Nature Protocols最新文献

{"title":"Tutorial: fluorescence lifetime microscopy of membrane mechanosensitive Flipper probes","authors":"Chloé Roffay, Juan Manuel García-Arcos, Pierrik Chapuis, Javier López-Andarias, Falk Schneider, Adai Colom, Caterina Tomba, Ilaria Di Meglio, Katia Barrett, Valentin Dunsing, Stefan Matile, Aurélien Roux, Vincent Mercier","doi":"10.1038/s41596-024-01027-6","DOIUrl":"10.1038/s41596-024-01027-6","url":null,"abstract":"Measuring forces within living cells remains a technical challenge. In this Tutorial, we cover the development of hydrophobic mechanosensing fluorescent probes called Flippers, whose fluorescence lifetime depends on lipid packing. Flipper probes can therefore be used as reporters for membrane tension via the measurement of changes in their fluorescence lifetime. We describe the technical optimization of the probe for imaging and provide working examples for their characterizations in a variety of biological and in vitro systems. We further provide a guideline to measure biophysical parameters of cellular membranes by fluorescence lifetime imaging microscopy using Flipper probes, providing evidence that flippers can report long range forces in cells, tissues and organs. A Tutorial review on the measurement of membrane mechanical forces reported via fluorescence lifetime variations induced by conformational changes of the Flipper probes.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 12","pages":"3457-3469"},"PeriodicalIF":13.1,"publicationDate":"2024-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142109660","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genome-wide analysis of the biophysical properties of chromatin and nuclear proteins in living cells with Hi-D","authors":"Cesar Augusto Valades-Cruz, Roman Barth, Marwan Abdellah, Haitham A. Shaban","doi":"10.1038/s41596-024-01038-3","DOIUrl":"10.1038/s41596-024-01038-3","url":null,"abstract":"To understand the dynamic nature of the genome, the localization and rearrangement of DNA and DNA-binding proteins must be analyzed across the entire nucleus of single living cells. Recently, we developed a computational light microscopy technique, called high-resolution diffusion (Hi-D) mapping, which can accurately detect, classify and map diffusion dynamics and biophysical parameters such as the diffusion constant, the anomalous exponent, drift velocity and model physical diffusion from the data at a high spatial resolution across the genome in living cells. Hi-D combines dense optical flow to detect and track local chromatin and nuclear protein motion genome-wide and Bayesian inference to characterize this local movement at nanoscale resolution. Here we present the Python implementation of Hi-D, with an option for parallelizing the calculations to run on multicore central processing units (CPUs). The functionality of Hi-D is presented to the users via user-friendly documented Python notebooks. Hi-D reduces the analysis time to less than 1 h using a multicore CPU with a single compute node. We also present different applications of Hi-D for live-imaging of DNA, histone H2B and RNA polymerase II sequences acquired with spinning disk confocal and super-resolution structured illumination microscopy. High-resolution diffusion uses a dense optical flow algorithm to quantify and classify the motion of nuclear macromolecules, assigning biophysical parameters such as the diffusion constant, the anomalous exponent and the drift velocity.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 1","pages":"163-179"},"PeriodicalIF":13.1,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142093563","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"STRAIGHT-IN: a platform for rapidly generating panels of genetically modified human pluripotent stem cell lines.","authors":"Albert Blanch-Asensio, Catarina Grandela, Christine L Mummery, Richard P Davis","doi":"10.1038/s41596-024-01039-2","DOIUrl":"https://doi.org/10.1038/s41596-024-01039-2","url":null,"abstract":"<p><p>Targeted integration of large DNA cargoes (>10 kb) or genomic replacements in mammalian cells, such as human pluripotent stem cells (hPS cells), remains challenging. Here we describe a platform termed serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation (STRAIGHT-IN) to circumvent this. First, a landing pad cassette is precisely inserted or used to replace specific genomic regions. The site-specific integrase Bxb1 then enables DNA constructs, including those >50 kb, to be integrated into the genome, while Cre recombinase excises auxiliary DNA sequences to prevent postintegrative silencing. Using a strategy whereby the positive selection marker is only expressed if the donor plasmid carrying the payload is correctly targeted, we can obtain 100% enrichment for cells containing the DNA payload. Procedures for expressing Cre efficiently also mean that a clonal isolation step is no longer essential to derive the required genetically modified hPS cells containing the integrated DNA, potentially reducing clonal variability. Furthermore, STRAIGHT-IN facilitates rapid and multiplexed generation of genetically matched hPS cells when multiple donor plasmids are delivered simultaneously. STRAIGHT-IN has various applications, which include integrating complex genetic circuits for synthetic biology, as well as creating panels of hPS cells lines containing, as necessary, hundreds of disease-linked variants for disease modeling and drug discovery. After establishing the hPS cell line containing the landing pad, the entire procedure, including donor plasmid synthesis, takes 1.5-3 months, depending on whether single or multiple DNA payloads are integrated. This protocol only requires the researcher to be skilled in molecular biology and standard cell culture techniques.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142046915","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fabrication of cyborg bacterial cells as living cell–material hybrids using intracellular hydrogelation","authors":"Ofelya Baghdasaryan,&nbsp;Luis E. Contreras-Llano,&nbsp;Shahid Khan,&nbsp;Aijun Wang,&nbsp;Che-Ming Jack Hu,&nbsp;Cheemeng Tan","doi":"10.1038/s41596-024-01035-6","DOIUrl":"10.1038/s41596-024-01035-6","url":null,"abstract":"The production of living therapeutics, cell-based delivery of drugs and gene-editing tools and the manufacturing of bio-commodities all share a common concept: they use either a synthetic or a living cell chassis to achieve their primary engineering or therapeutic goal. Live-cell chassis face limitations inherent to their auto-replicative nature and the complexity of the cellular context. This limitation highlights the need for a new chassis combining the engineering simplicity of synthetic materials and the functionalities of natural cells. Here, we describe a protocol to assemble a synthetic polymeric network inside bacterial cells, rendering them incapable of cell division and allowing them to resist environmental stressors such as high pH, hydrogen peroxide and cell-wall-targeting antibiotics that would otherwise kill unmodified bacteria. This cellular bioengineering protocol details how bacteria can be transformed into single-lifespan devices that are resistant to environmental stressors and possess programable functionality. We designate the modified bacteria as cyborg bacterial cells. This protocol expands the synthetic biology toolset, conferring precise control over living cells and creating a versatile cell chassis for biotechnology, biomedical engineering and living therapeutics. The protocol, including the preparation of gelation reagents and chassis strain, can be completed in 4 d. The implementation of the protocol requires expertise in microbiology techniques, hydrogel chemistry, fluorescence microscopy and flow cytometry. Further functionalization of the cyborg bacterial cells and adaptation of the protocol requires skills ranging from synthetic genetic circuit engineering to hydrogel polymerization chemistries. This protocol describes the generation of cyborg bacterial cells by using intracellular hydrogelation. These nondividing, stressor-resistant engineered bacteria chassis can find application as living environmental biosensors and in disease treatment.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 12","pages":"3613-3639"},"PeriodicalIF":13.1,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142036441","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis of N-heterocycles through alcohol dehydrogenative coupling","authors":"Bhaskar Paul,&nbsp;Dibyajyoti Panja,&nbsp;Sabuj Kundu","doi":"10.1038/s41596-024-01031-w","DOIUrl":"10.1038/s41596-024-01031-w","url":null,"abstract":"Nitrogen heterocycles are found in the structures of many biologically important compounds, as well as materials used in the synthesis of fine chemicals. Notably, ~59% of US Food and Drug Administration-approved small-molecule drugs contain nitrogen heterocycles. It is therefore meaningful to explore greener or more sustainable methods for their synthesis. The use of alcohols as reagents is attractive as they can be readily obtained from biomass derived natural resources. In the last two decades, alcohol dehydrogenative coupling reaction to synthesize various heterocycles were extensively explored which furnished hydrogen (H2) and water (H2O) as the two greener byproducts. In this protocol, we describe several efficient catalytic transformations to synthesize quinolines, 1,8-naphthyridines, quinoxalines, quinazolines, pyrimidines, benzimidazoles, pyrroles and pyridines, using alcohol as starting materials. We also describe the synthesis of several homogeneous iridium/ruthenium catalysts and heterogeneous cobalt/copper catalysts that can be used in these transformations. The reaction setup is simple; in a Schlenk/reaction tube with magnetic stir-bar, alcohol, corresponding coupling reagents (nucleophiles), catalyst, base and solvent (water or organic solvent such as toluene, dioxane or p-xylene) are added. The reaction mixture is refluxed at the specified temperature (110–150 °C)—either in air or under argon—to furnish these heterocycles. Synthesis of the catalysts takes 3–5 h and the coupling reactions take 4–5 h depending on the target product. The cobalt- and copper-based heterogeneous catalytic systems displayed an good catalyst recyclability. Nitrogen heterocycles are a large class of chemically and biologically relevant compounds. This protocol describes their synthesis from alcohols using homogeneous iridium/ruthenium catalysts and heterogeneous cobalt/copper catalysts.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 12","pages":"3640-3676"},"PeriodicalIF":13.1,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142036443","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Native MS-guided lipidomics to define endogenous lipid microenvironments of eukaryotic receptors and transporters","authors":"Di Wu,&nbsp;Haiping Tang,&nbsp;Xingyu Qiu,&nbsp;Siyuan Song,&nbsp;Siyun Chen,&nbsp;Carol V. Robinson","doi":"10.1038/s41596-024-01037-4","DOIUrl":"10.1038/s41596-024-01037-4","url":null,"abstract":"The mammalian membrane is composed of various eukaryotic lipids interacting with extensively post-translationally modified proteins. Probing interactions between these mammalian membrane proteins and their diverse and heterogeneous lipid cohort remains challenging. Recently, native mass spectrometry (MS) combined with bottom-up ‘omics’ approaches has provided valuable information to relate structural and functional lipids to membrane protein assemblies in eukaryotic membranes. Here we provide a step-by-step protocol to identify and provide relative quantification for endogenous lipids bound to mammalian membrane proteins and their complexes. Using native MS to guide our lipidomics strategies, we describe the necessary sample preparation steps, followed by native MS data acquisition, tailored lipidomics and data interpretation. We also highlight considerations for the integration of different levels of information from native MS and lipidomics and how to deal with the various challenges that arise during the experiments. This protocol begins with the preparation of membrane proteins from mammalian cells and tissues for native MS. The results enable not only direct assessment of copurified endogenous lipids but also determination of the apparent affinities of specific lipids. Detailed sample preparation for lipidomics analysis is also covered, along with comprehensive settings for liquid chromatography–MS analysis. This protocol is suitable for the identification and quantification of endogenous lipids, including fatty acids, sterols, glycerolipids, phospholipids and glycolipids and can be used to interrogate proteins from recombinant sources to native membranes. Native mass spectrometry can be combined with lipidomic experiments to determine the structural and functional lipids of receptor and transporter assemblies. This protocol describes how to use initial native mass spectrometry results to guide experimental design.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 1","pages":"1-25"},"PeriodicalIF":13.1,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142036442","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proximity sequencing for the detection of mRNA, extracellular proteins and extracellular protein complexes in single cells","authors":"Luke Vistain,&nbsp;Bijentimala Keisham,&nbsp;Junjie Xia,&nbsp;Hoang Van Phan,&nbsp;Savaş Tay","doi":"10.1038/s41596-024-01030-x","DOIUrl":"10.1038/s41596-024-01030-x","url":null,"abstract":"Complex cellular functions occur via the coordinated formation and dissociation of protein complexes. Functions such as the response to a signaling ligand can incorporate dozens of proteins and hundreds of complexes. Until recently, it has been difficult to measure multiple protein complexes at the single-cell level. Here, we present a step-by-step procedure for proximity sequencing, which enables the simultaneous measurement of proteins, mRNA and hundreds of protein complexes located on the outer membrane of cells. We guide the user through probe creation, sample preparation, staining, sequencing and computational quantification of protein complexes. This protocol empowers researchers to study, for example, the interplay between transcriptional states and cellular functions by coupling measurements of transcription to measurements of linked effector molecules, yet could be generalizable to other paired events. The protocol requires roughly 16 h spread over several days to complete by users with expertise in basic molecular biology and single-cell sequencing. Proximity sequencing uses a panel of antibodies to probe single cells for up to hundreds of targets simultaneously. The approach is capable of detecting targets that are located within 50–70 nm of each other and thus likely to form complexes.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 12","pages":"3568-3589"},"PeriodicalIF":13.1,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141988358","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimizing the standardized assays for determining the catalytic activity and kinetics of peroxidase-like nanozymes","authors":"Jia-Jia Zheng,&nbsp;Feiyan Zhu,&nbsp;Ningning Song,&nbsp;Fang Deng,&nbsp;Qi Chen,&nbsp;Chen Chen,&nbsp;Jiuyang He,&nbsp;Xingfa Gao,&nbsp;Minmin Liang","doi":"10.1038/s41596-024-01034-7","DOIUrl":"10.1038/s41596-024-01034-7","url":null,"abstract":"Nanozymes are nanomaterials with enzyme-like catalytic properties. They are attractive reagents because they do not have the same limitations of natural enzymes (e.g., high cost, low stability and difficult storage). To test, optimize and compare nanozymes, it is important to establish fundamental principles and systematic standards to fully characterize their catalytic performance. Our 2018 protocol describes how to characterize the catalytic activity and kinetics of peroxidase nanozymes, the most widely used type of nanozyme. This approach was based on Michaelis–Menten enzyme kinetics and is now updated to take into account the unique physicochemical properties of nanomaterials that determine the catalytic kinetics of nanozymes. The updated procedure describes how to determine the number of active sites as well as other physicochemical properties such as surface area, shape and size. It also outlines how to calculate the hydroxyl adsorption energy from the crystal structure using the density functional theory method. The calculations now incorporate these measurements and computations to better characterize the catalytic kinetics of peroxidase nanozymes that have different shapes, sizes and compositions. This updated protocol better describes the catalytic performance of nanozymes and benefits the development of nanozyme research since further nanozyme development requires precise control of activity by engineering the electronic, geometric structure and atomic configuration of the catalytic sites of nanozymes. The characterization of the catalytic activity of peroxidase nanozymes and the evaluation of their kinetics can be performed in 4 h. The procedure is suitable for users with expertise in nano- and materials technology. Developing optimal nanozymes requires standardized methods for measuring their catalytic activity and reaction kinetics. This protocol integrates enzyme based Michaelis–Menten kinetics with measured physical properties and computational methods.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 12","pages":"3470-3488"},"PeriodicalIF":13.1,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141988357","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP","authors":"Andrew Leduc,&nbsp;Luke Khoury,&nbsp;Joshua Cantlon,&nbsp;Saad Khan,&nbsp;Nikolai Slavov","doi":"10.1038/s41596-024-01033-8","DOIUrl":"10.1038/s41596-024-01033-8","url":null,"abstract":"Single-cell proteomics by mass spectrometry (MS) allows the quantification of proteins with high specificity and sensitivity. To increase its throughput, we developed nano-proteomic sample preparation (nPOP), a method for parallel preparation of thousands of single cells in nanoliter-volume droplets deposited on glass slides. Here, we describe its protocol with emphasis on its flexibility to prepare samples for different multiplexed MS methods. An implementation using the plexDIA MS multiplexing method, which uses non-isobaric mass tags to barcode peptides from different samples for data-independent acquisition, demonstrates accurate quantification of ~3,000–3,700 proteins per human cell. A separate implementation with isobaric mass tags and prioritized data acquisition demonstrates analysis of 1,827 single cells at a rate of &gt;1,000 single cells per day at a depth of 800–1,200 proteins per human cell. The protocol is implemented by using a cell-dispensing and liquid-handling robot—the CellenONE instrument—and uses readily available consumables, which should facilitate broad adoption. nPOP can be applied to all samples that can be processed to a single-cell suspension. It takes 1 or 2 d to prepare &gt;3,000 single cells. We provide metrics and software (the QuantQC R package) for quality control and data exploration. QuantQC supports the robust scaling of nPOP to higher plex reagents for achieving reliable and scalable single-cell proteomics. nPOP is a method for parallel preparation of thousands of single cells in nanoliter-volume droplets deposited on glass slides by using the CellenONE instrument. This protocol describes the liquid handling for multiplexed mass spectrometry proteomics.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 12","pages":"3750-3776"},"PeriodicalIF":13.1,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141907079","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Flux synthesis of two-dimensional covalent organic frameworks","authors":"Zhifang Wang,&nbsp;Yushu Zhang,&nbsp;Jinjin Liu,&nbsp;Yao Chen,&nbsp;Peng Cheng,&nbsp;Zhenjie Zhang","doi":"10.1038/s41596-024-01028-5","DOIUrl":"10.1038/s41596-024-01028-5","url":null,"abstract":"Covalent organic frameworks (COFs) are crystalline porous polymers constructed from organic building blocks into ordered two- or three-dimensional networks through dynamic covalent bonds. Attributed to their high porosity, well-defined structure, tailored functionality and excellent chemical stability, COFs have been considered ideal sorbents for various separation applications. The synthesis of COFs mainly employs the solvothermal method, which usually requires organic solvents in sealed Pyrex tubes, resulting in unscalable powdery products and environmental pollution that seriously limits their practical applications. Herein, our protocol focuses on an emerging synthesis method for COFs based on organic flux synthesis without adding solvents. The generality of this synthesis protocol has been applied in preparing various types of COFs, including olefin-linked, imide-linked, Schiff-based COFs on both gram and kilogram scales. Furthermore, organic flux synthesis avoids the disadvantages of solvothermal synthesis and enhances the crystallization and porosity of COFs. Typically, COF synthesis takes 3–5 d to complete, and subsequent washing procedures leading to pure COFs need 1 d. The procedure for kilogram-scale production of COFs with commercially available monomers is also provided. The resulting COFs are suitable for separation applications, particularly as adsorbent materials for industrial gas separation and water treatment applications. The protocol is suited for users with prior expertise in the synthesis of inorganic materials and porous organic materials. This protocol describes a flux synthesis approach for two-dimensional covalent organic frameworks. Compared with other approaches, this method does not use solvents, making it environmentally friendly, and is scalable up to the kilogram scale.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 12","pages":"3489-3519"},"PeriodicalIF":13.1,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141902358","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}