Current protocols最新文献

{"title":"CRISPR/Cas9-Mediated Gene Knockout in Cereal Crops","authors":"Dibyajyoti Pramanik,&nbsp;Kan Wang,&nbsp;Keunsub Lee","doi":"10.1002/cpz1.70210","DOIUrl":"10.1002/cpz1.70210","url":null,"abstract":"<p>High-precision genome editing tools, such as programmable nucleases, are poised to transform crop breeding and significantly impact fundamental plant research. Among these tools, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated 9) system is a programmable, RNA-guided nuclease that introduces targeted, site-specific double-stranded breaks in the target DNA loci. When these breaks are repaired, it often results in a frame-shift mutation via short insertion/deletion (indel), leading to gene knockout. Since its first successful use in plants, CRISPR/Cas9 has been widely adopted for targeting genes of agronomic and scientific importance in multiple crops, including rice, maize, wheat, and sorghum. These cereal crops ensure global food security, provide essential nutrition, and support economic stability. Additionally, such crops support biofuel production, livestock feed, and sustainable farming practices through crop rotation. This article outlines the strategies for implementing CRISPR/Cas9 genome editing in plants, including a step-by-step process of guide RNA target selection, oligonucleotide design, construct development, assembly, and analysis of genome edits. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: CRISPR/Cas9 guide RNA target selection</p><p><b>Support Protocol 1</b>: Genomic DNA extraction in-house protocol</p><p><b>Basic Protocol 2</b>: Construction of a binary plasmid vector</p><p><b>Support Protocol 2</b>: <i>Agrobacterium</i> transformation with a binary vector construct and stability check</p><p><b>Support Protocol 3</b>: Plant transformation</p><p><b>Basic Protocol 3</b>: Genotyping of edited events</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70210","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145139842","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":"Cover Image, Volume 5, Issue 9","authors":"","doi":"10.1002/cpz1.70222","DOIUrl":"https://doi.org/10.1002/cpz1.70222","url":null,"abstract":"<p>The cover image is based on the article <i>CRISPR/Cas9-mediated gene knockout in cereal crops</i> by Dibyajyoti Pramanik et al., https://doi.org/10.1002/cpz1.70210.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70222","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145146213","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":"Optimization of the Blocking and Signal Preservation Protocol in High-Parameter Flow Cytometry","authors":"Oliver T. Burton,&nbsp;James Dooley,&nbsp;Adrian Liston","doi":"10.1002/cpz1.70214","DOIUrl":"10.1002/cpz1.70214","url":null,"abstract":"<p>High quality input data is the key to successful interpretation of any scientific assay. In flow cytometry, fluorescently-conjugated antibodies allow us to simultaneously measure an incredible range of protein-based targets on single cells with a high degree of specificity. Limiting the quality of data generated, however, is the non-specific interaction that can occur between antibodies and off-target binders. Judicious use of blocking reagents can improve the specificity of the staining by reducing this non-specific binding to cells, improving the sensitivity of the assay to detect the authentic signal above assay noise. Additional beneficial effects include preventing interactions between dyes and even limiting the degradation of dyes, improving data quality. In this article, we provide a workflow for minimizing these unwanted effects, increasing specificity and sensitivity in highly multiplex flow cytometry. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Surface staining</p><p><b>Basic Protocol 2</b>: Intracellular staining</p><p><b>Basic Protocol 3</b>: Intracellular cytokine staining</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70214","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145139892","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":"Mouse Models and Experimental Protocols to Study Alloantibody-Mediated Transplant Rejection","authors":"Jason M. Zimmerer,&nbsp;Hatem Aldhahi,&nbsp;Ginny L. Bumgardner","doi":"10.1002/cpz1.70198","DOIUrl":"10.1002/cpz1.70198","url":null,"abstract":"<p>Transplantation is the definitive treatment for patients with end-stage organ failure. Following allogeneic transplant, the recipient's immune system recognizes transplanted cells or organs as foreign. The immune system recognizes and targets the foreign tissue for damage through cell-mediated rejection (CMR) and/or antibody-mediated rejection (AMR). Immunosuppressive agents are utilized to protect the transplant from rejection and extend transplant function and survival. Despite advances in immunosuppressive agents, AMR remains a critical barrier to the success of transplantation. AMR occurs when B cells produce alloantibodies that bind the allograft causing antibody-dependent, complement-mediated or immune cell-mediated cytotoxic damage. Continued research on AMR is required to develop novel and effective therapeutic strategies. Murine AMR models have been utilized to investigate mechanisms mediating the production of posttransplant alloantibodies and the pathology of damage to the transplanted allograft. These models facilitating the investigation of cellular and molecular mechanisms of alloantibody production and allograft damage are critical to the development of novel therapeutic strategies to prevent and treat AMR. This article describes the methodologies used to study AMR in animal transplant models. These include protocols to detect and measure alloantibodies, allograft survival, AMR pathology, and effector immune cell responses following transplantation. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Alloserum transfer into immune-incompetent recipient mice to determine transplant organ susceptibility to AMR</p><p><b>Basic Protocol 2</b>: Allogeneic transplantation into immune-deficient mice to study critical cellular and molecular pathways impacting AMR</p><p><b>Support Protocol 1</b>: Quantification of posttransplant alloantibody titer</p><p><b>Support Protocol 2</b>: Monitoring of transplant allograft survival</p><p><b>Support Protocol 3</b>: Assessment of immunopathology and severity of AMR</p><p><b>Basic Protocol 3</b>: Analysis of posttransplant immunologic responses</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70198","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145139827","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":"Tracking Protein Motions using Serial Femtosecond Crystallography with X-Ray Free-Electron Laser","authors":"Eiichi Mizohata,&nbsp;Eriko Nango,&nbsp;Takehiko Tosha,&nbsp;So Iwata,&nbsp;Minoru Kubo","doi":"10.1002/cpz1.70212","DOIUrl":"10.1002/cpz1.70212","url":null,"abstract":"<p>Since the birth of biochemistry, researchers have investigated the structure–function relationship of a wide variety of proteins. However, until recently, when X-ray free-electron lasers (XFELs) became available, it was not possible to visualize the motion of proteins from moment to moment with excellent temporal and spatial resolution. Here, we introduce practical methods to visualize protein motions at room temperature using serial femtosecond crystallography (SFX) using XFELs. With the development of this technology, it will be possible to visualize the entire reaction mechanism of many proteins in the future. We first outline a streamlined microcrystallization workflow for hen egg-white lysozyme, enabling rapid detector calibration and data-collection optimization. Next, we present a rotational seeding approach refined on copper-containing nitrite reductase that yields homogeneous microcrystals suitable for high-resolution SFX and readily adaptable to other challenging targets. Finally, we describe a time-resolved strategy combining microcrystals of fungal nitric-oxide reductase with photolabile caged substrates and synchronized UV triggering, capturing catalytic intermediates on the millisecond timescale. Together, these procedures enable investigators to progress from preparing samples to capturing dynamic structural snapshots. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Microcrystallization of lysozyme</p><p><b>Basic Protocol 2</b>: Microcrystallization of copper-containing nitrite reductase</p><p><b>Basic Protocol 3</b>: Time-resolved serial femtosecond crystallography</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70212","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145126824","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":"Enhancing Antimicrobial Susceptibility Testing for Acinetobacter baumannii Using Physiologically Relevant Culture Media and Biofilm Formation Assays","authors":"Nana Yaa P. Sakyi Opoku,&nbsp;Arunima Mishra,&nbsp;Hansel Fletcher,&nbsp;Victor Nizet,&nbsp;Jacinda C. Abdul-Mutakabbir","doi":"10.1002/cpz1.70207","DOIUrl":"10.1002/cpz1.70207","url":null,"abstract":"<p><i>Acinetobacter baumannii</i> is a high-risk pathogen associated with increased patient morbidity and mortality. Host-pathogen interactions amplify its virulence, in part by promoting biofilm formation—a crucial factor in antimicrobial resistance and persistence. Given the bacterium's strong propensity for acquiring resistance, antimicrobial susceptibility testing (AST) is essential for guiding effective therapeutic interventions. However, discrepancies have been observed between <i>in vitro</i> AST results and therapeutic outcomes, with some antimicrobials being deemed to show <i>in vivo</i> efficacy despite appearing ineffective <i>in vitro</i>. This discordance may stem from traditional AST protocols, which rely on bacteriological media such as Mueller Hinton broth (MHB) optimized for bacterial growth but not for mimicking the host environment. Moreover, conventional AST does not account for virulence traits such as biofilm formation, which further contribute to treatment failure. Incorporating physiologically relevant culture media, such as Roswell Park Memorial Institute (RPMI) 1640 medium, alongside assessment of biofilm formation may improve the predictive value of AST. This work outlines two complementary protocols for improving AST interpretation in <i>A. baumannii</i> infections. Basic Protocol 1 compares minimum inhibitory concentration (MIC) values generated using MHB and RPMI. Basic Protocol 2 evaluates biofilm formation in MHB, tryptic soy broth (TSB; control), and RPMI, with and without antimicrobial exposure. Together, these approaches aim to inform alternative AST strategies that better reflect <i>in vivo</i> conditions and optimize therapeutic decision-making. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Comparing <i>A. baumannii</i> minimum inhibitory concentration (MIC) results in bacteriological (MHB) versus physiological (RPMI) media</p><p><b>Basic Protocol 2</b>: Comparing <i>A. baumannii</i> isolate(s) biofilm formation following assays completed in bacteriological culture media (MHB and control TSB) and physiological medium (RPMI)</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70207","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145111199","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":"Monitoring the Energy Metabolisms of Mice with the Suprachiasmatic Nuclei Lesioned","authors":"Shaoying Lan,&nbsp;Xianpu Sun,&nbsp;Ximing Qin","doi":"10.1002/cpz1.70211","DOIUrl":"10.1002/cpz1.70211","url":null,"abstract":"<p>The central clock in mammals is located in the suprachiasmatic nucleus (SCN), which coordinates daily biological and behavioral rhythms. Lesioning the SCN is commonly used as a model to disrupt the circadian clock, with significant impacts on organisms. This protocol describes the complete SCN lesion experiment, from surgery to verification. This technology involves using a stereotactic device to locate the brain area and damaging the mouse SCN via electrophysiological instruments, followed by observing the external effects on basal metabolism caused by the loss of rhythm. After surgical damage to the SCN, the mice are allowed to recover for a period before being placed in running wheels to record their activity rhythms. The loss of rhythm in the mice is judged by a 7-day LD environment (light:dark = 12:12) and a 7-day DD environment (continuous darkness). Mice with confirmed rhythm loss are selected for metabolic cage experiments. Following the experiments, the mice are euthanized, and the SCN sections are prepared and stained for confirmation, verifying that rhythm loss is caused by SCN damage. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Mouse suprachiasmatic nuclei lesions</p><p><b>Basic Protocol 2</b>: Wheel-running tests, metabolic cage assays, SCN slicing and staining to confirm that SCN lesions induce rhythm loss.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145111310","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":"Isolation, Extraction, and Analysis of Cells After Confined Migration","authors":"Xu Gao,&nbsp;Yixuan Li,&nbsp;Jia Wen Nicole Lee,&nbsp;Jianxuan Zhou,&nbsp;Vaishnavi Rangaraj,&nbsp;Avery Rui Sun,&nbsp;Jennifer L. Young,&nbsp;Andrew W. Holle","doi":"10.1002/cpz1.70204","DOIUrl":"10.1002/cpz1.70204","url":null,"abstract":"<p>Cell migration through confined microenvironments is a critical biological process that underlies numerous physiological and pathological events, including immune cell trafficking, tissue morphogenesis, and cancer metastasis. Although polydimethylsiloxane-based microchannel devices have enabled detailed studies of confined migration, the efficient collection of cells post-migration for downstream molecular analyses remains a major challenge. Existing approaches often rely on harsh mechanical dissociation that compromises cell viability and integrity and do not permit <i>in situ</i> collection of cell lysates. To overcome these limitations, we have developed the Trap-based Recovery After Permeation (TRAP) chip, a pump-free microfluidic platform that integrates controlled confined migration with efficient post-migration cell or lysate collection. The TRAP chip incorporates microchannel arrays terminating in a precisely engineered trap region that enables gentle recovery of cells or cellular components without exposing them to high shear forces or requiring large buffer volumes. This innovation ensures the viability of recovered cells and expands the applicability of confined migration assays beyond imaging-based studies. We demonstrate that the TRAP chip facilitates the extraction of post-confinement cells for mechanical characterization, including measurement of Young's modulus, as well as the isolation of proteins and RNA suitable for downstream assays such as western blot and qPCR. The TRAP chip thus represents a significant advancement in microfluidic technologies, offering a robust, reproducible, and minimally invasive approach for studying the mechanobiology of confined migration, with broad potential for applications in basic research, cellular engineering, and translational studies where cell behavior under physical confinement is of critical importance. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Fabrication of TRAP and control chips</p><p><b>Basic Protocol 2</b>: Cell seeding and live cell isolation from TRAP chips</p><p><b>Alternate Protocol</b>: Biomolecular extraction from TRAP chips</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70204","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145111200","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":"Introducing the Dish Soap Protocol: A Unified Approach for Multi-Modal Intracellular Staining","authors":"Oliver T. Burton,&nbsp;James Dooley,&nbsp;Adrian Liston","doi":"10.1002/cpz1.70206","DOIUrl":"10.1002/cpz1.70206","url":null,"abstract":"<p>Recent advances in dyes and cytometers have seen an exponential increase in the ability to perform multidimensional flow cytometry. As we increase our capacity to extract information from cells, the need to acquire different types of data simultaneously from the same cells becomes limited by fixation buffer compatibility. Accessing the intracellular compartments for staining, particularly with large molecular weight structures such as antibody–fluorophore conjugates, requires a balance between solubilizing lipids to create entry points through the membranes, while using crosslinking fixatives to maintain structural integrity of the cell and prevent the loss of intracellular contents. Unfortunately, common fixation protocols result in a trade-off, where preservation of fluorescent proteins and accessibility of nuclear staining are often diametrically opposed. In this article, we detail the impact of various fix-perm reagents on key features, such as nuclear staining, green fluorescent protein (GFP) retention, intracellular cytokine staining, epitope retention, scatter profiles, cell recovery, and fluorophore stability. We provide a protocol for the use of “Burton's Best Buffer”, which is readily made at 100-fold lower cost than commercial buffers, which can be used to overcome the limitations of current fixation buffers and achieve simultaneous efficient detection of transcription factors, cytokines, and endogenous fluorescent proteins, among other uses. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Dish soap protocol</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70206","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145088664","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":"Profiling RNA G-Quadruplexes In vivo","authors":"Bibo Yang,&nbsp;Yiliang Ding,&nbsp;Yueying Zhang","doi":"10.1002/cpz1.70209","DOIUrl":"10.1002/cpz1.70209","url":null,"abstract":"<p>RNA G-quadruplexes (RG4s) are specific and complex tertiary structures that form in guanine-rich regions of RNA and can be detected <i>in vitro</i>. A recently developed transcriptome-wide technique, SHALiPE-Seq, enables the assessment of RG4 folding status within living cells in a quantitative manner. This method integrates chemical probing with high-throughput sequencing. SHALiPE-Seq relies on the property of 2-methylnicotinic acid imidazolide (NAI), which preferentially modifies the last guanine in G-tracts when RG4s are folded. To establish reference profiles, <i>in vitro</i> NAI modification patterns are generated under potassium ion (K⁺) conditions, which promote folding, and lithium ion (Li⁺) conditions, which maintain RG4s in an unfolded state. By comparing <i>in vivo</i> SHALiPE-Seq profiles with these <i>in vitro</i> benchmarks, it becomes possible to identify and evaluate the formation of RG4s in living cells. Although this protocol has been applied to <i>Arabidopsis thaliana</i> and rice, SHALiPE-Seq is broadly applicable to other systems and provides a valuable approach for investigating the <i>in vivo</i> dynamics of RG4s and their potential biological functions. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: <i>In vitro</i> and <i>in vivo</i> NAI probing of RNA</p><p><b>Basic Protocol 2</b>: Construction of the SHALiPE-Seq libraries</p><p><b>Basic Protocol 3</b>: Measurement of the RNA G-quadruplex folding status based on SHALiPE-Seq libraries</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70209","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145088595","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}