Current protocols最新文献

{"title":"Two-Photon Microscopy Functional Assays for Serial Imaging of Brain Microvessels and the Neurovascular Unit Through Disease Progression","authors":"Kareem El-Ghazawi,&nbsp;William A. Mills (III),&nbsp;Shayn M. Peirce,&nbsp;Ukpong B. Eyo","doi":"10.1002/cpz1.70167","DOIUrl":"https://doi.org/10.1002/cpz1.70167","url":null,"abstract":"<p>Microvascular impairments are a major issue in many diseases of the brain. In fact, they are often considered the earliest pathological phenomena in neurodegenerative diseases like Alzheimer's Disease or the locus of pathology as in stroke. Still, little is known about the mechanistic and cellular level events that contribute to these impairments. Given the importance of the neurovascular unit (NVU) in maintaining functional brain tissue, alterations to NVU cell types are important to study in the context of disease progression. With the use of two-photon microscopy, microvessels and cells can be imaged and evaluated throughout disease progression. Herein we aim to provide a comprehensive protocol for setting up and using two-photon microscopy for serial imaging of neurovascular unit cell types (i.e., pericytes, astrocytes, and microglia). We also describe interpreting results from cell and vessel changes based on disease or functional tests through multiple timepoints. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Mouse set up for serial, live 2-photon imaging</p><p><b>Basic Protocol 2</b>: Field of view acquisitions over multiple timepoints</p><p><b>Basic Protocol 3</b>: Line scan acquisitions for acquiring red blood cell metrics</p><p><b>Basic Protocol 4</b>: Quantitative measurements of vessel and cell changes</p><p><b>Basic Protocol 5</b>: Documenting changes in astrocyte morphology following induction of transient focal photothrombotic stroke using Rose Bengal</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70167","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144473119","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":"An Alternative Procedure for the Storage and Preservation of Vermamoeba vermiformis and Other Amoebae in the Laboratory","authors":"Nidia Esther Colquehuanca Arias,&nbsp;Matheus Felipe dos Reis Rodrigues,&nbsp;Bruna Nascimento Neiva,&nbsp;Juliana Miranda Tatara,&nbsp;Gabriel Magno de Freitas Almeida,&nbsp;Jônatas Santos Abrahão","doi":"10.1002/cpz1.70164","DOIUrl":"https://doi.org/10.1002/cpz1.70164","url":null,"abstract":"<p>Free-living amoebae (FLA), particularly <i>Vermamoeba vermiformis</i> and <i>Acanthamoeba</i> spp., have emerged as indispensable models for the study of giant viruses. These organisms provide a unique and efficient system for the isolation and replication of giant viruses, serving as known permissive hosts and thus playing a pivotal role in advancing research in this field. FLA can alternate between vegetative (trophozoite) and resistant (cyst) forms during their life cycle. Current methods for FLA storage and preservation face limitations, including prolonged excystment times and low rates of trophozoite viability. To address these challenges, these protocols provide instructions for the storage and preservation of <i>Vermamoeba vermiformis</i> at refrigeration temperatures, allowing feasible and rapid reactivation of cultures as an alternative to the more fastidious cyst-based storage methods. This method is also applicable to other FLA species, such as <i>Acanthamoeba castellanii</i> and <i>Acanthamoeba polyphaga</i> © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: <i>Vermamoeba vermiformis</i> propagation</p><p><b>Basic Protocol 2</b>: Preparation of <i>Vermamoeba vermiformis</i> stocks for cold room storage</p><p><b>Basic Protocol 3</b>: Reactivation of cold-stored <i>Vermamoeba vermiformis</i></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144367518","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mapping Motor Learning Stages: A Longitudinal fNIRS-Based Assessment of Cortical Activation","authors":"Xiaoli Li,&nbsp;Yongxin Zhu,&nbsp;Hongman Wei,&nbsp;Nan Zhang,&nbsp;LianHui Fu,&nbsp;Qi Qi","doi":"10.1002/cpz1.70147","DOIUrl":"https://doi.org/10.1002/cpz1.70147","url":null,"abstract":"<p>Here we describe a protocol to measure changes in cortical activation over stages of motor learning. Participants are recruited and assigned to either simple or complex motor tasks, performed using their non-dominant hand over 10 days. Motor performance is measured using the Minnesota Manual Dexterity Test, while cortical activation is assessed using functional near-infrared spectroscopy (fNIRS). The primary goal is to assess how varying levels of task complexity affect motor learning and to characterize the corresponding neural activity. Generalized estimating equations (GEE) are used to establish marginal models for the statistical analysis of factors influencing motor learning. This protocol has potential use for therapeutic applications, particularly in neurological rehabilitation contexts, where the findings could help inform recovery protocols for patients with motor impairments, such as stroke survivors. By establishing a clear understanding of motor learning stages and associated brain activity, this research could guide the development of noninvasive brain stimulation techniques, such as transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS), in rehabilitative treatments. Beyond its therapeutic applications, use of this protocol may contribute to the broader understanding of how task complexity influences motor learning in real-world settings. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Task design for motor learning and cortical activation</p><p><b>Basic Protocol 2</b>: fNIRS set up and data collection</p><p><b>Support Protocol</b>: Data analysis using GEE</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144315035","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Pocket Guide for the Experimenter Studying Daily Rhythms in Memory-Related Behavior","authors":"Matthew J. Hartsock,&nbsp;Ilia N. Karatsoreos,&nbsp;Robert L. Spencer","doi":"10.1002/cpz1.70157","DOIUrl":"https://doi.org/10.1002/cpz1.70157","url":null,"abstract":"<p>The study of circadian rhythms requires a deep understanding of how biological systems shift on a daily timescale. Similarly, the study of memory requires an appreciation of how memory mechanisms change across time. These complexities create serious challenges for examining circadian rhythms in memory-related behavior, an undertaking in which both sets of considerations must be integrated and assessed simultaneously. In this article, we present a pocket guide with an experimental design optimized to distinguish circadian influences on different stages of the memory process. Our approach distills ideas from a complex body of literature, offering a simple path forward in circadian memory research. © 2025 Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144308852","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":"Production of Functional miRNA Mimics Using In Vitro Transcription","authors":"Teja N. Sata,&nbsp;Md Ismail,&nbsp;Amrendra K. Sah,&nbsp;Senthil K. Venugopal","doi":"10.1002/cpz1.70163","DOIUrl":"https://doi.org/10.1002/cpz1.70163","url":null,"abstract":"<p>MicroRNAs (miRNAs) are nearly 22 nucleotide RNA species involved in modulating gene expression via post-transcriptional regulation. In almost all in vitro studies, miRNA mimics are used to overexpress them to understand their role in various cellular processes. These mimics are also utilized as therapeutics for various diseases, such as scleroderma, mesothelioma, and multiple solid tumors. Commercial miRNA mimics are chemically synthesized, followed by HPLC purification. This article describes a simple in vitro transcription (IVT) procedure to generate miRNA mimics from DNA templates using RNA polymerase, followed by purification using silica-based columns and annealing. The procedure is economical and quick. Produced miRNA mimics can be overexpressed in mammalian cells using transfection agents. A comparison between chemically synthesized miRNA mimics and IVT-synthesized miRNA mimics demonstrates similar efficiencies in post-transcriptional regulation. After poly(A) polymerase-mediated cDNA synthesis, validation is performed by qPCR expression analysis of target genes. Alternatively, miRNA mimics can be validated by immunoblotting target proteins. We present efficient, quick protocols to synthesize functional miRNA mimics using IVT, whose function can be validated by qPCR or immunoblotting. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Design of in vitro transcription templates, in vitro transcription, RNA purification, and RNA strand annealing</p><p><b>Basic Protocol 2</b>: Transfection of miRNA mimics, total RNA isolation, poly(A) polymerase-mediated cDNA synthesis, validation of miRNA mimic expression by qPCR, and functional validation by immunoblotting</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144308853","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":"Epigenetic Histone Deacetylases Activity Assay in the Brain and Peripheral Organ Tissues","authors":"Doodipala Samba Reddy,&nbsp;Hudson Boyd,&nbsp;Vinitha Karnati,&nbsp;Joon Kim,&nbsp;Sreevidhya Ramakrishnan,&nbsp;Xin Wu","doi":"10.1002/cpz1.70143","DOIUrl":"https://doi.org/10.1002/cpz1.70143","url":null,"abstract":"<p>Histone deacetylases (HDACs) are crucial epigenetic regulators involved in the modulation of gene expression and are promising therapeutic targets for treating various diseases, including central nervous system disorders and cancer. HDAC inhibitors exhibit neuroprotective, antiepileptogenic, and antidepressant properties in animal models, underscoring their clinical relevance. Quantifying HDAC activity is essential for identifying inhibitors and evaluating their effects under physiological or pathological conditions. This article outlines a comprehensive, sensitive, and robust assay to quantify HDAC activity in tissue lysates, with specific application to brain tissue. The assay is based on the catalytic removal of an acetyl group from the Boc-Lys(Ac)-AMC substrate by HDAC enzymes. Following nuclear protein extraction, tissue HDAC activity can be quantitatively assessed using a fluorometric Boc-Lys(Ac) HDAC activity kit. Adding a developer containing trypsin then converts the deacetylated product into a measurable fluorophore. This enables precise quantification of HDAC activity levels across different tissues, making the method suitable for screening putative HDAC inhibitors and assessing their effects on epigenetically modulated phenotypes. We validated these protocols using brain tissue samples from mice subjected to traumatic brain injury, a condition known to elevate HDAC activity levels. This assay provides an efficient, scalable tool for exploring HDAC function, evaluating therapeutic interventions, and advancing the understanding of HDAC-mediated mechanisms in animal models. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of nuclear protein from brain and other tissues</p><p><b>Support Protocol 1</b>: Harvesting and microdissection of brain and other tissues</p><p><b>Support Protocol 2</b>: Estimation of extracted protein using the Pierce bicinchoninic acid (BCA) assay</p><p><b>Basic Protocol 2</b>: HDAC activity fluorometric assay in the brain and other tissues</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70143","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144264612","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":"Methods to Observe Plant Tissue Colonization by Fusarium oxysporum","authors":"Domingo Martínez-Soto,&nbsp;Gengtan Li,&nbsp;Li-Jun Ma","doi":"10.1002/cpz1.70152","DOIUrl":"https://doi.org/10.1002/cpz1.70152","url":null,"abstract":"<p><i>Fusarium oxysporum</i>, an important soil-borne pathogen, causes vascular wilts in more than 100 plant species, leading to billions of dollars in annual yield losses. Controlling <i>Fusarium</i> wilt diseases is challenging due to the persistence of pathogen spores in infested fields and the growing resistance to available fungicides. Understanding the molecular interactions between <i>F. oxysporum</i> and its host plants is crucial for developing novel control strategies, but studying these interactions is difficult because <i>F. oxysporum</i> invades plant roots long before wilt symptoms can be detected in above-ground tissues. To illuminate the hidden interactions between <i>F. oxysporum</i> and its plant hosts, we present three confocal microscopy protocols for visualizing fungal colonization in plant tissues and the associated plant responses. The first protocol employs wheat germ agglutinin–Alexa Fluor 488 and propidium iodide to stain fungal cells and plant host tissues, respectively. The second uses sirofluor to detect deposition of callose, a (1,3)-β-glucan polymer found in plant cell walls that plays a significant role in plant defense. The third utilizes fluorescent protein–tagged fungal isolates and a stable transgenic <i>Arabidopsis thaliana</i> line, providing a clean and easily accessible system for visualizing early infection stages. The protocols described here will shed light on underground plant-pathogen interactions, aiding researchers in unraveling the complex dynamics between diverse <i>F. oxysporum</i> pathotypes and their plant hosts.© 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Observation of <i>F. oxysporum</i> cells in the tomato stem vasculature</p><p><b>Basic Protocol 2</b>: Observation of callose deposition in <i>F. oxysporum</i>–colonized tomato plant roots</p><p><b>Basic Protocol 3</b>: Observation of fungal colonization in an <i>F. oxysporum</i>–<i>A. thaliana</i> model system</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144244811","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 Genetic Association to Therapeutic Target: A Pipeline for Pleiotropic Gene Prioritization","authors":"Morgan Ewald,&nbsp;Erin Young,&nbsp;Michael Kuehn,&nbsp;Olivia Veatch","doi":"10.1002/cpz1.70148","DOIUrl":"https://doi.org/10.1002/cpz1.70148","url":null,"abstract":"<p>As our understanding of human health grows, we often see that similar biological dysfunction underlies the co-occurrence of various complex diseases. It remains difficult to determine if there are common genetic mechanisms contributing to clinically distinct conditions or if expression of both conditions relates to other shared risk factors. For example, in some situations, genetic variation may increase risk for one condition, and expression of this condition then increases risk for another disease. Identifying potentially pleiotropic genes is crucial for advancing the development of more effective treatment options, especially in instances where current therapies are insufficient. Genome-wide association studies (GWAS) provide cross-trait associations but do not provide the full functionality of how dysfunction in genes being tagged by GWAS hits are contributing to two or more distinct phenotypes. Fortunately, as other types of available data continue to grow exponentially (e.g., RNA-seq, mass spectrometry, mouse knock-out phenotype associations), these can be leveraged to help process GWAS results into meaningful information. The aim of this protocol is to provide clear instructions for using various databases and available software tools to identify key pleiotropic genes contributing to two distinct phenotypes of interest. The protocol uses information from various publicly available databases, including GWAS Catalog, Functional Mapping and Annotation (FUMA), Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT), International Mouse Phenotype Consortium (IMPC), STRINGdb, Pharos, and Cytoscape for network visualization. This pipeline, with code written in R and RStudio software, helps the user identify and generate hypotheses about shared genetic mechanisms contributing to their selected phenotypes of interest as well as prioritize genes of interest to functionally follow up in model systems that are more likely to be clinically relevant. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Pleiotropic gene prioritization pipeline for studies in model systems</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144244596","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":"Quantitative Assessment of Mitochondrial Volumetric Transitions in Arabidopsis thaliana","authors":"Bryan Ocampo-Hernández,&nbsp;Delia Luisa Rodríguez-Bustos,&nbsp;Javier Plasencia,&nbsp;Manuel Gutiérrez-Aguilar","doi":"10.1002/cpz1.70156","DOIUrl":"https://doi.org/10.1002/cpz1.70156","url":null,"abstract":"<p>Mitochondria in plants typically appear as discrete spherical or slightly tubular organelles, with their morphology and volume serving as indicators of metabolic state and dysfunction. Measuring changes in mitochondrial volume is relatively straightforward in organisms lacking plastids. However, in chlorophyll-rich tissues, such assessments often require purification protocols that may compromise accuracy. Here, we present protocols for the quantitative assessment of mitochondrial volume transitions in leaf mesophyll cells of <i>Arabidopsis thaliana</i>. The methods are simple and highly sensitive and offer a reliable approach for studying mitochondrial morphology transitions under both physiological and stress conditions. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Leaf mesophyll treatment and mitochondrial imaging</p><p><b>Basic Protocol 2</b>: Leaf mesophyll mitochondrial volume assessment</p><p><b>Basic Protocol 3</b>: Mitochondrial volume statistics</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70156","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144206343","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":"Peptide–Oligonucleotide Conjugates: Catalytic Preparation in Aqueous Solution or On-Column","authors":"Marion Gras,&nbsp;Michael Smietana,&nbsp;Pauline Adler","doi":"10.1002/cpz1.70154","DOIUrl":"https://doi.org/10.1002/cpz1.70154","url":null,"abstract":"<p>Peptide–oligonucleotide conjugates (POCs) are synthesized through a novel catalytic and sustainable approach. The amide coupling between peptide and oligonucleotide is facilitated by 1,4-diazabicyclo[2.2.2]octane (DABCO) as catalyst and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) as a stoichiometric activating agent. Two distinct methods are described for the POC synthesis: the first involves an on-column conjugation in an anhydrous environment, while the second enables a post-synthetic conjugation in an aqueous buffer. This last approach has been successfully extended to an iterative process, offering significant potential for the development of DNA-encoded libraries.© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: On-column conjugation of an amino acid or of a peptide to an oligonucleotide</p><p><b>Support Protocol</b>: Deprotection of the Boc-protected amine of the lysine</p><p><b>Basic Protocol 2</b>: In-solution conjugation of an amino acid or of a peptide on an oligonucleotide</p><p><b>Basic Protocol 3</b>: Iterative process for the preparation of a peptide–oligonucleotide conjugate</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70154","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144197140","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}