Proceedings of the National Academy of Sciences最新文献

{"title":"Mechanisms underlying drug-mediated regulation of membrane protein function","authors":"R. Rusinova, Changhao He, O. Andersen","doi":"10.1016/J.BPJ.2020.11.1515","DOIUrl":"https://doi.org/10.1016/J.BPJ.2020.11.1515","url":null,"abstract":"","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"30 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73599612","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 second S4 movement opens hyperpolarization-activated HCN channels","authors":"Xiaoan Wu, Rosamary Ramentol, Marta E. Perez, S. Noskov, H. Larsson","doi":"10.1016/J.BPJ.2020.11.1597","DOIUrl":"https://doi.org/10.1016/J.BPJ.2020.11.1597","url":null,"abstract":"","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82622893","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":"Electrical unfolding of cytochrome c during translocation through a nanopore constriction","authors":"Prabhat Tripathi, A. Benabbas, B. Mehrafrooz, Hirohito Yamazaki, A. Aksimentiev, P. Champion, M. Wanunu","doi":"10.1101/2021.02.10.430607","DOIUrl":"https://doi.org/10.1101/2021.02.10.430607","url":null,"abstract":"Significance Can localized electric fields drive the complete unfolding of a protein molecule? Protein unfolding prior to its translocation through a nanopore constriction is an important step in protein transport across biological membranes and also an important step in nanopore-based protein sequencing. We studied here the electric-field–driven translocation behavior of a model protein (cyt c) through nanopores of diameters ranging from 1.5 to 5.5 nm. These single-molecule measurements show that electric fields at the nanopore constriction can select both partially and fully unfolded protein conformations. Zero-field free energy gaps between these conformations, found using a simple thermodynamic model, are in remarkable agreement with previously reported studies of cyt c unfolding energetics. Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field–driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiNx) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of ∼30 to 100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field–induced deformability.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"118 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88337501","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":"Using time-use diaries to track changing behavior across successive stages of COVID-19 social restrictions","authors":"O. Sullivan, J. Gershuny, Almudena Sevilla, Francesca Foliano, Marga Vega-Rapun, Juana Lamote de Grignon, Teresa A. Harms, Pierre Walthery","doi":"10.1101/2021.01.29.21250766","DOIUrl":"https://doi.org/10.1101/2021.01.29.21250766","url":null,"abstract":"Significance What do people do during lockdown? This unique sequence of four time-use diary surveys provides real-time information on changing behavior in the United Kingdom during each major phase of social restrictions. We compare 24-h continuous and comprehensive information on the populations’ activities, their social context, and their location, assessing risk-related behavior during different phases of institutional response to the pandemic. Holding constant gender, age, and social grade, we show that the UK population spent on average 35 more minutes per day in high-risk activity combinations in the second UK lockdown in November 2020 than in the first lockdown (starting March 2020). This difference is shown to be associated with an increase in time spent doing paid work in the workplace. How did people change their behavior over the different phases of the UK COVID-19 restrictions, and how did these changes affect their risk of being exposed to infection? Time-use diary surveys are unique in providing a complete chronicle of daily behavior: 24-h continuous records of the populations’ activities, their social context, and their location. We present results from four such surveys, collected in real time from representative UK samples, both before and at three points over the course of the current pandemic. Comparing across the four waves, we find evidence of substantial changes in the UK population’s behavior relating to activities, locations, and social context. We assign different levels of risk to combinations of activities, locations, and copresence to compare risk-related behavior across successive “lockdowns.” We find evidence that during the second lockdown (November 2020), there was an increase in high-risk behaviors relative to the first (starting March 2020). This increase is shown to be associated with more paid work time in the workplace. At a time when capacity is still limited both in respect of immunization and track–trace technology, governments must continue to rely on changes in people’s daily behaviors to contain the spread of COVID-19 and similar viruses. Time-use diary information of this type, collected in real time across the course of the COVID-19 pandemic, can provide policy makers with information to assess and quantify changes in daily behaviors and the impact they are likely to have on overall behavioral-associated risks.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80968864","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":"Multiscale mechanics and temporal evolution of vimentin intermediate filament networks","authors":"Anna V. Schepers, C. Lorenz, P. Nietmann, A. Janshoff, S. Klumpp, S. Köster","doi":"10.1101/2021.01.30.428887","DOIUrl":"https://doi.org/10.1101/2021.01.30.428887","url":null,"abstract":"Significance The mechanical integrity of cells and their ability to adapt—for example, during wound healing or in metastasizing tumors—is largely determined by the cytoskeleton. The cytoskeleton is an intricate network of biopolymers and cross-linkers. Intermediate filaments, the softest and most extensible of the three filamentous proteins of the cytoskeleton, take the role of a safety belt for cells under strain. The mechanical properties of a network depend on several factors, such as the length and mechanical properties of the single filaments and, importantly, the interactions between filaments. Here, we use a multiscale approach to disentangle these effects, which allows for direct quantification of interaction kinetics. The cytoskeleton, an intricate network of protein filaments, motor proteins, and cross-linkers, largely determines the mechanical properties of cells. Among the three filamentous components, F-actin, microtubules, and intermediate filaments (IFs), the IF network is by far the most extensible and resilient to stress. We present a multiscale approach to disentangle the three main contributions to vimentin IF network mechanics—single-filament mechanics, filament length, and interactions between filaments—including their temporal evolution. Combining particle tracking, quadruple optical trapping, and computational modeling, we derive quantitative information on the strength and kinetics of filament interactions. Specifically, we find that hydrophobic contributions to network mechanics enter mostly via filament-elongation kinetics, whereas electrostatics have a direct influence on filament–filament interactions.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"75 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86374746","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":"Structure of Ycf1p reveals the transmembrane domain TMD0 and the regulatory region of ABCC transporters","authors":"S. Bickers, S. Benlekbir, J. Rubinstein, V. Kanelis","doi":"10.1101/2021.01.29.428621","DOIUrl":"https://doi.org/10.1101/2021.01.29.428621","url":null,"abstract":"Significance The Ycf1p structure provides an atomic model for the TMD0 domain of ABCC transporters and for two segments of the regulatory (R) region that links NBD1 to TMD2. The orientation of TMD0 in Ycf1p differs from that seen in SUR1, the regulatory ABCC protein in KATP channels, demonstrating flexibility in TMD0/ABC core contacts. The structure suggests how posttranslational modifications of the R region modulate ABC protein activity and provides a mechanistic understanding of several diseases that occur due to mutation of human homologs of Ycf1p. ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is composed of two transmembrane domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2). Some members of the C-subfamily of ABC (ABCC) proteins, including human multidrug resistance proteins (MRPs), also possess an N-terminal transmembrane domain (TMD0) that contains five transmembrane α-helices and is connected to the ABC core by the L0 linker. While TMD0 was resolved in SUR1, the atypical ABCC protein that is part of the hetero-octameric ATP-sensitive K+ channel, little is known about the structure of TMD0 in monomeric ABC transporters. Here, we present the structure of yeast cadmium factor 1 protein (Ycf1p), a homolog of human MRP1, determined by electron cryo-microscopy (cryo-EM). A comparison of Ycf1p, SUR1, and a structure of MRP1 that showed TMD0 at low resolution demonstrates that TMD0 can adopt different orientations relative to the ABC core, including a ∼145° rotation between Ycf1p and SUR1. The cryo-EM map also reveals that segments of the regulatory (R) region, which links NBD1 to TMD2 and was poorly resolved in earlier ABCC structures, interacts with the L0 linker, NBD1, and TMD2. These interactions, combined with fluorescence quenching experiments of isolated NBD1 with and without the R region, suggest how posttranslational modifications of the R region modulate ABC protein activity. Mapping known mutations from MRP2 and MRP6 onto the Ycf1p structure explains how mutations involving TMD0 and the R region of these proteins lead to disease.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"60 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77736121","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":"Complexity and graded regulation of neuronal cell-type–specific alternative splicing revealed by single-cell RNA sequencing","authors":"Huijuan Feng, Daniel F. Moakley, Shuonan Chen, Melissa G McKenzie, V. Menon, Chaolin Zhang","doi":"10.1101/2021.01.27.428525","DOIUrl":"https://doi.org/10.1101/2021.01.27.428525","url":null,"abstract":"Significance Alternative splicing (AS) is extensively used in the mammalian brain, but its contribution to the molecular and cellular diversity across neuronal cell types remains poorly understood. Through systematic and integrative analysis of over 100 transcriptomically defined cortical neuronal types, we found neuronal subclass-specific splicing-regulatory programs consist of overlapping alternative exons showing differential splicing at multiple hierarchical levels. Evidence is provided that this graded AS regulation is controlled by unique combinations of RNA-binding proteins (RBPs). Importantly, these RBPs also contribute to splicing dynamics across neuronal cell types that do not conform to the hierarchical taxonomy established based on transcriptional profiles, suggesting that the graded AS regulation may provide a molecular mechanism orthogonal to transcriptional regulation in specifying neuronal identity and function. The enormous cellular diversity in the mammalian brain, which is highly prototypical and organized in a hierarchical manner, is dictated by cell-type–specific gene-regulatory programs at the molecular level. Although prevalent in the brain, the contribution of alternative splicing (AS) to the molecular diversity across neuronal cell types is just starting to emerge. Here, we systematically investigated AS regulation across over 100 transcriptomically defined neuronal types of the adult mouse cortex using deep single-cell RNA-sequencing data. We found distinct splicing programs between glutamatergic and GABAergic neurons and between subclasses within each neuronal class. These programs consist of overlapping sets of alternative exons showing differential splicing at multiple hierarchical levels. Using an integrative approach, our analysis suggests that RNA-binding proteins (RBPs) Celf1/2, Mbnl2, and Khdrbs3 are preferentially expressed and more active in glutamatergic neurons, while Elavl2 and Qk are preferentially expressed and more active in GABAergic neurons. Importantly, these and additional RBPs also contribute to differential splicing between neuronal subclasses at multiple hierarchical levels, and some RBPs contribute to splicing dynamics that do not conform to the hierarchical structure defined by the transcriptional profiles. Thus, our results suggest graded regulation of AS across neuronal cell types, which may provide a molecular mechanism to specify neuronal identity and function that are orthogonal to established classifications based on transcriptional regulation.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"65 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76636680","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 a novel family of integral membrane siderophore reductases","authors":"I. Josts, K. Veith, Vincent Normant, I. Schalk, H. Tidow","doi":"10.1101/2021.01.28.428567","DOIUrl":"https://doi.org/10.1101/2021.01.28.428567","url":null,"abstract":"Significance Secretion of siderophores allows most microbes to assimilate ferric ions into their biological processes. Siderophores must be taken up into the cells, and chelated iron must be released. Here, we present the structure of an inner membrane siderophore reductase, FoxB, which is involved in the uptake of iron from ferrioxamine siderophores in Pseudomonas aeruginosa. Our structure reveals FoxB to be a di-heme membrane protein, which is able to reduce the iron in chelated ferric-siderophore complexes. In combination with in vivo uptake studies, these results offer insights into the function of this poorly characterized membrane protein family and its role in iron release from bacterial siderophores. Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from Pseudomonas aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a fold with two tightly bound heme molecules. In combination with in vitro reduction assays and in vivo iron uptake studies, these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"97 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80501755","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":"Obligate movements of an active site–linked surface domain control RNA polymerase elongation and pausing via a Phe pocket anchor","authors":"Yu Bao, R. Landick","doi":"10.1101/2021.01.27.428476","DOIUrl":"https://doi.org/10.1101/2021.01.27.428476","url":null,"abstract":"Significance RNA synthesis by cellular RNA polymerases depends on an active-site component called the trigger loop that oscillates between an unstructured loop that admits NTP substrates and a helical hairpin that positions the NTP in every round of nucleotide addition. In most bacteria, the trigger loop contains a large, surface-exposed insertion module that occupies different positions in different transcription complexes but whose function during active transcription is unknown. By developing and using a disulfide reporter system, we find the insertion module must also alternate between in and out positions for every nucleotide addition, must swivel to a paused position to support regulation, and in enterobacteria, evolved a “Phe pocket” that captures a key phenylalanine in the out and swivel positions. The catalytic trigger loop (TL) in RNA polymerase (RNAP) alternates between unstructured and helical hairpin conformations to admit and then contact the NTP substrate during transcription. In many bacterial lineages, the TL is interrupted by insertions of two to five surface-exposed, sandwich-barrel hybrid motifs (SBHMs) of poorly understood function. The 188-amino acid, two-SBHM insertion in Escherichia coli RNAP, called SI3, occupies different locations in elongating, NTP-bound, and paused transcription complexes, but its dynamics during active transcription and pausing are undefined. Here, we report the design, optimization, and use of a Cys-triplet reporter to measure the positional bias of SI3 in different transcription complexes and to determine the effect of restricting SI3 movement on nucleotide addition and pausing. We describe the use of H2O2 as a superior oxidant for RNAP disulfide reporters. NTP binding biases SI3 toward the closed conformation, whereas transcriptional pausing biases SI3 toward a swiveled position that inhibits TL folding. We find that SI3 must change location in every round of nucleotide addition and that restricting its movements inhibits both transcript elongation and pausing. These dynamics are modulated by a crucial Phe pocket formed by the junction of the two SBHM domains. This SI3 Phe pocket captures a Phe residue in the RNAP jaw when the TL unfolds, explaining the similar phenotypes of alterations in the jaw and SI3. Our findings establish that SI3 functions by modulating TL folding to aid transcriptional regulation and to reset secondary channel trafficking in every round of nucleotide addition.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80094663","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":"Mapping partner drug resistance to guide antimalarial combination therapy policies in sub-Saharan Africa","authors":"Hanna Y. Ehrlich, A. Bei, D. Weinberger, J. Warren, S. Parikh","doi":"10.2139/SSRN.3756808","DOIUrl":"https://doi.org/10.2139/SSRN.3756808","url":null,"abstract":"Significance Antimalarial resistance has emerged and spread with every antimalarial deployed to date. Currently, parasite genotypes associated with reduced artemisinin and partner drug sensitivity have been reported in Asia, South America, and, most recently, Africa. Analyzing spatial-temporal trends in molecular markers can help policymakers choose efficacious partner drugs and slow the emergence of artemisinin resistance and spread of multidrug-resistant parasites. We display evidence of a continent-wide increase in molecular markers associated with reduced lumefantrine susceptibility, the partner drug of the most widely used artemisinin-based combination therapy in sub-Saharan Africa. We also generate hypotheses for large-scale demographic and environmental risk factors implicated in the spread of antimalarial resistance. Our results can help identify regions of developing parasite resistance that may require enhanced surveillance. Resistance to artemisinin-based combination therapies (ACTs) threatens the global control of Plasmodium falciparum malaria. ACTs combine artemisinin-derived compounds with partner drugs to enable multiple mechanisms of clearance. Although ACTs remain widely effective in sub-Saharan Africa, long-standing circulation of parasite alleles associated with reduced partner drug susceptibility may contribute to the development of clinical resistance. We fitted a hierarchical Bayesian spatial model to data from over 500 molecular surveys to predict the prevalence and frequency of four key markers in transporter genes (pfcrt 76T and pfmdr1 86Y, 184F, and 1246Y) in first-level administrative divisions in sub-Saharan Africa from the uptake of ACTs (2004 to 2009) to their widespread usage (2010 to 2018). Our models estimated that the pfcrt 76T mutation decreased in prevalence in 90% of regions; the pfmdr1 N86 and D1246 wild-type genotypes increased in prevalence in 96% and 82% of regions, respectively; and there was no significant directional selection at the pfmdr1 Y184F locus. Rainfall seasonality was the strongest predictor of the prevalence of wild-type genotypes, with other covariates, including first-line drug policy and transmission intensity more weakly associated. We lastly identified regions of high priority for enhanced surveillance that could signify decreased susceptibility to the local first-line ACT. Our results can be used to infer the degree of molecular resistance and magnitude of wild-type reversion in regions without survey data to inform therapeutic policy decisions.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"88 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73681093","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}