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Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity 进化耦合饱和诱变：共同进化指导下鉴定影响纳加诺芽孢杆菌普鲁兰酶活性的远距离位点
IF 3.5 4区 生物学
FEBS Letters Pub Date : 2020-03-01 DOI: 10.1002/1873-3468.13652
Xinye Wang, Xiaoran Jing, Yi Deng, Y. Nie, F. Xu, Yan Xu, Yi-Lei Zhao, J. Hunt, G. Montelione, T. Szyperski
{"title":"Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity","authors":"Xinye Wang, Xiaoran Jing, Yi Deng, Y. Nie, F. Xu, Yan Xu, Yi-Lei Zhao, J. Hunt, G. Montelione, T. Szyperski","doi":"10.1002/1873-3468.13652","DOIUrl":"https://doi.org/10.1002/1873-3468.13652","url":null,"abstract":"Pullulanases are well‐known debranching enzymes hydrolyzing α‐1,6‐glycosidic linkages. To date, engineering of pullulanase is mainly focused on catalytic pocket or domain tailoring based on structure/sequence information. Saturation mutagenesis‐involved directed evolution is, however, limited by the low number of mutational sites compatible with combinatorial libraries of feasible size. Using Bacillus naganoensis pullulanase as a target protein, here we introduce the ‘evolutionary coupling saturation mutagenesis’ (ECSM) approach: residue pair covariances are calculated to identify residues for saturation mutagenesis, focusing directed evolution on residue pairs playing important roles in natural evolution. Evolutionary coupling (EC) analysis identified seven residue pairs as evolutionary mutational hotspots. Subsequent saturation mutagenesis yielded variants with enhanced catalytic activity. The functional pairs apparently represent distant sites affecting enzyme activity.","PeriodicalId":50454,"journal":{"name":"FEBS Letters","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/1873-3468.13652","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43284777","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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: 19
Hirudin or hirudin‐like factor ‐ that is the question: insights from the analyses of natural and synthetic HLF variants 水蛭素或水蛭素样因子——这是一个问题：来自天然和合成HLF变体分析的见解
IF 3.5 4区 生物学
FEBS Letters Pub Date : 2019-11-16 DOI: 10.1002/1873-3468.13683
Christian Müller, Phil Lukas, Michel Böhmert, J. Hildebrandt
{"title":"Hirudin or hirudin‐like factor ‐ that is the question: insights from the analyses of natural and synthetic HLF variants","authors":"Christian Müller, Phil Lukas, Michel Böhmert, J. Hildebrandt","doi":"10.1002/1873-3468.13683","DOIUrl":"https://doi.org/10.1002/1873-3468.13683","url":null,"abstract":"The hirudin‐like factor 1 (HLF1) of Hirudo medicinalis belongs to a new class of leech‐derived factors. In previous investigations, HLF1 did not exhibit anticoagulatory activities. Here, we describe the analysis of natural and synthetic variants of HLF1 and HLF‐Hyb, a yet uncharacterized member of the HLF family. Modifications within the N terminus of HLF1 have a strong impact on its activity. Some variants of HLF1 exhibit thrombin‐inhibiting activity comparable to hirudins, whereas others have reduced or no activity. The analyses of HLF‐Hyb variants revealed a strong impact of the central globular domain on activity. Our results indicate a comparable mode of action of hirudins and thrombin‐inhibiting HLF variants. Finally, we propose and discuss criteria for classifying hirudins and HLFs.","PeriodicalId":50454,"journal":{"name":"FEBS Letters","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2019-11-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/1873-3468.13683","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46528291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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: 12
Proteomic analysis of affinity-purified 26S proteasomes identifies a suite of assembly chaperones in Arabidopsis. 亲和纯化的 26S 蛋白质体的蛋白质组学分析确定了拟南芥中的一套组装伴侣。
4区 生物学
FEBS Letters Pub Date : 2019-11-15 Epub Date: 2019-09-27 DOI: 10.1074/jbc.RA119.010219
David C Gemperline, Richard S Marshall, Kwang-Hee Lee, Qingzhen Zhao, Weiming Hu, Fionn McLoughlin, Mark Scalf, Lloyd M Smith, Richard D Vierstra
{"title":"Proteomic analysis of affinity-purified 26S proteasomes identifies a suite of assembly chaperones in <i>Arabidopsis</i>.","authors":"David C Gemperline, Richard S Marshall, Kwang-Hee Lee, Qingzhen Zhao, Weiming Hu, Fionn McLoughlin, Mark Scalf, Lloyd M Smith, Richard D Vierstra","doi":"10.1074/jbc.RA119.010219","DOIUrl":"10.1074/jbc.RA119.010219","url":null,"abstract":"<p><p>The 26S proteasome is an essential protease that selectively eliminates dysfunctional and short-lived regulatory proteins in eukaryotes. To define the composition of this proteolytic machine in plants, we tagged either the core protease (CP) or the regulatory particle (RP) sub-complexes in <i>Arabidopsis</i> to enable rapid affinity purification followed by mass spectrometric analysis. Studies on proteasomes enriched from whole seedlings, with or without ATP needed to maintain the holo-proteasome complex, identified all known proteasome subunits but failed to detect isoform preferences, suggesting that <i>Arabidopsis</i> does not construct distinct proteasome sub-types. We also detected a suite of proteasome-interacting proteins, including likely orthologs of the yeast and mammalian chaperones Pba1, Pba2, Pba3, and Pba4 that assist in CP assembly; Ump1 that helps connect CP half-barrels; Nas2, Nas6, and Hsm3 that assist in RP assembly; and Ecm29 that promotes CP-RP association. Proteasomes from seedlings exposed to the proteasome inhibitor MG132 accumulated assembly intermediates, reflecting partially built proteasome sub-complexes associated with assembly chaperones, and the CP capped with the PA200/Blm10 regulator. Genetic analyses of <i>Arabidopsis</i> UMP1 revealed that, unlike in yeast, this chaperone is essential, with mutants lacking the major UMP1a and UMP1b isoforms displaying a strong gametophytic defect. Single <i>ump1</i> mutants were hypersensitive to conditions that induce proteotoxic, salt and osmotic stress, and also accumulated several proteasome assembly intermediates, consistent with its importance for CP construction. Insights into the chaperones reported here should enable study of the assembly events that generate the 26S holo-proteasome in <i>Arabidopsis</i> from the collection of 64 or more subunits.</p>","PeriodicalId":50454,"journal":{"name":"FEBS Letters","volume":"38 1","pages":"17570-17592"},"PeriodicalIF":0.0,"publicationDate":"2019-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873196/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74637357","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Proton transfer in uncoupled variants of cytochrome c oxidase 细胞色素c氧化酶非偶联变异体的质子转移
IF 3.5 4区 生物学
FEBS Letters Pub Date : 2019-11-14 DOI: 10.1002/1873-3468.13679
Jóhanna Vilhjálmsdóttir, Ingrid Albertsson, M. Blomberg, Pia Ädelroth, P. Brzezinski
{"title":"Proton transfer in uncoupled variants of cytochrome c oxidase","authors":"Jóhanna Vilhjálmsdóttir, Ingrid Albertsson, M. Blomberg, Pia Ädelroth, P. Brzezinski","doi":"10.1002/1873-3468.13679","DOIUrl":"https://doi.org/10.1002/1873-3468.13679","url":null,"abstract":"Cytochrome c oxidase is a membrane‐bound redox‐driven proton pump that harbors two proton‐transfer pathways, D and K, which are used at different stages of the reaction cycle. Here, we address the question if a D pathway with a modified energy landscape for proton transfer could take over the role of the K pathway when the latter is blocked by a mutation. Our data indicate that structural alterations near the entrance of the D pathway modulate energy barriers that influence proton transfer to the proton‐loading site. The data also suggest that during reduction of the catalytic site, its protonation has to occur via the K pathway and that this proton transfer to the catalytic site cannot take place through the D pathway.","PeriodicalId":50454,"journal":{"name":"FEBS Letters","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2019-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/1873-3468.13679","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43092912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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: 7
Imprinted X‐chromosome inactivation impacts primitive endoderm differentiation in mouse blastocysts 印迹X染色体失活影响小鼠囊胚原始内胚层分化
IF 3.5 4区 生物学
FEBS Letters Pub Date : 2019-11-13 DOI: 10.1002/1873-3468.13676
A. Fukuda, N. Motosugi, Mikiko Ando, M. Kimura, A. Umezawa, H. Akutsu
{"title":"Imprinted X‐chromosome inactivation impacts primitive endoderm differentiation in mouse blastocysts","authors":"A. Fukuda, N. Motosugi, Mikiko Ando, M. Kimura, A. Umezawa, H. Akutsu","doi":"10.1002/1873-3468.13676","DOIUrl":"https://doi.org/10.1002/1873-3468.13676","url":null,"abstract":"Epigenetic and transcriptome alterations are essential for lineage specification, represented by imprinted X‐chromosome inactivation (iXCI) in female mouse preimplantation embryos. However, how various factors affect transcriptome states and lineage commitment remains unclear. We found that in vitro culture duration strongly influences transcriptional variation compared to iXCI loss. Single‐cell analysis of the inner cell mass (ICM) for major transcription and epigenomic factors revealed that sex‐specific differences in expression are diminished by loss of iXCI in the primitive endoderm (PrE) but not in the epiblast. Females had a higher proportion of ICM compared to that in males, and PrE development was affected by iXCI states in female embryos. Our findings provide insight into sex differences and iXCI function in lineage specification.","PeriodicalId":50454,"journal":{"name":"FEBS Letters","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2019-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/1873-3468.13676","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42966439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
Tau (297‐391) forms filaments that structurally mimic the core of paired helical filaments in Alzheimer’s disease brain Tau蛋白(297‐391)在阿尔茨海默病大脑中形成细丝，在结构上模仿成对螺旋细丝的核心
IF 3.5 4区 生物学
FEBS Letters Pub Date : 2019-11-13 DOI: 10.1002/1873-3468.13675
Youssra K Al-Hilaly, B. E. Foster, Luca Biasetti, Liisa Lutter, S. Pollack, J. Rickard, J. Storey, C. Harrington, Wei-Feng Xue, C. Wischik, L. Serpell
{"title":"Tau (297‐391) forms filaments that structurally mimic the core of paired helical filaments in Alzheimer’s disease brain","authors":"Youssra K Al-Hilaly, B. E. Foster, Luca Biasetti, Liisa Lutter, S. Pollack, J. Rickard, J. Storey, C. Harrington, Wei-Feng Xue, C. Wischik, L. Serpell","doi":"10.1002/1873-3468.13675","DOIUrl":"https://doi.org/10.1002/1873-3468.13675","url":null,"abstract":"The constituent paired helical filaments (PHFs) in neurofibrillary tangles are insoluble intracellular deposits central to the development of Alzheimer’s disease (AD) and other tauopathies. Full‐length tau requires the addition of anionic cofactors such as heparin to enhance assembly. We have shown that a fragment from the proteolytically stable core of the PHF, tau 297‐391 known as ‘dGAE’, spontaneously forms cross‐β‐containing PHFs and straight filaments under physiological conditions. Here, we have analysed and compared the structures of the filaments formed by dGAE in vitro with those deposited in the brains of individuals diagnosed with AD. We show that dGAE forms PHFs that share a macromolecular structure similar to those found in brain tissue. Thus, dGAEs may serve as a model system for studying core domain assembly and for screening for inhibitors of tau aggregation.","PeriodicalId":50454,"journal":{"name":"FEBS Letters","volume":"594 1","pages":"944 - 950"},"PeriodicalIF":3.5,"publicationDate":"2019-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/1873-3468.13675","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46290861","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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: 43
How the protonation state of a phosphorylated amino acid governs molecular recognition: insights from classical molecular dynamics simulations 磷酸化氨基酸的质子化状态如何控制分子识别:来自经典分子动力学模拟的见解
IF 3.5 4区 生物学
FEBS Letters Pub Date : 2019-11-12 DOI: 10.1002/1873-3468.13674
Raiji Kawade, Daisuke Kuroda, K. Tsumoto
{"title":"How the protonation state of a phosphorylated amino acid governs molecular recognition: insights from classical molecular dynamics simulations","authors":"Raiji Kawade, Daisuke Kuroda, K. Tsumoto","doi":"10.1002/1873-3468.13674","DOIUrl":"https://doi.org/10.1002/1873-3468.13674","url":null,"abstract":"Physicochemical properties of proteins are controlled mainly by post‐translational modifications such as amino acid phosphorylation. Although molecular dynamics simulations have been shown to be a valuable tool for studying the effects of phosphorylation on protein structure and dynamics, most of the previous studies assumed that the phosphate group was in the unprotonated ( PO32- ) state, even though the protonation state could in fact vary at physiological pH. In this study, we performed molecular dynamics simulations of four different protein‐phosphorylated peptide complexes both in the PO32- and PO3H− states. Our simulations delineate different dynamics and energetics between the two states, suggesting importance of the protonation state of a phosphorylated amino acid in molecular recognition.","PeriodicalId":50454,"journal":{"name":"FEBS Letters","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2019-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/1873-3468.13674","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47106629","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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: 7
The Nbp35/ApbC homolog acts as a nonessential [4Fe‐4S] transfer protein in methanogenic archaea Nbp35/ApbC同源物在产甲烷古菌中作为非必需的[4Fe‐4S]转移蛋白
IF 3.5 4区 生物学
FEBS Letters Pub Date : 2019-11-11 DOI: 10.1002/1873-3468.13673
Cuiping Zhao, Zhe Lyu, Feng Long, Taiwo S Akinyemi, Kasidet Manakongtreecheep, D. Söll, W. Whitman, David J. Vinyard, Yuchen Liu
{"title":"The Nbp35/ApbC homolog acts as a nonessential [4Fe‐4S] transfer protein in methanogenic archaea","authors":"Cuiping Zhao, Zhe Lyu, Feng Long, Taiwo S Akinyemi, Kasidet Manakongtreecheep, D. Söll, W. Whitman, David J. Vinyard, Yuchen Liu","doi":"10.1002/1873-3468.13673","DOIUrl":"https://doi.org/10.1002/1873-3468.13673","url":null,"abstract":"The nucleotide binding protein 35 (Nbp35)/cytosolic Fe‐S cluster deficient 1 (Cfd1)/alternative pyrimidine biosynthetic protein C (ApbC) protein homologs have been identified in all three domains of life. In eukaryotes, the Nbp35/Cfd1 heterocomplex is an essential Fe‐S cluster assembly scaffold required for the maturation of Fe‐S proteins in the cytosol and nucleus, whereas the bacterial ApbC is an Fe‐S cluster transfer protein only involved in the maturation of a specific target protein. Here, we show that the Nbp35/ApbC homolog MMP0704 purified from its native archaeal host Methanococcus maripaludis contains a [4Fe‐4S] cluster that can be transferred to a [4Fe‐4S] apoprotein. Deletion of mmp0704 from M. maripaludis does not cause growth deficiency under our tested conditions. Our data indicate that Nbp35/ApbC is a nonessential [4Fe‐4S] cluster transfer protein in methanogenic archaea.","PeriodicalId":50454,"journal":{"name":"FEBS Letters","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2019-11-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/1873-3468.13673","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43426188","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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: 3
UvrA and UvrC subunits of the Mycobacterium tuberculosis UvrABC excinuclease interact independently of UvrB and DNA 结核分枝杆菌UvrABC切切酶的UvrA和UvrC亚基独立于UvrB和DNA相互作用
IF 3.5 4区 生物学
FEBS Letters Pub Date : 2019-11-09 DOI: 10.1002/1873-3468.13671
Manoj Thakur, S. Badugu, K. Muniyappa
{"title":"UvrA and UvrC subunits of the Mycobacterium tuberculosis UvrABC excinuclease interact independently of UvrB and DNA","authors":"Manoj Thakur, S. Badugu, K. Muniyappa","doi":"10.1002/1873-3468.13671","DOIUrl":"https://doi.org/10.1002/1873-3468.13671","url":null,"abstract":"The UvrABC excinuclease plays a vital role in bacterial nucleotide excision repair. While UvrA and UvrB subunits associate to form a UvrA2B2 complex, interaction between UvrA and UvrC has not been demonstrated or quantified in any bacterial species. Here, using Mycobacterium tuberculosis UvrA (MtUvrA), UvrB (MtUvrB) and UvrC (MtUvrC) subunits, we show that MtUvrA binds to MtUvrB and equally well to MtUvrC with submicromolar affinity. Furthermore, MtUvrA forms a complex with MtUvrC both in vivo and in vitro, independently of DNA and UvrB. Collectively, these findings reveal new insights into the pairwise relationships between the subunits of the UvrABC incision complex.","PeriodicalId":50454,"journal":{"name":"FEBS Letters","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2019-11-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/1873-3468.13671","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46755339","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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: 6
Molecular evidence for the evolution of the eukaryotic mitochondrial arginyl‐tRNA synthetase from the prokaryotic suborder Cystobacterineae 原核胞杆菌亚目真核线粒体精氨酸- tRNA合成酶进化的分子证据
IF 3.5 4区 生物学
FEBS Letters Pub Date : 2019-11-09 DOI: 10.1002/1873-3468.13665
G. Igloi
{"title":"Molecular evidence for the evolution of the eukaryotic mitochondrial arginyl‐tRNA synthetase from the prokaryotic suborder Cystobacterineae","authors":"G. Igloi","doi":"10.1002/1873-3468.13665","DOIUrl":"https://doi.org/10.1002/1873-3468.13665","url":null,"abstract":"The evolutionary origin of the family of eukaryotic aminoacyl‐tRNA synthetases that are essential to all living organisms is a matter of debate. In order to shed molecular light on the ancient source of arginyl‐tRNA synthetase, a total of 1347 eukaryotic arginyl‐tRNA synthetase sequences were mined from databases and analyzed. Their multiple sequence alignment reveals a signature sequence that is characteristic of the nuclear‐encoded enzyme, which is imported into mitochondria. Using this molecular beacon, the origins of this gene can be traced to modern prokaryotes. In this way, a previous phylogenetic analysis linking Myxococcus to the emergence of the eukaryotic mitochondrial arginyl‐tRNA synthetase is supported by the unique existence of the molecular signature within the suborder Cystobacterineae that includes Myxococcus.","PeriodicalId":50454,"journal":{"name":"FEBS Letters","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2019-11-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/1873-3468.13665","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45288534","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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: 4
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