bioRxiv - Biochemistry最新文献

{"title":"Cytosolic CRISPR RNA for RNA-targeting CRISPR-Cas systems","authors":"Ezra C.K. Cheng, S. Chul Kwon","doi":"10.1101/2024.09.18.613662","DOIUrl":"https://doi.org/10.1101/2024.09.18.613662","url":null,"abstract":"Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technologies have evolved rapidly over the past decade with the continuous discovery of new Cas systems. In particular, RNA-targeting CRISPR-Cas13 proteins are promising single-effector systems to regulate target mRNAs without altering genomic DNA, yet the current Cas13 systems are still restrained by suboptimal efficiencies. Here, we show that U1-driven CRISPR RNAs (crRNAs) can dramatically increase the efficiency of various applications, including RNA knockdown and editing, without modifying the Cas13 protein effectors. We confirm that U1-driven crRNAs are exported into the cytoplasm, while conventional U6 promoter-driven crRNAs are mostly confined in the nucleus. Furthermore, we reveal that the end positions of crRNAs expressed by the U1 promoter are consistent regardless of different guide sequences and lengths. We also demonstrate that U1-driven crRNAs, but not U6-driven crRNAs, can efficiently repress the translation of target genes in combination with catalytically inactive Cas13 proteins. Finally, we show that U1-driven crRNAs can counteract the inhibitory effect of miRNAs. Our simple and effective engineering enables unprecedented cytosolic RNA-targeting applications.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142268870","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural insights into terminal arabinosylation biosynthesis of the mycobacterial cell wall arabinan","authors":"Yaqi Liu, Chelsea M. Brown, Satchal Erramilli, Yi-Chia Su, Po-Sen Tseng, Yu-Jen Wang, Nam Ha Duong, Piotr Tokarz, Brian Kloss, Cheng-Ruei Han, Hung-Yu Chen, Jose Rodrigues, Margarida Archer, Todd L. Lowary, Anthony A. Kossiakoff, Phillip J. Stansfeld, Rie Nygaard, Filippo Mancia","doi":"10.1101/2024.09.17.613533","DOIUrl":"https://doi.org/10.1101/2024.09.17.613533","url":null,"abstract":"The emergence of drug-resistant strains exacerbates the global challenge of tuberculosis caused by Mycobacterium tuberculosis (Mtb). Central to the pathogenicity of Mtb is its complex cell envelope, which serves as a barrier against both immune system and pharmacological attacks. Two key components of this envelope, arabinogalactan (AG) and lipoarabinomannan (LAM) are complex polysaccharides that contain integral arabinan domains important for cell wall structural and functional integrity. The arabinofuranosyltransferase AftB terminates the synthesis of these arabinan domains by catalyzing the addition of the addition of β-(1→2)-linked terminal arabinofuranose residues. Here, we present the cryo-EM structures of Mycobacterium chubuense AftB in its apo and donor substrate analog-bound form, determined to 2.9 Å and 3.4 Å resolution, respectively. Our structures reveal that AftB has a GT-C fold transmembrane (TM) domain comprised of eleven TM helices and a periplasmic cap domain. AftB has an irregular tube-shaped cavity that bridges the two proposed substrate binding sites. By integrating structural analysis, biochemical assays, and molecular dynamics simulations, we elucidate the molecular basis of the reaction mechanism of AftB and propose a model for catalysis.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254856","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mechanistic studies of mycobacterial glycolipid biosynthesis by the mannosyltransferase PimE","authors":"Yaqi Liu, Chelsea M. Brown, Nuno Borges, Rodrigo N. Nobre, Satchal Erramilli, Meagan Belcher Dufrisne, Brian Kloss, Sabrina Giacometti, Ana M. Esteves, Cristina G. Timoteo, Piotr Tokarz, Rosemary Cater, Yasu S. Morita, Anthony A. Kossiakoff, Helena Santos, Phillip J. Stansfeld, Rie Nygaard, Filippo Mancia","doi":"10.1101/2024.09.17.613550","DOIUrl":"https://doi.org/10.1101/2024.09.17.613550","url":null,"abstract":"Tuberculosis (TB), exceeded in mortality only by COVID-19 among global infectious diseases, is caused by Mycobacterium tuberculosis (Mtb). The pathogenicity of Mtb is largely attributed to its complex cell envelope, which includes a class of glycolipids called phosphatidyl-myo-inositol mannosides (PIMs), found uniquely in mycobacteria and its related corynebacterineae. These glycolipids maintain the integrity of the mycobacterial cell envelope, regulate its permeability, and mediate host-pathogen interactions. PIMs consist of a phosphatidyl-myo-inositol core decorated with one to six mannose residues and up to four acyl chains. The mannosyltransferase PimE catalyzes the transfer of the fifth PIM mannose residue from a polyprenyl phosphate-mannose (PPM) donor. This step in the biosynthesis of higher-order PIMs contributes to the proper assembly and function of the mycobacterial cell envelope; however, the structural basis for substrate recognition and the catalytic mechanism of PimE remain poorly understood. Here, we present the cryo-electron microscopy (cryo-EM) structures of PimE from Mycobacterium abscessus captured in its apo form and in a product-bound complex with the reaction product Ac1PIM5 and the by-product polyprenyl phosphate (PP), determined at 3.0 Å and 3.5 Å, respectively. The structures reveal the active site within a distinctive binding cavity that accommodates both donor and acceptor substrates/products. Within the cavity, we identified residues involved in substrate coordination and catalysis, which we confirmed through in vitro enzymatic assays and further validated by in vivo complementation experiments. Molecular dynamics simulations were applied to identify the access pathways and the dynamics involved in substrate binding. Integrating structural, biochemical, genetic, and computational experiments, our study provides comprehensive insights into how PimE functions, opening potential avenues for development of novel anti-TB therapeutics.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"50 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142268873","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular activity mediates the composition and assembly of dissolved organic matter in lake sediments","authors":"Shuailong Wen, Ang Hu, Francisco Dini-Andreote, Lei Han, Shuyu Jiang, Kyoung-Soon Jiang, Jianjun Wang","doi":"10.1101/2024.09.17.613390","DOIUrl":"https://doi.org/10.1101/2024.09.17.613390","url":null,"abstract":"Lake sediments are hotspots for carbon transformation and burial, where dissolved organic matter (DOM) interacts with microorganisms to regulate global carbon cycling. The potential for individual DOM molecules to undergo biochemical transformations, i.e., their activity, is a critical molecular trait affecting DOM turnover in environment. However, the composition of sediment DOM and how its assembly mechanisms are related to molecular activity remains poorly understood. Here, 63 freshwater sediments were collected from tropical to cold temperate climatic zones in China. We explored the molecular composition and assembly of sediment DOM and the underlying mechanisms driven by climate, physicochemical factors, and microbes along the molecular activity gradient. Sediment DOM was dominated by lipid- (34.8%) and lignin-like compounds (33.01%), and the latter were enriched as molecular activity of DOM increased. Besides, DOM composed of more active molecules had greater compositional similarity across different climatic zones, and was inclined to assemble deterministically. This was supported by the fact that as potential transformations of molecular assemblages increased from 0.4 to 14, the assembly of these molecules was structured by a shift from stochastic to deterministic processes, with the latter accounting for ≥ 75% thereafter. Overall, the molecular assemblage was primarily structured by physicochemical factors, including sediment total organic carbon and electrical conductivity. As molecular activity increased, however, assemblage was increasingly affected by climate and bacterial communities, consistent with the enhanced complexity of bacterial-molecular networks. Collectively, our study highlights that the intrinsic activity of DOM molecules determines their fate through distinct biotic and abiotic mechanisms.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254855","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Affinity tag free purification of SARS-Cov-2 N protein and its crystal structure in complex with ssDNA","authors":"Atanu Maiti, Hiroshi Matsuo","doi":"10.1101/2024.09.16.613250","DOIUrl":"https://doi.org/10.1101/2024.09.16.613250","url":null,"abstract":"The nucleocapsid (N) protein is one of the four structural proteins in SARS-CoV-2, playing key roles in viral assembly, immune evasion, and stability. One of its primary functions is to protect viral RNA by forming the nucleocapsid. However, the precise mechanisms of how the N protein interacts with viral RNA and assembles into a nucleocapsid remain unclear. Compared to other SARS-CoV-2 components, the N protein has several advantages: higher sequence conservation, lower mutation rates, and stronger immunogenicity, making it an attractive target for antiviral drug development and diagnostics. Therefore, a detailed understanding of the N protein's structure is essential for deciphering its role in viral assembly and for developing effective therapeutics. In this study, we report the expression and purification of a soluble recombinant N protein, along with a 1.55Å resolution crystal structure of its nucleic acid-binding domain (N-NTD) in complex with ssDNA. Our structure reveals new insights into the conformation and interaction of the flexible N-arm, which could aid in understanding nucleocapsid assembly. Additionally, we identify residues that are critical for ssDNA interaction.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Adipocyte sphingosine kinase 1 regulates histone modifiers to disrupt circadian function","authors":"Andrea Anderson, Anna Kovilakath, Maryam Jamil, Johana M Lambert, Lauren Ashley Cowart","doi":"10.1101/2024.09.13.612486","DOIUrl":"https://doi.org/10.1101/2024.09.13.612486","url":null,"abstract":"Circadian rhythms align biological functions with the 24-hour day-night cycle, but modern artificial light disrupts these patterns, contributing to health issues like obesity and cardiovascular disease. The circadian clock operates through a transcriptional-translational feedback loop involving core components such as BMAL1 and CLOCK. Recent research has shown circadian variations in sphingolipid metabolism, specifically sphingosine-1-phosphate (S1P), which plays crucial signaling roles. This study investigates the sphingolipid enzyme, sphingosine kinase 1 (SphK1), which converts sphingosine to S1P, as a circadian-regulated gene in adipocytes. We find that SphK1 expression and activity follow a circadian rhythm, regulated by BMAL1 and CLOCK binding to its promoter. Adipocyte-specific SphK1 knockout mice exhibit disrupted circadian rhythms and impaired adipocyte function. Additionally, SphK1 deficiency leads to reduced histone acetylation and altered histone deacetylase (HDAC) localization, affecting gene regulation. These results highlight the critical role of SphK1 in linking lipid metabolism with circadian biology.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"190 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142268875","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The late onset Alzheimer's disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis","authors":"Georg Jocher, Stefan F. Lichtenthaler, Stephan A. Müller, Hung-En Hsia, Miranda Lastra-Osua, Gözde Özcelik, Laura Isabelle Hofmann, Marlene Aßfalg, Lina Dinkel, Kai Schlepckow, Michael Willem, Christian Haass, Sabina Tahirovic, Carl P Blobel, Xiao Feng","doi":"10.1101/2024.09.13.612888","DOIUrl":"https://doi.org/10.1101/2024.09.13.612888","url":null,"abstract":"The cell surface receptor TREM2 is a key genetic risk factor and drug target in Alzheimer's disease (AD). In the brain, TREM2 is expressed in microglia, where it undergoes proteolytic cleavage, linked to AD risk, but the responsible protease in microglia is still unknown. Another microglia-expressed AD risk factor is catalytically inactive rhomboid 2 (iRhom2, RHBDF2), which binds to and acts as a non-catalytic subunit of the metalloprotease ADAM17. A potential role in TREM2 proteolysis is not yet known. Using microglial-like BV2 cells, bone marrow-derived macrophages and primary murine microglia, we identify iRhom2 as a modifier of ADAM17-mediated TREM2 shedding. Loss of iRhom2 increased TREM2 in cell lysates and at the cell surface and enhanced TREM2 signaling and microglial phagocytosis of the amyloid β-peptide (Aβ). This study establishes ADAM17 as a physiological TREM2 protease in microglia, and suggests iRhom2 as a potential drug target for modulating TREM2 proteolysis in AD.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"39 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254889","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cryo-EM structures of PP2A:B55-Eya3 and PP2A:B55-p107 define PP2A:B55 substrate recruitment","authors":"Sathish KR Padi, Rachel J Godek, Wolfgang Peti, Rebecca Page","doi":"10.1101/2024.09.16.613291","DOIUrl":"https://doi.org/10.1101/2024.09.16.613291","url":null,"abstract":"The phosphoprotein phosphatase (PPP) family of ser/thr phosphatases are responsible for the majority of all ser/thr dephosphorylation in cells. However, unlike their kinase counterpart, they do not achieve specificity via phosphosite recognition sequences, but instead bind substrates and regulators using PPP-specific short linear and/or helical motifs (SLiMs, SHelMs). Protein phosphatase 2A (PP2A) is a highly conserved PPP that regulates cell signaling and is a tumor suppressor. Here, we investigate the mechanisms of substrate and regulator recruitment to the PP2A:B55 holoenzyme to define how substrates and regulators engage B55 and understand, in turn, how these interactions direct phosphosite dephosphorylation. Our cryo-EM structures of PP2A:B55 bound to p107 (substrate) and Eya3 (regulator), coupled with biochemical, biophysical and cell biology assays, show that while B55 associates using a common set of interaction pockets, the mechanisms of substrate and regulator binding can differ substantially. This shows that B55-mediated substrate recruitment is distinct from that observed for PP2A:B56 and other PPPs. It also allowed us to identify the core B55 recruitment motif in Eya3 proteins, a sequence we show is conserved amongst the Eya family. Finally, using NMR-based dephosphorylation assays, we also showed how B55 recruitment directs PP2A:B55 fidelity, via the selective dephosphorylation of specific phosphosites. Because of the key regulatory functions of PP2A:B55 in mitosis and DNA damage repair, these data provide a roadmap for pursuing new avenues to therapeutically target this complex by individually blocking a subset of regulators that use different B55 interaction sites.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"24 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254858","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Potential Method for Identifying Milk Adulteration and Pb(II) Contamination Scenarios Using Principal Component Analysis from Smartphone Photographs","authors":"Alicia Catelyn Chandra, Cheralyn Clarecia Lianto, Felicia Liem Sulimro, Gabriella Anna Santoso, Michelle Aiko Wang, Lie Miah, Norbertus Krisnu Prabowo","doi":"10.1101/2024.09.16.613186","DOIUrl":"https://doi.org/10.1101/2024.09.16.613186","url":null,"abstract":"Heavy metal contaminants and adulteration in cow milk products are major issues affecting milk safety and quality, posing health risks to consumers of all ages. These contaminants are sometimes difficult to detect with the naked eye and can potentially pass sensory tests, particularly in white cow milk. This research explores the detection of lead(II) poisoning in milk post-production and the adulteration of different milk samples using an alternative approach through chemometric techniques based on RGB and Grey Area image analysis. A controlled photography environment was used. We analyzed over 105 samples of control, adulterated, and lead(II)-added milk in this study using image processing software. Each photograph was analyzed to provide triplicate Regions of Interest (ROI), resulting in a total of 315 statistical datasets. We found that Principal Component Analysis (PCA) effectively clustered control white milk and Pb(II)-contaminated milk. Clusters of different adulterants were recognized simply by feeding RGB and Grey Area data into PCA. However, some clusters, such as mixed chocolate milk and white milk with lead(II) contamination, were not well distinguished. In this early-stage method, a comparison study with infrared spectra will be required in future research. This alternative method shows potential promise for deployment in limited settings for real-world food quality surveillance, regulation, and biochemistry experiments.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"48 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142268871","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering a Cell-Based Orthogonal Ubiquitin Transfer Cascade for Profiling the Substrates of RBR E3 Parkin","authors":"Shuai Fang, Li Zhou, Geng Chen, Xiaoyu Wang, In Ho Jeong, Savannah E Jacobs, Bradley R. Kossmann, Wei Wei, Jing Zhang, Geon Jeong, Ivaylo Ivanov, Angela M Mabb, Hiroaki Kiyokawa, Bo Zhao, Jun Yin","doi":"10.1101/2024.09.14.613079","DOIUrl":"https://doi.org/10.1101/2024.09.14.613079","url":null,"abstract":"The E3 ubiquitin (UB) ligase Parkin utilizes a Ring-Between-Ring (RBR) domain to mediate the transfer of UB to its substrates to regulate diverse cellular functions, including mitochondrial quality control, cell cycle progression, metabolism programming, and the establishment of synaptic functions. Mutations affecting the E3 ligase activity of Parkin are associated with cancer and Parkinson's disease (PD). An essential role of Parkin is to synthesize UB chains on the surface of damaged mitochondria to initiate mitophagy. Still, it is not clear how Parkin carries out other biological functions through the ubiquitination of its downstream targets in the cell. We hypothesized that a comprehensive substrate profile of Parkin would facilitate the discovery of ubiquitination pathways underpinning its multifaceted roles in cell regulation and reveal mechanistic linkages between Parkin malfunction and disease development. Here, we used phage display to assemble an orthogonal ubiquitin transfer (OUT) cascade of Parkin that can exclusively deliver an engineered UB mutant (xUB) to Parkin and its substrates in living cells. We then generated a substrate profile of Parkin by purifying xUB-conjugated proteins from cells and identifying them by proteomics. The OUT screen identified Parkin substrates involved in DNA replication, protein translation, intracellular protein transport, and rhythmic regulation. Based on previous literature implicating alterations in membrane vesicle trafficking in PD, we verified Parkin-catalyzed ubiquitination of Rab GTPases (Rab1a, Rab5a, Rab5c, Rab7a, Rab8a, Rab10, an Rab13) as well as CDK5, with reconstituted ubiquitination reactions in vitro and in cells. We also found chemical-induced stimulation of mitophagy enhanced Parkin-mediated ubiquitination of Rab proteins. These findings demonstrate that the OUT cascade of Parkin can serve as an empowering tool for identifying Parkin substrates to elucidate its cellular functions.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254890","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}