Jove-Journal of Visualized Experiments最新文献

{"title":"Elucidating Tissue- and Plasma-Specific Proteomic Alterations in Health and Disease.","authors":"Lily S Neff, James C DeMar, Nicholas C Gary, Mital Y Patel, Burook Misganaw, Aarti Gautam, Rasha Hammamieh","doi":"10.3791/67240","DOIUrl":"https://doi.org/10.3791/67240","url":null,"abstract":"<p><p>Proteomics is the large-scale study of expressed proteins, focusing on their structure, function, interactions, abundance, and post-translational modifications within a biological system. For example, phosphoproteomics, a vital subset of total proteomics, is the study of phosphorylation patterns and alterations on proteins. The methodology described herein illustrates a total proteomics approach to elucidate alterations in overall protein expression profiles, including phosphorylation patterns, in the plasma, brain, lung, spleen, and liver tissues collected from research animal models (i.e., ferrets and mice). This technique is applicable for other tissue types, as well as almost any bio-sample containing proteins (e.g., cultured cells). Following dissection, tissues of interest were flash frozen and stored at -80 °C. Tissues were then homogenized using a mortar and pestle with liquid nitrogen to preserve protein integrity and phosphorylation changes. Total protein was extracted from the tissue homogenates using a lysis buffer with a universal nuclease and protease/phosphatase inhibitors. Proteins extracted from tissue and equivalent plasma were converted to total peptides by controlled protease digestion using a commercial mass spectrometer sample preparation kit. Peptides were directly analyzed using ultra-high performance liquid chromatography/Orbitrap-tribrid tandem mass spectrometry. Identities of constituent proteins were reconstructed from the peptide mass spectral data, using proteomics bioinformatics software, as matched to a species-specific amino acid sequence library. Following this, the analysis should include strict cutoff criteria, especially using only high-confidence protein identifications. Statistically significant proteomic differences (>2-fold change; p < 0.05) can be determined between control and experimental groups.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 223","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145287331","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Acupuncture Strategies in a Mouse Model of Alzheimer's Disease.","authors":"Xin Hao, Ning Ding, Yue Zhang, Meng Wu, Yu Ning, Zidong Wang, Zhigang Li","doi":"10.3791/68809","DOIUrl":"https://doi.org/10.3791/68809","url":null,"abstract":"<p><p>Alzheimer's disease (AD) is a progressive neurodegenerative disease. Numerous studies have demonstrated that alterations in intestinal flora can influence the central nervous system (CNS) through multiple pathways, ultimately contributing to the onset and progression of AD. Recent research suggests the limitations of pharmacological therapies, emphasizing the need for multi-targeted interventions in AD treatment. Traditional Chinese medicine (TCM) highlights the physiological and pathological relationship between the brain and the intestine. Therefore, therapeutic strategies targeting gastrointestinal regulation to enhance brain function are of great importance for delaying the pathological progression of AD. This protocol introduces a brain-intestine coordination acupuncture method. The experimental results showed that this acupuncture protocol could modulate intestinal flora, suppress intestinal inflammation and neuroinflammation in AD model mice, thereby achieving bidirectional therapeutic effects on brain-intestine regulation. Moreover, a mouse bag fixation device for acupuncture treatment was described in this study, which can reduce stress reaction and improve experimental efficiency.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 223","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145287343","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Frailty Assessment in an Aging Mouse Model.","authors":"Braydon A Crum, Heather M Gransee, Carlos B Mantilla","doi":"10.3791/69076","DOIUrl":"https://doi.org/10.3791/69076","url":null,"abstract":"<p><p>A frailty index (FI) is a powerful method of assessing animal models of aging, serving as a proxy measure of biologic age and health span. Frailty represents a state of increased vulnerability to adverse outcomes due to the accumulation of health deficits that limit independence. The concept is valuable for animal studies that inform human geroscience. For example, using FI scores, animals with a similar chronological age can be differentiated by their biological age. Thus, FI can be used in preclinical studies to measure biological age, track frailty longitudinally, and assess health span. A murine frailty index was recently developed using 31 health-related deficits. However, its length and requirement for specialized equipment pose limitations in practical use. Here, we present an adapted 16 item frailty index that excludes parameters requiring special instrumentation or invasive testing. The selected deficits are easily observable or measurable and evaluate integument, musculoskeletal, neuromuscular, sensory, urogenital, and respiratory systems. Body weight is measured for longitudinal tracking but excluded from the FI calculation. Each deficit is scored as 0, 0.5, or 1, based on severity, with the FI computed as the mean of the 16 deficit scores. This streamlined FI allows rapid assessment of large cohorts, can be applied longitudinally to the same animals, and requires no specialized equipment. We demonstrate its feasibility in C57BL/6×129 mice, showing age-associated increases in frailty, variation among individual mice, and the ability to detect deficits across multiple systems. This tool enables laboratories to incorporate frailty into diverse experimental designs, provides a practical approach for assessing health span, and enhances translational relevance in geroscience. This frailty index is adapted from previously validated mouse clinical frailty methodologies and is intended for straightforward, longitudinal use alongside ongoing studies.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 223","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145287366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vitro Isolation and Culturing of Mouse Primary Chondrocytes for Cartilage Biology.","authors":"Qingxin Song, Yangwei Xu, Yajie Xie, Xinyu Huo, Jian Sun, Yitong Zhao, Wenfang Wang","doi":"10.3791/68454","DOIUrl":"https://doi.org/10.3791/68454","url":null,"abstract":"<p><p>Articular cartilage destruction leads to altered chondrocyte activity, which is a major causative factor in a variety of cartilage diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). As the most abundant cell type in cartilage, an in-depth study of the biological properties of chondrocytes is essential for the development of effective disease treatment strategies. Isolation and culturing of primary chondrocytes provide important experimental materials for the investigation of cartilage disease and contribute to unraveling the mechanisms of cartilage injury and repair. This protocol provides a detailed description of the in vitro isolation and culturing methods for primary chondrocytes derived from mice. By using the optimized collagenase II protocol, chondrocyte isolation can be completed within 8 h for neonatal mouse knee joint cartilage specimens. The cell yield is approximately 1-2 × 10³ cells/mg of cartilage tissue, varying with tissue freshness and initial cell density; the isolated cells exhibit >90% viability. This protocol uses mouse primary chondrocytes as an example, showing the morphological characteristics and adhesion status of cells cultured for 1, 2, and 5 days after isolation. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting showed that interleukin-1β (IL-1β) treatment reduced Collagen Type II Alpha 1 (Col2a1) and increased Matrix Metallopeptidase 13 (Mmp13) expression, confirming that isolated chondrocytes respond to inflammatory stimuli. Compared with conventional methods, this protocol employs a higher collagenase concentration to reduce the impact of prolonged isolation time on cell viability, while avoiding potential cell damage from trypsin treatment. The obtained primary chondrocytes are pure and viable, suitable for in-depth studies on cartilage injury-related mechanisms, which have important implications for in vitro research and the clinical treatment of cartilage disease.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 223","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145287304","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Age-dependent Dynamics of Locomotion in Caenorhabditis elegans: A Lyapunov Exponent Analysis.","authors":"Olivia Trader, Dimitrios Tzepos, Hugo Nicolas Reyes Cardozo, Jenny Magnes","doi":"10.3791/68955","DOIUrl":"https://doi.org/10.3791/68955","url":null,"abstract":"<p><p>This study investigates the influence of age on the mobility of Caenorhabditis elegans (C. elegans) by employing Dynamic Optical Diffraction (DOD) to estimate the Largest Lyapunov Exponent (LLE). The LLE, a key metric in dynamical systems, quantifies the rate of divergence or convergence of trajectories in phase space, indicating the predictability and chaos in the system's dynamics, in this case, the locomotive behaviors of the worm. 632 nm laser light diffracts off the swimming worm in a water column, forming a diffraction pattern. A photodiode detects the light at a single point within the diffraction pattern, capturing a one-dimensional time series as the worm undulates. This time series serves as a composite representation of the entire worm's motion, encapsulating its locomotion dynamics since one point in the diffraction pattern is a superposition of all points on the worm. The time series is then embedded into a higher-dimensional phase space to calculate the LLE. C. elegans typically live for around 14 days, following a pattern of increasing and declining motor control with age. To isolate age-specific effects, worms were transferred to fresh agar plates containing E. coli every two days, ensuring they were appropriately aged (3 to 12 days old). Analysis of a cohort of 13 C. elegans revealed that the LLE peaked at five days post-hatching, at a rate of 1.34 ± 0.03 1/s. This peak signifies a critical point in development where the worms' locomotion displays the highest complexity and chaotic behavior. The observed LLE values align with the Moore equation, a well-established model that describes age-related changes in voluntary activity, linking decreasing motor control and activity levels with increasing age in C. elegans.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 223","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145287371","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Neonatal Mouse Model of Necrotizing Enterocolitis and Lamina Propria Isolation for Immune Cell Profiling.","authors":"Gergely Mozes, Lauren Bolzan, Corey M Jania, Vikram Puri, Yukihiro Yamaguchi, Ahmad Salah Sami, Catherine R Rizzuto, Danielle E Sklar, Journie N Roper, Natalia S Akopyants, Misty Good","doi":"10.3791/69233","DOIUrl":"https://doi.org/10.3791/69233","url":null,"abstract":"<p><p>Necrotizing enterocolitis (NEC) is a severe inflammatory disease of the neonatal intestine, primarily affecting preterm infants. NEC is characterized by epithelial injury, microbial dysbiosis, and a dysregulated immune response, often resulting in intestinal necrosis and systemic inflammation. Experimental murine models that recapitulate the key aspects of human disease have been essential in advancing our understanding of the mechanisms that drive disease and inform therapeutic development. This protocol describes a well-established neonatal mouse model of necrotizing enterocolitis (NEC) utilizing formula feeding, intermittent hypoxia, oral administration of lipopolysaccharide (LPS), and enteric bacteria cultured from an infant with NEC. This combination reliably induces histological, transcriptional, and immunological features consistent with NEC in human infants and has been foundational in multiple key discoveries in the field. Additionally, this protocol describes the isolation of immune cells from the small intestinal lamina propria of neonatal mice. Lamina propria cell isolation enables detailed immune cell profiling via flow cytometry, facilitating analysis of both innate and adaptive cell populations within the intestine.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 223","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145239930","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detection of Mycoplasma pneumoniae Nucleic Acid and Drug Resistance Gene.","authors":"Yunxia Wang, Chunling Xue, Qiuying Luo, Jianhong Ma, Xiangxing Zeng","doi":"10.3791/68500","DOIUrl":"https://doi.org/10.3791/68500","url":null,"abstract":"<p><p>Mycoplasma pneumoniae (Mp) represents one of the most prevalent pathogens responsible for community-acquired pneumonia (CAP) in pediatric populations. The detection of Mycoplasma pneumoniae resistance genes provides robust technical support and a theoretical foundation for the precise clinical diagnosis and treatment of MP infections. Accurate diagnosis and evaluation of drug resistance in Mp infection are essential for clinical treatment. Common detection methods for Mycoplasma pneumoniae include culture, antibody detection, and molecular biology-based assays. Mp culture is the gold standard for diagnosis but requires a specific medium, is time-consuming, and has low sensitivity. Antibody-based detection of Mp is widely used; however, false negatives may occur in the early stage of infection. Molecular biology-based detection provides rapid turnaround, low risk of contamination, high sensitivity, high specificity, and is not limited by the timing of sample collection or immune status. It has therefore been recognized as a new gold standard for diagnosing Mycoplasma pneumoniae infection. Macrolide agents are the first-choice treatment for Mp infection in children. However, with the extensive use of macrolides in respiratory tract infections in children, the incidence of drug-resistant Mycoplasma pneumoniae infection has been increasing. Patients infected with resistant strains experience significantly longer fever duration, extended hospitalization, and prolonged fever after administration compared with those infected with sensitive strains. Mutation sites identified to date include 2063, 2064, 2067, and 2617. In China, point mutations have been documented only at positions 2063 and 2064, with an A-to-G substitution at position 2063 being the most common. For these sites, polymerase chain reaction (PCR) combined with fluorescent probe technology has been employed to detect the nucleic acids and drug resistance mutations of Mycoplasma pneumoniae in human sputum samples. Detection of Mycoplasma pneumoniae resistance genes offers crucial technical support and a theoretical basis for accurate diagnosis and effective treatment of Mp infection.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 223","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145240056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preparation and Evaluation of Mouse Premature Ovarian Insufficiency Model.","authors":"Diandian Jiao, Ping Zhou","doi":"10.3791/69000","DOIUrl":"https://doi.org/10.3791/69000","url":null,"abstract":"<p><p>Premature ovarian insufficiency (POI) is a critical condition leading to female infertility, necessitating reliable animal models for mechanistic and therapeutic research. Here, we present a standardized protocol for establishing and evaluating a cyclophosphamide (CTX)-induced POI mouse model. Six-to-eight-week-old female mice with regular estrous cycles were selected and subjected to intraperitoneal CTX injections: an initial dose of 100 mg/kg on day 1, followed by daily doses of 20 mg/kg for the subsequent 14 days. Dynamic changes in estrous cycles were monitored via vaginal smear cytology with Wright staining. Serum levels of estradiol (E2), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) were quantified using ELISA to assess endocrine alterations. Ovarian histopathology was evaluated through hematoxylin-eosin (H&E) staining of paraffin-embedded sections to quantify follicular atresia, while immunohistochemical analysis of cleaved caspase-3 was performed to detect granulosa cell apoptosis. Results demonstrated disrupted estrous cyclicity, significantly reduced E2 and AMH levels, elevated FSH concentrations, increased follicular atresia, and enhanced granulosa cell apoptosis in CTX-treated mice, confirming successful POI modeling. This model-building method can highly mimic the mechanism of chemotherapy-induced ovarian damage, presenting typical pathological features such as follicle reserve depletion and sex hormone disorders. It provides a reliable experimental platform for revealing the reproductive toxicity mechanism of chemotherapy, screening ovarian-protecting drugs, and optimizing fertility preservation strategies. Moreover, this model is relatively simple to operate, low-cost, and has a short production cycle, making it easy to carry out and popularize. The methodology aligns with the requirements of JoVE for visualizable, step-by-step experimental demonstrations.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 223","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145240209","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Using the Droplet Transfer Method to Reliably Prepare Giant Unilamellar Vesicles.","authors":"Dario Cecchi, Elisa Roberti, Eugenia De Remigis, Stefano Palagi","doi":"10.3791/68340","DOIUrl":"https://doi.org/10.3791/68340","url":null,"abstract":"<p><p>Over the past two decades, the droplet transfer method, also known as inverted emulsion, double emulsion, phase transfer, or emulsion transfer, has proven advantageous for the preparation of Giant Unilamellar Vesicles (GUVs) and particularly, for loading them with different cargoes, thus playing a crucial role in synthetic biology. Because of the efficiency of encapsulation and the simplicity of execution, it has been broadly used for the development of artificial cells. A large variability in several parameters is observed in the literature, which leads to extremely variable outcomes. This is partially due to the adjustments required for different needs and applications of this versatile method, yet it may prove disorienting for researchers approaching this technique for the first time. To provide beginners with a basic understanding of the method and the role of critical parameters, a protocol is presented alongside hints on the fundamental physicochemical principles underlying GUV formation through droplet transfer. This step-by-step guide for the preparation of GUVs thus includes considerations and practical suggestions based on literature and direct experience. Considering possible sources of variability, a few aspects are identified as critical: the sensitivity of phospholipids to light, oxidation, and hydrolysis; the choice of the oil to dissolve phospholipids (the lipid solution or LS); and the recovery of GUVs after centrifugation. Cost-effective measures are proposed to minimize the interference of atmospheric oxygen and humidity. To help identify suitable combinations of oil and phospholipids, the general protocol is complemented with a discussion on the relevant physicochemical properties of the LS. Finally, to avoid impractical procedures, a straightforward and safe method is proposed to completely remove the oil phase at once and recover a clean GUV dispersion. These measures speed up the implementation of adjustments to new GUV compositions, potentially widening their adoption in synthetic biology and neighboring fields, including microrobotics.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 223","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145240214","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"FRET Dilution Assay for Analyzing Dynamic Exchange Between Protein Assemblies.","authors":"Reid Gordon, Daniel E Morse","doi":"10.3791/68678","DOIUrl":"https://doi.org/10.3791/68678","url":null,"abstract":"<p><p>As the molecular driver for tunable iridescence in cephalopods, the tunable phase behavior of reflectin A1 protein continues to be a focus of biomaterial engineering. Modulating salt concentration and protein net charge density of reflectin A1 drives the protein to form dynamic assemblies as intermediates to liquid-liquid phase separation. Reflectin assemblies, while limited in size by the extent of charge neutralization of the protein's cationic, Coulombic repulsion, are initially in dynamic exchange with monomers or oligomers from the surrounding solution. A novel fluorescence resonance energy transfer (FRET) dilution assay was used, in conjunction with dynamic light scattering (DLS) and protein concentration assays, to characterize the two-way flux of protein between reflectin A1 assemblies and a dilute phase as a function of assembly age. This FRET dilution assay distinguishes between one-way and two-way flux of protein into and out of protein assemblies and, therefore, can be applied during assembly formation. Differentiating between dynamic and kinetically arrested protein assemblies is crucial to understanding their biophysical origins, and this novel FRET dilution assay can be adapted to supplement biophysical investigations of other protein assemblies.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 223","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145240097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}