Glycobiology最新文献_第2页

Glyco-Forum.

IF 3.3 3区生物学

Glycobiology Pub Date : 2025-09-03 DOI: 10.1093/glycob/cwaf052

引用次数: 0

Modeling glycans with AlphaFold 3: capabilities, caveats, and limitations. 用AlphaFold 3建模聚糖：功能、注意事项和限制。

IF 3.3 3区生物学

Glycobiology Pub Date : 2025-09-03 DOI: 10.1093/glycob/cwaf048

Chin Huang, Natarajan Kannan, Kelley W Moremen

{"title":"Modeling glycans with AlphaFold 3: capabilities, caveats, and limitations.","authors":"Chin Huang, Natarajan Kannan, Kelley W Moremen","doi":"10.1093/glycob/cwaf048","DOIUrl":"10.1093/glycob/cwaf048","url":null,"abstract":"Glycans are complex carbohydrates that exhibit extraordinary structural complexity and stereochemical diversity while playing essential roles in many biological processes, including immune regulation, pathogen recognition, and cell communication. In humans, more than half of all proteins are glycosylated, particularly those in secretory and membrane-associated pathways, highlighting the importance of glycans in health and disease. The recent release of the AlphaFold 3 source code enables customizable modeling not only of proteins but also glycan-containing biomolecular complexes. We assessed the capacity of AlphaFold 3 to model glycans using several input formats and identified a hybrid syntax employing Chemical Component Dictionary (CCD)-based molecular building blocks linked by \"bondedAtomPairs\" (BAP) as most effective in generating stereochemically valid glycan models. This workflow was used to create a library of AlphaFold 3 input templates and corresponding structural models for various glycan classes. We further explored capabilities, limitations, and remediation strategies for modeling problematic structures. Glycan interactions were also modeled with glycosylation enzymes and lectins with benchmarking and validation against known crystal structures. This protocol-driven approach is valuable for generating stereochemically valid, static models of glycan-protein interactions to support hypothesis development and subsequent structural and functional validation. However, caution should be observed in overinterpretation of the static models since glycans are known to exhibit considerable conformational dynamics that can be further captured by equilibrium sampling using molecular dynamics-based approaches. By sharing benchmarked examples using the BAP syntax we aim to support broader evaluation of AlphaFold 3 in studying glycan-related mechanisms in biosynthesis, signaling, infection, and disease.","PeriodicalId":12766,"journal":{"name":"Glycobiology","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12448869/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144951416","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

O-fucosylation affects abundance but not localization of select nucleocytoplasmic proteins in toxoplasma gondii. O-聚焦影响刚地弓形虫核浆蛋白的丰度，但不影响其定位。

IF 3.3 3区生物学

Glycobiology Pub Date : 2025-09-03 DOI: 10.1093/glycob/cwaf051

Megna Tiwari, Elisabet Gas-Pascual, Janice Teal-Urquides, John Samuelson, Christopher M West

{"title":"O-fucosylation affects abundance but not localization of select nucleocytoplasmic proteins in toxoplasma gondii.","authors":"Megna Tiwari, Elisabet Gas-Pascual, Janice Teal-Urquides, John Samuelson, Christopher M West","doi":"10.1093/glycob/cwaf051","DOIUrl":"10.1093/glycob/cwaf051","url":null,"abstract":"Toxoplasma gondii is a highly successful intracellular mammalian and avian pathogen that must adapt to a wide range of intracellular and extracellular environments. A mechanism that may support this is the modification of hydroxyamino acid rich sequences of nucleocytoplasmic proteins with O-fucose. O-fucosylation of possibly hundreds of proteins is mediated by a single highly conserved nucleocytoplasmic enzyme. Deletion of the SPY O-fucosyltransferase gene is tolerated but inhibits parasite proliferation in fibroblasts and their accumulation in mouse brains. A prior ectopic expression study suggested that O-fucose is required to detect proteins considered essential. To distinguish whether the SPY requirement was specific to the method or for protein expression per se, GPN1, an RNA polymerase chaperone, was epitope-tagged at its endogenous locus in both normal and SPYΔ strains. GPN1 was shown to be substantially and quantitatively O-fucosylated and exhibited a modest 24% reduction in level in SPYΔ cells. Proteomic analysis of its interactome indicated that fucosylation did not affect its association with RNA polymerase subunits. GPN1 was mostly cytoplasmic based on super-resolution immunofluorescence microscopy, and this localization was not affected by O-Fuc. A fusion of its O-fucosylated serine-rich domain to yellow fluorescent protein behaved similarly. In comparison, the abundance of a Zn-finger containing protein also depended on SPY, whereas the abundance and localization of ERK7 were not affected nor were levels of two other proteins. Thus O-fucose directly but modestly promotes the accumulation of select targets, but it does not enforce their localization in nuclear assemblies that are highlighted by immunofluorescence studies.","PeriodicalId":12766,"journal":{"name":"Glycobiology","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12449179/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144951473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Serum N-glycosylation is altered in Nephropathic Cystinosis. 肾病型胱氨酸病患者血清n -糖基化改变。

IF 3.3 3区生物学

Glycobiology Pub Date : 2025-09-03 DOI: 10.1093/glycob/cwaf047

Andreea Cislaru, Radka Saldova, Alessandra Heggenstaller, Peter A Nigrovic, Emily Harlin, Gordon Greville, Rafael De Andrade Moral, Daniel Bojar, Atif Awan, Róisín O'Flaherty

{"title":"Serum N-glycosylation is altered in Nephropathic Cystinosis.","authors":"Andreea Cislaru, Radka Saldova, Alessandra Heggenstaller, Peter A Nigrovic, Emily Harlin, Gordon Greville, Rafael De Andrade Moral, Daniel Bojar, Atif Awan, Róisín O'Flaherty","doi":"10.1093/glycob/cwaf047","DOIUrl":"10.1093/glycob/cwaf047","url":null,"abstract":"Changes in glycosylation can serve as markers for rare genetic disorders, including lysosomal storage diseases (LSDs). Nephropathic Cystinosis (NC), caused by mutations in the CTNS gene, is characterised by cystine accumulation in lysosomes due to dysfunctional cystinosin, a heavily N-glycosylated lysosomal transporter. We analysed total serum and IgG N-glycosylation using hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC) to explore the diagnostic biomarker capabilities and their pathophysiological relevance in NC. In this double-blind study (n = 12), we examined N-glycosylation of total serum and serum IgG from Irish participants with and without NC. Dimensionality reduction methods were used applying their glycan data to predict NC status, yet only modest predictive power was observed (66.6% for serum and 50% for IgG N-glycosylation). However, upon unblinding the data, we identified significant differences in specific serum N-glycosylation in NC, particularly in sialylation. These findings provide the first evidence that serum N-glycosylation is altered in NC. These changes may indicate disease-associated systemic alteration including dysregulation in N-glycosylation pathway. It provides justification for the need for a larger validation study and invites further exploration of its role in NC pathophysiology. We provide key recommendations for age stratification for studying serum, plasma and IgG N-glycans in juvenile cohorts as they display unique profiles compared to adult populations, an important consideration for all juvenile studies, even beyond the scope of rare diseases.","PeriodicalId":12766,"journal":{"name":"Glycobiology","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12406699/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144951414","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development of Lamprey-derived Antibodies Against Human Blood Group Antigens. 七鳃鳗源抗人血型抗原抗体的研制。

IF 3.3 3区生物学

Glycobiology Pub Date : 2025-08-12 DOI: 10.1093/glycob/cwaf043

Pascal B Kunz, Ea Kristine Clarisse Tulin, Akul Y Mehta, Tianwei Jia, Jamie Heimburg-Molinaro, Vivianne I Otto, Sean R Stowell, Richard D Cummings

{"title":"Development of Lamprey-derived Antibodies Against Human Blood Group Antigens.","authors":"Pascal B Kunz, Ea Kristine Clarisse Tulin, Akul Y Mehta, Tianwei Jia, Jamie Heimburg-Molinaro, Vivianne I Otto, Sean R Stowell, Richard D Cummings","doi":"10.1093/glycob/cwaf043","DOIUrl":"https://doi.org/10.1093/glycob/cwaf043","url":null,"abstract":"A major challenge in the glycosciences is the scarcity of sensitive and specific glycan-binding reagents, such as monoclonal antibodies, for detecting and isolating glycans. Here we report the development and characterization of new monoclonal antibodies (mAbs) that bind carbohydrate-based red blood cell (RBC) antigens including the ABO(H) antigens. This approach exploits the immune system of the sea lamprey (Petromyzon marinus), which strongly responds to human glycans to enable the generation of high affinity antibodies. To develop these mAbs, we immunized the lamprey with RBCs and designed a targeted antibody enrichment and screening process using intact RBCs and a custom microarray displaying blood group antigens. Through multiple rounds of enrichment and testing we identified two mAbs; A_25 and A_39. Glycan binding analysis of the mAbs using glycan microarrays, the Luminex platform and western blot analysis revealed their binding to H antigens and terminal N-acetyllactosamine Galβ1-4GlcNAc (LacNAc, a type 2 sequence). Mechanistic insights into antigen specificity were gained through glycan inhibition assays, sequence homology analysis, and nanomolar-range affinity measurements. mAb binding to RBCs was determined using flow cytometry. Both mAbs bound RBCs of all ABO blood groups, whereas strongest binding was observed for blood group O RBCs. Our findings highlight the efficacy of the lamprey system to develop glycan-specific mAbs. These reagents allow investigation of expression of the H antigen and LacNAc-containing glycans in human tissues. In the future, they could also be modified using molecular engineering techniques to generate mAbs specific to other understudied blood group antigens.","PeriodicalId":12766,"journal":{"name":"Glycobiology","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144834850","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Evaluation of Multiplexed Liquid Glycan Array (LiGA) for Serological Detection of Glycanbinding Antibodies. 多重液体聚糖阵列（LiGA）用于糖结合抗体血清学检测的评价。

IF 3.3 3区生物学

Glycobiology Pub Date : 2025-08-12 DOI: 10.1093/glycob/cwaf042

Revathi Reddy, Eric Carpenter, Anne Halpin, Mirat Sojitra, Chuanhao Peng, Guilherme Meira Lima, Xiaochao Xue, Kejia Yan, Jean Pearcy, Maria Ellis, Bruce Motyka, Todd L Lowary, Lori West, Ratmir Derda

{"title":"Evaluation of Multiplexed Liquid Glycan Array (LiGA) for Serological Detection of Glycanbinding Antibodies.","authors":"Revathi Reddy, Eric Carpenter, Anne Halpin, Mirat Sojitra, Chuanhao Peng, Guilherme Meira Lima, Xiaochao Xue, Kejia Yan, Jean Pearcy, Maria Ellis, Bruce Motyka, Todd L Lowary, Lori West, Ratmir Derda","doi":"10.1093/glycob/cwaf042","DOIUrl":"https://doi.org/10.1093/glycob/cwaf042","url":null,"abstract":"We test the performance of the multiplexed liquid glycan array (LiGA) technology in serological assays. Specifically, we use LiGA to detect ABO blood group antibodies in human serum. This LiGA, which we name ABO-LiGA, contains ABO blood group trisaccharide glycans with an ethylazido aglycone conjugated to groups of ten multi-barcoded M13 particles carrying dibenzocyclooctyne (DBCO) on p8 proteins. ELISA clonal binding assays to anti-A/B antibodies confirmed the functional performance of ABO-clones and aligned with next-generation sequencing (NGS) of the mixed clones. Multiple DNA-barcoded technical replicates in LiGA allow for quantification of reproducibility and robustness as determined by the Z'-score using NGS. We then tested ABO-LiGA for specific detection of IgG and IgM anti-A and anti-B IgG and IgM antibodies in human serum samples. Comparison of antibody binding responses in sera from 31 healthy donors to ABO-LiGA with an ABO-Luminex-based method revealed consistent responses to LiGA-ABO but also minor deficiencies of ABO-LiGA such as low robustness of the current assay format and a limited ability to detect low intensity antibody responses. Some results point to undesired interactions of serum antibodies with small-footprint glycans conjugated to phage via the bulky DBCO moiety. This report illuminates the path for future development of LiGA-based serological assays and suggests the need to develop alternative methods for conjugating glycans to phage to avoid liabilities of the hydrophobic DBCO moiety.","PeriodicalId":12766,"journal":{"name":"Glycobiology","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144834851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Glyco-Forum.

IF 3.3 3区生物学

Glycobiology Pub Date : 2025-08-11 DOI: 10.1093/glycob/cwaf045

引用次数: 0

Evidence for functional regulation of the KLHL3/WNK pathway by O-GlcNAcylation. o - glcn酰化对KLHL3/WNK通路功能调控的证据。

IF 3.3 3区生物学

Glycobiology Pub Date : 2025-08-11 DOI: 10.1093/glycob/cwaf046

Jimin Hu, Duc T Huynh, Denise E Dunn, Jianli Wu, Cindy Manriquez-Rodriguez, Qianyi E Zhang, Gabrielle A Hirschkorn, Tetsuya Hirata, George R Georgiou, Samuel A Myers, Scott R Floyd, Jen-Tsan Chi, Michael Boyce

{"title":"Evidence for functional regulation of the KLHL3/WNK pathway by O-GlcNAcylation.","authors":"Jimin Hu, Duc T Huynh, Denise E Dunn, Jianli Wu, Cindy Manriquez-Rodriguez, Qianyi E Zhang, Gabrielle A Hirschkorn, Tetsuya Hirata, George R Georgiou, Samuel A Myers, Scott R Floyd, Jen-Tsan Chi, Michael Boyce","doi":"10.1093/glycob/cwaf046","DOIUrl":"10.1093/glycob/cwaf046","url":null,"abstract":"The 42-member Kelch-like (KLHL) protein family are adaptors for ubiquitin E3 ligase complexes, governing the stability of a wide range of substrates. KLHL proteins are critical for maintaining proteostasis in a variety of tissues and are mutated in human diseases, including cancer, neurodegeneration, and familial hyperkalemic hypertension. However, the regulation of KLHL proteins remains incompletely understood. Previously, we reported that two KLHL family members, KEAP1 and gigaxonin, are regulated by O-linked β-N-acetylglucosamine (O-GlcNAc), an intracellular form of glycosylation. Interestingly, some ubiquitination targets of KEAP1 and gigaxonin are themselves also O-GlcNAcylated, suggesting that multi-level control by this post-translational modification may influence many KLHL pathways. To test this hypothesis, we examined KLHL3, which ubiquitinates with-no-lysine (WNK) kinases to modulate downstream ion channel activity. Our biochemical and glycoproteomic data demonstrate that human KLHL3 and all four WNK kinases (WNK1-4) are O-GlcNAcylated. Moreover, our results suggest that O-GlcNAcylation affects WNK4 function in both osmolarity control and ferroptosis, with potential implications ranging from blood pressure regulation to neuronal health and survival. This work demonstrates the functional regulation of the KLHL3/WNK axis by O-GlcNAcylation and supports a broader model of O-GlcNAc serving as a general regulator of KLHL signaling and proteostasis.","PeriodicalId":12766,"journal":{"name":"Glycobiology","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12360702/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144834852","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Glycoengineering of nematode antigens using insect cells: a promising approach for producing bioactive vaccine antigens of the barber's pole worm Haemonchus contortus. 利用昆虫细胞对线虫抗原进行糖工程：一种有前途的生产理发师杆状虫弯曲血蜱生物活性疫苗抗原的方法。

IF 3.3 3区生物学

Glycobiology Pub Date : 2025-08-11 DOI: 10.1093/glycob/cwaf044

Isabella Adduci, Floriana Sajovitz-Grohmann, Licha N Wortha, Zuzanna Dutkiewicz, Hugo Weidinger, Anja Joachim, Thomas Wittek, Dirk Werling, Iain B H Wilson, Katharina Lichtmannsperger, Shi Yan

{"title":"Glycoengineering of nematode antigens using insect cells: a promising approach for producing bioactive vaccine antigens of the barber's pole worm Haemonchus contortus.","authors":"Isabella Adduci, Floriana Sajovitz-Grohmann, Licha N Wortha, Zuzanna Dutkiewicz, Hugo Weidinger, Anja Joachim, Thomas Wittek, Dirk Werling, Iain B H Wilson, Katharina Lichtmannsperger, Shi Yan","doi":"10.1093/glycob/cwaf044","DOIUrl":"10.1093/glycob/cwaf044","url":null,"abstract":"The H11 antigens, located on the intestinal microvilli of Haemonchus contortus, comprise a group of homologous aminopeptidases essential for the parasite's digestion of blood meals. Native H11 proteins are promising vaccine antigens, capable of eliciting robust protective immunity against H. contortus in sheep and goats. However, recombinant forms of H11, produced either in conventional expression systems or in transgenic Caenorhabditis elegans, failed to replicate the protective efficacy of the native form, most likely due to two critical factors: improper glycosylation and protein misfolding. To address these limitations, we developed a novel strategy to produce recombinant Haemonchus antigens in glycoengineered insect cells. By introducing three C. elegans genes that alter the native N-glycosylation pathways of Hi5 insect cells we successfully expressed soluble H11 and GA1 antigens featuring nematode-specific glycan epitopes, including tri-fucosylated structures and the Galβ1,4Fuc motif. The glycoengineered H11 proteins retained aminopeptidase activity and stimulated cytokine secretion from ovine peripheral blood mononuclear cells in vitro. These findings establish a platform for producing bioactive vaccine antigens against the parasitic nematode H. contortus.","PeriodicalId":12766,"journal":{"name":"Glycobiology","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12343074/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144775249","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Protein engineering strategies to develop lectins by design. 设计开发凝集素的蛋白质工程策略。

IF 3.4 3区生物学

Glycobiology Pub Date : 2025-07-22 DOI: 10.1093/glycob/cwaf041

Ryoma Hombu, Lauren E Beatty, Sriram Neelamegham

{"title":"Protein engineering strategies to develop lectins by design.","authors":"Ryoma Hombu, Lauren E Beatty, Sriram Neelamegham","doi":"10.1093/glycob/cwaf041","DOIUrl":"https://doi.org/10.1093/glycob/cwaf041","url":null,"abstract":"Glycans regulate a wide array of biological processes, making them central to studies of cell biology. Thus, it is essential to characterize the spatiotemporal dynamics of glycans on cells and tissues, and to elucidate how glycan structures affect protein and cell function. Among the available molecular tools, glycan-binding proteins (GBPs), including naturally occurring lectins, are uniquely suited to provide this information at single-cell resolution. However, the diversity of cell-surface glycans far exceeds the number of readily available GBPs. Moreover, conventional lectins often possess shallow binding pockets that limit their recognition to terminal glycan epitopes, and such recognition often proceeds with low binding affinity. Protein engineering offers a promising strategy to expand GBP specificity, enhance affinity, and introduce novel binding capabilities. Currently large gaps remain between the available protein design principles and their application to GBP engineering. This has somewhat slowed progress in the development of glycan-targeted tools. In this review, we outline recent efforts that use rational design to inform GBP engineering for specific tasks. We also present methods to select suitable protein scaffolds and the application of directed evolution for optimizing lectin design. This includes our recent efforts to modify glycosyltransferases into GBPs, which potentially offers a predictive strategy to design lectins based on desired properties. Together, the presentation offers a roadmap for developing next-generation glycan binding proteins capable of decoding the complex glycan landscape of cells.","PeriodicalId":12766,"journal":{"name":"Glycobiology","volume":" ","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144690027","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0