Metabolomics最新文献

{"title":"Critical assessment of quenching and extraction/sample preparation methods for microorganisms in metabolomics.","authors":"Hossein Sedighikamal, Shohreh Mashayekhan","doi":"10.1007/s11306-025-02228-0","DOIUrl":"https://doi.org/10.1007/s11306-025-02228-0","url":null,"abstract":"<p><strong>Background: </strong>Advancements in the research of intracellular metabolome have the potential to affect our understanding of biological processes. The applications and findings of intracellular metabolome analysis are useful in understanding cellular pathways, microbial interactions, and the detection of secreted metabolites and their functions.</p><p><strong>Aim of review: </strong>This work focuses on the analysis of intracellular metabolomes in microorganisms. The techniques used for analyzing the intracellular metabolomes including metabolomics approaches such as mass spectrometry, nuclear magnetic resonance, liquid chromatography, and gas chromatography are discussed.</p><p><strong>Key scientific concepts of review: </strong>Challenges such as sample preparation, data analysis, metabolite extraction, sample storage and collection, and processing techniques were investigated, as they can highlight emerging technologies and advancements in metabolome analysis, future applications in drug discovery, personalized medicine, systems biology, and the limitations and challenges in studying the metabolome of microorganisms.</p>","PeriodicalId":18506,"journal":{"name":"Metabolomics","volume":"21 2","pages":"40"},"PeriodicalIF":3.5,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143625526","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PLD2 is a marker for MASLD-HCC with early-stage fibrosis: revealed by lipidomic and gene expression analysis.","authors":"Jihan Sun, Fatima Dahboul, Estelle Pujos-Guillot, Mélanie Petera, Emeline Chu-Van, Benoit Colsch, Delphine Weil, Vincent Di Martino, Aicha Demidem, Armando Abergel","doi":"10.1007/s11306-025-02226-2","DOIUrl":"https://doi.org/10.1007/s11306-025-02226-2","url":null,"abstract":"<p><strong>Introduction: </strong>Metabolic steatotic liver disease (MASLD) can progress to hepatocellular carcinoma (HCC). 25% of MASLD-HCCs occur in the absence of fibrosis.</p><p><strong>Objectives: </strong>This study aimed to explore lipid metabolic pathways through \"omics\" and to identify biomarkers of MASLD-HCC based on the degree of fibrosis.</p><p><strong>Methods: </strong>Our cohort included 79 pairs of MASLD-HCC tumor tissues (TT) and adjacent non-tumor human liver tissues (NTT), which were divided into two groups according to fibrosis degree (F0F2 n = 45 and F3F4 n = 34). Lipidomic analysis (n = 52) using liquid chromatography high-resolution mass spectrometry (LC-HRMS/MS) and gene expression analysis (n = 79) using RT-qPCR were performed. For each group, TT was compared with NTT. Five healthy liver tissues were used as calibrators in gene expression analysis.</p><p><strong>Results: </strong>Using LC-HRMS/MS, 130 lipids were putatively annotated, 30 of which showed a significant difference between TT and NTT. In MASLD-HCC-F0F2, ceramide levels decreased. While sphingomyelin, most phosphatidylcholine and phosphatidylethanolamine species were increased. In contrast, in MASLD-HCC-F3F4, most lipid contents decreased. Based on lipidomic data, a panel of 18 genes related to lipid metabolism was analyzed. The expression of six genes, ACAT2, DGAT2, ACOX1, CHKA, PLD1, and PLD2, was exclusively upregulated in MASLD-HCC-F0F2. Taken together, these data support the existence of two MASLD-HCC lipid metabolic phenotypes, according to the degree of fibrosis.</p><p><strong>Conclusion: </strong>In conclusion, our results allow: (1) discriminate two phenotypes of MASLD-HCC according to fibrosis level; (2) propose PLD as a potential drug target for MASLD-HCC-F0F2 patients, and suggest that PLD inhibitor could be evaluated in combination with immunotherapy treatment.</p>","PeriodicalId":18506,"journal":{"name":"Metabolomics","volume":"21 2","pages":"39"},"PeriodicalIF":3.5,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143597379","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Differential metabolic profiles by Hispanic ethnicity among male Tucson firefighters.","authors":"Tuo Liu, Melissa A Furlong, Justin M Snider, Malak M Tfaily, Christian Itson, Shawn C Beitel, John J Gulotta, Krishna Parsawar, Kristen Keck, James Galligan, Douglas I Walker, Jaclyn M Goodrich, Jefferey L Burgess","doi":"10.1007/s11306-024-02198-9","DOIUrl":"10.1007/s11306-024-02198-9","url":null,"abstract":"<p><strong>Introduction: </strong>Firefighters face regular exposure to known and probable human carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), benzene, and formaldehyde, leading to an increased risk of various cancers compared to the general population. Hispanic and black firefighters are at increased risk of additional cancers not elevated in non-Hispanic white firefighters, yet biological pathways underlying these differences are unknown.</p><p><strong>Objectives: </strong>The study objectives were to evaluate differences in the urinary metabolome between Hispanic and non-Hispanic firefighters, pre-and post-fireground exposure.</p><p><strong>Methods: </strong>To investigate the metabolic patterns, we employed a comprehensive metabolomics pipeline that leveraged liquid chromatography coupled with high-resolution mass spectrometry. We applied linear mixed effects regression to identify the differential metabolites at an FDR < 0.05 among 19 Hispanic and 81 non-Hispanic firefighters. We also performed overrepresentation analysis using Mummichog to identify enriched pathways at FDR < 0.05.</p><p><strong>Results: </strong>Out of 175 features in HILIC(-) mode and 1847 features in RP(+) mode, we found 26 and 276 differential urinary features, respectively, when comparing Hispanic and non-Hispanic firefighters. We noted pathway enrichment in tryptophan and galactose metabolism. However, post-exposure, we did not observe differences in the metabolomic response by ethnicity despite differing fireground exposures.</p><p><strong>Conclusion: </strong>Dysregulation in the tryptophan and galactose pathway is an important contributor to cancer risks and may explain the increased cancer risk among Hispanic firefighters.</p>","PeriodicalId":18506,"journal":{"name":"Metabolomics","volume":"21 2","pages":"37"},"PeriodicalIF":3.5,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11885328/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143567535","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction: Metabolic response of Klebsiella oxytoca to ciprofloxacin exposure: a metabolomics approach.","authors":"Shwan Ahmed, Sahand Shams, Dakshat Trivedi, Cassio Lima, Rachel McGalliard, Christopher M Parry, Enitan D Carrol, Howbeer Muhamadali, Royston Goodacre","doi":"10.1007/s11306-025-02234-2","DOIUrl":"10.1007/s11306-025-02234-2","url":null,"abstract":"","PeriodicalId":18506,"journal":{"name":"Metabolomics","volume":"21 2","pages":"38"},"PeriodicalIF":3.5,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11885347/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143567533","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolomic analysis to study the effect of foliar copper supplementation on sulfur-containing compounds of garlic bulb by LC-MS.","authors":"Natália Reis de Almeida, Brena Rodrigues Mota Ikehara, Camila Cristina Calácio, Thaísa Fernanda Oliveira, Frederico Garcia Pinto","doi":"10.1007/s11306-025-02237-z","DOIUrl":"https://doi.org/10.1007/s11306-025-02237-z","url":null,"abstract":"<p><strong>Introduction: </strong>Garlic (Allium sativum L.) is renowned for its health-promoting properties, largely due to its sulfur-rich compounds. While copper is essential for plant growth and metabolism, excessive levels can disrupt cellular processes and lead to oxidative stress.</p><p><strong>Objectives: </strong>This study aims to investigate the impact of copper supplementation on the metabolic profile of garlic, with a particular focus on changes in sulfur metabolism.</p><p><strong>Methods: </strong>Ito garlic cloves were harvested in 2020 on Red-Yellow Latosol soil. Copper chelate fertilizer was applied foliarly at 300 mL/ha, 30, 20, and 10 days before harvest. After harvesting, cloves were refrigerated and analyzed. Using LC-MS metabolomics, the metabolic profile of garlic was analyzed after copper supplementation to assess changes, specifically in sulfur-containing compounds.</p><p><strong>Results: </strong>Copper supplementation led to a significant reduction in key sulfur-containing metabolites critical for the health-promoting properties of garlic, including allicin (FC = 0.0947), alliin (FC = 0.0147), and γ-glutamyl-S-allylcysteine (FC = 0.0076). Enrichment analysis identified alterations in pathways related to glutamine, glutamate, alanine, and aspartate metabolism. Additionally, precursors of glutathione (GSH) were depleted, likely as a result of GSH sparing efforts to counteract copper-induced oxidative stress. This redirection may increase susceptibility to ferroptosis, a form of cell death linked to oxidative damage.</p><p><strong>Conclusion: </strong>The metabolomic analysis of copper-supplemented Ito garlic cloves showed a significant reduction in sulfur compounds allicin, alliin, and γ-glutamyl-S-allylcysteine, important for organoleptic and medicinal properties. This decrease indicates a metabolic shift towards antioxidant defenses, with glutathione being redirected to defense pathways rather than secondary metabolites. Future studies should explore oxidative stress and ferroptosis markers, and lipidomics for a deeper understanding of garlic response to copper exposure.</p>","PeriodicalId":18506,"journal":{"name":"Metabolomics","volume":"21 2","pages":"36"},"PeriodicalIF":3.5,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143557255","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of gangliosides and ceramides as biomarkers for mucopolysaccharidosis type II (hunter syndrome) through untargeted lipidomic analysis.","authors":"Asma Farjallah, Bruno Maranda, Roberto Giugliani, Christiane Auray-Blais","doi":"10.1007/s11306-025-02233-3","DOIUrl":"https://doi.org/10.1007/s11306-025-02233-3","url":null,"abstract":"<p><strong>Introduction: </strong>Mucopolysaccharidosis type II (Hunter syndrome) is an X-linked recessive disorder caused by iduronate-2-sulfatase deficiency, affecting mainly male patients. The lack of its enzyme activity causes the accumulation of the glycosaminoglycans heparan sulfate and dermatan sulfate in all body tissues and leads to a secondary accumulation of gangliosides and ceramides.</p><p><strong>Objective and methods: </strong>We conducted a lipidomic study to investigate the dysregulation of lipid pathways in neuronopathic MPS type II. A modified liquid extraction was performed for untargeted lipid analysis. A reverse phase ultraperformance liquid chromatography coupled to quadrupole time-of-flight (UPLC-QTOF) mass spectrometry allowed the identification of upregulated ganglioside and ceramide biomarkers in the plasma and urine of a MPS II patient group compared to a healthy control group.</p><p><strong>Results: </strong>The altered pathways, including those related to glycerophospholipid metabolism and fatty acid oxidation, highlight the essential role of lipid metabolism in the progression of the disease.</p><p><strong>Conclusion: </strong>The accumulation of gangliosides and ceramides could be associated with the neuropathology in various lysosomal storage diseases including MPS II.</p>","PeriodicalId":18506,"journal":{"name":"Metabolomics","volume":"21 2","pages":"35"},"PeriodicalIF":3.5,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537343","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolomic profiling of plasma from glioma and meningioma patients based on two complementary mass spectrometry techniques.","authors":"Adrian Godlewski, Patrycja Mojsak, Tomasz Pienkowski, Tomasz Lyson, Zenon Mariak, Joanna Reszec, Karol Kaminski, Marcin Moniuszko, Adam Kretowski, Michal Ciborowski","doi":"10.1007/s11306-025-02231-5","DOIUrl":"10.1007/s11306-025-02231-5","url":null,"abstract":"<p><strong>Introduction: </strong>Extracranial and intracranial tumors are a diverse group of malignant and benign neoplasms, influenced by multiple factors. Given the complex nature of these tumors and usually late or accidental diagnosis, minimally invasive, rapid, early, and accurate diagnostic methods are urgently required. Metabolomics offers promising insights into central nervous system tumors by uncovering distinctive metabolic changes linked to tumor development.</p><p><strong>Objectives: </strong>This study aimed to elucidate the role of altered metabolites and the associated biological pathways implicated in the development of gliomas and meningiomas.</p><p><strong>Methods: </strong>The study was conducted on 95 patients with gliomas, 68 patients with meningiomas, and 71 subjects as a control group. The metabolic profiling of gliomas and meningiomas achieved by integrating untargeted metabolomic analysis based on GC-MS and targeted analysis performed using LC-MS/MS represents the first comprehensive study. Three comparisons (gliomas or meningiomas vs. controls as well as gliomas vs. meningiomas) were performed to reveal statistically significant metabolites.</p><p><strong>Results: </strong>Comparative analysis revealed 97, 56, and 27 significant metabolites for gliomas vs. controls, meningiomas vs. controls and gliomas vs. meningiomas comparison, respectively. Moreover, among above mentioned comparisons unique metabolites involved in arginine biosynthesis and metabolism, the Krebs cycle, and lysine degradation pathways were found. Notably, 2-aminoadipic acid has been identified as a metabolite that can be used in distinguishing two tumor types.</p><p><strong>Conclusions: </strong>Our results provide a deeper understanding of the metabolic changes associated with brain tumor development and progression.</p>","PeriodicalId":18506,"journal":{"name":"Metabolomics","volume":"21 2","pages":"33"},"PeriodicalIF":3.5,"publicationDate":"2025-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143476530","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolic and proteomic analysis of a medicinal morel (Morchella elata) from Western Himalayas, Kashmir.","authors":"Tariq Saiff Ullah, Syeda Sadiqa Firdous, Hamayun Shaheen, Muhamamd Manzoor, Syed Waseem Gillani, Wayne Thomas Shier, Baber Ali, Tabarak Malik, Sezai Ercisli, Reem M Aljowaie, Mohamed S Elshikh","doi":"10.1007/s11306-025-02222-6","DOIUrl":"https://doi.org/10.1007/s11306-025-02222-6","url":null,"abstract":"<p><p>Morels are edible fungi growing naturally in the wild and cultivated for food and medicines worldwide. They have been collected and consumed by people since ancient times. In the present study, fruiting bodies of Morchella elata were collected from the field during the years 2020-22 through consecutive field visits. Identification was carried out through a morpho-anatomical and phylogenetic study that confirmed the collected morel species as Morchella elata. The metabolic analysis was conducted using Ultra High-Performance Liquid Chromatography/Mass Spectrometry (UHPLC/MS) and FTICR/orbitrap techniques. The study revealed the presence of 159 organic compounds and 435 peptide sequences in the ascocarp. Different bioactive and significant compounds have been identified in the fruiting bodies of M. elata. This mushroom is highly nutritious, and the presence of these bioactive compounds contributes to its health benefits, making it a potential functional food in nutraceuticals. From the current study, it is concluded that M. elata is an edible, highly nutritive fungus and contains many bioactive contents. It could be used in the screening of bioactive substances useful in the preparation of anticancer drugs.</p>","PeriodicalId":18506,"journal":{"name":"Metabolomics","volume":"21 2","pages":"34"},"PeriodicalIF":3.5,"publicationDate":"2025-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143476527","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of metabolite-disease associations based on knowledge graph.","authors":"Fuheng Xiao, Canling Huang, Ali Chen, Wei Xiao, Zhanchao Li","doi":"10.1007/s11306-025-02227-1","DOIUrl":"10.1007/s11306-025-02227-1","url":null,"abstract":"<p><strong>Background: </strong>Despite the insights that metabolite analysis can provide into the onset, development, and progression of diseases-thus offering new concepts and methodologies for prevention, diagnosis, and treatment-traditional wet lab experiments are often time-consuming and labor-intensive. Consequently, this study aimed to develop a machine learning model named COM-RAN, which is based on a knowledge graph and random forest algorithm, to identify potential associations between metabolites and diseases.</p><p><strong>Methods: </strong>Firstly, we integrated the known associations between diseases and metabolites. Secondly, we provided a synthesis of the extant data regarding diseases and metabolites, accompanied by supplementary information pertinent to these entities. Thirdly, knowledge graph-based embedded features were used to characterize disease-metabolite associations. Finally, a random forest algorithm was employed to construct a model for identifying potential disease-metabolite associations.</p><p><strong>Results: </strong>The experimental results demonstrated that the proposed model achieved an Area Under the Receiver Operating Characteristic Curve (AUC) of 0.968 in 5-fold cross-validations, while the Area Under the Precision-Recall Curve (AUPR) was 0.901, outperforming the vast majority of existing prediction methods. The case studies corroborated the majority of the novel associations identified by COM-RAN, thereby further demonstrating the reliability of the current method in predicting the potential relationship between metabolites and diseases.</p><p><strong>Conclusion: </strong>The COM-RAN model demonstrated promise in predicting associations between diseases and metabolites, suggesting that integrating knowledge graphs with machine learning methodologies can significantly improve the accuracy and reliability of predictions related to disease-associated metabolites.</p>","PeriodicalId":18506,"journal":{"name":"Metabolomics","volume":"21 2","pages":"32"},"PeriodicalIF":3.5,"publicationDate":"2025-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143476522","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of solutions for stabilizing feces in metabolomics studies using GC × GC-TOFMS.","authors":"Seo Lin Nam, Kieran S Tarazona Carrillo, A Paulina de la Mata, Ryland T Giebelhaus, Olle M de Bruin, Evgueni Doukhanine, James J Harynuk","doi":"10.1007/s11306-025-02232-4","DOIUrl":"https://doi.org/10.1007/s11306-025-02232-4","url":null,"abstract":"<p><strong>Introduction: </strong>Fecal metabolomics studies have garnered interest in recent years due to the potential for these samples to provide unique information about an individual. Stool is a dynamic mixture of human excrement, microbiota, and enzymes that yields a constantly changing metabolite profile. The main challenge in a fecal metabolomics study is ensuring that the metabolite profile changes as little as possible between sample collection and sample processing/analysis.</p><p><strong>Objectives: </strong>This study aimed to evaluate the efficacy of five solutions in preserving human fecal metabolites over a seven-day storage period at ambient temperature, enabling at-home collection, cost-effective ambient transport and sample storage.</p><p><strong>Method: </strong>Five solutions with varying chemical compositions were evaluated for their ability to stabilize fecal metabolites. Samples were stored at ambient temperature for seven days, and metabolites were analyzed using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-TOFMS). The stabilizing efficacy of the solutions was assessed using total useful peak area (TUPA), absolute relative change (ARC) and compound class-based analyses, comparing the initial, stabilized, and unstabilized samples.</p><p><strong>Results: </strong>Different solutions demonstrated varied efficiencies for different compound classes. Overall, the results indicated that the use of stabilization solutions significantly minimized changes in the fecal metabolite profile compared to unstabilized samples left at room temperature for one week.</p><p><strong>Conclusion: </strong>This study demonstrates that stabilization solutions are effective in preserving fecal metabolites during storage at ambient temperature, supporting the feasibility of at-home sample collection.</p>","PeriodicalId":18506,"journal":{"name":"Metabolomics","volume":"21 2","pages":"31"},"PeriodicalIF":3.5,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143468129","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}