Glycoconjugate JournalPub Date : 2024-10-01Epub Date: 2024-08-20DOI: 10.1007/s10719-024-10162-x
Marilica Zemkollari, Colin Ruprecht, Markus Blaukopf, Reingard Grabherr, Erika Staudacher
{"title":"Cloning, expression and characterisation of a novel mollusc α-1,2-Fucosyltransferase from Crassostrea gigas (CgFUT2).","authors":"Marilica Zemkollari, Colin Ruprecht, Markus Blaukopf, Reingard Grabherr, Erika Staudacher","doi":"10.1007/s10719-024-10162-x","DOIUrl":"10.1007/s10719-024-10162-x","url":null,"abstract":"<p><p>Glycans containing fucose play crucial roles in cell biology, particularly in recognition processes. In humans, fucose found in H-blood group antigens is recognized by various pathogens, thereby influencing host-pathogen interactions. However, in invertebrate biology the specific functions of these modifications and the corresponding glycosyltransferases are not fully elucidated. Therefore, cloning these glycosyltransferases from different model systems will provide valuable insights into this process. Little is known about fucosyltransferases in molluscs. For this study, a sequence of the Pacific oyster, Crassostrea gigas, based on amino acid sequence homologies with rabbit and human α-1,2-fucosyltransferases, was chosen. The recombinant enzyme (350 amino acids) was able to transfer fucose from GDP-fucose to the galactose residue of type II disaccharides, terminal galactoses in complex N-glycan structures and several linear and branched galactans which were tested using a glycan microarray. The α-1,2-linkage formed was confirmed by NMR analysis. The enzyme was active in a broad pH-range, it was relatively stable upon storage conditions and its activity was not dependent on the presence of divalent cations. In this study, we were able to clone, express and characterise a novel α-1,2-fucosyltrasferase from Crassostrea gigas (CgFUT2).</p>","PeriodicalId":12762,"journal":{"name":"Glycoconjugate Journal","volume":" ","pages":"255-265"},"PeriodicalIF":2.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522050/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142004068","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Binayak Acharya, Marthe Tofthagen, Marissa L. Maciej-Hulme, Michal Rachel Suissa, Niclas G. Karlsson
{"title":"Limited support for a direct connection between prebiotics and intestinal permeability – a systematic review","authors":"Binayak Acharya, Marthe Tofthagen, Marissa L. Maciej-Hulme, Michal Rachel Suissa, Niclas G. Karlsson","doi":"10.1007/s10719-024-10165-8","DOIUrl":"https://doi.org/10.1007/s10719-024-10165-8","url":null,"abstract":"<p>The intestinal barrier is a selective interface between the body´s external and the internal environment. Its layer of epithelial cells is joined together by tight junction proteins. In intestinal permeability (IP), the barrier is compromised, leading to increased translocation of luminal contents such as large molecules, toxins and even microorganisms. Numerous diseases including Inflammatory Bowel Disease (IBD), Coeliac disease (CD), autoimmune disorders, and diabetes are believed to be associated with IP. Dietary interventions, such as prebiotics, may improve the intestinal barrier. Prebiotics are non-digestible food compounds, that promote the growth and activity of beneficial bacteria in the gut. This systematic review assesses the connection between prebiotic usage and IP. PubMed and Trip were used to identify relevant studies conducted between 2010–2023. Only six studies were found, which all varied in the characteristics of the population, study design, and types of prebiotics interventions. Only one study showed a statistically significant effect of prebiotics on IP. Alteration of intestinal barrier function was measured by lactulose/mannitol, chromium-labelled Ethylenediaminetetraacetic acid (<sup>51</sup>Cr-EDTA), lactulose/rhamnose, and sucralose/erythritol excretion as well as zonulin and glucagon-like peptide 2 levels. Three studies also conducted gut microbiota assessment, and one of them showed statistically significant improvement of the gut microbiome. This study also reported a decrease in zonulin level. The main conclusion from this review is that there is a lack of human studies in this important field. Futhermore, large population studies and using standardized protocols, would be required to properly assess the impact of prebiotic intervention and improvement on IP.</p>","PeriodicalId":12762,"journal":{"name":"Glycoconjugate Journal","volume":"65 1","pages":""},"PeriodicalIF":3.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250596","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Glycoconjugate JournalPub Date : 2024-06-01Epub Date: 2024-07-03DOI: 10.1007/s10719-024-10157-8
Jianguo Gu, Tomoya Isaji
{"title":"Specific sialylation of N-glycans and its novel regulatory mechanism.","authors":"Jianguo Gu, Tomoya Isaji","doi":"10.1007/s10719-024-10157-8","DOIUrl":"10.1007/s10719-024-10157-8","url":null,"abstract":"<p><p>Altered glycosylation is a common feature of cancer cells. Some subsets of glycans are found to be frequently enriched on the tumor cell surface and implicated in different tumor phenotypes. Among these, changes in sialylation have long been associated with metastatic cell behaviors such as invasion and enhanced cell survival. Sialylation typically exists in three prominent linkages: α2,3, α2,6, and α2,8, catalyzed by a group of sialyltransferases. The aberrant expression of all three linkages has been related to cancer progression. The increased α2,6 sialylation on N-glycans catalyzed by β-galactoside α2,6 sialyltransferase 1 (ST6Gal1) is frequently observed in many cancers. In contrast, functions of α2,3 sialylation on N-glycans catalyzed by at least three β-galactoside α2,3-sialyltransferases, ST3Gal3, ST3Gal4, and ST3Gal6 remain elusive due to a possibility of compensating for one another. In this minireview, we briefly describe functions of sialylation and recent findings that different α2,3 sialyltransferases specifically modify target proteins, as well as sialylation regulatory mechanisms vis a complex formation among integrin α3β1, Golgi phosphoprotein 3 (GOLPH3), phosphatidylinositol 4-kinase IIα (PI4KIIα), focal adhesion kinase (FAK) and sialyltransferase, which suggests a new concept for the regulation of glycosylation in cell biology.</p>","PeriodicalId":12762,"journal":{"name":"Glycoconjugate Journal","volume":" ","pages":"175-183"},"PeriodicalIF":2.7,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11329402/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141491704","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Glycosylated SARs Cov 2 interaction with plant lectins.","authors":"Oinam Sangita Devi, Senjam Sunil Singh, Rana Kamei, Hanjabam Joykishan Sharma, Maharabam Anandi Devi, Nidhi Brahmacharimayum","doi":"10.1007/s10719-024-10154-x","DOIUrl":"10.1007/s10719-024-10154-x","url":null,"abstract":"<p><p>Lectins are non-immune carbohydrate-binding proteins/glycoproteins that are found everywhere in nature, from bacteria to human cells. They have also been a valuable biological tool for the purification and subsequent characterisation of glycoproteins due to their carbohydrate binding recognition capacity. Antinociceptive, antiulcer, anti-inflammatory activities and immune modulatory properties have been discovered in several plant lectins, with these qualities varying depending on the lectin carbohydrate-binding site. The Coronavirus of 2019 (COVID-19) is a respiratory disease that has swept the globe, killing millions and infecting millions more. Despite the availability of COVID-19 vaccinations and the vaccination of a huge portion of the world's population, viral infection rates continue to rise, causing major concern. Part of the reason for the vaccine's ineffectiveness has been attributed to repeated mutations in the virus's epitope determinant elements. The surface of the Coronavirus envelope is heavily glycosylated, with approximately sixty N-linked oligomannose, composite, and hybrid glycans covering the core of Man3GlcNAc2Asn. Some O-linked glycans have also been discovered. Many of these glyco-chains have also been subjected to multiple mutations, with only a few remaining conserved. As a result, numerous plant lectins with specificity for these viral envelope sugars have been discovered to interact preferentially with them and are being investigated as a potential future tool to combat coronaviruses such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by preventing viral attachment to the host. The review will discuss the possible applications of plant lectins as anti-coronaviruses including SARS-CoV-2, antinociceptive, anti-inflammation and its immune modulating effect.</p>","PeriodicalId":12762,"journal":{"name":"Glycoconjugate Journal","volume":" ","pages":"185-199"},"PeriodicalIF":2.7,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140921821","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Structural elucidation a complex galactosyl and glucosyl-rich pectin from the pericarp of immature fruits of Juglans mandshurica Maxim.","authors":"Xi-Zhe Sun, Qing-Yu Zhang, Si-Liang Jiang, Rong-Jian Zhu, Jun-Hong Chai, Jun Liang, Hai-Xue Kuang, Yong-Gang Xia","doi":"10.1007/s10719-024-10156-9","DOIUrl":"10.1007/s10719-024-10156-9","url":null,"abstract":"<p><p>A glucosyl-rich pectin, JMMP-3 (M<sub>w</sub>, 2.572 × 10<sup>4</sup> g/mol, O-methyl % = 3.62%), was isolated and purified from the pericarp of the immature fruit of Juglans mandshurica Maxim. (QingLongYi). The structure of JMMP-3 was studied systematically by infrared spectroscopy, monosaccharide compositions, methylation analysis, partial acid hydrolysis, and 1/2D-NMR. The backbone of JMMP-3 possessed a smooth region (→ <sup>4</sup>GalA<sup>1</sup> →) and a hairy region (→ <sup>4</sup>GalA<sup>1</sup> → <sup>2</sup>Rha<sup>1</sup> →) with a molar ratio of 2: 5. The substitution of four characteristic side chains (R<sub>1</sub>-R<sub>4</sub>) occurs at C-4 of → 2,4)-α-Rhap-(1→, where R<sub>1</sub> is composed of → 5)-α-Araf-(1→, R<sub>2</sub> is composed of → 4)-β-Galp-(1 → and β-Galp-(1→, R<sub>3</sub> is composed of α-Glcp-(1→, →4)-α-Glcp-(1 → and → 4,6)-α-Glcp-(1→, and R<sub>4</sub> is composed of → 5)-α-Araf-(1→, β-Galp-(1→, → 4)-β-Galp-(1→, → 3,4)-β-Galp-(1→, → 4,6)-β-Galp-(1 → and → 2,4)-β-Galp-(1 → . In addition, the antitumor activity of JMMP-3 on HepG2 cells was preliminarily investigated.</p>","PeriodicalId":12762,"journal":{"name":"Glycoconjugate Journal","volume":" ","pages":"201-216"},"PeriodicalIF":2.7,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141491705","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Retraction Note: 9-O-acetylated sialic acids differentiating normal haematopoietic precursors from leukemic stem cells with high aldehyde dehydrogenase activity in children with acute lymphoblastic leukaemia.","authors":"Suchandra Chowdhury, Sarmila Chandra, Chitra Mandal","doi":"10.1007/s10719-024-10159-6","DOIUrl":"10.1007/s10719-024-10159-6","url":null,"abstract":"","PeriodicalId":12762,"journal":{"name":"Glycoconjugate Journal","volume":" ","pages":"227"},"PeriodicalIF":2.7,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141619822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ingrid A V Wolin, Ana Paula M Nascimento, Rodrigo Seeger, Gabriela G Poluceno, Alfeu Zanotto-Filho, Claudia B Nedel, Carla I Tasca, Sarah Elizabeth Gomes Correia, Messias Vital Oliveira, Vanir Reis Pinto-Junior, Vinicius Jose Silva Osterne, Kyria Santiago Nascimento, Benildo Sousa Cavada, Rodrigo Bainy Leal
{"title":"Retraction Note: The lectin DrfL inhibits cell migration, adhesion and triggers autophagy-dependent cell death in glioma cells.","authors":"Ingrid A V Wolin, Ana Paula M Nascimento, Rodrigo Seeger, Gabriela G Poluceno, Alfeu Zanotto-Filho, Claudia B Nedel, Carla I Tasca, Sarah Elizabeth Gomes Correia, Messias Vital Oliveira, Vanir Reis Pinto-Junior, Vinicius Jose Silva Osterne, Kyria Santiago Nascimento, Benildo Sousa Cavada, Rodrigo Bainy Leal","doi":"10.1007/s10719-024-10158-7","DOIUrl":"10.1007/s10719-024-10158-7","url":null,"abstract":"","PeriodicalId":12762,"journal":{"name":"Glycoconjugate Journal","volume":" ","pages":"225"},"PeriodicalIF":2.7,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141491703","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Glycoconjugate JournalPub Date : 2024-06-01Epub Date: 2024-05-23DOI: 10.1007/s10719-024-10155-w
Ravneet K Grewal, Priyanka Basu, Sandeep Kaur, Akshdeep Singh
{"title":"Aberrant glycosylation of secretory mucin from the oral cavity in tobacco consumers: a pilot study.","authors":"Ravneet K Grewal, Priyanka Basu, Sandeep Kaur, Akshdeep Singh","doi":"10.1007/s10719-024-10155-w","DOIUrl":"10.1007/s10719-024-10155-w","url":null,"abstract":"<p><p>Mucins are a family of high-molecular-weight O-linked glycoproteins which are the primary structural components of mucus and maintain homeostasis in the oral cavity. The present study was conducted as the first step towards establishing a correlation of aberrant mucin glycosylation with tobacco-associated clinical conditions. Tobacco habituates for the study were identified on the basis of type, duration, amount, and frequency of using tobacco products. The secretory mucin and its saccharides were determined from the saliva collected from smokers, smokeless tobacco habituates, and healthy, nonsmoking individuals. On the one hand, the salivary mucin content was markedly reduced in smokeless tobacco habituates with respect to smokers. On the other hand, the amount of sialic acid and fucose moieties of salivary mucin was increased in both smokers and smokeless tobacco habituates compared to the healthy cohort. Furthermore, the duration of tobacco exposure have been identified as the main factor influencing the extent of damage to the oral mucosa in terms of mucin secretion. The reduced secretory mucin content with aberrant glycosylation in the oral cavity may have a significant role in the further development or progression of oral diseases.</p>","PeriodicalId":12762,"journal":{"name":"Glycoconjugate Journal","volume":" ","pages":"217-224"},"PeriodicalIF":2.7,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141081230","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Peng He, Yuefan Song, Weihua Jin, Yunran Li, Ke Xia, Seon Beom Kim, Rohini Dwivedi, Marwa Farrag, John Bates, Vitor H. Pomin, Chunyu Wang, Robert J. Linhardt, Jonathan S. Dordick, Fuming Zhang
{"title":"Marine sulfated glycans inhibit the interaction of heparin with S-protein of SARS-CoV-2 Omicron XBB variant","authors":"Peng He, Yuefan Song, Weihua Jin, Yunran Li, Ke Xia, Seon Beom Kim, Rohini Dwivedi, Marwa Farrag, John Bates, Vitor H. Pomin, Chunyu Wang, Robert J. Linhardt, Jonathan S. Dordick, Fuming Zhang","doi":"10.1007/s10719-024-10150-1","DOIUrl":"https://doi.org/10.1007/s10719-024-10150-1","url":null,"abstract":"<p>The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide COVID-19 pandemic, leading to 6.8 million deaths. Numerous variants have emerged since its outbreak, resulting in its significantly enhanced ability to spread among humans. As with many other viruses, SARS‑CoV‑2 utilizes heparan sulfate (HS) glycosaminoglycan (GAG) on the surface of host cells to facilitate viral attachment and initiate cellular entry through the ACE2 receptor. Therefore, interfering with virion-HS interactions represents a promising target to develop broad-spectrum antiviral therapeutics. Sulfated glycans derived from marine organisms have been proven to be exceptional reservoirs of naturally existing HS mimetics, which exhibit remarkable therapeutic properties encompassing antiviral/microbial, antitumor, anticoagulant, and anti-inflammatory activities. In the current study, the interactions between the receptor-binding domain (RBD) of S-protein of SARS-CoV-2 (both WT and XBB.1.5 variants) and heparin were applied to assess the inhibitory activity of 10 marine-sourced glycans including three sulfated fucans, three fucosylated chondroitin sulfates and two fucoidans derived from sea cucumbers, sea urchin and seaweed <i>Saccharina japonica</i>, respectively. The inhibitory activity of these marine derived sulfated glycans on the interactions between RBD of S-protein and heparin was evaluated using Surface Plasmon Resonance (SPR). The RBDs of S-proteins from both Omicrion XBB.1.5 and wild-type (WT) were found to bind to heparin, which is a highly sulfated form of HS. All the tested marine-sourced sulfated glycans exhibited strong inhibition of WT and XBB.1.5 S-protein binding to heparin. We believe the study on the molecular interactions between S-proteins and host cell glycosaminoglycans provides valuable insight for the development of marine-sourced, glycan-based inhibitors as potential anti-SARS-CoV-2 agents.</p>","PeriodicalId":12762,"journal":{"name":"Glycoconjugate Journal","volume":"204 1","pages":""},"PeriodicalIF":3.0,"publicationDate":"2024-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140629595","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Emanuela Andretta, Stefania De Chiara, Chiara Pagliuca, Roberta Cirella, Elena Scaglione, Martina Di Rosario, Maxim S. Kokoulin, Olga I. Nedashkovskaya, Alba Silipo, Paola Salvatore, Antonio Molinaro, Flaviana Di Lorenzo
{"title":"Increasing outer membrane complexity: the case of the lipopolysaccharide lipid A from marine Cellulophaga pacifica","authors":"Emanuela Andretta, Stefania De Chiara, Chiara Pagliuca, Roberta Cirella, Elena Scaglione, Martina Di Rosario, Maxim S. Kokoulin, Olga I. Nedashkovskaya, Alba Silipo, Paola Salvatore, Antonio Molinaro, Flaviana Di Lorenzo","doi":"10.1007/s10719-024-10149-8","DOIUrl":"https://doi.org/10.1007/s10719-024-10149-8","url":null,"abstract":"<p>Gram-negative bacteria living in marine waters have evolved peculiar adaptation strategies to deal with the numerous stress conditions that characterize aquatic environments. Among the multiple mechanisms for efficient adaptation, these bacteria typically exhibit chemical modifications in the structure of the lipopolysaccharide (LPS), which is a fundamental component of their outer membrane. In particular, the glycolipid anchor to the membrane of marine bacteria LPSs, i.e. the lipid A, frequently shows unusual chemical structures, which are reflected in equally singular immunological properties with potential applications as immune adjuvants or anti-sepsis drugs. In this work, we determined the chemical structure of the lipid A from <i>Cellulophaga pacifica</i> KMM 3664<sup>T</sup> isolated from the Sea of Japan. This bacterium showed to produce a heterogeneous mixture of lipid A molecules that mainly display five acyl chains and carry a single phosphate and a D-mannose disaccharide on the glucosamine backbone. Furthermore, we proved that <i>C. pacifica</i> KMM 3664<sup>T</sup> LPS acts as a weaker activator of Toll-like receptor 4 (TLR4) compared to the prototypical enterobacterial <i>Salmonella typhimurium</i> LPS. Our results are relevant to the future development of novel vaccine adjuvants and immunomodulators inspired by marine LPS chemistry.</p>","PeriodicalId":12762,"journal":{"name":"Glycoconjugate Journal","volume":"31 1","pages":""},"PeriodicalIF":3.0,"publicationDate":"2024-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140623823","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}