Bio-protocol最新文献

{"title":"Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin","authors":"Eider Núñez, Arantza Muguruza-Montero, Sara Alicante, Alvaro Villarroel","doi":"10.21769/BioProtoc.4963","DOIUrl":"https://doi.org/10.21769/BioProtoc.4963","url":null,"abstract":"The assessment of peptide–protein interactions is a pivotal aspect of studying the functionality and mechanisms of various bioactive peptides. In this context, it is essential to employ methods that meet specific criteria, including sensitivity, biocompatibility, versatility, simplicity, and the ability to offer real-time monitoring. In cellular contexts, only a few proteins naturally possess inherent fluorescence, specifically those containing aromatic amino acids, particularly tryptophan. Nonetheless, by covalently attaching fluorescent markers, almost all proteins can be modified for monitoring purposes. Among the early extrinsic fluorescent probes designed for this task, dansyl chloride (DNSC) is a notable option due to its versatile nature and reliable performance. DNSC has been the primary choice as a fluorogenic derivatizing reagent for analyzing amino acids in proteins and peptides for an extended period of time. In our work, we have effectively utilized the distinctive properties of dansylated-calmodulin (D-CaM) for monitoring the interaction dynamics between proteins and peptides, particularly in the context of their association with calmodulin (CaM), a calcium-dependent regulatory protein. This technique not only enables us to scrutinize the affinity of diverse ligands but also sheds light on the intricate role played by calcium in these interactions. Key features • Dynamic fluorescence and real-time monitoring: dansyl-modified CaM enables sensitive, real-time fluorescence, providing valuable insights into the dynamics of molecular interactions and ligand binding. • Selective interaction and stable fluorescent adducts: DNSC selectively interacts with primary amino groups, ensuring specific detection and forming stable fluorescent sulfonamide adducts. • Versatility in research and ease of identification: D-CaM is a versatile tool in biological research, facilitating identification, precise quantification, and drug assessment for therapeutic development. • Sensitivity to surrounding alterations: D-CaM exhibits sensitivity to its surroundings, particularly ligand-induced changes, offering subtle insights into molecular interactions and environmental influences.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140740406","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":"Purification of Native Dentilisin Complex from Treponema denticola by Preparative Continuous Polyacrylamide Gel Electrophoresis and Functional Analysis by Gelatin Zymography","authors":"P. Kamarajan, John C. Timm, M. Goetting-Minesky, Erin Malone, Sean Ganther, A. Radaic, Christian Tafolla, J. Fenno, Yvonne L Kapila","doi":"10.21769/BioProtoc.4970","DOIUrl":"https://doi.org/10.21769/BioProtoc.4970","url":null,"abstract":"Periodontal disease is characterized by the destruction of the hard and soft tissues comprising the periodontium. This destruction translates to a degradation of the extracellular matrices (ECM), mediated by bacterial proteases, host-derived matrix metalloproteinases (MMPs), and other proteases released by host tissues and immune cells. Bacterial pathogens interact with host tissue, triggering adverse cellular functions, including a heightened immune response, tissue destruction, and tissue migration. The oral spirochete Treponema denticola is highly associated with periodontal disease. Dentilisin, a T. denticola outer membrane protein complex, contributes to the chronic activation of pro-MMP-2 in periodontal ligament (PDL) cells and triggers increased expression levels of activators and effectors of active MMP-2 in PDL cells. Despite these advances, no mechanism for dentilisin-induced MMP-2 activation or PDL cytopathic behaviors leading to disease is known. Here, we describe a method for purification of large amounts of the dentilisin protease complex from T. denticola and demonstrate its ability to activate MMP-2, a key regulator of periodontal tissue homeostasis. The T. denticola dentilisin and MMP-2 activation model presented here may provide new insights into the dentilisin protein and identify potential therapeutic targets for further research. Key features • This protocol builds upon a method described by Cunningham et al. [1] for selective release of Treponema outer membrane proteins. • We adapted the protocol for the purification of biologically active, detergent-stable outer membrane protein complexes from large batch cultures of T. denticola. • The protocol involves large-scale preparative electrophoresis using a Model 491 Prep Cell. • We then use gelatin zymography to demonstrate the activity of the purified dentilisin complex by its ability to activate matrix metalloproteinase 2 (MMP-2).","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140736078","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":"In-house Extraction and Purification of Pfu-Sso7d, a High-processivity DNA Polymerase","authors":"Aisha Mahboob, Nishat Fatma, Afzal Husain","doi":"10.21769/BioProtoc.4967","DOIUrl":"https://doi.org/10.21769/BioProtoc.4967","url":null,"abstract":"The polymerase chain reaction (PCR) is an extensively used technique to quickly and accurately make many copies of a specific segment of DNA. In addition to naturally existing DNA polymerases, PCR utilizes a range of genetically modified recombinant DNA polymerases, each characterized by varying levels of processivity and fidelity. Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA-binding protein, with Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity but is sold at a sumptuously high price under various trade names and commercial variants. We recently reported a quick and easy purification protocol that utilizes ethanol or acetone to precipitate Pfu-Sso7d from heat-cleared lysates. We also optimized a PCR buffer solution that outperforms commercial buffers when used with Pfu-Sso7d. Here, we provide a step-by-step guide on how to purify recombinant Pfu-Sso7d. This purification protocol and the buffer system will offer researchers cost-efficient access to fusion polymerase. Key features • We detail a precipitation-based protocol utilizing ethanol and acetone for purifying Pfu-Sso7d. • Despite ethanol and acetone displaying effective precipitation efficiency, acetone is preferred for its superior performance. • Furthermore, we present a PCR buffer that outperforms commercially available PCR buffers. • The Pfu-Sso7d purified in-house and the described PCR buffer exhibit excellent performance in PCR applications.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140740529","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":"Purification of Photorhabdus Virulence Cassette (PVC) Protein Complexes from Escherichia coli for Artificial Translocation of Heterologous Cargo Proteins","authors":"Yueying Wang, Xinting Zhang, Xiao Feng, Xia Wang, Qi Jin, Feng Jiang","doi":"10.21769/BioProtoc.4966","DOIUrl":"https://doi.org/10.21769/BioProtoc.4966","url":null,"abstract":"Contractile injection systems (CISs), one of the most important bacterial secretion systems that transport substrates across the membrane, are a collection of diverse but evolutionarily related macromolecular devices. Numerous effector proteins can be loaded and injected by this secretion complex to their specific destinations. One group of CISs called extracellular CIS (eCIS) has been proposed as secretory molecules that can be released from the bacterial cytoplasm and attack neighboring target cells from the extracellular environment. This makes them a potential delivery vector for the transportation of various cargos without the inclusion of bacterial cells, which might elicit certain immunological responses from hosts. We have demonstrated that the Photorhabdus virulence cassette (PVC), which is a typical eCIS, could be applied as an ideal vector for the translocation of proteinaceous cargos with different physical or chemical properties. Here, we describe the in-depth purification protocol of this mega complex from Escherichia coli. The protocol provided is a simpler, faster, and more productive way of generating the eCIS complexes than available methodologies reported previously, which can facilitate the subsequent applications of these nanodevices and other eCIS in different backgrounds.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140737759","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":"Method for Large-scale Production of hIPSC Spheroids","authors":"Lucas Lemarié, Edwin-Joffrey Courtial, Jérôme Sohier","doi":"10.21769/BioProtoc.4965","DOIUrl":"https://doi.org/10.21769/BioProtoc.4965","url":null,"abstract":"Stem cell spheroids are rapidly becoming essential tools for a diverse array of applications ranging from tissue engineering to 3D cell models and fundamental biology. Given the increasing prominence of biotechnology, there is a pressing need to develop more accessible, efficient, and reproducible methods for producing these models. Various techniques such as hanging drop, rotating wall vessel, magnetic levitation, or microfluidics have been employed to generate spheroids. However, none of these methods facilitate the easy and efficient production of a large number of spheroids using a standard 6-well plate. Here, we present a novel method based on pellet culture (utilizing U-shaped microstructures) using a silicon mold produced through 3D printing, along with a detailed and illustrated manufacturing protocol. This technique enables the rapid production of reproducible and controlled spheroids (for 1× 106 cells, spheroids = 130 ± 10 μm) from human induced pluripotent stem cells (hIPSCs) within a short time frame (24 h). Importantly, the method allows the production of large quantities (2 × 104 spheroids for 1 × 106 cells) in an accessible and cost-effective manner, thanks to the use of a reusable mold. The protocols outlined herein are easily implementable, and all the necessary files for the method replication are freely available. Key features • Provision of 3D mold files (STL) to produce silicone induction device of spheroids using 3D printing. • Cost-effective, reusable, and autoclavable device capable of generating up to 1.2 × 104 spheroids of tunable diameters in a 6-well plate. • Spheroids induction with multiple hIPSC cell lines. • Robust and reproducible production method suitable for routine laboratory use.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140737360","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":"Agrobacterium-Mediated Transient Gene Expression Optimized for the Bioenergy Crop Camelina sativa","authors":"Pawan Kumar, Z. Banday, John Riley, Jean T. Greenberg","doi":"10.21769/BioProtoc.4964","DOIUrl":"https://doi.org/10.21769/BioProtoc.4964","url":null,"abstract":"Camelina sativa, a Brassicaceae family crop, is used for fodder, human food, and biofuels. Its relatively high resistance to abiotic and biotic stresses, as well as being a climate-resilient oilseed crop, has contributed to its popularity. Camelina's seed yield and oil contents have been improved using various technologies like RNAi and CRISPR/Cas9 genome editing. A stable transformation system for protein localization and other cell autonomous investigations, on the other hand, is tedious and time consuming. This study describes a transient gene expression protocol for Camelina sativa cultivar DH55 leaves using Agrobacterium strain C58C1. The method is suitable for subcellular protein localization and colocalization studies and can be used with both constitutive and chemically induced genes. We report the subcellular localization of the N-terminal ER membrane signal anchor region (1–32 aa) of the At3G28580 gene-encoded protein from Arabidopsis in intact leaves and the expression and localization of other known organelle markers. This method offers a fast and convenient way to study proteins in the commercially important Camelina crop system. Key features • This method is based on the approach of Zhang et al. [1] and has been optimized for bioenergy crop Camelina species. • A constitutive and inducible transient gene expression in the hexaploid species Camelina sativa cultivar DH55. • Requires only 16–18 days to complete with high efficacy. Graphical overview","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140739343","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":"From Llama to Nanobody: A Streamlined Workflow for the Generation of Functionalised VHHs.","authors":"Lauren E-A Eyssen, Siva Ramadurai, Sahar Abdelkarim, Imogen Buckle, Katy Cornish, Hong Lin, A K Jones, Gary J Stephens, Raymond J Owens","doi":"10.21769/BioProtoc.4962","DOIUrl":"10.21769/BioProtoc.4962","url":null,"abstract":"<p><p>Nanobodies are recombinant antigen-specific single domain antibodies (VHHs) derived from the heavy chain-only subset of camelid immunoglobulins. Their small molecular size, facile expression, high affinity, and stability have combined to make them unique targeting reagents with numerous applications in the biomedical sciences. From our work in producing nanobodies to over sixty different proteins, we present a standardised workflow for nanobody discovery from llama immunisation, library building, panning, and small-scale expression for prioritisation of binding clones. In addition, we introduce our suites of mammalian and bacterial vectors, which can be used to functionalise selected nanobodies for various applications such as in imaging and purification. Key features • Standardise the process of building nanobody libraries and finding nanobody binders so that it can be repeated in any lab with reasonable equipment. • Introduce two suites of vectors to functionalise nanobodies for production in either bacterial or mammalian cells.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10958182/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141263159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing.","authors":"Beverly Naigles, Jan Soroczynski, Nan Hao","doi":"10.21769/BioProtoc.4960","DOIUrl":"10.21769/BioProtoc.4960","url":null,"abstract":"<p><p>CRISPR/Cas9 genome editing is a widely used tool for creating genetic knock-ins, which allow for endogenous tagging of genes. This is in contrast with random insertion using viral vectors, where expression of the inserted transgene changes the total copy number of a gene in a cell and does not reflect the endogenous chromatin environment or any trans-acting regulation experienced at a locus. There are very few protocols for endogenous fluorescent tagging in macrophages. Here, we describe a protocol to design and test CRISPR guide RNAs and donor plasmids, to transfect them into RAW 264.7 mouse macrophage-like cells using the Neon transfection system and to grow up clonal populations of cells containing the endogenous knock-in at various loci. We have used this protocol to create endogenous fluorescent knock-ins in at least six loci, including both endogenously tagging genes and inserting transgenes in the Rosa26 and Tigre safe harbor loci. This protocol uses circular plasmid DNA as the donor template and delivers the sgRNA and Cas9 as an all-in-one expression plasmid. We designed this protocol for fluorescent protein knock-ins; it is best used when positive clones can be identified by fluorescence. However, it may be possible to adapt the protocol for non-fluorescent knock-ins. This protocol allows for the fairly straightforward creation of clonal populations of macrophages with tags at the endogenous loci of genes. We also describe how to set up imaging experiments in 24-well plates to track fluorescence in the edited cells over time. Key features • CRISPR knock-in of fluorescent proteins in RAW 264.7 mouse macrophages at diverse genomic loci. • This protocol is optimized for the use of the Neon transfection system. • Includes instructions for growing up edited clonal populations from single cells with one single-cell sorting step and efficient growth in conditioned media after cell sorting. • Designed for knocking in fluorescent proteins and screening transfected cells by FACS, but modification for non-fluorescent knock-ins may be possible.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10958172/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141263192","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mesenchymal Stromal Cell (MSC) Functional Analysis-Macrophage Activation and Polarization Assays.","authors":"Hazel Y Stevens, Angela C Jimenez, Bryan Wang, Ye Li, Shivaram Selvam, Annie C Bowles-Welch","doi":"10.21769/BioProtoc.4957","DOIUrl":"10.21769/BioProtoc.4957","url":null,"abstract":"<p><p>Stem cell-based therapies have evolved to become a key component of regenerative medicine approaches to human pathologies. Exogenous stem cell transplantation takes advantage of the potential of stem cells to self-renew, differentiate, home to sites of injury, and sufficiently evade the immune system to remain viable for the release of anti-inflammatory cytokines, chemokines, and growth factors. Common to many pathologies is the exacerbation of inflammation at the injury site by proinflammatory macrophages. An increasing body of evidence has demonstrated that mesenchymal stromal cells (MSCs) can influence the immunophenotype and function of myeloid lineage cells to promote therapeutic effects. Understanding the degree to which MSCs can modulate the phenotype of macrophages within an inflammatory environment is of interest when considering strategies for targeted cell therapies. There is a critical need for potency assays to elucidate these intercellular interactions in vitro and provide insight into potential mechanisms of action attributable to the immunomodulatory and polarizing capacities of MSCs, as well as other cells with immunomodulatory potential. However, the complexity of the responses, in terms of cell phenotypes and characteristics, timing of these interactions, and the degree to which cell contact is involved, have made the study of these interactions challenging. To provide a research tool to study the direct interactions between MSCs and macrophages, we developed a potency assay that directly co-cultures MSCs with naïve macrophages under proinflammatory conditions. Using this assay, we demonstrated changes in the macrophage secretome and phenotype, which can be used to evaluate the abilities of the cell samples to influence the cell microenvironment. These results suggest the immunomodulatory effects of MSCs on macrophages while revealing key cytokines and phenotypic changes that may inform their efficacy as potential cellular therapies. Key features • The protocol uses monocytes differentiated into naïve macrophages, which are loosely adherent, have a relatively homogeneous genetic background, and resemble peripheral blood mononuclear cells-derived macrophages. • The protocol requires a plate reader and a flow cytometer with the ability to detect six fluorophores. • The protocol provides a quantitative measurement of co-culture conditions by the addition of a fixed number of freshly thawed or culture-rescued MSCs to macrophages. • This protocol uses assessment of the secretome and cell harvest to independently verify the nature of the interactions between macrophages and MSCs.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10958173/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141263211","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient Genetic Transformation and Suicide Plasmid-mediated Genome Editing System for Non-model Microorganism <i>Erwinia persicina</i>.","authors":"Tingfeng Cheng, Tongling Ge, Xinyue Zhao, Zhu Liu, Lei Zhao","doi":"10.21769/BioProtoc.4956","DOIUrl":"10.21769/BioProtoc.4956","url":null,"abstract":"<p><p><i>Erwinia</i> persicina is a gram-negative bacterium that causes diseases in plants. Recently, E. persicina BST187 was shown to exhibit broad-spectrum antibacterial activity due to its inhibitory effects on bacterial acetyl-CoA carboxylase, demonstrating promising potential as a biological control agent. However, the lack of suitable genetic manipulation techniques limits its exploitation and industrial application. Here, we developed an efficient transformation system for E. persicina. Using pET28a as the starting vector, the expression cassette of the red fluorescent protein-encoding gene with the strong promoter J23119 was constructed and transformed into BST187 competent cells to verify the overexpression system. Moreover, suicide plasmid-mediated genome editing systems was developed, and lacZ was knocked out of BST187 genome by parental conjugation transfer using the recombinant suicide vector pKNOCK-sacB-km-lacZ. Therefore, both the transformation and suicide plasmid-mediated genome editing system will greatly facilitate genetic manipulations in E. persicina and promote its development and application. Key features • Our studies establish a genetic manipulation system for <i>Erwinia persicina</i>, providing a versatile tool for studying the gene function of non-model microorganisms. • Requires approximately 6-10 days to complete modification of a chromosome locus.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10958168/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141263157","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}