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Ion-Mediated Effects of Glycosylation on the Disordered Mucin Domain: Insights from Coarse-Grained Simulations. 离子介导的糖基化对无序粘蛋白结构域的影响:来自粗粒度模拟的见解。
IF 3.4 3区 生物学
Biophysical journal Pub Date : 2025-07-23 DOI: 10.1016/j.bpj.2025.07.021
Gargi Biswas,Yaakov Levy
{"title":"Ion-Mediated Effects of Glycosylation on the Disordered Mucin Domain: Insights from Coarse-Grained Simulations.","authors":"Gargi Biswas,Yaakov Levy","doi":"10.1016/j.bpj.2025.07.021","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.07.021","url":null,"abstract":"Mucins are essential glycoproteins that form the backbone of mucus, a hydrogel protecting epithelial surfaces throughout the body. Their biophysical properties are governed by the densely glycosylated and highly disordered proline-threonine-serine (PTS) mucin domain, which becomes negatively charged by the addition of terminal sialic acid and sulfate groups to its glycans. The properties of mucins are further modulated by their interactions with cations, particularly sodium and calcium, which influence mucus expansion and viscoelasticity. Alterations in mucin glycosylation patterns or cation interactions contribute to the development of various pathological conditions. Modulating mucin's functional relationships to ameliorate these conditions requires first obtaining a detailed understanding of its structure, however, the large size, extensive disorder, and polymeric nature of mucins present significant challenges to their structural characterization. Here, we employed a coarse-grained modeling approach to investigate the effects of glycosylation, glycan charge, and salt concentration on mucin PTS organization. Using two different glycan structures, we explored how their interactions with monovalent (Na+) and divalent (Ca2+) cations affect the structural organization and ionic interactions of the PTS region. Our findings revealed that the presence of cations promoted tighter mucin conformations, with divalent cations inducing greater structural compaction than monovalent cations, consistently with their greater charge-shielding efficacy. Higher glycosylation levels and greater glycan charge densities enhance local glycan clustering and overall PTS structural expansion through their effects on cation binding and electrostatic repulsion. Furthermore, we examined the diffusion of charged peptides within mucin, demonstrating that peptide net charge affects both penetration and mobility. This study provides a comprehensive understanding of the structural organization and ionic interactions of mucin PTS, offering valuable insights into the molecular basis of mucin's protective properties and its role in health and disease.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"121 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144693483","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
Transmembrane coupling drives the growth of liquid-like protein condensates. 跨膜耦合驱动液体样蛋白凝聚物的生长。
IF 3.2 3区 生物学
Biophysical journal Pub Date : 2025-07-23 DOI: 10.1016/j.bpj.2025.07.022
Yohan Lee, Feng Yuan, Jerry L Cabriales, Jeanne C Stachowiak
{"title":"Transmembrane coupling drives the growth of liquid-like protein condensates.","authors":"Yohan Lee, Feng Yuan, Jerry L Cabriales, Jeanne C Stachowiak","doi":"10.1016/j.bpj.2025.07.022","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.07.022","url":null,"abstract":"<p><p>Timely and precise assembly of protein complexes on membrane surfaces is essential to the physiology of living cells. Recently, protein phase separation has been observed at cellular membranes, suggesting it may play a role in the assembly of protein complexes. Inspired by these findings, we observed that two-dimensional protein condensates on one side of a planar suspended membrane spontaneously colocalized with those on the opposite side. How might this phenomenon contribute to the assembly of stable transmembrane complexes? To address this question, we examined the diffusion and growth of two-dimensional protein condensates on both sides of membranes. Our results reveal that transmembrane coupling of protein condensates on opposite sides of the membrane slows down condensate diffusion while promoting condensate growth. How can the condensate growth be driven simultaneously with a decrease in the rate of condensate diffusion? We provide insights into these seemingly contradictory observations by distinguishing between diffusion-limited and coupling-driven growth processes. While transmembrane coupling slows down diffusion, it also locally concentrates condensates within a confined area. This confinement increases the probability of condensate coalescence and thereby promotes the growth of coupled condensates. These findings suggest that transmembrane coupling could play a role in the assembly of diverse membrane-bound structures by promoting the localization and growth of protein complexes on both membrane surfaces. This phenomenon could help to promote the assembly of transmembrane structures in diverse cellular contexts.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144706177","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
Collagen as a stress test and a tool for improvement of Glycine and Proline conformations in biomolecular force fields. 胶原蛋白在生物分子力场中作为压力测试和改善甘氨酸和脯氨酸构象的工具。
IF 3.4 3区 生物学
Biophysical journal Pub Date : 2025-07-22 DOI: 10.1016/j.bpj.2025.07.018
Kenno Vanommeslaeghe
{"title":"Collagen as a stress test and a tool for improvement of Glycine and Proline conformations in biomolecular force fields.","authors":"Kenno Vanommeslaeghe","doi":"10.1016/j.bpj.2025.07.018","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.07.018","url":null,"abstract":"","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"109 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144693278","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
Crumbs2 acts as a dynamic cellular sensor triggering stable podocyte cell-matrix anchorage. crums2作为一个动态的细胞传感器,触发稳定的足细胞-基质锚定。
IF 3.4 3区 生物学
Biophysical journal Pub Date : 2025-07-22 DOI: 10.1016/j.bpj.2025.07.020
Rohan Bhatia,Annika Möller-Kerutt,Sven Gerlach,Yannick Herfs,Rebecca Rixen,Hermann Pavenstädt,Bernd Hoffmann,Rudolf Merkel,Ulrich Kubitscheck,Thomas Weide,Jan Peter Siebrasse
{"title":"Crumbs2 acts as a dynamic cellular sensor triggering stable podocyte cell-matrix anchorage.","authors":"Rohan Bhatia,Annika Möller-Kerutt,Sven Gerlach,Yannick Herfs,Rebecca Rixen,Hermann Pavenstädt,Bernd Hoffmann,Rudolf Merkel,Ulrich Kubitscheck,Thomas Weide,Jan Peter Siebrasse","doi":"10.1016/j.bpj.2025.07.020","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.07.020","url":null,"abstract":"Mammalian crumbs proteins (CRB) 2 and 3 are expressed in the kidney, with CRB2 mainly found within podocytes. Here, we focused on the function of CRB2 in podocytes. We investigated the CRB2/CRB3A membrane dynamics at the podocyte-podocyte contact interface in a cell culture model. Traction force microscopy was employed to clarify whether the homophilic CRB2 interactions occur in cis or trans. Live-cell imaging revealed the effects of CRB2 and CRB3A expression on cell migration from the single cell level to large cellular networks at long space and time scales. We found that CRB2 interacts in trans, but surprisingly does not form stable interconnections. Instead, CRB2 acts as a dynamic sensor for the cellular environment and triggers a massive reorganisation of focal adhesions and the actin cytoskeleton upon contact of CRB2-positive cells resulting in a strongly increased cell-matrix anchoring. Cytoskeleton reorganization can also be triggered by CRB3A, however, the increased cell-matrix anchorage is CRB2-specific.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"67 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144693435","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
The Membrane Properties Determine Protein Function - A New Aspect of Allosteric Modulation. 膜性质决定蛋白质功能——变构调节的新视角。
IF 3.4 3区 生物学
Biophysical journal Pub Date : 2025-07-22 DOI: 10.1016/j.bpj.2025.07.019
Daniel Huster
{"title":"The Membrane Properties Determine Protein Function - A New Aspect of Allosteric Modulation.","authors":"Daniel Huster","doi":"10.1016/j.bpj.2025.07.019","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.07.019","url":null,"abstract":"","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"23 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144693436","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
3D multiscale shape analysis of nuclei and in-vivo elastic stress sensors allows force inference. 核的三维多尺度形状分析和体内弹性应力传感器允许力推断。
IF 3.4 3区 生物学
Biophysical journal Pub Date : 2025-07-22 DOI: 10.1016/j.bpj.2025.07.015
Alejandro Jurado,Jonas Isensee,Arne Hofemeier,Lea Johanna Krüger,Raphael Wittkowski,Ramin Golestanian,Philip Bittihn,Timo Betz
{"title":"3D multiscale shape analysis of nuclei and in-vivo elastic stress sensors allows force inference.","authors":"Alejandro Jurado,Jonas Isensee,Arne Hofemeier,Lea Johanna Krüger,Raphael Wittkowski,Ramin Golestanian,Philip Bittihn,Timo Betz","doi":"10.1016/j.bpj.2025.07.015","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.07.015","url":null,"abstract":"The measurement of stresses and forces at the tissue level has proven to be an indispensable tool for the understanding of complex biological phenomena such as cancer invasion, embryo development or wound healing. One of the most versatile tools for force inference at the cell and tissue level are elastic force sensors, whose biocompatibility and tunable material properties make them suitable for many different experimental scenarios. The evaluation of those forces, however, is still a bottleneck due to the numerical methods seen in literature until now, which are usually slow and render low experimental yield. Here we present BeadBuddy, a ready-to-use platform for the evaluation of deformation and stresses from fluorescently labelled sensors within seconds. The strengths of BeadBuddy lie in the pre-computed analytical solutions of the elastic problem, the abstraction of data into Spherical Harmonics, and a simple user interface that creates a smooth workflow for force inference.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"11 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144693277","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
Allometric cell spreading and the geometrical control of focal adhesion collective organization. 异速细胞扩张与黏附集体组织的几何控制。
IF 3.4 3区 生物学
Biophysical journal Pub Date : 2025-07-21 DOI: 10.1016/j.bpj.2025.07.016
Célian Bimbard,Ali-Alhadi Wahhod,Démosthéne Mitrossilis,Joseph Vermeil,Rémi Bousquet,Alain Richert,David Pereira,Pauline Durand-Smet,Sophie Asnacios,Jocelyn Étienne,Atef Asnacios,Jonathan Fouchard
{"title":"Allometric cell spreading and the geometrical control of focal adhesion collective organization.","authors":"Célian Bimbard,Ali-Alhadi Wahhod,Démosthéne Mitrossilis,Joseph Vermeil,Rémi Bousquet,Alain Richert,David Pereira,Pauline Durand-Smet,Sophie Asnacios,Jocelyn Étienne,Atef Asnacios,Jonathan Fouchard","doi":"10.1016/j.bpj.2025.07.016","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.07.016","url":null,"abstract":"Focal adhesions are protein complexes that transmit actin cytoskeleton forces to the extracellular matrix and serve as signaling hubs that regulate cell physiology. While their growth is achieved through a local force-dependent process, the requirement of sustaining stress at the cell scale suggests a global regulation of the collective organization of focal adhesions. To investigate evidence of such large-scale regulation, we compared changes in cell shape and the organization of focal adhesion-like structures during the early spreading of fibroblasts either on a two-dimensional substrate or confined between two parallel plates, and for cells of different volumes. In this way, we reveal that the areal density of focal adhesions is conserved regardless of cell size or third-dimensional confinement, despite different absolute values of the surface covered by adhesion clusters. In particular, the width of the focal adhesions ring, which fills the flat lamella at the cell front, adapts to cell size and third-dimensional confinement and scales with cell-substrate contact radius. We find that this contact radius also adapts in the parallel-plate geometry so that the cumulated area of cell-substrate contact is conserved at the cell scale. We suggest that this behavior is the result of 3D cell shape changes which govern spreading transitions. Indeed, because of volume conservation constraints, the evolution of cell-body contact angle, adjusts according to cell size and confinement, whereas the rate of early spreading at the cell-substrate contact is not affected by third-dimensional geometry. Overall, our data suggest that a coordination between global and local scales mediates the adaptation of cell-substrate contacts and focal adhesions distribution to large scale geometrical constraints, which allows an invariant cell-substrate adhesive energy.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"47 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144684243","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
Role of Dynamical Instability in QT Interval Variability and Early Afterdepolarization Propensity. 动态不稳定性在QT间期变异性和早期后去极化倾向中的作用。
IF 3.2 3区 生物学
Biophysical journal Pub Date : 2025-07-21 DOI: 10.1016/j.bpj.2025.07.017
Daisuke Sato, Bence Hegyi, Crystal M Ripplinger, Donald M Bers
{"title":"Role of Dynamical Instability in QT Interval Variability and Early Afterdepolarization Propensity.","authors":"Daisuke Sato, Bence Hegyi, Crystal M Ripplinger, Donald M Bers","doi":"10.1016/j.bpj.2025.07.017","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.07.017","url":null,"abstract":"<p><p>Beat-to-beat variability of the QT interval (QTV) is a well-established marker of cardiac health, with increased QTV (> 5 ms) often associated with a higher risk of arrhythmias. However, the underlying mechanisms contributing to this phenomenon remain poorly understood. Recently, we showed that cardiac instability is a major cause of QTV. Early afterdepolarizations (EADs) are abnormal electrical oscillations that occur during the plateau phase of the cardiac action potential (AP), often arising when the membrane potential becomes unstable. In this study, we use a physiologically detailed computational model of rabbit ventricular myocytes with stochastic ion channel gating to investigate the relationship between QTV and EAD propensity. We found that increased AP duration (APD) variability, which serves as a surrogate for QTV on the ECG at the single-cell level, can arise even in the absence of apparent EADs, driven by intrinsic dynamical instability. As the cellular state approaches the threshold for EAD generation, small perturbations in membrane voltage are amplified, leading to increased APD variability. The phase-plane analysis in the voltage-calcium channel inactivation space demonstrates that proximity to the EAD-generating basin of attraction strongly influences repolarization variability, establishing a mechanistic link between QTV and EAD propensity. Furthermore, we observed that QTV increases at longer pacing cycle lengths (PCLs), distinguishing it from alternans-associated APD variability, which increases at shorter PCLs. These findings suggest that increased QTV may serve as an early indicator of arrhythmic risk before the manifestation of EADs, potentially offering a critical window for preventive intervention. Our results provide novel insights into the fundamental mechanisms underlying QTV and its potential role in arrhythmia prediction.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144688790","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
Comparative Analysis of the Bending Modulus for Phosphatidylcholine, Phosphatidylethanolamine, and Sphingomyelin Membranes Based on Coarse-Grained Molecular Dynamics Simulations. 基于粗粒度分子动力学模拟的磷脂酰胆碱、磷脂酰乙醇胺和鞘磷脂膜弯曲模量的比较分析。
IF 3.4 3区 生物学
Biophysical journal Pub Date : 2025-07-16 DOI: 10.1016/j.bpj.2025.07.014
Sam Brown,Jessica Pallarez,Marat R Talipov
{"title":"Comparative Analysis of the Bending Modulus for Phosphatidylcholine, Phosphatidylethanolamine, and Sphingomyelin Membranes Based on Coarse-Grained Molecular Dynamics Simulations.","authors":"Sam Brown,Jessica Pallarez,Marat R Talipov","doi":"10.1016/j.bpj.2025.07.014","DOIUrl":"https://doi.org/10.1016/j.bpj.2025.07.014","url":null,"abstract":"Bilayer membranes are essential biological structures with complex and largely unexplored mechanical properties. Using coarse-grained molecular dynamics simulations, we evaluated the bending modulus across diverse lipid compositions, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM). Three computational techniques were employed to calculate the bending modulus from thermal fluctuations of the simulated bilayers: the Fourier transform of the lipid height function (q-4 fitting), Bedeaux-Weeks Density Correlation Functions (BW-DCF) method, and Real Space Fluctuations (RSF). The analysis revealed substantial variations in bending modulus values across methods, underscoring the inherent complexities and discrepancies in computational assessments. These findings advance our understanding of membrane dynamics and provide valuable insights into bilayer structural behavior. The results support the broader application of computational approaches to study biological systems and inspire the development of biomimetic materials with tailored mechanical properties.","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"153 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144652905","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
Thermal adaptation of extremozymes: Temperature-sensitive contact analysis of serine proteases. 极端酶的热适应:丝氨酸蛋白酶的温度敏感接触分析。
IF 3.2 3区 生物学
Biophysical journal Pub Date : 2025-07-15 Epub Date: 2025-06-05 DOI: 10.1016/j.bpj.2025.06.001
Dulitha P Kulathunga, Davit A Potoyan
{"title":"Thermal adaptation of extremozymes: Temperature-sensitive contact analysis of serine proteases.","authors":"Dulitha P Kulathunga, Davit A Potoyan","doi":"10.1016/j.bpj.2025.06.001","DOIUrl":"10.1016/j.bpj.2025.06.001","url":null,"abstract":"<p><p>Enzyme thermal adaptation reflects a delicate interplay between the sequence, structure, and dynamics of proteins which are fined tuned to meet environmental demands of organisms. Understanding these evolutionary relationships can drive advances in bioengineering, including the design of industrial enzymes and the development of novel therapeutics. This work explores sequence-to-dynamics connections in subtilisin-like serine protease homologs using a recently developed computational methodology that employs expanded ensemble simulations and temperature-sensitive contact analysis. We reveal that thermophilic enzymes achieve thermal stability through extensive salt bridges and hydrophobic networks, whereas psychrophilic enzymes rely on the stability of targeted interactions for cold adaptation. An unsupervised cluster analysis of residue conservation, flexibility, and hydrophobic interactions provides a comprehensive view of residue-specific contributions to thermal adaptation. These findings highlight the coordinated roles of conserved and variable regions in enzyme stability, offering a framework for tailoring enzymes to specific thermal properties for biotechnological applications.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":" ","pages":"2291-2302"},"PeriodicalIF":3.2,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144246246","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
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