Genomics, proteomics & bioinformatics最新文献

Evolution of Plant Genome Size and Composition. 植物基因组大小和组成的进化。

Genomics, proteomics & bioinformatics Pub Date : 2024-11-05 DOI: 10.1093/gpbjnl/qzae078

Bing He, Wanfei Liu, Jianyang Li, Siwei Xiong, Jing Jia, Qiang Lin, Hailin Liu, Peng Cui

引用次数: 0

Enzymes Repertoires and Genomic Insights into Lycium Barbarum Pectin Polysaccharides Biosynthesis. 枸杞果胶多糖生物合成的酵素再现和基因组洞察。

Genomics, proteomics & bioinformatics Pub Date : 2024-11-04 DOI: 10.1093/gpbjnl/qzae079

Haiyan Yue, Yiheng Tang, Aixuan Li, Lili Zhang, Yiwei Niu, Yiming Zhang, Hao Wang, Jianjun Luo, Yi Zhao, Shunmin He, Chang Chen, Runsheng Chen

{"title":"Enzymes Repertoires and Genomic Insights into Lycium Barbarum Pectin Polysaccharides Biosynthesis.","authors":"Haiyan Yue, Yiheng Tang, Aixuan Li, Lili Zhang, Yiwei Niu, Yiming Zhang, Hao Wang, Jianjun Luo, Yi Zhao, Shunmin He, Chang Chen, Runsheng Chen","doi":"10.1093/gpbjnl/qzae079","DOIUrl":"https://doi.org/10.1093/gpbjnl/qzae079","url":null,"abstract":"Lycium barbarum, a member of the Solanaceae family, represents an important eudicot lineage with homology of food and medicine. Lycium barbarum pectin polysaccharides (LBPPs) are key bioactive ingredients of Lycium barbarum, and are among the few polysaccharides with both biocompatibility and biomedical activity. While previous studies have primarily focused on the functional properties of LBPPs, the mechanisms of biosynthesis and transport by key enzymes remain poorly understood. Here, we reported the completion of a 2.18-gigabase reference genome of Lycium barbarum, reconstructed the first entire biosynthesis of pectin polysaccharides and sugar transport, and characterized the important genes responsible for backbone extending, sidechain synthesis, and modification of pectin polysaccharides. Additionally, we characterized long non-coding RNAs (lncRNAs) associated with polysaccharide metabolism and identified a specific rhamnogalacturonan I (RG-I) rhamnosyltransferase, RRT3020, which enhances RG-I biosynthesis in LBPPs. These newly identified enzymes and pivotal genes endow L. barbarum with specific pectin biosynthesis capabilities, distinguishing it from other Solanaceae species. Our findings provide a foundation for evolutionary studies and molecular breeding to enhance the diverse applications of L. barbarum.","PeriodicalId":94020,"journal":{"name":"Genomics, proteomics & bioinformatics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142570796","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}

引用次数: 0

Multi-omics Mediated Wide Association Studies: Novel Approaches for Understanding Diseases. 多组学介导的广泛关联研究：了解疾病的新方法。

Genomics, proteomics & bioinformatics Pub Date : 2024-10-29 DOI: 10.1093/gpbjnl/qzae077

Mengting Shao, Kaiyang Chen, Shuting Zhang, Min Tian, Yan Shen, Chen Cao, Ning Gu

{"title":"Multi-omics Mediated Wide Association Studies: Novel Approaches for Understanding Diseases.","authors":"Mengting Shao, Kaiyang Chen, Shuting Zhang, Min Tian, Yan Shen, Chen Cao, Ning Gu","doi":"10.1093/gpbjnl/qzae077","DOIUrl":"https://doi.org/10.1093/gpbjnl/qzae077","url":null,"abstract":"The rapid development of multi-omics (transcriptome, proteome, cistrome, imaging, and regulome) mediated wide association studies methods have opened new avenues for biologists to understand the susceptibility genes underlying complex diseases. Thorough comparisons of these methods are essential for selecting the most appropriate tool for a given research objective. This review provides a detailed categorization and summary of the statistical models, use cases, and advantages of recent multi-omics mediated wide association studies. In addition, to illustrate gene-disease association studies based on transcriptome-wide association studies (TWAS), we collected 478 disease entries across 22 categories from 235 manually reviewed publications. Our analysis reveals that mental disorders are the most frequently studied by TWAS, indicating its potential to deepen our understanding of the genetic architecture of complex diseases. In summary, this review underscores the importance of multi-omics mediated wide association studies in elucidating complex diseases and highlights the significance of selecting the appropriate method for each study.","PeriodicalId":94020,"journal":{"name":"Genomics, proteomics & bioinformatics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142549772","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}

引用次数: 0

Centromere Landscapes Resolved from Hundreds of Human Genomes. 从数百个人类基因组中解析中心粒景观

Genomics, proteomics & bioinformatics Pub Date : 2024-10-18 DOI: 10.1093/gpbjnl/qzae071

Shenghan Gao, Yimeng Zhang, Stephen J Bush, Bo Wang, Xiaofei Yang, Kai Ye

{"title":"Centromere Landscapes Resolved from Hundreds of Human Genomes.","authors":"Shenghan Gao, Yimeng Zhang, Stephen J Bush, Bo Wang, Xiaofei Yang, Kai Ye","doi":"10.1093/gpbjnl/qzae071","DOIUrl":"https://doi.org/10.1093/gpbjnl/qzae071","url":null,"abstract":"High-fidelity (HiFi) sequencing has facilitated the assembly and analysis of the most repetitive region of the genome, the centromere. Nevertheless, our current understanding of human centromeres is based on a relatively small number of telomere-to-telomere assemblies, which has not yet captured its full diversity. In this study, we investigated the genomic diversity of human centromere higher order repeats (HORs) via both HiFi reads and haplotype-resolved assemblies from hundreds of samples drawn from ongoing pangenome-sequencing projects and reprocessed them via a novel HOR annotation pipeline, HiCAT-human. We used this wealth of data to provide a global survey of the centromeric HOR landscape; in particular, we found that 23 HORs presented significant copy number variability between populations. We detected three centromere genotypes with unbalanced population frequencies on chromosomes 5, 8, and 17. An inter-assembly comparison of HOR loci further revealed that while HOR array structures are diverse, they nevertheless tend to form a number of specific landscapes, each exhibiting different levels of HOR subunit expansion and possibly reflecting a cyclical evolutionary transition from homogeneous to nested structures and back.","PeriodicalId":94020,"journal":{"name":"Genomics, proteomics & bioinformatics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142484010","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}

引用次数: 0

Bioinformatic Resources for Exploring Human-virus Protein-protein Interactions Based on Binding Modes. 基于结合模式探索人类-病毒蛋白质-蛋白质相互作用的生物信息资源。

Genomics, proteomics & bioinformatics Pub Date : 2024-10-15 DOI: 10.1093/gpbjnl/qzae075

Huimin Chen, Jiaxin Liu, Gege Tang, Gefei Hao, Guangfu Yang

引用次数: 0

MitoSort: Robust Demultiplexing of Pooled Single-cell Genomics Data Using Endogenous Mitochondrial Variants. MitoSort：利用内源性线粒体变异对汇集的单细胞基因组学数据进行稳健的解复用。

Genomics, proteomics & bioinformatics Pub Date : 2024-10-15 DOI: 10.1093/gpbjnl/qzae073

Zhongjie Tang, Weixing Zhang, Peiyu Shi, Sijun Li, Xinhui Li, Yueming Li, Yicong Xu, Yaqing Shu, Zheng Hu, Jin Xu

{"title":"MitoSort: Robust Demultiplexing of Pooled Single-cell Genomics Data Using Endogenous Mitochondrial Variants.","authors":"Zhongjie Tang, Weixing Zhang, Peiyu Shi, Sijun Li, Xinhui Li, Yueming Li, Yicong Xu, Yaqing Shu, Zheng Hu, Jin Xu","doi":"10.1093/gpbjnl/qzae073","DOIUrl":"https://doi.org/10.1093/gpbjnl/qzae073","url":null,"abstract":"Multiplexing across donors has emerged as a popular strategy to increase throughput, reduce costs, overcome technical batch effects, and improve doublet detection in single-cell genomic studies. To eliminate additional experimental steps, endogenous nuclear genome variants are used for demultiplexing pooled single-cell RNA sequencing (scRNA-seq) data by several computational tools. However, these tools have limitations when applied to single-cell sequencing methods that do not cover nuclear genomic regions well, such as single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq). Here, we demonstrate that mitochondrial germline variants are an alternative, robust, and computationally efficient endogenous barcode for sample demultiplexing. We propose MitoSort, a tool that uses mitochondrial germline variants to assign cells to their donor of origin and identify cross-genotype doublets in single-cell genomics datasets. We evaluate its performance by using in silico pooled mitochondrial scATAC-seq (mtscATAC-seq) libraries and experimentally multiplexed data with cell hashtags. MitoSort achieves high accuracy and efficiency in genotype clustering and doublet detection for mtscATAC-seq data, addressing the limitations of current computational techniques tailored for scRNA-seq data. Moreover, MitoSort exhibits versatility and can be applied to various single-cell sequencing approaches beyond mtscATAC-seq, provided the mitochondrial variants are reliably detected. Furthermore, we demonstrate the application of MitoSort in a case study where B cells from eight donors were pooled and assayed by single-cell multi-omics sequencing. Altogether, our results demonstrate the accuracy and efficiency of MitoSort, which enables reliable sample demultiplexing in various single-cell genomic applications. MitoSort is available at https://github.com/tangzhj/MitoSort.","PeriodicalId":94020,"journal":{"name":"Genomics, proteomics & bioinformatics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142484015","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}

引用次数: 0

The Role of N6-methyladenosine Modification in Gametogenesis and Embryogenesis: Impact on Fertility. N6-甲基腺苷修饰在配子发生和胚胎发生中的作用：对生育能力的影响

Genomics, proteomics & bioinformatics Pub Date : 2024-10-15 DOI: 10.1093/gpbjnl/qzae050

Yujie Wang, Chen Yang, Hanxiao Sun, Hui Jiang, Pin Zhang, Yue Huang, Zhenran Liu, Yaru Yu, Zuying Xu, Huifen Xiang, Chengqi Yi

{"title":"The Role of N6-methyladenosine Modification in Gametogenesis and Embryogenesis: Impact on Fertility.","authors":"Yujie Wang, Chen Yang, Hanxiao Sun, Hui Jiang, Pin Zhang, Yue Huang, Zhenran Liu, Yaru Yu, Zuying Xu, Huifen Xiang, Chengqi Yi","doi":"10.1093/gpbjnl/qzae050","DOIUrl":"10.1093/gpbjnl/qzae050","url":null,"abstract":"The most common epigenetic modification of messenger RNAs (mRNAs) is N6-methyladenosine (m6A), which is mainly located near the 3' untranslated region of mRNAs, near the stop codons, and within internal exons. The biological effect of m6A is dynamically modulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers). By controlling post-transcriptional gene expression, m6A has a significant impact on numerous biological functions, including RNA transcription, translation, splicing, transport, and degradation. Hence, m6A influences various physiological and pathological processes, such as spermatogenesis, oogenesis, embryogenesis, placental function, and human reproductive system diseases. During gametogenesis and embryogenesis, genetic material undergoes significant changes, including epigenomic modifications such as m6A. From spermatogenesis and oogenesis to the formation of an oosperm and early embryogenesis, m6A changes occur at every step. m6A abnormalities can lead to gamete abnormalities, developmental delays, impaired fertilization, and maternal-to-zygotic transition blockage. Both mice and humans with abnormal m6A modifications exhibit impaired fertility. In this review, we discuss the dynamic biological effects of m6A and its regulators on gamete and embryonic development and review the possible mechanisms of infertility caused by m6A changes. We also discuss the drugs currently used to manipulate m6A and provide prospects for the prevention and treatment of infertility at the epigenetic level.","PeriodicalId":94020,"journal":{"name":"Genomics, proteomics & bioinformatics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11514847/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473856","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}

引用次数: 0

Integrative Omics Uncovers Low Tumorous Magnesium Content as A Driver Factor of Colorectal Cancer. 整合分子生物学发现肿瘤镁含量低是结直肠癌的一个驱动因素

Genomics, proteomics & bioinformatics Pub Date : 2024-10-15 DOI: 10.1093/gpbjnl/qzae053

Rou Zhang, Meng Hu, Yu Liu, Wanmeng Li, Zhiqiang Xu, Siyu He, Ying Lu, Yanqiu Gong, Xiuxuan Wang, Shan Hai, Shuangqing Li, Shiqian Qi, Yuan Li, Yang Shu, Dan Du, Huiyuan Zhang, Heng Xu, Zongguang Zhou, Peng Lei, Hai-Ning Chen, Lunzhi Dai

{"title":"Integrative Omics Uncovers Low Tumorous Magnesium Content as A Driver Factor of Colorectal Cancer.","authors":"Rou Zhang, Meng Hu, Yu Liu, Wanmeng Li, Zhiqiang Xu, Siyu He, Ying Lu, Yanqiu Gong, Xiuxuan Wang, Shan Hai, Shuangqing Li, Shiqian Qi, Yuan Li, Yang Shu, Dan Du, Huiyuan Zhang, Heng Xu, Zongguang Zhou, Peng Lei, Hai-Ning Chen, Lunzhi Dai","doi":"10.1093/gpbjnl/qzae053","DOIUrl":"10.1093/gpbjnl/qzae053","url":null,"abstract":"Magnesium (Mg) deficiency is associated with increased risk and malignancy in colorectal cancer (CRC), yet the underlying mechanisms remain elusive. Here, we used genomic, proteomic, and phosphoproteomic data to elucidate the impact of Mg deficiency on CRC. Genomic analysis identified 160 genes with higher mutation frequencies in Low-Mg tumors, including key driver genes such as KMT2C and ERBB3. Unexpectedly, initiation driver genes of CRC, such as TP53 and APC, displayed higher mutation frequencies in High-Mg tumors. Additionally, proteomic and phosphoproteomic data indicated that low Mg content in tumors may activate epithelial-mesenchymal transition (EMT) by modulating inflammation or remodeling the phosphoproteome of cancer cells. Notably, we observed a negative correlation between the phosphorylation of DBN1 at S142 (DBN1S142p) and Mg content. A mutation in S142 to D (DBN1S142D) mimicking DBN1S142p up-regulated MMP2 and enhanced cell migration, while treatment with MgCl2 reduced DBN1S142p, thereby reversing this phenotype. Mechanistically, Mg2+ attenuated the DBN1-ACTN4 interaction by decreasing DBN1S142p, which in turn enhanced the binding of ACTN4 to F-actin and promoted F-actin polymerization, ultimately reducing MMP2 expression. These findings shed new light on the crucial role of Mg deficiency in CRC progression and suggest that Mg supplementation may be a promising preventive and therapeutic strategy for CRC.","PeriodicalId":94020,"journal":{"name":"Genomics, proteomics & bioinformatics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11514849/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141763491","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}

引用次数: 0

Characterization of Cancer Stem Cells in Laryngeal Squamous Cell Carcinoma by Single-cell RNA Sequencing. 通过单细胞 RNA 测序鉴定喉鳞状细胞癌中癌症干细胞的特征

Genomics, proteomics & bioinformatics Pub Date : 2024-10-15 DOI: 10.1093/gpbjnl/qzae056

Yanguo Li, Chen Lin, Yidian Chu, Zhengyu Wei, Qi Ding, Shanshan Gu, Hongxia Deng, Qi Liao, Zhisen Shen

{"title":"Characterization of Cancer Stem Cells in Laryngeal Squamous Cell Carcinoma by Single-cell RNA Sequencing.","authors":"Yanguo Li, Chen Lin, Yidian Chu, Zhengyu Wei, Qi Ding, Shanshan Gu, Hongxia Deng, Qi Liao, Zhisen Shen","doi":"10.1093/gpbjnl/qzae056","DOIUrl":"10.1093/gpbjnl/qzae056","url":null,"abstract":"Cancer stem cells (CSCs) constitute a pivotal element within the tumor microenvironment (TME), driving the initiation and progression of cancer. However, the identification of CSCs and their underlying molecular mechanisms in laryngeal squamous cell carcinoma (LSCC) remains a formidable challenge. Here, we employed single-cell RNA sequencing of matched primary tumor tissues, paracancerous tissues, and local lymph nodes from three LSCC patients to comprehensively characterize the CSCs in LSCC. Two distinct clusters of stem cells originating from epithelial populations were delineated and verified as CSCs and normal stem cells (NSCs), respectively. CSCs were abundant in the paracancerous tissues compared to those in the tumor tissues. CSCs showed high expression of stem cell marker genes such as PROM1, ALDH1A1, and SOX4, and increased the activity of tumor-related hypoxia, Wnt/β-catenin, and Notch signaling pathways. We then explored the intricate crosstalk between CSCs and the TME cells and identified targets within the TME that related with CSCs. We also found eight marker genes of CSCs that were correlated significantly with the prognosis of LSCC patients. Furthermore, bioinformatics analyses showed that drugs such as erlotinib, OSI-027, and ibrutinib selectively targeted the CSC-specifically expressed genes. In conclusion, our results represent the first comprehensive characterization of CSC properties in LSCC at the single-cell level.","PeriodicalId":94020,"journal":{"name":"Genomics, proteomics & bioinformatics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522873/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141899231","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}

引用次数: 0

Comprehensive Characterization of the Integrin Family Across 32 Cancer Types. 32 种癌症类型中整合素家族的综合特征。

Genomics, proteomics & bioinformatics Pub Date : 2024-10-15 DOI: 10.1093/gpbjnl/qzae035

Cheng Zou, Jinwei Zhu, Jiangling Xiong, Yu Tian, Yousong Peng, Edwin Cheung, Dingxiao Zhang

{"title":"Comprehensive Characterization of the Integrin Family Across 32 Cancer Types.","authors":"Cheng Zou, Jinwei Zhu, Jiangling Xiong, Yu Tian, Yousong Peng, Edwin Cheung, Dingxiao Zhang","doi":"10.1093/gpbjnl/qzae035","DOIUrl":"10.1093/gpbjnl/qzae035","url":null,"abstract":"Integrin genes are widely involved in tumorigenesis. Yet, a comprehensive characterization of integrin family members and their interactome at the pan-cancer level is lacking. Here, we systematically analyzed integrin family in approximately 10,000 tumors across 32 cancer types. Globally, integrins represent a frequently altered and misexpressed pathway, with alteration and dysregulation overall being protumorigenic. Expression dysregulation, better than mutational landscape, of integrin family successfully identifies a subgroup of aggressive tumors with a high level of proliferation and stemness. The results reveal that several molecular mechanisms collectively regulate integrin expression in a context-dependent manner. For potential clinical usage, we constructed a weighted scoring system, integrinScore, to measure integrin signaling patterns in individual tumors. Remarkably, integrinScore was consistently correlated with predefined molecular subtypes in multiple cancers, with integrinScore-high tumors being more aggressive. Importantly, integrinScore was cancer-dependent and closely associated with proliferation, stemness, tumor microenvironment, metastasis, and immune signatures. IntegrinScore also predicted patients' response to immunotherapy. By mining drug databases, we unraveled an array of compounds that may modulate integrin signaling. Finally, we built a user-friendly database, Pan-cancer Integrin Explorer (PIExplorer; http://computationalbiology.cn/PIExplorer), to facilitate researchers to explore integrin-related knowledge. Collectively, we provide a comprehensive characterization of integrins across cancers and offer gene-specific and cancer-specific rationales for developing integrin-targeted therapy.","PeriodicalId":94020,"journal":{"name":"Genomics, proteomics & bioinformatics","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142484011","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}

引用次数: 0