Journal of Mass Spectrometry最新文献

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Tentative identification of phytochelatins, their derivatives, and Cd-phytochelatin complexes in Ocimum basilicum L. roots by Paper Spray Mass Spectrometry 利用纸喷雾质谱法初步鉴定 Ocimum basilicum L. 根中的植物螯合素、其衍生物和镉-植物螯合素复合物。
IF 1.9 3区 化学
Journal of Mass Spectrometry Pub Date : 2024-07-02 DOI: 10.1002/jms.5063
Vânia de Lourdes das G. Teles, Giselle V. de Sousa, Rodinei Augusti, Letícia M. Costa
{"title":"Tentative identification of phytochelatins, their derivatives, and Cd-phytochelatin complexes in Ocimum basilicum L. roots by Paper Spray Mass Spectrometry","authors":"Vânia de Lourdes das G. Teles,&nbsp;Giselle V. de Sousa,&nbsp;Rodinei Augusti,&nbsp;Letícia M. Costa","doi":"10.1002/jms.5063","DOIUrl":"10.1002/jms.5063","url":null,"abstract":"<p>An unprecedented and direct PS-MS (paper spray ionization mass spectrometry) method was proposed for the detection of native peptides, that is, glutathiones (GSHs), homoglutathiones (hGSHs), and phytochelatins (PCs), in basil (<i>Ocimum basilicum</i> L.) roots before and after cadmium exposure. The roots were submitted to cold maceration followed by sonication with formic acid as the extractor solvent for sample preparation. PS-MS was used to analyze such extracts in the positive mode, and the results allowed for the detection of several GSHs, hGSHs, and PCs. Some of these PCs were not distinguished in the control samples, that is, basil roots not exposed to cadmium. Other PCs were noticed in both types of roots, uncontaminated and cadmium-contaminated, but the intensities were higher in the former samples. Moreover, long-time exposure to cadmium stimulated the formation of some of these PCs and their cadmium complexes. The results, therefore, provided some crucial insights into the defense mechanism of plants against an external stress condition due to exposure to a toxic heavy metal. The present study represents a promising alternative to investigate other crucial physiological processes in plants submitted to assorted stress conditions.</p>","PeriodicalId":16178,"journal":{"name":"Journal of Mass Spectrometry","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141492327","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Recent advances in gas phase unfolding: Instrumentation and applications 气相展开的最新进展:仪器和应用。
IF 2.3 3区 化学
Journal of Mass Spectrometry Pub Date : 2024-06-18 DOI: 10.1002/jms.5059
Rowan Matney, Varun V. Gadkari
{"title":"Recent advances in gas phase unfolding: Instrumentation and applications","authors":"Rowan Matney,&nbsp;Varun V. Gadkari","doi":"10.1002/jms.5059","DOIUrl":"10.1002/jms.5059","url":null,"abstract":"<p>Broader adoption of native mass spectrometry (MS) and ion mobility-mass spectrometry (IM-MS) has propelled the development of several techniques which take advantage of the selectivity, sensitivity, and speed of MS to make measurements of complex biological molecules in the gas phase. One such method, collision induced unfolding (CIU), has risen to prominence in recent years, due to its well documented capability to detect shifts in structural stability of biological molecules in response to external stimuli (e.g., mutations, stress, non-covalent interactions, sample conditions etc.). This increase in reported CIU measurements is enabled partly due to advances in IM-MS instrumentation by vendors, and also innovative method development by researchers. This perspective highlights a few of these advances and concludes with a look forward toward the future of the gas phase unfolding field.</p>","PeriodicalId":16178,"journal":{"name":"Journal of Mass Spectrometry","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jms.5059","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141419460","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
An innovative method to identify structural change through ion-molecule collision, making use of Time-Of-Flight measurements and SIMION simulations 利用飞行时间测量和 SIMION 模拟,采用创新方法识别离子-分子碰撞带来的结构变化。
IF 2.3 3区 化学
Journal of Mass Spectrometry Pub Date : 2024-06-18 DOI: 10.1002/jms.5066
Nicolas Solem, Claire Romanzin, Christian Alcaraz, Roland Thissen
{"title":"An innovative method to identify structural change through ion-molecule collision, making use of Time-Of-Flight measurements and SIMION simulations","authors":"Nicolas Solem,&nbsp;Claire Romanzin,&nbsp;Christian Alcaraz,&nbsp;Roland Thissen","doi":"10.1002/jms.5066","DOIUrl":"10.1002/jms.5066","url":null,"abstract":"<p>Structural change of ions induced by collision with a neutral has been studied in a guided ion beam tandem mass spectrometer, using Time-Of-Flight measurements and SIMION simulation. The exothermic catalytic isomerization of HOC<sup>+</sup> to HCO<sup>+</sup> is used to explore the new methodology. Isomerization is catalyzed via a proton transport mechanism through the interplay of a neutral molecule, the catalyst. Four different potential catalysts, Ne, D<sub>2</sub>, CH<sub>4,</sub> and C<sup>18</sup>O, were studied at different collision energies. SIMION simulation of the ion path and collision in the instrument leads to the highlight of a specific signature related to the catalytic isomerization in the time-of-flight spectra. This signature is used to identify the experimental conditions where isomerization takes place. Only C<sup>18</sup>O, at low collision energies, gives a clear signature of catalytic isomerization, and a quantitative estimate of the catalyzed isomerization cross-section and rate constant is derived. This new methodology is sensitive to clear presence of catalyzed isomerization and can be used in instruments designed for cross-section measurements, provided low collision energy is used and ion bunching is available.</p>","PeriodicalId":16178,"journal":{"name":"Journal of Mass Spectrometry","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jms.5066","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141419459","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
The path forward for protein footprinting, covalent labeling, and mass spectrometry-based protein conformational analyses 蛋白质足迹、共价标记和基于质谱的蛋白质构象分析的未来之路。
IF 2.3 3区 化学
Journal of Mass Spectrometry Pub Date : 2024-06-14 DOI: 10.1002/jms.5064
Nicholas B. Borotto
{"title":"The path forward for protein footprinting, covalent labeling, and mass spectrometry-based protein conformational analyses","authors":"Nicholas B. Borotto","doi":"10.1002/jms.5064","DOIUrl":"10.1002/jms.5064","url":null,"abstract":"<p>Mass spectrometry-based approaches to assess protein conformation have become widely utilized due to their sensitivity, low sample requirements, and broad applicability to proteins regardless of size and environment. Their wide applicability and sensitivity also make these techniques suitable for the analysis of complex mixtures of proteins, and thus, they have been applied at the cell and even the simple organism levels. These works are impressive, but they predominately employ “bottom-up” workflows and require proteolytic digestion prior to analysis. Once digested, it is not possible to distinguish the proteoform from which any single peptide is derived and therefore, one cannot associate distal—in primary structure—concurrent post-translational modifications (PTMs) or covalent labels, as they would be found on separate peptides. Thus, analyses via bottom-up proteomics report the average PTM status and higher-order structure (HOS) of all existing proteoforms. Second, these works predominately employ promiscuous reagents to probe protein HOS. While this does lead to improved conformational resolution, the formation of many products can divide the signal associated with low-copy number proteins below signal-to-noise thresholds and complicate the bioinformatic analysis of these already challenging systems. In this perspective, I further detail these limitations and discuss the positives and negatives of top-down proteomics as an alternative.</p>","PeriodicalId":16178,"journal":{"name":"Journal of Mass Spectrometry","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141317544","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Hardware and software solutions for implementing nanospray desorption electrospray ionization (nano-DESI) sources on commercial mass spectrometers 在商用质谱仪上实施纳米喷雾解吸电喷雾离子源(nano-DESI)的硬件和软件解决方案。
IF 2.3 3区 化学
Journal of Mass Spectrometry Pub Date : 2024-06-12 DOI: 10.1002/jms.5065
Li-Xue Jiang, Ryan T. Hilger, Julia Laskin
{"title":"Hardware and software solutions for implementing nanospray desorption electrospray ionization (nano-DESI) sources on commercial mass spectrometers","authors":"Li-Xue Jiang,&nbsp;Ryan T. Hilger,&nbsp;Julia Laskin","doi":"10.1002/jms.5065","DOIUrl":"10.1002/jms.5065","url":null,"abstract":"<p>Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization mass spectrometry imaging (MSI) approach that enables spatial mapping of biological and environmental samples with high spatial resolution and throughput. Because nano-DESI has not yet been commercialized, researchers develop their own sources and interface them with different commercial mass spectrometers. Previously, several protocols focusing on the fabrication of nano-DESI probes have been reported. In this tutorial, we discuss different hardware requirements for coupling the nano-DESI source to commercial mass spectrometers, such as the safety interlock, inlet extension, and contact closure. In addition, we describe the structure of our custom software for controlling the nano-DESI MSI platform and provide detailed instructions for its usage. With this tutorial, interested researchers should be able to implement nano-DESI experiments in their labs.</p>","PeriodicalId":16178,"journal":{"name":"Journal of Mass Spectrometry","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jms.5065","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310902","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Analyzing noncovalent interactions between notoginseng saponins and lysozyme by deposition scanning intensity fading MALDI-TOF mass spectrometry 通过沉积扫描强度衰减 MALDI-TOF 质谱分析田七皂甙与溶菌酶之间的非共价相互作用
IF 2.3 3区 化学
Journal of Mass Spectrometry Pub Date : 2024-06-06 DOI: 10.1002/jms.5058
Xintong Zhao, Juan Ren, Ze Wang, Xiangfeng Chen
{"title":"Analyzing noncovalent interactions between notoginseng saponins and lysozyme by deposition scanning intensity fading MALDI-TOF mass spectrometry","authors":"Xintong Zhao,&nbsp;Juan Ren,&nbsp;Ze Wang,&nbsp;Xiangfeng Chen","doi":"10.1002/jms.5058","DOIUrl":"10.1002/jms.5058","url":null,"abstract":"<p>Analysis of noncovalent interactions between natural products and proteins is important for rapid screening of active ingredients and understanding their pharmacological activities. In this work, the intensity fading MALDI-TOF mass spectrometry (IF-MALDI-MS) method with improved reproducibility was implemented to investigate the binding interactions between saponins from <i>Panax notoginseng</i> and lysozyme. The benchmark IF-MALDI-MS experiment was established using <i>N</i>,<i>N</i>′,<i>N</i>″-triacetylchitotriose-lysozyme as a model system. The reproducibility of ion intensities in IF-MALDI-MS was improved by scanning the whole sample deposition with a focused laser beam. The relative standard deviation (RSD) of deposition scanning IF-MALDI-MS is 5.7%. Similar decay trends of the relative intensities of notoginseng saponins against increasing amounts of lysozyme were observed for all six notoginseng saponins. The half-maximal fading concentration (FC<sub>50</sub>) was calculated to quantitatively characterize the binding affinity of each ligand based on the decay curve. According to the FC<sub>50</sub> values obtained, the binding affinities of the six notoginseng saponins were evaluated in the following order: notoginsenoside S &gt; notoginsenoside Fc &gt; ginsenoside Rb1 &gt; ginsenoside Rd &gt; notoginsenoside Ft1 &gt; ginsenoside Rg1. The binding order was in accordance with molecular docking studies, which showed hydrogen bonding might play a key role in stabilizing the binding interaction. Our results demonstrated that deposition scanning IF-MALDI-MS can provide valuable information on the noncovalent interactions between ligands and proteins.</p>","PeriodicalId":16178,"journal":{"name":"Journal of Mass Spectrometry","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141261142","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Mass spectrometry imaging-based multi-omics approaches to understand drug metabolism and disposition 以质谱成像为基础的多组学方法了解药物代谢和处置。
IF 2.3 3区 化学
Journal of Mass Spectrometry Pub Date : 2024-06-05 DOI: 10.1002/jms.5042
Nav Raj Phulara, Herana Kamal Seneviratne
{"title":"Mass spectrometry imaging-based multi-omics approaches to understand drug metabolism and disposition","authors":"Nav Raj Phulara,&nbsp;Herana Kamal Seneviratne","doi":"10.1002/jms.5042","DOIUrl":"10.1002/jms.5042","url":null,"abstract":"","PeriodicalId":16178,"journal":{"name":"Journal of Mass Spectrometry","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141261258","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Phytochemical investigation and characterisation of methanolic extract of Glycine max seeds using LCMS/MS and in silico studies for wound healing activity 利用 LCMS/MS 对 Glycine max 种子甲醇提取物进行植物化学调查和表征,并对其伤口愈合活性进行硅学研究
IF 2.3 3区 化学
Journal of Mass Spectrometry Pub Date : 2024-06-05 DOI: 10.1002/jms.5045
Madhusmita Sahu,  Kamakshi, Jyotirmaya Sahoo, Sudhansu Ranjan Swain, Manisha Chauhan, Riya Goyal, Sakshi Gupta, Komalpreet Kaur
{"title":"Phytochemical investigation and characterisation of methanolic extract of Glycine max seeds using LCMS/MS and in silico studies for wound healing activity","authors":"Madhusmita Sahu,&nbsp; Kamakshi,&nbsp;Jyotirmaya Sahoo,&nbsp;Sudhansu Ranjan Swain,&nbsp;Manisha Chauhan,&nbsp;Riya Goyal,&nbsp;Sakshi Gupta,&nbsp;Komalpreet Kaur","doi":"10.1002/jms.5045","DOIUrl":"https://doi.org/10.1002/jms.5045","url":null,"abstract":"<p>Soybean is scientifically known as <i>Glycine max</i>. It belongs to the Fabaceae family. It consists of a lot of bioactive phytochemicals like saponin, phenolic acid, flavonoid, sphingolipids and phytosterols. It also owns excellent immune-active effects in the physiological system. Soy and its phytochemicals have been found to have pharmacological properties that include anticancer, antioxidant, anti-hypercholesterolaemic, anti-diabetic, oestrogenic, anti-hyperlipidaemic, anti-inflammatory, anti-obesity, anti-hypertensive, anti-mutagenic, immunomodulatory, anti-osteoporotic, antiviral, hepatoprotective, antimicrobial, goitrogenic anti-skin ageing, wound healing, neuroprotective and anti-photoageing activities. Present study has been designed to set standard pharmacognostical extraction method, complexation of compounds, qualitative evaluation through phytochemical screening, identification by TLC, physicochemical properties, solubility profile, total phenolic, flavonoid content as well as analytical evaluation or characterisation like UV and FT-IR of methanolic extract of <i>G. max</i>. The final observations like physicochemical properties such as total ash value, LOD and pH were recorded. Phytochemical screenings show the presence of flavonoid, alkaloid, saponin, carbohydrate, tannins, protein, gums and mucilage, fixed oils and fats. The results were found significant. Further in silico studies proved creatinine and euparin to be potent wound healing agents.</p>","PeriodicalId":16178,"journal":{"name":"Journal of Mass Spectrometry","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141251467","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Glycoproteomics: Charting new territory in mass spectrometry and glycobiology By Stacy A. Malaker 糖蛋白组学:开拓质谱和糖生物学的新领域 作者:Stacy A. Malaker
IF 2.3 3区 化学
Journal of Mass Spectrometry Pub Date : 2024-06-03 DOI: 10.1002/jms.5061
Malaker
{"title":"Glycoproteomics: Charting new territory in mass spectrometry and glycobiology By Stacy A. Malaker","authors":"Malaker","doi":"10.1002/jms.5061","DOIUrl":"https://doi.org/10.1002/jms.5061","url":null,"abstract":"<p>\u0000 \u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":16178,"journal":{"name":"Journal of Mass Spectrometry","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jms.5061","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141245535","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Electrospray ionization tandem mass spectrometry of 4-aryl-3,4-dihydrocoumarins 4- 芳基-3,4-二氢香豆素的电喷雾离子化串联质谱法。
IF 2.3 3区 化学
Journal of Mass Spectrometry Pub Date : 2024-06-03 DOI: 10.1002/jms.5062
Herbert J. Dias, William H. Santos, Luis C. S. Filho, Eduardo J. Crevelin, J. Scott McIndoe, Ricardo Vessecchi, Antônio E. M. Crotti
{"title":"Electrospray ionization tandem mass spectrometry of 4-aryl-3,4-dihydrocoumarins","authors":"Herbert J. Dias,&nbsp;William H. Santos,&nbsp;Luis C. S. Filho,&nbsp;Eduardo J. Crevelin,&nbsp;J. Scott McIndoe,&nbsp;Ricardo Vessecchi,&nbsp;Antônio E. M. Crotti","doi":"10.1002/jms.5062","DOIUrl":"10.1002/jms.5062","url":null,"abstract":"<p>We have investigated the gas-phase fragmentation reactions of 11 synthetic 4-aryl-3,4-dihydrocoumarins by electrospray ionization tandem mass spectrometry (ESI-MS/MS) on a quadrupole-time-of flight (Q-TOF) hybrid mass spectrometer. We have also estimated thermochemical data for the protonated coumarins (precursor ion <b>A</b>) and product ion structures by computational chemistry at a B3LYP level of theory to establish the ion structures and to rationalize the fragmentation pathways. The most abundant ions in the product ion spectra of coumarins <b>1–11</b> resulted from C<sub>8</sub>H<sub>8</sub>O<sub>2</sub>, CO<sub>2</sub>, C<sub>4</sub>H<sub>4</sub>O<sub>3</sub>, C<sub>8</sub>H<sub>10</sub>O<sub>3</sub>, C<sub>8</sub>H<sub>8</sub>O<sub>2</sub>, and CH<sub>3</sub>OH eliminations through retro-Diels–Alder (RDA) reactions, remote hydrogen rearrangements (β-eliminations), and β-lactone ring contraction. Although the investigated coumarins shared most of the fragmentation pathways, formation of a benzylic product ion and its corresponding tropylium ion was diagnostic of the substituents at ring C. The thermochemical data revealed that the nature and position of the substituents at ring C played a key role in the formation of this product ion and determined its relative intensity in the product ion spectrum. The results of this study contribute to knowledge of the gas-phase ion chemistry of this important class of organic compounds.</p>","PeriodicalId":16178,"journal":{"name":"Journal of Mass Spectrometry","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141237714","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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