Biochemical Journal最新文献

Phosphorus-specific, liquid chromatography inductively coupled plasma mass-spectrometry for analysis of inositol phosphate and inositol pyrophosphate metabolism. 磷酸特异性液相色谱-电感耦合等离子体质谱法分析磷酸肌醇和焦磷酸肌醇代谢。

IF 4.1 3区生物学

Biochemical Journal Pub Date : 2025-10-02 DOI: 10.1042/bcj20253151

Colleen Sprigg,Hayley L Whitfield,Philip T Leftwich,Hui-Fen Kuo,Tzyy-Jen Chiou,Adolfo Saiardi,Megan L Shipton,Andrew M Riley,Barry V L Potter,Dawn Scholey,Emily Burton,Mike R Bedford,Charles A Brearley

{"title":"Phosphorus-specific, liquid chromatography inductively coupled plasma mass-spectrometry for analysis of inositol phosphate and inositol pyrophosphate metabolism.","authors":"Colleen Sprigg,Hayley L Whitfield,Philip T Leftwich,Hui-Fen Kuo,Tzyy-Jen Chiou,Adolfo Saiardi,Megan L Shipton,Andrew M Riley,Barry V L Potter,Dawn Scholey,Emily Burton,Mike R Bedford,Charles A Brearley","doi":"10.1042/bcj20253151","DOIUrl":"https://doi.org/10.1042/bcj20253151","url":null,"abstract":"Inositol phosphate (InsP) and diphosphoinositol phosphate (PP-InsP) analysis in tissues is plagued by multiple difficulties of sensitivity, regioisomer resolution and the need for radiolabeling with metabolic precursors. We describe a liquid chromatography (LC) inductively coupled plasma (ICP) mass spectrometry (MS) method (LC-ICP-MS) that addresses all such issues and use LC-ICP-MS to analyse InsPs in avian tissues. The highly sensitive technique tolerates complex matrices and, by powerful chromatography, resolves in a single run multiple non-enantiomeric myo-inositol tetrakisphosphates, myo-inositol pentakisphosphates and all inositol hexakisphosphates, including myo-inositol 1,2,3,4,5,6-hexakisphosphate (phytate), known in nature. It also separates and quantifies diphospho myo-inositol pentakisphosphate (PP-InsP5) isomers from their biological precursors and from 1,5-bis-diphospho myo-inositol 2,3,4,6 tetrakisphosphate (1,5-[PP]2-InsP4). Gut tissue inositol phosphates, belonging to a non-canonical, lipid-independent pathway, are shown to differ from phytate digestion products and to be responsive to diet.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"22 1","pages":""},"PeriodicalIF":4.1,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145235869","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Proteostasis of immune checkpoint receptors. 免疫检查点受体的蛋白质停滞。

IF 4.3 3区生物学

Biochemical Journal Pub Date : 2025-10-01 DOI: 10.1042/BCJ20253299

Pei Yee Tey, Sylvie Urbé, Michael J Clague

{"title":"Proteostasis of immune checkpoint receptors.","authors":"Pei Yee Tey, Sylvie Urbé, Michael J Clague","doi":"10.1042/BCJ20253299","DOIUrl":"https://doi.org/10.1042/BCJ20253299","url":null,"abstract":"Immunotherapy relies on the targeting of immune checkpoint receptors and their respective ligands by specific antibodies that bind to the cell surface proteins. The pace of this highly successful clinical advancement has outstripped our cell biological understanding of these receptors. Here, we discuss what is known about their intracellular trafficking itineraries, which determine the bioavailability of these proteins for clinical targeting. Some of them are amongst the shortest-lived membrane proteins (CTLA-4), whilst others can be very stable (PD-L1). We highlight the ubiquitin system, which is key to determining their turnover, as it plays a key role in disposing of misfolded newly synthesised proteins via the ERAD pathway and generating a key signal for endosomal sorting towards lysosomes. In some cases, ubiquitylation can modulate the signalling function of the immune checkpoint receptor, as seen for LAG-3. Immune checkpoint proteins can evade lysosomal degradation by effective recycling to the plasma membrane using highly specialised factors, including CMTM6 (for PD-L1) and LRBA (for CTLA-4). Lastly, we consider how reprogramming the ubiquitin system emerges as an alternative modality in targeting immune checkpoint receptors.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"482 19","pages":"1489-1516"},"PeriodicalIF":4.3,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145197856","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Functional analysis and transcriptional profiling of non-coding RNAs in yeast. 酵母非编码rna的功能分析和转录谱分析。

IF 4.1 3区生物学

Biochemical Journal Pub Date : 2025-09-30 DOI: 10.1042/bcj20253069

Tanda Qi,Daniela Delneri,Soukaina Timouma

{"title":"Functional analysis and transcriptional profiling of non-coding RNAs in yeast.","authors":"Tanda Qi,Daniela Delneri,Soukaina Timouma","doi":"10.1042/bcj20253069","DOIUrl":"https://doi.org/10.1042/bcj20253069","url":null,"abstract":"Advanced transcriptomic technology has identified a great number of non-coding RNAs (ncRNAs) that are pervasively transcribed in the yeast genome. ncRNAs can be classified into short ncRNAs (<200 nt) and long ncRNAs (lncRNAs; >200 nt). Those transcripts are strictly regulated through transcription and degradation mechanisms to maintain proper cellular homeostasis and prevent aberrant expression. It has been revealed that ncRNAs can play roles in various regulatory processes, particularly in transcriptional regulation. While short ncRNAs are well characterised, the function of lncRNAs remains poorly understood. Both functional and transcriptional profiling have been applied to fill the gap in the lncRNA functions landscape. It has been proven by functional profiling that these long transcripts can serve important cellular roles in gene regulation, RNA metabolism, sexual differentiation and telomeric overhang homeostasis. In addition, transcriptional profiling allowed the characterisation of ncRNAs involved in the cell cycle, colony subpopulation dynamics, virulence and regulatory networks. In this review, we introduce the classification, the cellular fate, the evolution and conservation, the mechanisms of action, and the profiling of yeast ncRNAs.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"1 1","pages":""},"PeriodicalIF":4.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145194920","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Histone supply: a precious commodity for cell identity. 组蛋白供应：细胞身份的宝贵商品。

IF 4.1 3区生物学

Biochemical Journal Pub Date : 2025-09-25 DOI: 10.1042/bcj20253163

Sara Gonske,Thelma M Escobar,Alejandra Loyola

引用次数: 0

Membrane disruption attenuates agonist potency in prostanoid receptors. 膜破坏减弱前列腺素受体的激动剂效力。

IF 4.1 3区生物学

Biochemical Journal Pub Date : 2025-09-25 DOI: 10.1042/bcj20253332

Uurtuya Hochban,Imke Wallenstein,Michaela Ulrich,Alwina Bittner,Lisa Spänig,Katharina Klingelhöfer,Sebastian Neumann,Torsten Steinmetzer,Moritz Bünemann,Michael Kurz

{"title":"Membrane disruption attenuates agonist potency in prostanoid receptors.","authors":"Uurtuya Hochban,Imke Wallenstein,Michaela Ulrich,Alwina Bittner,Lisa Spänig,Katharina Klingelhöfer,Sebastian Neumann,Torsten Steinmetzer,Moritz Bünemann,Michael Kurz","doi":"10.1042/bcj20253332","DOIUrl":"https://doi.org/10.1042/bcj20253332","url":null,"abstract":"G protein-coupled receptors (GPCRs) are key signal transducers and the target of about one-third of all FDA-approved drugs. Many structural and pharmacological studies rely on disrupted membrane conditions, such as purified receptors in artificial systems or radioligand binding assays using membrane fragments, even though it had not been systematically studied whether membrane integrity affects GPCR function. To address this, we developed Förster resonance energy transfer (FRET)-based GPCR conformation sensors to directly measure receptor activation in both intact and disrupted membranes. Our results show that while some GPCRs remain unaffected, prostanoid receptor conformation sensors exhibit a strong dependence on membrane integrity: their agonist and antagonist potencies decrease up to 30-fold upon membrane disruption, revealing a crucial role of the membrane integrity in ligand-receptor affinity. Validation with wild-type receptors in functional signaling assays confirmed that these effects reflect genuine receptor characteristics rather than unspecific signals from the sensor design. We ruled out several factors that could explain the loss of affinity, but were unable to fully elucidate the mechanism behind this phenomenon. Nevertheless, this effect may introduce bias into structural and pharmacological studies. It is therefore essential to account for membrane integrity and to employ optimized experimental strategies to ensure robust and reliable data interpretation.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"29 1","pages":""},"PeriodicalIF":4.1,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145182665","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

And… cut! - how conformational regulation of CRISPR-Cas effectors directs nuclease activity. 和……切!- CRISPR-Cas效应物的构象调控如何指导核酸酶活性。

IF 4.1 3区生物学

Biochemical Journal Pub Date : 2025-09-25 DOI: 10.1042/bcj20240481

Roland W Calvert,Gavin J Knott

{"title":"And… cut! - how conformational regulation of CRISPR-Cas effectors directs nuclease activity.","authors":"Roland W Calvert,Gavin J Knott","doi":"10.1042/bcj20240481","DOIUrl":"https://doi.org/10.1042/bcj20240481","url":null,"abstract":"Controlling the conformation of dynamic protein, RNA and DNA molecules underpins many biological processes, from the activation of enzymes and induction of signalling cascades to cellular replication. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) effectors are enzymes tightly controlled by conformational steps that gate activation of nuclease domains core to their function in bacterial adaptive immunity. These precise conformational checkpoints combined with programmable activation specified by RNA guides have driven the success of CRISPR-Cas tools in biotechnology, medicine and beyond. To illustrate the importance of conformation in controlling CRISPR-Cas activity, we review the discrete conformational checkpoints at play in class 2 CRISPR-Cas systems. Using Cas9, Cas12a and Cas13a as examples, we describe how protein and nucleic acid conformations precisely control the loading of guide RNA, the selection of target nucleic acids and the activation of nuclease domains. Much like a director controls the timing of transitions between scenes in a movie, CRISPR effectors use conformational checkpoints to precisely direct their enzymatic activity.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"94 1","pages":""},"PeriodicalIF":4.1,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145134092","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Unconventional binding of Calmodulin to CHK2 kinase inhibits catalytic activity. 钙调素与CHK2激酶的非常规结合抑制了催化活性。

IF 4.3 3区生物学

Biochemical Journal Pub Date : 2025-09-23 DOI: 10.1042/BCJ20253431

Christopher R Horne, Tingting Wang, Samuel N Young, Toby A Dite, Hunter G Nyvall, Sushant Suresh, Katherine A Davies, Abner Gonzalez Castro, Vineet Vaibhav, Lucy Mather J, Laura F Dagley, Matthew J Belousoff, Gerard Manning, Anthony R Means, John E Burke, Janni Petersen, John W Scott, James M Murphy

{"title":"Unconventional binding of Calmodulin to CHK2 kinase inhibits catalytic activity.","authors":"Christopher R Horne, Tingting Wang, Samuel N Young, Toby A Dite, Hunter G Nyvall, Sushant Suresh, Katherine A Davies, Abner Gonzalez Castro, Vineet Vaibhav, Lucy Mather J, Laura F Dagley, Matthew J Belousoff, Gerard Manning, Anthony R Means, John E Burke, Janni Petersen, John W Scott, James M Murphy","doi":"10.1042/BCJ20253431","DOIUrl":"https://doi.org/10.1042/BCJ20253431","url":null,"abstract":"Calmodulin (CaM) serves an essential role in eukaryotic cells as a Ca2+ sensor. Ca2+ binding leads to conformation changes in CaM that enable engagement of a repertoire of enzymes and the regulation of their catalytic activities. Classically, Ca2+-CaM binds to an inhibitory pseudosubstrate sequence C-terminal to the kinase domain in members of the Ca2+-CaM dependent protein kinase (CAMK) family, and relieves inhibition to promote catalytic activity. Here, we report an unexpected mechanism by which CaM can bind CHK2 kinase to inhibit its kinase activity. Using biochemical, biophysical, and structural mass spectrometry, we identify a direct interaction of Ca2+-CaM with the CHK2 kinase domain that suppresses CHK2 catalytic activity in vitro and identify K373 in CHK2 as crucial for cell proliferation in human cells following DNA damage. Our findings add direct suppression of kinase activity to the repertoire of CaM's functions, complementing the paradigmatic mechanism of promoting kinase activity through autoinhibitory domain sequestration.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145147546","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Characterisation of an allosteric site in PLCγ enzymes and implications for development of their specific inhibitors. PLCγ酶变构位点的特征及其特异性抑制剂开发的意义。

IF 4.1 3区生物学

Biochemical Journal Pub Date : 2025-09-23 DOI: 10.1042/bcj20253358

Tom D Bunney,Hunter G Nyvall,Calum Macrae,Damjan Lalović,Ashley Gregory,Kyle I P Le Huray,Nikita Harvey,Nikos Pinotsis,Antreas C Kalli,Christopher Waudby,John E Burke,Matilda Katan

{"title":"Characterisation of an allosteric site in PLCγ enzymes and implications for development of their specific inhibitors.","authors":"Tom D Bunney,Hunter G Nyvall,Calum Macrae,Damjan Lalović,Ashley Gregory,Kyle I P Le Huray,Nikita Harvey,Nikos Pinotsis,Antreas C Kalli,Christopher Waudby,John E Burke,Matilda Katan","doi":"10.1042/bcj20253358","DOIUrl":"https://doi.org/10.1042/bcj20253358","url":null,"abstract":"PLCγ enzymes are key components of intracellular signal transduction processes and are involved in disease development, including immune dysregulation, specific cancer types, and neurodegeneration. Although recognised as important targets for intervention, validated pharmacological tools are lacking. Here, we demonstrate that inhibitory nucleotides bind directly to an allosteric site at the interface between the PLC-core and regulatory-array unique for PLCγ, underlying their specificity for the PLCγ family. This binding site overlaps with the PLCγ autoinhibitory interface, suggesting that the inhibitory impact of nucleotides involves stabilization of autoinhibition. We have also analysed disease-linked variants of PLCγ1 and PLCγ2 to show that multiple mechanisms could underpin their gain-of-function phenotype. While sensitivity of these variants to physiological nucleotide inhibition is reduced, we identified artificial nucleotide compounds that can inhibit such variants not only in vitro but also in cell-based assays. Therefore, our findings suggest a route for development of isozyme specific PLCγ inhibitors allowing further studies of their roles in health and disease.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"41 1","pages":""},"PeriodicalIF":4.1,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145140137","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Anionic lipids modulate the membrane localization and conformational dynamics of KirBac1.1 slide helix during lipid-dependent activation. 阴离子脂质在脂质依赖激活过程中调节KirBac1.1滑动螺旋的膜定位和构象动力学。

IF 4.1 3区生物学

Biochemical Journal Pub Date : 2025-09-17 DOI: 10.1042/bcj20253215

Arpan Bysack,Chandrima Jash,H Raghuraman

{"title":"Anionic lipids modulate the membrane localization and conformational dynamics of KirBac1.1 slide helix during lipid-dependent activation.","authors":"Arpan Bysack,Chandrima Jash,H Raghuraman","doi":"10.1042/bcj20253215","DOIUrl":"https://doi.org/10.1042/bcj20253215","url":null,"abstract":"Inward-rectifier potassium (Kir) channels are essential for regulating various physiological processes and are implicated in several life-threatening diseases, making them key drug targets. KirBac1.1, a well-characterized prokaryotic homolog of Kir channels, is known to undergo anionic lipid-dependent gating. Although the slide helix is an important structural component in the gating mechanism of KirBac1.1, its structural dynamics associated with the anionic lipid-driven activation is not well-understood. Here, we have reconstituted KirBac1.1 in zwitterionic POPC and anionic POPC/POPG membranes to stabilize the inactive and active conformations of the channel, respectively. Our liposome K+ flux assay results show that all the slide helix single-cysteine mutants display PG-driven gating, and increasing the PG from 25 to 40 mol% does not have any linear dependency on both the activation and K+ flux rates. Site-directed NBD fluorescence results suggest that the structural dynamics of the slide helix is significantly altered upon PG-induced activation. For instance, we observe significant changes in hydration dynamics and rotational mobility of slide helix residues between functional states. MEM-based lifetime distribution analysis suggests that the conformational heterogeneity of the slide helix is functional-state dependent. Importantly, membrane penetration depth measurements reveal that the slide helix in the active KirBac1.1 is located ~3 Å deeper within the membrane interface, well supported by increased fluorescence lifetimes. Notably, the non-linear relationship between structural dynamics and PG content highlights the critical role of lipid-protein interactions and membrane surface charge in PG-mediated KirBac1.1 activation. These findings provide valuable insights into Kir channel gating mechanisms, and lipid-dependent gating of other channels.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"16 1","pages":""},"PeriodicalIF":4.1,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145083617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

De novo talin-1 variant L353F connects multifaceted clinical symptoms to alterations in talin-1 function. 新生talin-1变体L353F将多方面的临床症状与talin-1功能的改变联系起来。

IF 4.1 3区生物学

Biochemical Journal Pub Date : 2025-09-17 DOI: 10.1042/bcj20253128

Muktesh Athale,Neil Ball,Latifeh Azizi,Irene Valenzuela,Marta Codina,Andrea Martin-Nalda,Vasyl V Mykuliak,Rolle Rahikainen,Benjamin T Goult,Paula Turkki,Vesa P Hytönen

{"title":"De novo talin-1 variant L353F connects multifaceted clinical symptoms to alterations in talin-1 function.","authors":"Muktesh Athale,Neil Ball,Latifeh Azizi,Irene Valenzuela,Marta Codina,Andrea Martin-Nalda,Vasyl V Mykuliak,Rolle Rahikainen,Benjamin T Goult,Paula Turkki,Vesa P Hytönen","doi":"10.1042/bcj20253128","DOIUrl":"https://doi.org/10.1042/bcj20253128","url":null,"abstract":"Talin-1 is a central integrin adapter protein connecting cytoplasmic domains of integrins to the cytoskeleton. These talin-1-mediated mechanical linkages are crucial for cellular functions such as cell movement and connections with other cells. Here, we report a patient carrying a missense variant, L353F, in the talin-1 head which is associated with a complex set of symptoms, including skin lesions, blood cell abnormalities, and congenital cataracts. We conducted structural and cellular characterization of this variant. Recombinant talin-1 F2F3 fragment with the corresponding mutation showed a decrease in thermal stability and decreased solubility. Reconstitution of talin-deficient cells with L353F talin-1 revealed decreased cell migration velocity, defects in wound healing capacity, and changes in recruitment of the focal adhesion complex protein paxillin. We also observed decreased levels of activated integrin in cells expressing the talin-1 variant, while integrin-binding affinity was preserved as determined biochemically. These observations suggest that changes in integrin adhesion complex dynamics reflect cellular processes and the multifaceted patient phenotype.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"37 1","pages":"1337-1352"},"PeriodicalIF":4.1,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145071744","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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