Jove-Journal of Visualized Experiments最新文献

{"title":"Individualized Periprocedural Management of Congenital Dysfibrinogenemia during Transvaginal Oocyte Retrieval: A Case Report.","authors":"Anran Wang, Li Zhou, Shujia Wang, Jie Li, Shiwen Zhang, Xiaoran Li, Xuehong Zhu, Xiao Teng, Juanbao Peng, Sien Mo","doi":"10.3791/70815","DOIUrl":"https://doi.org/10.3791/70815","url":null,"abstract":"<p><p>Congenital dysfibrinogenemia is a qualitative fibrinogen disorder with phenotypes ranging from isolated laboratory abnormalities to bleeding or thrombosis. Transvaginal ultrasound-guided oocyte retrieval is minimally invasive but may cause pelvic bleeding, and peri-procedural management in patients with congenital dysfibrinogenemia is not standardized. We report a 34-year-old woman with tubal-factor infertility and persistently low functional fibrinogen who pursued in vitro fertilization. Targeted sequencing of coagulation-related genes identified a heterozygous fibrinogen gamma chain gene (FGG) variant (NM_021870.3:c.902G>A, p.Arg301His), which was considered potentially contributory to the laboratory phenotype. She had no personal history of abnormal bleeding or thrombosis and had previously tolerated laparoscopic salpingotomy without transfusion or fibrinogen supplementation. A multidisciplinary team involving reproductive medicine, anesthesia, laboratory medicine, transfusion medicine, and internal medicine implemented a conservative, trigger-based plan without routine prophylactic transfusion, with prearranged availability of fibrinogen-containing products and predefined escalation criteria based on symptoms, hemodynamics, hemoglobin trends, and ultrasound findings. Controlled ovarian stimulation was performed using a gonadotropin-releasing hormone antagonist protocol, and oocyte retrieval was completed under standard intravenous sedation by an experienced operator. Ten oocytes were retrieved with an estimated blood loss of <5 mL. Postprocedure monitoring with serial vital signs, complete blood counts, coagulation tests, and transvaginal ultrasound showed hemodynamic stability and no clinically significant hemoperitoneum. This case illustrates the feasibility of transvaginal oocyte retrieval in a carefully selected, low-risk patient with congenital dysfibrinogenemia and provides a practical framework for individualized, risk-stratified peri-procedural management.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844921","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":"Metagenomic Next-Generation Sequencing of Cerebrospinal Fluid for the Detection of Central Nervous System Pathogens.","authors":"Sumanth P Reddy, Jarod Morgenroth-Rebin, Shiyin Wang, Michael R Wilson","doi":"10.3791/70075","DOIUrl":"https://doi.org/10.3791/70075","url":null,"abstract":"<p><p>Metagenomic next-generation sequencing (mNGS) has emerged as a powerful tool for unbiased pathogen detection and host transcriptional profiling in clinical and research settings. While its utility in diagnosing central nervous system (CNS) infections is increasingly recognized, cerebrospinal fluid (CSF) samples pose unique challenges due to low nucleic acid abundance and susceptibility to degradation. This study presents an optimized research-based protocol for Illumina sequencing platforms tailored to CSF mNGS, spanning sample handling, nucleic acid extraction, library preparation, sequencing, and bioinformatic analysis. Quality control approaches adaptable to both high and low-resource settings are also provided, including alternatives to capillary electrophoresis. This study demonstrates the protocol's robustness through two representative cohorts: a high-depth, NovaSeq-based workflow and a cost-conscious NextSeq-based workflow. Across these cohorts, pathogens were detected in over 50% of cases, underscoring the method's diagnostic potential even with resource-constrained adaptations. This protocol facilitates reproducible CSF mNGS, providing a foundation for diverse applications in neuroinfectious disease research and diagnostics.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844929","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":"Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model.","authors":"Bo-Wen Zhang, Xin Zhong, Chu-Yi Cao, Fan Wu, Hui Wang, Jin-Xiang Zhang","doi":"10.3791/69321","DOIUrl":"https://doi.org/10.3791/69321","url":null,"abstract":"<p><p>Ubiquitination, a post-translational modification, is a critical regulator of intracellular protein function. Ubiquitination modulates protein functions by promoting proteasomal degradation or altering subcellular localization through ubiquitin chain-dependent signaling. Here, we describe two cell lysis methods for detecting SMAD2 ubiquitination levels in HEK293T cells and compare their effectiveness in analyzing protein ubiquitination levels. Protein overexpression in cells was induced by transient transfection. The plasmids (HA-Ub, FLAG-SMAD2, and MYC-SMURF2) and transfection reagents were separately added to basal medium, mixed, and the mixture was added to the cells. Prior to harvest, MG132 was added to inhibit proteasomal degradation and enhance ubiquitinated protein accumulation. The primary divergence between the two experimental approaches is their cell lysis methods-the ice-bath method (performed at 4 °C) and the heat-treatment method (involving incubation at 95 °C), which substantially affects the efficiency of protein lysis. After cell lysis was completed, the cell lysate, agarose beads, and FLAG antibody were mixed and incubated at 4 °C overnight. Ubiquitinated proteins were then detected by western blot analysis. Before detecting ubiquitinated proteins, a light-chain antibody was used for secondary antibody incubation. Then, ubiquitination bands were detected. The results show that both the ice-bath method and the heat-treatment method can be used to detect ubiquitination levels, while the heat-treatment method may make it easier to detect ubiquitination of SMAD2. This study delineates and compares two cell lysis methods for measuring ubiquitination levels in mammalian cells, using SMURF2/SMAD2 as a model, to assist researchers in selecting more appropriate methods for detecting the ubiquitination levels of substrate proteins.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844783","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":"Visualization and Quantitative Analysis of Genotoxin-induced PARP1/PARP2 Activation in Cells using a Fluorescent Fusion Protein-based Reporter.","authors":"Wynand P Roos, Rasha Q Al-Rahahleh, Christopher A Koczor, Libby A Beers, Louis Boyang, Robert W Sobol","doi":"10.3791/70497","DOIUrl":"https://doi.org/10.3791/70497","url":null,"abstract":"<p><p>Poly(ADP-ribose) (PAR) is a polymer of ADP-ribose synthesized by four members of the ADP-ribose polymerase family of enzymes-PARP1, PARP2, PARP5a, and PARP5b. However, only PARP1 and PARP2 synthesize PAR in response to DNA breaks. PAR is defined as a protein post-translational modification, but it is also shown to exist as a DNA or RNA modification. Levels of PAR are further regulated by PARG, a PAR glycohydrolase that, together with PARP1 and PARP2, modulates the cellular level of DNA damage-induced PAR. The dynamic synthesis and degradation of PAR is critical to its regulatory role in DNA repair, and the DNA damage response, which in turn affects chromatin reorganization, replication, transcription, and cell death. PARP1/PARP2 activation and the accumulation of PAR can be considered sites of ongoing base excision repair or DNA single-strand break repair; however, numerous PARP1/PARP2 activators are also associated with replication stress and other DNA metabolic processes. Once formed, PAR chains facilitate the recruitment of DNA repair and DNA damage response (DDR) factors to sites of DNA damage or genomic insult via their PAR-binding domains (PBDs). Ten different PBDs recognize various regions of the PAR molecule, including the PAR binding motif, PAR binding Zinc finger, the WWE domain, and the macrodomain, among other PBDs. To facilitate cellular analysis of PAR dynamics, we used PBDs fused to enhanced green fluorescent protein (EGFP) to optimize cell-based quantitation of PAR foci. We describe an assay that uses a fragment of RNF146 encoding the PBD/WWE domain, linked to EGFP, to visualize and quantify PAR accumulation at sites of genomic insult and ongoing BER or SSBR. We describe experimental steps, including the production of lentiviral particles, transduction of the target cell line, treatment of mammalian cells to induce genomic DNA damage, acquisition of confocal fluorescence micrographs, and semi-automated quantification of the data.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844693","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":"Artificial Intelligence-Driven Personalized Learning Improves Operating Room Instrument Training: A Prospective Observational Study.","authors":"Jing Cai, Luping Li, Jianshu Cai, Xiaoling Huang","doi":"10.3791/70487","DOIUrl":"https://doi.org/10.3791/70487","url":null,"abstract":"<p><p>Traditional operating room instrument training often relies on one-size-fits-all teaching, produces poor long-term skill retention, and contributes to preventable surgical errors. The present study hypothesized that an artificial intelligence (AI) powered personalized learning system could improve technical competency, patient safety, and training efficiency by tailoring practice to individual learner profiles. An AI-powered personalized learning system (APLS) was developed that combines data-driven learner phenotyping, deep-learning-based instrument recognition, ensemble prediction of competency trajectories, and reinforcement learning to deliver adaptive multimodal feedback during simulation-based training. In a prospective observational study with cluster-based departmental allocation (introducing quasi-experimental elements) at a tertiary hospital, 107 multidisciplinary operating room staff completed either the AI-driven curriculum or standard instructor-led training. The primary outcome was 12-month retention of instrument-handling competency, measured by the perioperative instrument proficiency scale (PIPS); secondary outcomes included operating room safety incidents, training time, and cost per competent professional. Compared with traditional training, the personalized system was associated with substantially higher 12-month competency scores (APLS 84.1 ± 9.2 vs. Control 58.9 ± 18.7, p < 0.001), fewer safety-related events in clinical practice, and nearly half the training time while reducing overall training costs by 38.3%. The AI ensemble also outperformed its individual machine-learning components when predicting learner performance and selecting feedback strategies. These findings suggest that integrating unsupervised phenotype discovery, supervised prediction, and reinforcement learning into a unified platform can meaningfully enhance operating room instrument training and may offer a scalable framework for data-driven workforce development in perioperative care.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844791","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":"Electroacupuncture-Based Cerebral Electric Field Therapy for Unilateral Vocal Cord Paralysis with Secondary Dysphagia Post Upper Respiratory Infection.","authors":"Wei Zhao, Weibin Gao, Ruiou Chen, Yuwei Ben, Qiang Tang","doi":"10.3791/70030","DOIUrl":"https://doi.org/10.3791/70030","url":null,"abstract":"<p><p>Unilateral vocal fold paralysis secondary to peripheral nerve injury presents limited treatment options and inconsistent outcomes. This protocol describes an electroacupuncture-based cerebral electric field therapy designed to target neuroanatomically relevant pathways through combined scalp, posterior cervical, and anterior neck electroacupuncture. The procedure includes standardized point selection, stimulation parameters, and treatment sequencing, followed by objective functional assessment using laryngoscopy and videofluoroscopic swallowing study. In a representative case, the application of this protocol was associated with observable improvements in voice quality and swallowing function. Patients with unilateral vocal fold paralysis frequently experience hoarseness, dysphagia, aspiration, and reduced laryngeal sensation, leading to substantial functional limitations and decreased quality of life. This protocol demonstrates the feasibility of electroacupuncture-based cerebral electric field therapy as a structured intervention for unilateral vocal fold paralysis and provides a reproducible framework for future clinical exploration. Additional investigation may help clarify its potential role as a supportive therapeutic option. Future controlled studies may further evaluate its clinical effectiveness and applicability.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844801","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":"Exploring the Mechanism of San Jie Tong Mai Formula Against Atherosclerosis via Network Pharmacology and Proteomics.","authors":"Huize Han, Hongyu Li, Zhilin Zhou, Aidong Liu, Congbo Zhong, Ye Sun","doi":"10.3791/70080","DOIUrl":"https://doi.org/10.3791/70080","url":null,"abstract":"<p><p>This study aimed to elucidate the mechanism of San Jie Tong Mai Formula (SJTMF) against atherosclerosis (AS), a leading cause of cardiovascular morbidity. Using ApoE^-/- mouse models, we demonstrated that SJTMF significantly inhibits AS plaque progression. Through an integrated network pharmacology and proteomics strategy, five core bioactive components were identified: beta-sitosterol, naringenin, luteolin, isorhamnetin, and 3beta-hydroxy-24-methylene-8-lanostene-21-oic acid. Concurrently, proteomics revealed 129 AS-related proteins that were differentially expressed. Molecular docking confirmed high-affinity binding interactions between these components and the key target Hsd11b1, with their binding energies all below -5 kcal/mol. Mechanistic investigations further revealed that SJTMF may regulate Hsd11b1-mediated glucocorticoid metabolism. This regulation contributes to significant amelioration of both dyslipidemia and vascular inflammation, thereby suppressing AS development. Collectively, this work demonstrates, for the first time, the innovative mechanism by which a traditional Chinese medicine formula exerts anti-AS effects through multi-component synergistic regulation of the Hsd11b1 target, offering new insights for therapeutic intervention.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844883","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":"Traditional Chinese Medicine Modulation of the Gut-Liver Axis: From Barrier Disruption to Inflammasome Activation in MASLD/MASH.","authors":"Lianjie Ren, Qin He, Zhuyuan Hu, Jinjin Jin, Feibao Tao","doi":"10.3791/70259","DOIUrl":"https://doi.org/10.3791/70259","url":null,"abstract":"<p><p>Metabolic dysfunction-associated steatotic liver disease (MASLD) and its inflammatory-fibrotic phenotype (MASH) exhibit pronounced immunometabolic coupling. This review synthesizes evidence along the gut-liver axis, from epithelial tight-junction and gut vascular barrier (GVB) failure to TLR4-MyD88/TRIF-NF-κB amplification and NLRP3 inflammasome activation and outlines the logic of the gut-derived exposure spectrum-lipopolysaccharide (LPS), endogenous ethanol, bile acids (BAs), short-chain fatty acids (SCFAs), and trimethylamine N-oxide (TMAO). We position BA-FXR/TGR5, SCFAs-GPR41/43, and AMPK/Nrf2 as upstream/downstream modulators that reset inflammatory thresholds and confer stage-dependent druggability. Based on node-to-pathway mapping, we summarize mechanisms and translational signals for berberine (BBR), Qushihuayu (QSHY), Da-Chai-Hu Decoction (DCHD), and polysaccharides (e.g., Astragalus, Ganoderma), emphasizing pharmacokinetic and site-of-exposure constraints that support \"gut-first\" actions. We propose a minimal companion biomarker set-LBP/sCD14, BA profiles with FGF19-C4 dynamics, and IL-1β/GSDMD-N-paired with hierarchical imaging gates (≥30% relative MRI-PDFF decline; MRE/ELF) to underpin response typing and go/no-go decisions. Finally, we highlight critical gaps (direct human GVB readouts; longitudinal multi-omics bridged to clinical outcomes) and outline a biomarker-driven multi-arm multi-stage (MAMS) pathway for adaptive, stratified development of multi-target traditional medicine interventions in MASLD/MASH.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844565","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":"Wire-Calibrated Induction of Segmental Ureteral Strictures in Rats via Midline Laparotomy.","authors":"Alexander Studier-Fischer, Paula Grützner, Berkin Özdemir, Caelan Max Haney, Samuel Knoedler, Luisa Renner, Nicolas Carl, Tilmann Clanget, Gabriel Alexander Salg, Niklas Westhoff, Maurice Stephan Michel, Karl-Friedrich Kowalewski","doi":"10.3791/70726","DOIUrl":"https://doi.org/10.3791/70726","url":null,"abstract":"<p><p>Ureteral strictures remain a major challenge in reconstructive urology and can arise from iatrogenic injury, chronic inflammation, trauma, congenital anomalies, or radiation.​ These lesions frequently lead to progressive obstructive uropathy and loss of renal function. Despite advances in endoscopic and surgical techniques, ureteral strictures remain challenging to address, and their remodeling effects on the renal parenchyma remain the subject of ongoing research, underscoring the need for robust preclinical models. This study establishes a reproducible rat model for controlled induction of various degrees of segmental ureteral strictures using wire‑calibrated partial ligation via midline laparotomy. The protocol enables segmental, non‑transecting constriction of the ureter, intended to approximate fibrotic narrowing at a functional and anatomical level while minimizing evident ischemic damage and maintaining macroscopic tissue viability with ureteral continuity. By providing a reliable experimental platform with low inter‑animal and inter-surgeon variability, this approach supports translational research on biomolecular mechanisms, biomaterials, imaging modalities, and reconstructive techniques for ureteral stricture disease.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844687","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":"Submucosal Tunnel Endoscopic Resection of Giant Leiomyoma of the Esophagus Combined with Circular Arc Incision and 3D Volume Rendering.","authors":"Jia Wang, Manli Cui, Wei Hu, Hua Wen, Mingxin Zhang","doi":"10.3791/68916","DOIUrl":"https://doi.org/10.3791/68916","url":null,"abstract":"<p><p>Esophageal leiomyoma is a common benign tumor of the esophagus. Traditionally, surgical resection is performed for large lesions. In this case, a 19-year-old male patient with a GEL underwent three-dimensional volume rendering to assess the surrounding structures, followed by submucosal tunneling and resection (STER). A modified arc-shaped mucosal incision was designed to provide sufficient exposure for dissection and complete removal of the lesion. Postoperative pathology confirmed leiomyoma. The patient resumed oral intake on postoperative day 2 and was discharged on day 5. STER creates a submucosal tunnel between the mucosal and muscular layers of the digestive tract and is mainly used for removing small esophageal and cardia submucosal tumors originating from the muscularis propria. It offers several advantages, including a short operation time, minimal trauma, rapid recovery, and no visible scars. In this case, STER was successfully applied to remove a large lesion, indicating that endoscopic treatment of GEL is both safe and feasible. However, further experience is required to evaluate its long-term efficacy.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844956","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}