulfation of mucin O-glycans, increased the expression of four core type 2 O-glycans (F1H2N2, F2H2N2, S1F2H2N2, and A1F1H2N2) significantly. This is the first comprehensive study of the anti-allergic activity of GMOs, and the results lay the foundation for the development of GMOs-based natural anti-allergic components.

Glycans have been proven to play special roles in keeping human health as a class of nutritional and bioactive ingredients in many food materials. However, their broad use in the food industry is hindered by the lack of comprehensive analytical methods for high-quality food glycomics studies and large-quantity raw materials for their production. This study focuses on structural identification and quantitative comparison of bioactive N-glycans in seven species of livestock and poultry plasma as potential natural glycan resources by a novel comprehensive relative quantification strategy based on stable isotope labeling with nondeuterated and deuterated 4-methyl-1-(2-hydrazino-2-oxoethyl)-pyridinium bromide (d0/d7-HMP) in combination with linkage-specific derivatization of sialic acid residues. Methodological validation of the method in terms of detection sensitivity, signal resolution, quantification linearity, precision, and accuracy on model neutral and complicated sialylated glycans demonstrated its advantages over the existing methods. Based on this method, a series of bioactive N-glycans were found in seven species of livestock and poultry plasma, and their differences in structure, abundance percentages, and relative contents of N-glycans were revealed, demonstrating their excellent applicability for comprehensive food glycomics analysis and great exploitation potential of these plasma samples as large-quantity raw materials in producing bioactive N-glycans for application in food and pharmaceutical industries.

Understanding how dietary polysaccharides affect mucin O-glycosylation and gut microbiota could provide various nutrition-based treatments. Here, the O-glycan profile of the colonic mucosa and gut microbiome were investigated in C57BL/6J mice fed six structurally diverse dietary polysaccharides and a mixture of six fibers. Dietary polysaccharides increased total O-glycans, mainly by stimulating neutral glycans. Highly branched arabinogalactan promoted terminally fucosylated core 1 O-glycans; whereas linear polysaccharides, including pectin, konjac glucomannan, inulin, and the fiber mixture, favored terminally di-fucosylated O-glycans. The last three polysaccharides also lowered the level of sulfated O-glycans and sialylated mono-fucosylated O-glycans. Varied monosaccharide composition in mixed polysaccharides had a synergistic beneficial effect, boosting fucosylated neutral glycans, decreasing acidic glycans, and stimulating microbial richness and diversity. Dietary polysaccharides containing arabinose and sulfate groups enhanced the relative abundances of Akkermansia and Muribaculaceae, respectively. The present comparison reveals the relationship between dietary polysaccharide structure, mucin O-glycan composition, and intestinal microorganisms.

Amide compounds are important organic compounds, which play an important role in biomedical chemistry, materials science, life science, and other fields. The synthesis of α-CF3 amides, especially compounds containing 3-(trifluoromethyl)-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepine-2-one, has long been a challenge due to the tensile properties and instability of the rings. Here, we report an example of palladium-catalyzed carbonylation of CF3-containing olefin to form α-CF3 acrylamide. By controlling the ligands, we can get different amide compounds as products. This method has good substrate adaptability and functional group tolerance.

Human milk is important for infant growth, and oligosaccharides are one of its main functional nutrients. To enable a systematic comparison of free oligosaccharide and glycoconjugate content in milk from different species, the phenol-sulfuric acid and resorcinol assays were combined to determine the content. Using real samples, the method revealed that human milk contained the highest amount of total, neutral (9.84 ± 0.31 g/L), and sialylated (3.21 ± 0.11 g/L) free oligosaccharides, followed by goat milk, with neutral (0.135 ± 0.015 g/L) and sialylated (0.192 ± 0.016 g/L) free oligosaccharides and at a distance by bovine and yak milk. The highest total glycoconjugate content was detected in yak milk (0.798 ± 0.011 g/L), followed by human, bovine, and goat milk. These findings suggest that goat milk is the best source of free oligosaccharides in infant formula and functional dairy products and yak milk is the best source of glycoconjugates.

All solid-state lithium metal batteries (ASSLMBs) are regarded as one favorable future technology for energy storage. Composite solid-state electrolytes (CSEs) exploit the strengths of both solid polymer electrolytes (SPEs) and inorganic ceramic electrolytes (ICEs), which commonly exhibit enhanced ionic conductivity, as well as excellent cycling stability, and favorable interfacial resistance. However, rational structure design for better CSE/electrodes interface contact is necessary for the future practical applications. Herein, an asymmetrical composite solid electrolyte is constructed by introducing the electro-spun polyacrylonitrile (PAN) into the casting membrane. Oxidation-resistant PAN shows strong adsorption of Li salt anions, providing long range Li+ transmission path and extended electrochemical window and protections for bottom oxidative polyethylene oxide (PEO)-based layer. Meanwhile, the bottom PEO/garnet Li6.4La3Zr1.4Ta0.6O12 (LLZTO) matrix obtains good ion conductivity and stability to Li metal anode, and successfully protects the antioxidative PAN for possible severe side reactions with anodes. Obtained bilayer CSE shows high ionic conductivity (6.0 x 10(-4) S cm(-1) at 60 degrees C), broad electrochemical window (5.2 V, vs . Li/Li (+)), and good mechanical properties. The battery displays 159.9 mA h g(-1) and remained 153.5 mA h g(-1) after 90 cycles at 0.2 C rate and good inhibition to lithium dendrites growth and side reactions. This work indicates that the asymmetric designed CSE film provides a possible directions towards future high energy density ASSLMBs. (C) 2021 Elsevier Ltd. All rights reserved.

The structure of chondroitin sulfate oligosaccharides (CSOs), especially their sulfation pattern, has been found to be closely related with many biological pathways and diseases. However, detailed functional analysis such as their interaction with glycan binding proteins (GBPs) has been lagging, presumably due to the unavailability of well-defined, diverse structures. Besides challenging chemical and enzymatic synthesis, this is also due to the challenges in their purification at the isomer level and structural analysis owing to their instability, structural complexity, and low mass spectrometry detection sensitivity. Herein, we first used recycling preparative HPLC to separate and purify shark CS tetrasaccharide component labeled by a bifunctional fluorescent linker 2-amino-N-(2-aminoethyl)benzamide (AEAB) at the isomer level. Then, each isomer was derivatized through a multistage procedure including N-acetylation, carboxyl amidation, permethylation, and desulfation with silylating reagent. Structural analysis of each derivatized isomer was performed with ESI-MSn in positive ion mode. A total of 16 isomers of CSO-AEAB were isolated, with a minimum mass component of 0.007 mg and a maximum mass component of 17.53 mg, of which 10 isomers (>90 μg) were structurally analyzed. This preparation and structure analysis of CSOs lay the foundation for further study of the structure-activity relationship of CSOs.

A palladium(II)-catalyzed direct 2-arylation of indoles by tetraarylstannanes with oxygen (balloon) as the oxidant at room temperature has been developed. Various tetraarylstannanes can be employed as aryl sources for 2-arylation of indoles in up to 89% yield, providing a practical and efficient catalytic protocol for accessing 2-arylindoles.

Previous studies have shown that crude polysaccharides from the Lycium barbarum fruit could inhibit cancer cell growth, but the major effective constituents are yet to be identified. In this study, we compared the effects of L. barbarum fruit polysaccharide fractions on the growth of hepatoma cells (SMMC-7721 and HepG2), cervical cancer cells (HeLa), gastric carcinoma cells (SGC-7901), and human breast cancer cells (MCF-7). LBGP-I-3 showed stronger inhibitory effects on MCF-7 cells (cell viability of 48.96%) than SMMC-7721 (cell viability of 78.91%) and HeLa cells (cell viability of 55.94%), and had no effect on HepG2 and SGC-7901 cells. In addition, LBGP-I-3 had no inhibitory effect on normal liver cells (L02, cell viability of 115.58%). Investigation of the underlying mechanism suggested that LBGP-I-3 inhibited the growth of cancer cells by cell cycle arrest and apoptosis. LBGP-I-3 arrested the cell cycle at the G0/G1 phase, altered mitochondrial function, activated oxidative stress, and regulated the MAPK signaling pathway to induce apoptosis. Thus, LBGP-I-3 may be a potential functional food ingredient for the prevention of cancer without toxicity to normal cells in vitro. These results could help further elucidate the structure–activity relationship of L. barbarum fruit polysaccharides and functional food development.

A platelet-like CuS material with twin crystals is successfully constructed. The twin crystal structure inside the material provides more channels and reaction sites for Na ions intercalation and conversion.

Polysaccharides are a major active component of Porphyra haitanensis, which is an important food source in many countries. Four different molecular-weight fractions, namely PHPD-I (329 kDa), PHPD-II (203 kDa), PHPD-III (128 kDa), and PHPD-IV (10 kDa), were obtained from P. haitanensis polysaccharides by degradation using the H2O2/ascorbic acid system. PHPD-IV elicited the highest level of antioxidant and immunostimulatory activity among the four fractions. PHPD-IV was purified by DEAE-cellulose column and five fractions were obtained, designated PHPD-IV-1-PHPD-IV-5. PHPD-IV-4 displayed the greatest biological activity by up-regulating the phosphorylation of MAPK signalling molecules. PHPD-IV-4 was further purified, and its structure was characterized by monosaccharide composition and 1/2D-NMR analysis. The result revealed that PHPD-IV-4 was repeated units of -> 3) beta-D-galactose (1 -> 4) 3, 6-anhydro-alpha-L-galactose (1 ->, and -> 3) beta-D-galactose (1 -> 4) alpha-L-galactose-6-S (1 -> This study provides a theoretical basis for the utilisation and structure-activity assessment of P. haitanensis polysaccharides.

Biological functions of chondroitin sulfate, including anti-oxidation and anti-inflammation, are associated with its molecular weight. This study aimed to evaluate the correlation between antioxidant activity and molecular weights of chondroitin sulfate derived frombovine nasal cartilage (BCS). BCS extracted by compound enzymatic method was further purified via DEAE-cellulose column separation to obtain BCS-II (129.4 kDa), which was further degraded by H2O2-Vc to obtain four subfractions: BCS-II-1 (92.7 kDa), BCS-II-2 (54.1 kDa), BCS-II-3 (26.3 kDa), and BCS-II-4 (19.7 kDa). Changes in the physicochemical properties of BCS-II before and after degradation were compared via FT-IR, NMR and monosaccharide composition analysis. Finally, antioxidant activities of BCS-II and its subfractions BCS-II-1-4 were compared. Our results showed that the H2O2-Vc system did not disrupt the primary functional group of BCS-II, with no significant change in sulfate content between BCSII and its degraded fractions; however, uronic acid levels increased in degraded fractions when compared with BCS-II. In vitro, BCS-II-4 displayed the lowest molecular weight and had the strongest antioxidant activity. Therefore, the antioxidant activity of chondroitin sulfate in vitro is robustly associated with its molecular weight, and low-molecular-weight chondroitin sulfate can be used as an antioxidant in the food and pharmaceutical industries and other sectors. (C) 2019 Elsevier B.V. All rights reserved.