EMBO Reports最新文献_第2页

Schizophrenia-related Xpo7 haploinsufficiency leads to behavioral and nuclear transport pathologies. 精神分裂症相关的Xpo7单倍不足导致行为和核转运病理。

IF 6.5 1区生物学

EMBO Reports Pub Date : 2025-01-07 DOI: 10.1038/s44319-024-00362-9

Saori Toyoda, Masataka Kikuchi, Yoshifumi Abe, Kyosei Tashiro, Takehisa Handa, Shingo Katayama, Yukiko Motokawa, Kenji F Tanaka, Hidehiko Takahashi, Hiroki Shiwaku

{"title":"Schizophrenia-related Xpo7 haploinsufficiency leads to behavioral and nuclear transport pathologies.","authors":"Saori Toyoda, Masataka Kikuchi, Yoshifumi Abe, Kyosei Tashiro, Takehisa Handa, Shingo Katayama, Yukiko Motokawa, Kenji F Tanaka, Hidehiko Takahashi, Hiroki Shiwaku","doi":"10.1038/s44319-024-00362-9","DOIUrl":"https://doi.org/10.1038/s44319-024-00362-9","url":null,"abstract":"Recent genetic studies by the Schizophrenia Exome Sequencing Meta-Analysis (SCHEMA) consortium have identified that protein-truncating variants of exportin 7 (XPO7) can increase the risk of schizophrenia (odds ratio, 28.1). Here we show that mice with Xpo7 haploinsufficiency (Xpo7+/- mice) present with cognitive and social behavioral impairments. Through proteome analysis using immunoprecipitation and frontal cortex nuclear isolation of Xpo7+/- mice, we identify 45 molecules interacting with Xpo7, including CutC, Rbfox3, and Gria3. Through single-nucleus RNA sequencing of the frontal cortex and striatum of Xpo7+/- mice differentiating between the onset and progressive stages, we also identify 284 gene expression changes that correlate with these stages. These genes encompass high-odds risk genes of schizophrenia identified by SCHEMA, including Gria3, Grin2A, Herc1, and Trio. Furthermore, our approach reveals 15 gene expression changes in the frontal cortex that correlate with the progressive stages. Our findings indicate the importance of investigating whether the interactions among the high-risk genes identified by SCHEMA contribute to a common schizophrenia pathology and underscore the significance of stage-dependent analysis.","PeriodicalId":11541,"journal":{"name":"EMBO Reports","volume":" ","pages":""},"PeriodicalIF":6.5,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142946546","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Dbi1 is an oxidoreductase and an assembly chaperone for mitochondrial inner membrane proteins. Dbi1是一种氧化还原酶，是线粒体内膜蛋白的组装伴侣。

IF 6.5 1区生物学

EMBO Reports Pub Date : 2025-01-03 DOI: 10.1038/s44319-024-00349-6

Soraya Badrie, Kai Hell, Dejana Mokranjac

{"title":"Dbi1 is an oxidoreductase and an assembly chaperone for mitochondrial inner membrane proteins.","authors":"Soraya Badrie, Kai Hell, Dejana Mokranjac","doi":"10.1038/s44319-024-00349-6","DOIUrl":"https://doi.org/10.1038/s44319-024-00349-6","url":null,"abstract":"Import and assembly of mitochondrial proteins into multimeric complexes are essential for cellular function. Yet, many steps of these processes and the proteins involved remain unknown. Here, we identify a novel pathway for disulfide bond formation and assembly of mitochondrial inner membrane (IM) proteins. Dbi1, a previously uncharacterized IM protein, interacts with an unassembled pool of Tim17, the central subunit of the presequence translocase of the IM, and is upregulated in cells with increased levels of unassembled Tim17. In the absence of Dbi1, the conformation of the presequence translocase is affected and stability of Tim17 is reduced. Furthermore, Dbi1, through its conserved CxxC motif, is involved in the formation of the disulfide bond in Tim17 in a manner independent of the disulfide relay system, the major oxidation-driven protein import pathway into mitochondria. The substrate spectrum of Dbi1 is not limited to Tim17 but includes at least two more IM proteins, Tim22 and Cox20. We conclude that Dbi1 is a novel oxidoreductase in mitochondria which introduces disulfide bonds into IM proteins and chaperones their assembly into multimeric protein complexes.","PeriodicalId":11541,"journal":{"name":"EMBO Reports","volume":" ","pages":""},"PeriodicalIF":6.5,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142925977","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Pro-inflammatory macrophage activation does not require inhibition of oxidative phosphorylation. 促炎巨噬细胞的激活不需要氧化磷酸化的抑制。

IF 6.5 1区生物学

EMBO Reports Pub Date : 2025-01-03 DOI: 10.1038/s44319-024-00351-y

Andréa B Ball, Anthony E Jones, Kaitlyn B Nguyễn, Amy Rios, Nico Marx, Wei Yuan Hsieh, Krista Yang, Brandon R Desousa, Kristen K O Kim, Michaela Veliova, Zena Marie Del Mundo, Orian S Shirihai, Cristiane Benincá, Linsey Stiles, Steven J Bensinger, Ajit S Divakaruni

{"title":"Pro-inflammatory macrophage activation does not require inhibition of oxidative phosphorylation.","authors":"Andréa B Ball, Anthony E Jones, Kaitlyn B Nguyễn, Amy Rios, Nico Marx, Wei Yuan Hsieh, Krista Yang, Brandon R Desousa, Kristen K O Kim, Michaela Veliova, Zena Marie Del Mundo, Orian S Shirihai, Cristiane Benincá, Linsey Stiles, Steven J Bensinger, Ajit S Divakaruni","doi":"10.1038/s44319-024-00351-y","DOIUrl":"https://doi.org/10.1038/s44319-024-00351-y","url":null,"abstract":"Pro-inflammatory macrophage activation is a hallmark example of how mitochondria serve as signaling organelles. Oxidative phosphorylation sharply decreases upon classical macrophage activation, as mitochondria are thought to shift from ATP production towards accumulating signals that amplify effector function. However, evidence is conflicting regarding whether this collapse in respiration is essential or dispensable. Here we systematically examine this question and show that reduced oxidative phosphorylation is not required for pro-inflammatory macrophage activation. Different pro-inflammatory stimuli elicit varying effects on bioenergetic parameters, and pharmacologic and genetic models of electron transport chain inhibition show no causative link between respiration and macrophage activation. Furthermore, the signaling metabolites succinate and itaconate can accumulate independently of characteristic breaks in the TCA cycle in mouse and human macrophages, and peritoneal macrophages can be activated in vivo without inhibition of oxidative phosphorylation. The results indicate there is plasticity in the metabolic phenotypes that can support pro-inflammatory macrophage activation.","PeriodicalId":11541,"journal":{"name":"EMBO Reports","volume":" ","pages":""},"PeriodicalIF":6.5,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142926581","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA. 转录因子p65 NF-κB/RELA的邻近蛋白相互作用及其调控逻辑。

IF 6.5 1区生物学

EMBO Reports Pub Date : 2025-01-03 DOI: 10.1038/s44319-024-00339-8

Lisa Leib, Jana Juli, Liane Jurida, Christin Mayr-Buro, Jasmin Priester, Hendrik Weiser, Stefanie Wirth, Simon Hanel, Daniel Heylmann, Axel Weber, M Lienhard Schmitz, Argyris Papantonis, Marek Bartkuhn, Jochen Wilhelm, Uwe Linne, Johanna Meier-Soelch, Michael Kracht

{"title":"The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA.","authors":"Lisa Leib, Jana Juli, Liane Jurida, Christin Mayr-Buro, Jasmin Priester, Hendrik Weiser, Stefanie Wirth, Simon Hanel, Daniel Heylmann, Axel Weber, M Lienhard Schmitz, Argyris Papantonis, Marek Bartkuhn, Jochen Wilhelm, Uwe Linne, Johanna Meier-Soelch, Michael Kracht","doi":"10.1038/s44319-024-00339-8","DOIUrl":"https://doi.org/10.1038/s44319-024-00339-8","url":null,"abstract":"The protein interactome of p65/RELA, the most active subunit of the transcription factor (TF) NF-κB, has not been previously determined in living cells. Using p65-miniTurbo fusion proteins and biotin tagging, we identify >350 RELA interactors from untreated and IL-1α-stimulated cells, including many TFs (47% of all interactors) and >50 epigenetic regulators belonging to different classes of chromatin remodeling complexes. A comparison with the interactomes of two point mutants of p65 reveals that the interactions primarily require intact dimerization rather than DNA-binding properties. A targeted RNAi screen for 38 interactors and subsequent functional transcriptome and bioinformatics studies identify gene regulatory (sub)networks, each controlled by RELA in combination with one of the TFs ZBTB5, GLIS2, TFE3/TFEB, or S100A8/A9. The large, dynamic and versatile high-resolution interactome of RELA and its gene regulatory logics provides a rich resource and a new framework for explaining how RELA cooperativity determines gene expression patterns.","PeriodicalId":11541,"journal":{"name":"EMBO Reports","volume":" ","pages":""},"PeriodicalIF":6.5,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142926559","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Viral oncogene EBNALP regulates YY1 DNA binding and alters host 3D genome organization. 病毒癌基因EBNALP调节YY1 DNA结合并改变宿主三维基因组组织。

IF 6.5 1区生物学

EMBO Reports Pub Date : 2025-01-02 DOI: 10.1038/s44319-024-00357-6

Chong Wang, Merrin Manlong Leong, Weiyue Ding, Yohei Narita, Xiang Liu, Hongbo Wang, Stefanie P T Yiu, Jessica Lee, Katelyn R S Zhao, Amy Cui, Benjamin Gewurz, Wolfgang Hammerschmidt, Mingxiang Teng, Bo Zhao

{"title":"Viral oncogene EBNALP regulates YY1 DNA binding and alters host 3D genome organization.","authors":"Chong Wang, Merrin Manlong Leong, Weiyue Ding, Yohei Narita, Xiang Liu, Hongbo Wang, Stefanie P T Yiu, Jessica Lee, Katelyn R S Zhao, Amy Cui, Benjamin Gewurz, Wolfgang Hammerschmidt, Mingxiang Teng, Bo Zhao","doi":"10.1038/s44319-024-00357-6","DOIUrl":"https://doi.org/10.1038/s44319-024-00357-6","url":null,"abstract":"The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNALP) is essential for the immortalization of naive B lymphocytes (NBLs). However, the mechanisms remain elusive. To understand EBNALP's role in B-cell transformation, we compare NBLs infected with wild-type EBV and an EBNALP-null mutant EBV using multi-omics techniques. EBNALP inactivation alters enhancer-promoter interactions, resulting in decreased CCND2 and increased CASP1 and BCL2L11 expression. Mechanistically, EBNALP interacts with and colocalizes with the looping factor YY1. Depletion of EBNALP reduces YY1 DNA-binding and enhancer-promoter interactions, similar to effects observed with YY1 depletion. Furthermore, EBNALP colocalizes with DPF2, a protein that binds to H3K14ac and H4K16ac. CRISPR depletion of DPF2 reduces both EBNALP and YY1 DNA binding, suggesting that the DPF2/EBNALP complex may tether YY1 to DNA to increase enhancer-promoter interactions. EBNALP inactivation also increases enhancer-promoter interactions at the CASP1 and BCL2L11 loci, along with elevated DPF2 and YY1 binding and DNA accessibility. Our data suggest that EBNALP regulates YY1 to rewire the host genome, which might facilitate naive B-cell transformation.","PeriodicalId":11541,"journal":{"name":"EMBO Reports","volume":" ","pages":""},"PeriodicalIF":6.5,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142921165","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

E3 ligase FBXW7 suppresses brown fat expansion and browning of white fat. E3连接酶FBXW7抑制棕色脂肪的扩张和白色脂肪的褐变。

IF 6.5 1区生物学

EMBO Reports Pub Date : 2025-01-02 DOI: 10.1038/s44319-024-00337-w

Jian Yu, Xuejiang Gu, Yingying Guo, Mingyuan Gao, Shimiao Cheng, Meiyao Meng, Xiangdi Cui, Zhe Zhang, Wenxiu Guo, Dandan Yan, Maozheng Sheng, Linhui Zhai, Jing Ji, Xinhui Ma, Yu Li, Yuxiang Cao, Xia Wu, Jiejie Zhao, Yepeng Hu, Minjia Tan, Yan Lu, Lingyan Xu, Bin Liu, Cheng Hu, Xinran Ma

{"title":"E3 ligase FBXW7 suppresses brown fat expansion and browning of white fat.","authors":"Jian Yu, Xuejiang Gu, Yingying Guo, Mingyuan Gao, Shimiao Cheng, Meiyao Meng, Xiangdi Cui, Zhe Zhang, Wenxiu Guo, Dandan Yan, Maozheng Sheng, Linhui Zhai, Jing Ji, Xinhui Ma, Yu Li, Yuxiang Cao, Xia Wu, Jiejie Zhao, Yepeng Hu, Minjia Tan, Yan Lu, Lingyan Xu, Bin Liu, Cheng Hu, Xinran Ma","doi":"10.1038/s44319-024-00337-w","DOIUrl":"https://doi.org/10.1038/s44319-024-00337-w","url":null,"abstract":"Thermogenic fat, including brown and beige fat, dissipates heat via thermogenesis and enhances energy expenditure. Thus, its activation represents a therapeutic strategy to combat obesity. Here, we demonstrate that levels of F-box and WD repeat domain-containing 7 (FBXW7), an E3 ubiquitin protein ligase, negatively correlate with thermogenic fat functionality. FBXW7 overexpression in fat suppresses energy expenditure and thermogenesis, thus aggravates obesity and metabolic dysfunctions in mice. Conversely, FBXW7 depletion in fat leads to brown fat expansion and browning of white fat, and protects mice from diet induced obesity, hepatic steatosis, and hyperlipidemia. Mechanistically, FBXW7 binds to S6K1 and promotes its ubiquitination and proteasomal degradation, which in turn impacts glycolysis and brown preadipocyte proliferation via lactate. Besides, the beneficial metabolic effects of FBXW7 depletion in fat are attenuated by fat-specific knockdown of S6K1 in vivo. In summary, we provide evidence that adipose FBXW7 acts as a major regulator for thermogenic fat biology and energy homeostasis and serves as potential therapeutic target for obesity and metabolic diseases.","PeriodicalId":11541,"journal":{"name":"EMBO Reports","volume":" ","pages":""},"PeriodicalIF":6.5,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142921055","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

HDAC6 deacetylates TRIM56 to negatively regulate cGAS-STING-mediated type I interferon responses. HDAC6使TRIM56脱乙酰，负调控cgas - sting介导的I型干扰素反应。

IF 6.5 1区生物学

EMBO Reports Pub Date : 2025-01-02 DOI: 10.1038/s44319-024-00358-5

Qiongzhen Zeng, Zixin Chen, Shan Li, Ziwei Huang, Zhe Ren, Cuifang Ye, Xiao Wang, Jun Zhou, Kaisheng Liu, Kai Zheng, Yifei Wang

引用次数: 0

Distinct mechanisms control the specific synaptic functions of Neuroligin 1 and Neuroligin 2. 不同的机制控制着神经胶质素1和神经胶质素2的特定突触功能。

IF 6.5 1区生物学

EMBO Reports Pub Date : 2025-01-02 DOI: 10.1038/s44319-024-00286-4

Jinzhao Wang, Thomas Sudhof, Marius Wernig

{"title":"Distinct mechanisms control the specific synaptic functions of Neuroligin 1 and Neuroligin 2.","authors":"Jinzhao Wang, Thomas Sudhof, Marius Wernig","doi":"10.1038/s44319-024-00286-4","DOIUrl":"https://doi.org/10.1038/s44319-024-00286-4","url":null,"abstract":"Neuroligins are postsynaptic cell-adhesion molecules that regulate synaptic function with a remarkable isoform specificity. Although Nlgn1 and Nlgn2 are highly homologous and biochemically interact with the same extra- and intracellular proteins, Nlgn1 selectively functions in excitatory synapses whereas Nlgn2 functions in inhibitory synapses. How this excitatory/inhibitory (E/I) specificity arises is unknown. Using a comprehensive structure-function analysis, we here expressed wild-type and mutant neuroligins in functional rescue experiments in cultured hippocampal neurons lacking all endogenous neuroligins. Electrophysiology confirmed that Nlgn1 and Nlgn2 selectively restored excitatory and inhibitory synaptic transmission, respectively, in neuroligin-deficient neurons, aligned with their synaptic localizations. Chimeric Nlgn1-Nlgn2 constructs reveal that the extracellular neuroligin domains confer synapse specificity, whereas their intracellular sequences are exchangeable. However, the cytoplasmic sequences of Nlgn2, including its Gephyrin-binding motif that is identically present in the Nlgn1, is essential for its synaptic function whereas they are dispensable for Nlgn1. These results demonstrate that although the excitatory vs. inhibitory synapse specificity of Nlgn1 and Nlgn2 are both determined by their extracellular sequences, these neuroligins enable normal synaptic connections via distinct intracellular mechanisms.","PeriodicalId":11541,"journal":{"name":"EMBO Reports","volume":" ","pages":""},"PeriodicalIF":6.5,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142920944","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The relevance of the history of biotechnology for healthcare : Teaching students how biotechnology and medicine have been closely entwined during the past century highlights how both fields have inspired and driven each other. 医疗保健生物技术历史的相关性：教授学生生物技术和医学如何在过去的一个世纪里紧密地交织在一起，突出了这两个领域是如何相互启发和推动的。

IF 6.5 1区生物学

EMBO Reports Pub Date : 2025-01-02 DOI: 10.1038/s44319-024-00355-8

Maurizio Bifulco, Erika Di Zazzo, Alessandra Affinito, Cristina Pagano

引用次数: 0

A phosphate-binding pocket in cyclin B3 is essential for XErp1/Emi2 degradation in meiosis I. 细胞周期蛋白B3中的磷酸盐结合袋对于减数分裂I中XErp1/Emi2的降解是必不可少的。

IF 6.5 1区生物学

EMBO Reports Pub Date : 2025-01-02 DOI: 10.1038/s44319-024-00347-8

Rebecca Schunk, Marc Halder, Michael Schäfer, Elijah Johannes, Andreas Heim, Andreas Boland, Thomas U Mayer

{"title":"A phosphate-binding pocket in cyclin B3 is essential for XErp1/Emi2 degradation in meiosis I.","authors":"Rebecca Schunk, Marc Halder, Michael Schäfer, Elijah Johannes, Andreas Heim, Andreas Boland, Thomas U Mayer","doi":"10.1038/s44319-024-00347-8","DOIUrl":"https://doi.org/10.1038/s44319-024-00347-8","url":null,"abstract":"To ensure the correct euploid state of embryos, it is essential that vertebrate oocytes await fertilization arrested at metaphase of meiosis II. This MII arrest is mediated by XErp1/Emi2, which inhibits the ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome). Cyclin B3 in complex with Cdk1 (cyclin-dependent kinase 1) is essential to prevent an untimely arrest of vertebrate oocytes in meiosis I by targeting XErp1/Emi2 for degradation. Yet, the molecular mechanism of XErp1/Emi2 degradation in MI is not well understood. Here, by combining TRIM-Away in oocytes with egg extract and in vitro studies, we demonstrate that a hitherto unknown phosphate-binding pocket in cyclin B3 is essential for efficient XErp1/Emi2 degradation in meiosis I. This pocket enables Cdk1/cyclin B3 to bind pre-phosphorylated XErp1/Emi2 facilitating further phosphorylation events, which ultimately target XErp1/Emi2 for degradation in a Plk1- (Polo-like kinase 1) dependent manner. Key elements of this degradative mechanism are conserved in frog and mouse. Our studies identify a novel, evolutionarily conserved determinant of Cdk/cyclin substrate specificity essential to prevent an untimely oocyte arrest at meiosis I with catastrophic consequences upon fertilization.","PeriodicalId":11541,"journal":{"name":"EMBO Reports","volume":" ","pages":""},"PeriodicalIF":6.5,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142920942","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0