Current topics in membranes最新文献

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Ion channels and the regulation of myogenic tone in peripheral arterioles. 离子通道与外周小动脉肌张力的调节。
4区 生物学
Current topics in membranes Pub Date : 2020-01-01 Epub Date: 2020-02-25 DOI: 10.1016/bs.ctm.2020.01.002
William F Jackson
{"title":"Ion channels and the regulation of myogenic tone in peripheral arterioles.","authors":"William F Jackson","doi":"10.1016/bs.ctm.2020.01.002","DOIUrl":"https://doi.org/10.1016/bs.ctm.2020.01.002","url":null,"abstract":"<p><p>Myogenic tone is a hall-mark feature of arterioles in the microcirculation. This pressure-induced, contractile activation of vascular smooth muscle cells (VSMCs) in the wall of these microvessels importantly contributes to the regulation and maintenance of blood pressure; blood flow to and within organs and tissues; and capillary pressure and fluid balance. Ion channels play a central role in the genesis and maintenance of myogenic tone. Mechanosensitive ion channels such as TRPC6 may serve as one of the sensors of pressure-induced membrane stress/strain, and TRPC6 along with TRPM4 channels are responsible pressure-induced VSMC depolarization that may be bolstered by the activity of Ca<sup>2+</sup>-activated Cl<sup>-</sup> channels and inhibition of voltage-gated K<sup>+</sup> (K<sub>V</sub>) channels, inwardly-rectifying K<sup>+</sup> (K<sub>IR</sub>) channels and ATP-sensitive K<sup>+</sup> (K<sub>ATP</sub>) channels. Membrane potential depolarization activates voltage-gated Ca<sup>2+</sup> channels (VGCCs), with CaV1.2 channels playing a central role. Calcium entry through CaV1.2 channels, which is amplified by Ca<sup>2+</sup> release through IP<sub>3</sub> receptors in the form of Ca<sup>2+</sup> waves in some arterioles, provides the major source of activator calcium responsible for arteriolar myogenic tone. Stabilizing negative-feedback comes from depolarization- and Ca<sup>2+</sup>-induced activation of large-conductance Ca<sup>2+</sup>-activated K<sup>+</sup> channels and depolarization-induced activation of K<sub>V</sub> channels. Myogenic tone also is dampened by tonic activity of K<sub>IR</sub> and K<sub>ATP</sub> channels. While much has been learned about ion channel expression and function in myogenic tone, additional studies are required to fill in our knowledge gaps due to significant regional differences in ion channel expression and function and a lack of data specifically from VSMCs in arterioles.</p>","PeriodicalId":11029,"journal":{"name":"Current topics in membranes","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/bs.ctm.2020.01.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37931605","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: 9
Preface. 前言。
4区 生物学
Current topics in membranes Pub Date : 2020-01-01 DOI: 10.1016/S1063-5823(20)30020-X
William F Jackson
{"title":"Preface.","authors":"William F Jackson","doi":"10.1016/S1063-5823(20)30020-X","DOIUrl":"https://doi.org/10.1016/S1063-5823(20)30020-X","url":null,"abstract":"","PeriodicalId":11029,"journal":{"name":"Current topics in membranes","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S1063-5823(20)30020-X","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37931609","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
Lipid bilayers: Phase behavior and nanomechanics. 脂质双层:相行为和纳米力学。
4区 生物学
Current topics in membranes Pub Date : 2020-01-01 Epub Date: 2020-09-16 DOI: 10.1016/bs.ctm.2020.08.005
Lorena Redondo-Morata, Patricia Losada-Pérez, Marina Inés Giannotti
{"title":"Lipid bilayers: Phase behavior and nanomechanics.","authors":"Lorena Redondo-Morata,&nbsp;Patricia Losada-Pérez,&nbsp;Marina Inés Giannotti","doi":"10.1016/bs.ctm.2020.08.005","DOIUrl":"https://doi.org/10.1016/bs.ctm.2020.08.005","url":null,"abstract":"<p><p>Lipid membranes are involved in many physiological processes like recognition, signaling, fusion or remodeling of the cell membrane or some of its internal compartments. Within the cell, they are the ultimate barrier, while maintaining the fluidity or flexibility required for a myriad of processes, including membrane protein assembly. The physical properties of in vitro model membranes as model cell membranes have been extensively studied with a variety of techniques, from classical thermodynamics to advanced modern microscopies. Here we review the nanomechanics of solid-supported lipid membranes with a focus in their phase behavior. Relevant information obtained by quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM) as complementary techniques in the nano/mesoscale interface is presented. Membrane morphological and mechanical characterization will be discussed in the framework of its phase behavior, phase transitions and coexistence, in simple and complex models, and upon the presence of cholesterol.</p>","PeriodicalId":11029,"journal":{"name":"Current topics in membranes","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/bs.ctm.2020.08.005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25578642","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: 10
Mechanisms of endothelial stiffening in dyslipidemia and aging: Oxidized lipids and shear stress. 血脂异常和老化导致内皮僵化的机制:氧化脂质和剪切应力。
4区 生物学
Current topics in membranes Pub Date : 2020-01-01 Epub Date: 2020-09-24 DOI: 10.1016/bs.ctm.2020.08.006
Elizabeth Le Master, Sang Joon Ahn, Irena Levitan
{"title":"Mechanisms of endothelial stiffening in dyslipidemia and aging: Oxidized lipids and shear stress.","authors":"Elizabeth Le Master, Sang Joon Ahn, Irena Levitan","doi":"10.1016/bs.ctm.2020.08.006","DOIUrl":"10.1016/bs.ctm.2020.08.006","url":null,"abstract":"<p><p>Vascular stiffening of the arterial walls is well-known as a key factor in aging and the development of cardiovascular disease; however, the role of endothelial stiffness in vascular dysfunction is still an emerging topic. In this review, the authors discuss the impact of dyslipidemia, oxidized lipids, substrate stiffness, age and pro-atherogenic disturbed flow have on endothelial stiffness. Furthermore, we investigate several mechanistic pathways that are key contributors in endothelial stiffness and discuss their physiological effects in the onset of atherogenesis in the disturbed flow regions of the aortic vasculature. The findings in this chapter describe a novel paradigm of synergistic interaction of plasma dyslipidemia/oxidized lipids and pro-atherogenic disturbed shear stress, as well as aging has on endothelial stiffness and vascular dysfunction.</p>","PeriodicalId":11029,"journal":{"name":"Current topics in membranes","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168803/pdf/nihms-1706109.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25578645","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
Introduction to ion channels and calcium signaling in the microcirculation. 介绍微循环中的离子通道和钙信号。
4区 生物学
Current topics in membranes Pub Date : 2020-01-01 Epub Date: 2020-03-13 DOI: 10.1016/bs.ctm.2020.01.001
William F Jackson
{"title":"Introduction to ion channels and calcium signaling in the microcirculation.","authors":"William F Jackson","doi":"10.1016/bs.ctm.2020.01.001","DOIUrl":"https://doi.org/10.1016/bs.ctm.2020.01.001","url":null,"abstract":"<p><p>The microcirculation is the network of feed arteries, arterioles, capillaries and venules that supply and drain blood from every tissue and organ in the body. It is here that exchange of heat, oxygen, carbon dioxide, nutrients, hormones, water, cytokines, and immune cells takes place; essential functions necessary to maintenance of homeostasis throughout the life span. This chapter will outline the structure and function of each microvascular segment highlighting the critical roles played by ion channels in the microcirculation. Feed arteries upstream from the true microcirculation and arterioles within the microcirculation contribute to systemic vascular resistance and blood pressure control. They also control total blood flow to the downstream microcirculation with arterioles being responsible for distribution of blood flow within a tissue or organ dependent on the metabolic needs of the tissue. Terminal arterioles control blood flow and blood pressure to capillary units, the primary site of diffusional exchange between blood and tissues due to their large surface area. Venules collect blood from capillaries and are important sites for fluid exchange and immune cell trafficking. Ion channels in microvascular smooth muscle cells, endothelial cells and pericytes importantly contribute to all of these functions through generation of intracellular Ca<sup>2+</sup> and membrane potential signals in these cells.</p>","PeriodicalId":11029,"journal":{"name":"Current topics in membranes","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/bs.ctm.2020.01.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37929669","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: 2
Ion channels and myogenic activity in retinal arterioles. 视网膜小动脉中的离子通道和肌生成活动。
4区 生物学
Current topics in membranes Pub Date : 2020-01-01 Epub Date: 2020-02-26 DOI: 10.1016/bs.ctm.2020.01.008
Peter Barabas, Josy Augustine, José A Fernández, J Graham McGeown, Mary K McGahon, Tim M Curtis
{"title":"Ion channels and myogenic activity in retinal arterioles.","authors":"Peter Barabas,&nbsp;Josy Augustine,&nbsp;José A Fernández,&nbsp;J Graham McGeown,&nbsp;Mary K McGahon,&nbsp;Tim M Curtis","doi":"10.1016/bs.ctm.2020.01.008","DOIUrl":"https://doi.org/10.1016/bs.ctm.2020.01.008","url":null,"abstract":"<p><p>Retinal pressure autoregulation is an important mechanism that protects the retina by stabilizing retinal blood flow during changes in arterial or intraocular pressure. Similar to other vascular beds, retinal pressure autoregulation is thought to be mediated largely through the myogenic response of small arteries and arterioles which constrict when transmural pressure increases or dilate when it decreases. Over recent years, we and others have investigated the signaling pathways underlying the myogenic response in retinal arterioles, with particular emphasis on the involvement of different ion channels expressed in the smooth muscle layer of these vessels. Here, we review and extend previous work on the expression and spatial distribution of the plasma membrane and sarcoplasmic reticulum ion channels present in retinal vascular smooth muscle cells (VSMCs) and discuss their contribution to pressure-induced myogenic tone in retinal arterioles. This includes new data demonstrating that several key players and modulators of the myogenic response show distinctively heterogeneous expression along the length of the retinal arteriolar network, suggesting differences in myogenic signaling between larger and smaller pre-capillary arterioles. Our immunohistochemical investigations have also highlighted the presence of actin-containing microstructures called myobridges that connect the retinal VSMCs to one another. Although further work is still needed, studies to date investigating myogenic mechanisms in the retina have contributed to a better understanding of how blood flow is regulated in this tissue. They also provide a basis to direct future research into retinal diseases where blood flow changes contribute to the pathology.</p>","PeriodicalId":11029,"journal":{"name":"Current topics in membranes","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/bs.ctm.2020.01.008","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37930119","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
Vascular smooth muscle stiffness and its role in aging. 血管平滑肌僵硬及其在衰老中的作用。
4区 生物学
Current topics in membranes Pub Date : 2020-01-01 Epub Date: 2020-10-12 DOI: 10.1016/bs.ctm.2020.08.008
Andreea Trache, Michael P Massett, Christopher R Woodman
{"title":"Vascular smooth muscle stiffness and its role in aging.","authors":"Andreea Trache,&nbsp;Michael P Massett,&nbsp;Christopher R Woodman","doi":"10.1016/bs.ctm.2020.08.008","DOIUrl":"https://doi.org/10.1016/bs.ctm.2020.08.008","url":null,"abstract":"<p><p>Vascular smooth muscle cells (VSMC) are now considered important contributors to the pathophysiological and biophysical mechanisms underlying arterial stiffening in aging. Here, we review mechanisms whereby VSMC stiffening alters vascular function and contributes to the changes in vascular stiffening observed in aging and cardiovascular disease. Vascular stiffening in arterial aging was historically associated with changes in the extracellular matrix; however, new evidence suggests that endothelial and vascular smooth muscle cell stiffness also contribute to overall blood vessel stiffness. Furthermore, VSMC play an integral role in regulating matrix deposition and vessel wall contractility via interaction between the actomyosin contractile unit and adhesion structures that anchor the cell within the extracellular matrix. Aged-induce phenotypic modulation of VSMC from a contractile to a synthetic phenotype is associated with decreased cellular contractility and increased cell stiffness. Aged VSMC also display reduced mechanosensitivity and adaptation to mechanical signals from their microenvironment due to impaired intracellular signaling. Finally, evidence for decreased contractility in arteries from aged animals demonstrate that changes at the cellular level result in decreased functional properties at the tissue level.</p>","PeriodicalId":11029,"journal":{"name":"Current topics in membranes","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/bs.ctm.2020.08.008","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25578644","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
The interplay of membrane cholesterol and substrate on vascular smooth muscle biomechanics. 膜胆固醇和基质对血管平滑肌生物力学的相互作用
4区 生物学
Current topics in membranes Pub Date : 2020-01-01 Epub Date: 2020-09-28 DOI: 10.1016/bs.ctm.2020.08.003
Hanna J Sanyour, Alex P Rickel, Zhongkui Hong
{"title":"The interplay of membrane cholesterol and substrate on vascular smooth muscle biomechanics.","authors":"Hanna J Sanyour, Alex P Rickel, Zhongkui Hong","doi":"10.1016/bs.ctm.2020.08.003","DOIUrl":"10.1016/bs.ctm.2020.08.003","url":null,"abstract":"<p><p>Cardiovascular disease (CVD) remains the primary cause of death worldwide. Specifically, atherosclerosis is a CVD characterized as a slow progressing chronic inflammatory disease. During atherosclerosis, vascular walls accumulate cholesterol and cause fatty streak formation. The progressive changes in vascular wall stiffness exert alternating mechanical cues on vascular smooth muscle cells (VSMCs). The detachment of VSMCs in the media layer of the vessel and migration toward the intima is a critical step in atherosclerosis. VSMC phenotypic switching is a complicated process that modifies VSMC structure and biomechanical function. These changes affect the expression and function of cell adhesion molecules, thus impacting VSMC migration. Accumulating evidence has shown cholesterol is capable of regulating cellular migration, proliferation, and spreading. However, the interaction and coordinated effects of both cellular cholesterol and the extracellular matrix (ECM) stiffness/composition on VSMC biomechanics remains to be elucidated.</p>","PeriodicalId":11029,"journal":{"name":"Current topics in membranes","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041049/pdf/nihms-1668351.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25578648","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
Intrinsic regulation of microvascular tone by myoendothelial feedback circuits. 肌内皮反馈回路对微血管张力的内在调节。
4区 生物学
Current topics in membranes Pub Date : 2020-01-01 Epub Date: 2020-03-06 DOI: 10.1016/bs.ctm.2020.01.004
Hamish A L Lemmey, Christopher J Garland, Kim A Dora
{"title":"Intrinsic regulation of microvascular tone by myoendothelial feedback circuits.","authors":"Hamish A L Lemmey, Christopher J Garland, Kim A Dora","doi":"10.1016/bs.ctm.2020.01.004","DOIUrl":"10.1016/bs.ctm.2020.01.004","url":null,"abstract":"<p><p>The endothelium is an important regulator of arterial vascular tone, acting to release nitric oxide (NO) and open Ca<sup>2+</sup>-activated K<sup>+</sup> (K<sub>Ca</sub>) channels to relax vascular smooth muscle cells (VSMCs). While agonists acting at endothelial cell (EC) receptors are widely used to assess the ability of the endothelium to reduce vascular tone, the intrinsic EC-dependent mechanisms are less well characterized. In small resistance arteries and arterioles, the presence of heterocellular gap junctions termed myoendothelial gap junctions (MEGJs) allows the passage of not only current, but small molecules including Ca<sup>2+</sup> and inositol trisphosphate (IP<sub>3</sub>). When stimulated to contract, the increase in VSM Ca<sup>2+</sup> and IP<sub>3</sub> can therefore potentially pass through MEGJs to activate adjacent ECs. This activation releases NO and opens K<sub>Ca</sub> channels, which act to limit contraction. This myoendothelial feedback (MEF) is amplified by EC Ca<sup>2+</sup> influx and release pathways, and is dynamically modulated by processes regulating gap junction conductance. There is a remarkable localization of key signaling and regulatory proteins within the EC projection toward VSM, and the intrinsic EC-dependent signaling pathways occurring with this highly specialized microdomain are reviewed.</p>","PeriodicalId":11029,"journal":{"name":"Current topics in membranes","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37931606","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
Endothelial inwardly-rectifying K+ channels as a key component of shear stress-induced mechanotransduction. 内皮向内纠偏的K+通道是剪应力诱导的机械传导的关键组成部分。
4区 生物学
Current topics in membranes Pub Date : 2020-01-01 Epub Date: 2020-03-23 DOI: 10.1016/bs.ctm.2020.02.002
Ibra S Fancher, Irena Levitan
{"title":"Endothelial inwardly-rectifying K<sup>+</sup> channels as a key component of shear stress-induced mechanotransduction.","authors":"Ibra S Fancher,&nbsp;Irena Levitan","doi":"10.1016/bs.ctm.2020.02.002","DOIUrl":"https://doi.org/10.1016/bs.ctm.2020.02.002","url":null,"abstract":"<p><p>It has been recognized for decades that fluid shear stress plays a major role in vascular function. Acting on the endothelium shear stress induces vasorelaxation of resistance arteries and plays a major role in the propensity of the major arteries to atherosclerosis. Many elements of shear-induced signaling have been identified yet we are just beginning to decipher the roles that mechanosensitive ion channels may play in the signaling pathways initiated by shear stress. Endothelial inwardly-rectifying K<sup>+</sup> channels were identified as potential primary mechanosensors in the late 1980s yet until our recent works, highlighted in the forthcoming chapter, the functional effect of a shear-activated K<sup>+</sup> current was completely unknown. In this chapter, we present the physiological effects of shear stress in arteries in health and disease and highlight the most prevalent of today's investigated mechanosensitive ion channels. Ultimately, we focus on Kir2.1 channels and discuss in detail our findings regarding the downstream signaling events that are induced by shear-activated endothelial Kir2.1 channels. Most importantly, we examine our findings regarding hypercholesterolemia-induced inhibition of Kir channel shear-sensitivity and the impact on endothelial function in the context of flow (shear)-mediated vasodilation and atherosclerosis.</p>","PeriodicalId":11029,"journal":{"name":"Current topics in membranes","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/bs.ctm.2020.02.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37931607","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: 5
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