Nature Protocols最新文献

{"title":"Construction of complex bacteriogenic protocells from living material assembly.","authors":"Can Xu, Mei Li, Nicolas Martin, Stephen Mann","doi":"10.1038/s41596-025-01148-6","DOIUrl":"https://doi.org/10.1038/s41596-025-01148-6","url":null,"abstract":"<p><p>Protocell research offers diverse opportunities to understand cellular processes and the foundations of life and holds attractive potential applications across various fields. However, it is still a formidable task to construct a true-to-life synthetic cell with high organizational and functional complexity. Here we present a protocol for constructing bacteriogenic protocells by employing prokaryotes as on-site repositories of compositional, functional and structural building blocks to address this challenge. This approach is based on the capture and processing of two spatially segregated bacterial colonies within individual coacervate microdroplets to produce membrane-bounded, molecularly crowded, compositionally, structurally and functionally complex synthetic cells. The bacteriogenic protocells inherit sufficient biological components from their bacterial building units to exhibit highly integrated life-like properties, including biocatalysis, glycolysis and gene expression. The protocells can be endogenously remodeled to acquire diverse proto-organelles including a spatially partitioned nucleus-like DNA/histone-based condensate to store genetic material, membrane-bounded water vacuoles to adjust cellular osmotic pressure, a three-dimensional network of F-actin proto-cytoskeleton to support structural stability and proto-mitochondria to generate endogenous ATP as source of energy. The protocells ultimately develop a nonspherical morphology due to the continuous biogeneration of metabolic products by implanted living bacteria cells. This protocol provides a novel living material assembly strategy for the construction of functional protoliving microdevices and offers opportunities for potential applications in engineered synthetic biology and biomedicine. The protocol takes ~27 d to complete and requires expertise in microbiology, phase separation, biochemistry and molecular biology related techniques.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143567652","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":"Unraveling cell-cell communication with NicheNet by inferring active ligands from transcriptomics data.","authors":"Chananchida Sang-Aram, Robin Browaeys, Ruth Seurinck, Yvan Saeys","doi":"10.1038/s41596-024-01121-9","DOIUrl":"https://doi.org/10.1038/s41596-024-01121-9","url":null,"abstract":"<p><p>Ligand-receptor interactions constitute a fundamental mechanism of cell-cell communication and signaling. NicheNet is a well-established computational tool that infers ligand-receptor interactions that potentially regulate gene expression changes in receiver cell populations. Whereas the original publication delves into the algorithm and validation, this paper describes a best practices workflow cultivated over four years of experience and user feedback. Starting from the input single-cell expression matrix, we describe a 'sender-agnostic' approach that considers ligands from the entire microenvironment and a 'sender-focused' approach that considers ligands only from cell populations of interest. As output, users will obtain a list of prioritized ligands and their potential target genes, along with multiple visualizations. We include further developments made in NicheNet v2, in which we have updated the data sources and implemented a downstream procedure for prioritizing cell type-specific ligand-receptor pairs. Although a standard NicheNet analysis takes <10 min to run, users often invest additional time in making decisions about the approach and parameters that best suit their biological question. This paper serves to aid in this decision-making process by describing the most appropriate workflow for common experimental designs like case-control and cell-differentiation studies. Finally, in addition to the step-by-step description of the code, we also provide wrapper functions that enable the analysis to be run in one line of code, thus tailoring the workflow to users at all levels of computational proficiency.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143557451","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":"Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.","authors":"Srishti U Sahu, Madalena Castro, Joseph J Muldoon, Kunica Asija, Stacia K Wyman, Netravathi Krishnappa, Lorena de Oñate, Justin Eyquem, David N Nguyen, Ross C Wilson","doi":"10.1038/s41596-025-01154-8","DOIUrl":"10.1038/s41596-025-01154-8","url":null,"abstract":"<p><p>Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in and base editing. The protocol involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) homology-directed repair template (HDRT) DNA may be included. In contrast to electroporation, PERC is appealing because it needs no dedicated hardware and has less impact on cell phenotype and viability. Because of the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Editing efficiencies can surpass 90% when using either Cas9 or Cas12a in primary T cells or HSPCs. After 3 h dedicated to reagent preparation, the PERC delivery step can be completed in 1 h, with the associated cell culture steps taking 3-7 d total. Because the protocol calls for only three readily available reagents (protein, RNA and peptide) and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes the protocol appealing for rapid deployment in research and clinical settings.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143541536","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":"A guide to reverse metabolomics-a framework for big data discovery strategy.","authors":"Vincent Charron-Lamoureux, Helena Mannochio-Russo, Santosh Lamichhane, Shipei Xing, Abubaker Patan, Paulo Wender Portal Gomes, Prajit Rajkumar, Victoria Deleray, Andrés Mauricio Caraballo-Rodríguez, Kee Voon Chua, Lye Siang Lee, Zhao Liu, Jianhong Ching, Mingxun Wang, Pieter C Dorrestein","doi":"10.1038/s41596-024-01136-2","DOIUrl":"https://doi.org/10.1038/s41596-024-01136-2","url":null,"abstract":"<p><p>Untargeted metabolomics is evolving into a field of big data science. There is a growing interest within the metabolomics community in mining tandem mass spectrometry (MS/MS)-based data from public repositories. In traditional untargeted metabolomics, samples to address a predefined question are collected and liquid chromatography with MS/MS data are generated. We then identify metabolites associated with a phenotype (for example, disease versus healthy) and elucidate or validate their structural details (for example, molecular formula, structural classification, substructure or complete structural annotation or identification). In reverse metabolomics, we start with MS/MS spectra for known or unknown molecules. These spectra are used as search terms to search public data repositories to discover phenotype-relevant information such as organ/biofluid distribution, disease condition, intervention status (for example, pre- and postintervention), organisms (for example, mammals versus others), geography and any other biologically relevant associations. Here we guide the reader through a four-part process: (1) obtaining the MS/MS spectra of interest (Universal Spectrum Identifier) and (2) Mass Spectrometry Search Tool searches to find the files associated with the MS/MS that are in available databases, (3) using the Reanalysis Data User Interface framework to link the files with their metadata and (4) validating the observations. Parts 1-3 could take from hours to days depending on the method used for collecting MS/MS spectra. For example, we use MS/MS spectra from three small molecules: phenylalanine-cholic acid (a microbially conjugated bile acid), phenylalanine-C4:0 and histidine-C4:0 (two N-acyl amides). We leverage the Global Natural Products Social Molecular Networking-based framework to explore the microbial producers of these molecules and their associations with health conditions and organ distributions in humans and rodents.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143531663","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":"'Back-to-base' combined hypothermic and normothermic machine perfusion of human donor livers.","authors":"Otto B van Leeuwen, Veerle A Lantinga, Bianca Lascaris, Adam M Thorne, Silke B Bodewes, Maarten W Nijsten, Vincent E de Meijer, Robert J Porte","doi":"10.1038/s41596-024-01130-8","DOIUrl":"10.1038/s41596-024-01130-8","url":null,"abstract":"<p><p>The shortage of suitable donor organs has resulted in the use of suboptimal, high-risk, extended-criteria donor (ECD) livers, which are at an increased risk of failure after transplantation. Compared with traditional static cold storage, dynamic preservation by ex situ machine perfusion reduces the risks associated with the transplantation of ECD organs. Ex situ machine perfusion strategies differ in timing (that is, speed of procurement and transport), perfusion duration and perfusion temperature. For 'back-to-base' protocols, the donor liver is statically cold stored during transportation to the recipient hospital (the 'base') and then perfused, instead of transporting the liver using a portable perfusion system. While dual hypothermic (8-12 °C) oxygenated machine perfusion (DHOPE) allows safe prolongation of preservation duration and reduces ischemia-reperfusion injury-related complications, including post-transplant cholangiopathy, normothermic machine perfusion (NMP) at 35-37 °C facilitates ex situ viability testing of both liver parenchyma and bile ducts. Here, we describe a clinical protocol for 'back-to-base' combined DHOPE and NMP, linked by a period of controlled oxygenated rewarming (COR), which we call the DHOPE-COR-NMP protocol. This protocol enables restoration of mitochondrial function after static ischemic preservation and minimizes both ischemia-reperfusion and temperature-shift-induced injury during the start of NMP. The NMP phase allows viability assessment before final donor liver acceptance for transplantation. Sequential DHOPE and COR-NMP may reduce the risks associated with transplantation of ECD livers and facilitate enhanced utilization, thereby helping to alleviate the organ shortage.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143516242","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":"Retina-specific laminin-based generation of photoreceptor progenitors from human pluripotent stem cells under xeno-free and chemically defined conditions.","authors":"Wei Sheng Tan, Yixin Lai, Yingying Chung, Swarnaseetha Adusumalli, Xin Yi Lee, Karl Tryggvason, Hwee Goon Tay","doi":"10.1038/s41596-025-01142-y","DOIUrl":"https://doi.org/10.1038/s41596-025-01142-y","url":null,"abstract":"<p><p>Photoreceptor cell replacement therapy for retinal degenerative diseases is a promising approach. Presently, most protocols aimed at generating clinically safe and functional cells for retinal diseases face challenges such as low efficiency, poor reproducibility, and time-consuming and complex procedures. These could be due to the dependency on animal-derived components in cell culture media and substrates that support the cell differentiation process. Such conditions are poorly defined chemically, which could affect the robustness of the method and hinder clinical translation of cell therapy in retinal diseases. Here, we describe a simple protocol that is xenogen free and chemically defined to differentiate human embryonic stem cells to photoreceptor progenitors. Human recombinant extracellular matrix laminin 523 and 521 isoforms were used to mimic the inter-photoreceptor matrix niche environment to promote the retinal cell differentiation process. This was also accomplished by the unique combination of two cell differentiation media that recapitulates the retinal development signaling processes. In comparison to other protocols, our protocol does not require any mechanical dissection, which can be technically subjective and tedious. Our directed differentiation method generates photoreceptor progenitors that express ~17% cone-rod homeobox (CRX) transcript based on single-cell transcriptomic analyses by day 32. These day 32 photoreceptor progenitors can be cryopreserved and still maintain high cell viability after thawing for cell transplantation. This protocol can be easily reproduced and performed by researchers with basic cell culture experience, which is particularly important for retinal research progress and clinical cell manufacturing in a Good Manufacturing Practice facility.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143502413","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":"Circular Vectors as an efficient, fully synthetic, cell-free approach for preparing small circular DNA as a plasmid substitute for guide RNA expression in CRISPR-Cas9 genome editing.","authors":"Roman Teo Oliynyk, George M Church","doi":"10.1038/s41596-024-01138-0","DOIUrl":"https://doi.org/10.1038/s41596-024-01138-0","url":null,"abstract":"<p><p>Robust expression of guide RNA (gRNA) is essential for successful implementation of CRISPR-Cas9 genome-editing methods. The gRNA components, such as an RNA polymerase promoter followed by the gRNA coding sequence and an RNA polymerase terminator sequence, and the Cas9 protein are expressed either via an all-in-one plasmid or separate dedicated plasmids. The preparation of such plasmids involves a laborious multi-day process of DNA assembly, bacterial cloning, validation, purification and sequencing. Our Circular Vector (CV) protocol introduces an efficient, fully synthetic, cell-free approach for preparing gRNA expression templates suitable for transfection, marking a significant advancement over traditional plasmid-based approaches. This protocol consists of the circularization and purification of linear double-stranded DNA (dsDNA) containing gRNA expression elements into compact, bacterial-backbone-free circular DNA expression vectors in as little as 3 h. We provide a guide to the design of the dsDNA template coding for gRNA elements for CRISPR-Cas9 base and prime editing, along with step-by-step instructions for the efficient preparation of gRNA-expressing CVs. In addition to rapid preparation, CVs created via this protocol offer several key advantages: a compact size, absence of a bacterial backbone, absence of bacterial endotoxins and no contamination by bacterial RNA or DNA fragments. These features make gRNA-expressing CVs a superior choice over plasmid-based gRNA expression templates.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492717","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":"Linking single-cell transcriptomes with secretion using SEC-seq.","authors":"Justin Langerman, Sevana Baghdasarian, Rene Yu-Hong Cheng, Richard G James, Kathrin Plath, Dino Di Carlo","doi":"10.1038/s41596-024-01112-w","DOIUrl":"https://doi.org/10.1038/s41596-024-01112-w","url":null,"abstract":"<p><p>Cells secrete numerous proteins and other biomolecules into their surroundings to achieve critical functions-from communicating with other cells to blocking the activity of pathogens. Secretion of cytokines, growth factors, extracellular vesicles and even recombinant biologic drugs defines the therapeutic potency of many cell therapies. However, gene expression states that drive specific secretory phenotypes are largely unknown. We provide a protocol that enables the secretion amount of a target protein encoded (SEC) by oligonucleotide barcodes to be linked with transcriptional sequencing (seq) for thousands of single cells. SEC-seq leverages microscale hydrogel particles called Nanovials to isolate cells and capture their secretions in close proximity, oligonucleotide-labeled antibodies to tag secretions on Nanovials and flow cytometry and single-cell RNA-sequencing (scRNA-seq) platforms for readout. Cells on Nanovials can be sorted on the basis of viability, secretion amount or other surface markers without fixation or permeabilization, and cell- and secretion-containing Nanovials are directly introduced into microfluidic droplets-in-oil emulsions for single-cell barcoding of cell transcriptomes and secretions. We have used SEC-seq to link T cell receptor sequences to the relative amount of associated cytokine secretions, surface marker gene expression with a highly secreting and potential regenerative population of mesenchymal stromal cells and the transcriptome with high immunoglobulin secretion from plasma cells. Nanovial modification and cell loading takes <4 h, and once the desired incubation time is over, staining, cell sorting and emulsion generation for scRNA-seq can also be completed in <4 h. Compared to related techniques that link secretions to a cell's surface, SEC-seq provides a general solution across any secretion target because of the ease with which biotinylated Nanovials can be modified. By linking gene expression and secretory strength, SEC-seq can expand our understanding of cell secretion, how it is regulated and how it can be engineered to make better therapies.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143468606","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":"Measuring plasma membrane fluidity using confocal microscopy.","authors":"Pablo Carravilla, Luca Andronico, Jan Schlegel, Yagmur B Urem, Ellen Sjule, Franziska Ragaller, Florian Weber, Cenk O Gurdap, Yavuz Ascioglu, Taras Sych, Joseph Lorent, Erdinc Sezgin","doi":"10.1038/s41596-024-01122-8","DOIUrl":"https://doi.org/10.1038/s41596-024-01122-8","url":null,"abstract":"<p><p>Membrane fluidity is a crucial parameter for cellular physiology. Recent evidence suggests that fluidity varies between cell types and states and in diseases. As membrane fluidity has gradually become an important consideration in cell biology and biomedicine, it is essential to have reliable and quantitative ways to measure it in cells. In the past decade, there has been substantial progress both in chemical probes and in imaging tools to make membrane fluidity measurements easier and more reliable. We have recently established a robust pipeline, using confocal imaging and new environment-sensitive probes, that has been successfully used for several studies. Here we present our detailed protocol for membrane fluidity measurement, from labeling to imaging and image analysis. The protocol takes ~4 h and requires basic expertise in cell culture, wet lab and microscopy.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143458667","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":"Construction of RNA reference materials for improving the quantification of transcriptomic data.","authors":"Ying Yu, Wanwan Hou, Qingwang Chen, Xiaorou Guo, Leqing Sang, Hao Xue, Duo Wang, Jinming Li, Xiang Fang, Rui Zhang, Lianhua Dong, Leming Shi, Yuanting Zheng","doi":"10.1038/s41596-024-01111-x","DOIUrl":"https://doi.org/10.1038/s41596-024-01111-x","url":null,"abstract":"<p><p>RNA reference materials and their corresponding reference datasets act as the 'ground truth' for the normalization of experimental values and are indispensable tools for reliably measuring intrinsically small differences in RNA-sequencing data, such as those between molecular subtypes of diseases in clinical samples. However, the variability in 'absolute' expression profiles measured across different batches, methods or platforms limits the use of conventional RNA reference datasets. We recently proposed a ratio-based method for constructing reference datasets. The ratio for a gene is defined as the normalized expression levels between two sample groups and produces more reliable values than the 'absolute' values obtained across diverse transcriptomic technologies and batches. Our gene ratios have been used for the successful generation of omics-wide reference datasets. Here, we describe a step-by-step process for establishing RNA reference materials and reference datasets, covering three stages: (1) reference materials, including material preparation, homogeneity testing and stability testing; (2) ratio-based reference datasets, including characterization, uncertainty estimation and orthogonal validation; and (3) applications, including definition of performance metrics, performing proficiency tests and diagnosing and correcting batch effects. This approach established the Quartet RNA reference materials and reference datasets (chinese-quartet.org) that have been approved as the first suite of nationally certified RNA reference materials by China's State Administration for Market Regulation. The protocol can be utilized to establish and apply reference materials to improve RNA-sequencing data quality in diverse clinical settings. The procedure can be completed in 2 d and requires expertise in molecular biology and bioinformatics.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449527","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}