ORCID Profile
0000-0002-7591-0864
Current Organisations
University of Sydney
,
Heart Research Institute
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Biomechanical Engineering | Other Physical Sciences | Biomedical Instrumentation | Biochemistry and Cell Biology | Optical Properties of Materials | Biomedical Engineering | Fluidisation and Fluid Mechanics | Synchrotrons; Accelerators; Instruments and Techniques | Receptors and Membrane Biology
Expanding Knowledge in the Biological Sciences | Scientific Instruments | Expanding Knowledge in Technology | Expanding Knowledge in the Physical Sciences | Expanding Knowledge in Engineering |
Publisher: American Society of Hematology
Date: 11-05-2023
DOI: 10.1182/BLOODADVANCES.2022008650
Abstract: von Willebrand factor (VWF) is the protective carrier of procoagulant factor VIII (FVIII) in the shear forces of the circulation, prolonging its half-life and delivering it to the developing thrombus. Using force spectroscopy, VWF-FVIII complex formation is characterized by catch-bond behavior in which force first decelerates then accelerates bond dissociation. Patients with mutations in VWF at the FVIII binding site phenocopies hemophilia A and the most common mutations are of cysteine residues involving multiple disulfide bonds. From differential cysteine alkylation and mass spectrometry experiments, 13 VWF disulfide bonds at the FVIII binding site were found to exist in formed and unformed states, and binding of FVIII results in partial formation of 12 of the VWF bonds. Force spectroscopy studies indicate that the VWF-FVIII bond stiffens in response to force and this feature of the interaction is ablated when VWF disulfide bonds are prevented from forming, resulting in slip-only bond behavior. Exposure of VWF to pathological fluid shear forces ex vivo and in vivo causes partial cleavage of all 13 disulfide bonds, further supporting their malleable nature. These findings demonstrate that FVIII binding to VWF involves dynamic changes in the covalent states of several VWF disulfides that are required for productive interaction in physiological shear forces.
Publisher: American Chemical Society (ACS)
Date: 12-06-2020
Publisher: Springer New York
Date: 2017
DOI: 10.1007/978-1-4939-6881-7_15
Abstract: Upon engagement with a specific ligand, a cell surface receptor transduces intracellular signals to activate various cellular functions. This chapter describes a set of biomechanical methods for analyzing the characteristics of cross-junctional receptor-ligand interactions at the surface of living cells. These methods combine the characterization of kinetics of receptor-ligand binding with real-time imaging of intracellular calcium fluxes, which allow researchers to assess how the signal initiated from single receptor-ligand engagement is transduced across the cell membrane. A major application of these methods is the analysis of antigen recognition by triggering of the T cell receptor (TCR). Three related methods are described in this chapter: (1) the micropipette adhesion assay, (2) the biomembrane force probe (BFP) assay, and (3) combining BFP with fluorescence microscopy (fBFP). In all cases, an ultrasoft human red blood cell (RBC) is used as an ultrasensitive mechanical force probe. The micropipette assay detects binding events visually. The BFP uses a high-speed camera and real-time image tracking techniques to measure mechanical variables on a single molecular bond with up to ~1 pN (10
Publisher: Authorea, Inc.
Date: 23-02-2023
DOI: 10.22541/AU.167715770.06231875/V1
Abstract: Mechanical forces play a crucial role in biological processes at the molecular and cellular levels. Recent advancements in dynamic force spectroscopies (DFS) have enabled the application and measurement of forces and displacements with high resolutions, providing insights into the mechanical pathways involved in various diseases, including cancer, cardiovascular disease, and COVID-19. Among the various DFS techniques, biomembrane force probe (BFP) advancements have improved our ability to measure bond kinetics and cellular mechanosensing with pico-newton and nano-meter resolutions. In this review, we provide a comprehensive overview of the classical BFP-DFS setup and highlight key advancements, including the development of dual biomembrane force probe (dBFP) and fluorescence biomembrane force probe (fBFP). BFP-DFS not only enables the investigation of dynamic bond behaviors on living cells, but also contributed significantly to our understanding of the specific ligand–receptor axes mediated cell mechanosensing. Besides, we explore the contribution of discoveries made possible by BFP-DFS in cancer biology, thrombosis, and inflammation, as well as predict future BFP upgrades to improve output and feasibility. Although BFP-DFS is still a niche research modality, its contribution to the growing field of cell mechanobiology is unparalleled, and its potential to elucidate novel therapeutic discoveries is significant.
Publisher: Elsevier BV
Date: 12-2019
Publisher: BMJ
Date: 15-12-2019
Abstract: Arterial thrombosis is in part contributed by excessive platelet aggregation, which can lead to blood clotting and subsequent heart attack and stroke. Platelets are sensitive to the haemodynamic environment. Rapid haemodynamcis and disturbed blood flow, which occur in vessels with growing thrombi and atherosclerotic plaques or is caused by medical device implantation and intervention, promotes platelet aggregation and thrombus formation. In such situations, conventional antiplatelet drugs often have suboptimal efficacy and a serious side effect of excessive bleeding. Investigating the mechanisms of platelet biomechanical activation provides insights distinct from the classic views of agonist-stimulated platelet thrombus formation. In this work, we review the recent discoveries underlying haemodynamic force-reinforced platelet binding and mechanosensing primarily mediated by three platelet receptors: glycoprotein Ib (GPIb), glycoprotein IIb/IIIa (GPIIb/IIIa) and glycoprotein VI (GPVI), and their implications for development of antithrombotic ‘mechano-medicine’ .
Publisher: Springer Science and Business Media LLC
Date: 24-02-2021
Publisher: American Society of Hematology
Date: 05-09-2013
DOI: 10.1182/BLOOD-2013-03-484550
Abstract: CIP4 affects the remodeling of both plasma membrane and cortical cytoskeleton in megakaryocytes. CIP4 in platelet biogenesis involves cortical tension, as does WASP, and WASP-independent plasma membrane reorganization.
Publisher: Computers, Materials and Continua (Tech Science Press)
Date: 2019
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C8SC01023D
Abstract: Ligand competition directs heterogeneous bio-chemistry surface and self-assembly for upconversion nanoparticles.
Publisher: American Society for Cell Biology (ASCB)
Date: 07-11-2017
Abstract: Mechanosensing describes the ability of a cell to sense mechanical cues of its microenvironment, including not only all components of force, stress, and strain but also substrate rigidity, topology, and adhesiveness. This ability is crucial for the cell to respond to the surrounding mechanical cues and adapt to the changing environment. Ex les of responses and adaptation include (de)activation, proliferation/apoptosis, and (de)differentiation. Receptor-mediated cell mechanosensing is a multistep process that is initiated by binding of cell surface receptors to their ligands on the extracellular matrix or the surface of adjacent cells. Mechanical cues are presented by the ligand and received by the receptor at the binding interface but their transmission over space and time and their conversion into biochemical signals may involve other domains and additional molecules. In this review, a four-step model is described for the receptor-mediated cell mechanosensing process. Platelet glycoprotein Ib, T-cell receptor, and integrins are used as ex les to illustrate the key concepts and players in this process.
Publisher: Research Square Platform LLC
Date: 07-08-2023
DOI: 10.21203/RS.3.RS-3218836/V1
Abstract: Microgeometry profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains enigmatic. We introduce a fluorescent micropipette aspiration assay to concurrently observe intracellular calcium mobilization and cytoskeletal restructuring in real-time under distinct microscale geometric alterations. Utilizing finite element analyses, coupled with PIEZO1-specific transgenic models, we discern that steeper micropipette tip angles markedly enhance PIEZO1-mediated calcium influx. This mechanical stress on the aspirated cell induces a significant F-actin reorganization, resulting in a "mechanical buckle" that lifies PIEZO1 activity within the aspirated region. Remarkably, when this F-actin network is disrupted, PIEZO1 gating is significantly inhibited, signifying its indispensable role in mechanosensing under geometrical changes. This mechanobiology study illuminates the profound relationship between biomechanical microenvironment, cytoskeletal adaptation, and PIEZO1 activation, inspiring future bioengineering applications.
Publisher: Frontiers Media SA
Date: 25-11-2021
Abstract: Understanding how platelets can sense and respond to hemodynamic forces in disturbed blood flow and complexed vasculature is crucial to the development of more effective and safer antithrombotic therapeutics. By incorporating erse structural and functional designs, microfluidic technologies have emerged to mimic microvascular anatomies and hemodynamic microenvironments, which open the floodgates for fascinating platelet mechanobiology investigations. The latest endothelialized microfluidics can even recapitulate the crosstalk between platelets and the circulatory system, including the vessel walls and plasma proteins such as von Willebrand factor. Hereby, we highlight these exciting microfluidic applications to platelet mechanobiology and platelet–circulatory system interplay as implicated in thrombosis. Last but not least, we discuss the need for microfluidic standardization and summarize the commercially available microfluidic platforms for researchers to obtain reproducible and consistent results in the field.
Publisher: Springer Science and Business Media LLC
Date: 06-09-2018
DOI: 10.1038/S41467-018-05806-0
Abstract: Platelet αIIbβ3 integrin and its ligands are essential for thrombosis and hemostasis, and play key roles in myocardial infarction and stroke. Here we show that apolipoprotein A-IV (apoA-IV) can be isolated from human blood plasma using platelet β3 integrin-coated beads. Binding of apoA-IV to platelets requires activation of αIIbβ3 integrin, and the direct apoA-IV-αIIbβ3 interaction can be detected using a single-molecule Biomembrane Force Probe. We identify that aspartic acids 5 and 13 at the N-terminus of apoA-IV are required for binding to αIIbβ3 integrin, which is additionally modulated by apoA-IV C-terminus via intra-molecular interactions. ApoA-IV inhibits platelet aggregation and postprandial platelet hyperactivity. Human apoA-IV plasma levels show a circadian rhythm that negatively correlates with platelet aggregation and cardiovascular events. Thus, we identify apoA-IV as a novel ligand of αIIbβ3 integrin and an endogenous inhibitor of thrombosis, establishing a link between lipoprotein metabolism and cardiovascular diseases.
Publisher: Bio-Protocol, LLC
Date: 2019
Publisher: Royal Society of Chemistry (RSC)
Date: 2022
DOI: 10.1039/D2CB00010E
Abstract: The N-AIM of VWF-A1 forms a Rotini-like structure, therefore partially autoinhibit VWF-A1–GPIbα interaction. The N-AIM acts as a defending sword to protect and stabilize the VWF-A1 structure under harsh environments.
Publisher: Royal Society of Chemistry (RSC)
Date: 2022
DOI: 10.1039/D2AN00270A
Abstract: These studies demonstrate the versatility and relevance of a novel ‘platelet post’ model to examine the adhesive interactions between platelets and neutrophils under 3D disturbed flow conditions relevant to thromboinflammation.
Publisher: MyJove Corporation
Date: 20-11-2021
DOI: 10.3791/62490
Publisher: Oxford University Press (OUP)
Date: 18-04-2013
DOI: 10.1093/BIOINFORMATICS/BTT180
Abstract: Motivation: Abrupt reduction/resumption of thermal fluctuations of a force probe has been used to identify association/dissociation events of protein–ligand bonds. We show that off-rate of molecular dissociation can be estimated by the analysis of the bond lifetime, while the on-rate of molecular association can be estimated by the analysis of the waiting time between two neighboring bond events. However, the analysis relies heavily on subjective judgments and is time-consuming. To automate the process of mapping out bond events from thermal fluctuation data, we develop a hidden Markov model (HMM)-based method. Results: The HMM method represents the bond state by a hidden variable with two values: bound and unbound. The bond association/dissociation is visualized and pinpointed. We apply the method to analyze a key receptor–ligand interaction in the early stage of hemostasis and thrombosis: the von Willebrand factor (VWF) binding to platelet glycoprotein Ibα (GPIbα). The numbers of bond lifetime and waiting time events estimated by the HMM are much more than those estimated by a descriptive statistical method from the same set of raw data. The kinetic parameters estimated by the HMM are in excellent agreement with those by a descriptive statistical analysis, but have much smaller errors for both wild-type and two mutant VWF-A1 domains. Thus, the computerized analysis allows us to speed up the analysis and improve the quality of estimates of receptor–ligand binding kinetics. Contact: jeffwu@isye.gatech.edu or cheng.zhu@bme.gatech.edu
Publisher: Wiley
Date: 28-03-2023
Abstract: The Vein‐Chip recapitulates CVST Virchow's triad and enables systematic characterization of venous thrombogenesis with respect to fibrin formation and platelet aggregation. Distinct from the arterial setting, platelets universally adhere across the entire CVS Vein‐Chip independent of stenotic geometry and flow disturbance. Intriguingly, fibrin propagates along with the flow direction, but exclusively deposits to the inner vessel wall. Upon inflammatory endothelial injury, fibrin deposition mirrors to the outer vessel wall, but still not in the lumen. Together, the Vein‐Chip promises future applications for personalized thrombotic assessment and monitoring.
Publisher: IOP Publishing
Date: 27-07-2023
Abstract: During the final stage of cancer metastasis, tumor cells embed themselves in distant capillary beds, from where they extravasate and establish secondary tumors. Recent findings underscore the pivotal roles of blood/lymphatic flow and shear stress in this intricate tumor extravasation process. Despite the increasing evidence, there is a dearth of systematic and biomechanical methodologies that accurately mimic intricate 3D microtissue interactions within a controlled hydrodynamic microenvironment. Addressing this gap, we introduce an easy-to-operate 3D spheroid-microvasculature-on-a-chip (SMAC) model. Operating under both static and regulated flow conditions, the SMAC model facilitates the replication of the biomechanical interplay between heterogeneous tumor spheroids and endothelium in a quantitative manner. Serving as an in vitro model for metastasis mechanobiology, our model unveils the phenomena of 3D spheroid-induced endothelial compression and cell-cell junction degradation during tumor migration and expansion. Furthermore, we investigated the influence of shear stress on endothelial orientation, polarization, and tumor spheroid expansion. Collectively, our SMAC model provides a compact, cost-efficient, and adaptable platform for probing the mechanobiology of metastasis.
Publisher: Wiley
Date: 19-07-2023
DOI: 10.1002/AGT2.386
Abstract: Cerebral venous sinus thrombosis (CVST) is a type of stroke associated with COVID‐19 vaccine‐induced immune thrombotic thrombocytopenia. The precise etiology of CVST often remains elusive due to the highly heterogeneous nature of its governing mechanisms, specifically, Virchow's triad that involves altered blood flow, endothelial dysfunction, and hypercoagulability, which varies substantially amongst in iduals. Existing diagnostic and monitoring approaches lack the capability to reflect the combination of these patient‐specific thrombotic determinants. In response to this challenge, we introduce a Vein‐Chip platform that recapitulates the CVST vascular anatomy from magnetic resonance venography and the associated hemodynamic flow profile using the “Chinese Movable Type‐like” soft stereolithography technique. The resultant full‐lumen personalized Vein‐Chips, functionalized with endothelial cells, enable in‐vitro thrombosis assays that can elucidate distinct thrombogenic scenarios between normal vascular conditions and those of endothelial dysfunction. The former displayed minimal platelet aggregation and negligible fibrin deposition, while the latter presented significant fibrin extrusion from platelet aggregations. The low‐cost movable typing technique further enhances the potential for commercialization and broader utilization of personalized Vein‐Chips in surgical labs and at‐home monitoring. Future research and development in this direction will pave the way for improved management and prevention of CVST, ultimately benefiting both patients and healthcare systems.
Publisher: Springer New York
Date: 2019
DOI: 10.1007/978-1-4939-9187-7_7
Abstract: An emerging concept in chemical biology is that protein function that can be regulated by the redox state of disulphide bonds. This chapter describes the dynamic force spectroscopy method for analyzing redox regulation of receptor-ligand interactions at the surface of living cells. The main method described in this chapter is the biomembrane force probe (BFP), in which an ultrasoft human red blood cell is used as an ultrasensitive mechanical force probe. The BFP uses a high-speed camera and real-time imaging tracking techniques to characterize a single molecular bond with ~1 pN (10
Publisher: Elsevier BV
Date: 04-2015
Publisher: Elsevier
Date: 2018
Publisher: Springer Science and Business Media LLC
Date: 27-10-2017
DOI: 10.1038/S41598-017-13793-3
Abstract: Conventional approaches for studying receptor-mediated cell signaling, such as the western blot and flow cytometry, are limited in three aspects: 1) The perturbing preparation procedures often alter the molecules from their native state on the cell 2) Long processing time before the final readout makes it difficult to capture transient signaling events ( min) 3) The experimental environments are force-free, therefore unable to visualize mechanical signals in real time. In contrast to these methods in biochemistry and cell biology that are usually population-averaged and non-real-time, here we introduce a novel single-cell based nanotool termed dual biomembrane force probe (dBFP). The dBFP provides precise controls and quantitative readouts in both mechanical and chemical terms, which is particularly suited for juxtacrine signaling and mechanosensing studies. Specifically, the dBFP allows us to analyze dual receptor crosstalk by quantifying the spatiotemporal requirements and functional consequences of the up- and down-stream signaling events. In this work, the utility and power of the dBFP has been demonstrated in four important dual receptor systems that play key roles in immunological synapse formation, shear-dependent thrombus formation, and agonist-driven blood clotting.
Publisher: Springer Science and Business Media LLC
Date: 09-05-2019
Publisher: MDPI AG
Date: 06-01-2023
DOI: 10.3390/BIOS13010095
Abstract: Cancer cells interacting with the extracellular matrix (ECM) in the tumor microenvironment is pivotal for tumorigenesis, invasion, and metastasis. Cell–ECM adhesion has been intensively studied in cancer biology in the past decades to understand the molecular mechanisms underlying the adhesion events and extracellular mechanosensing, as well as develop therapeutic strategies targeting the cell adhesion molecules. Many methods have been established to measure the cell–ECM adhesion strength and correlate it with the metastatic potential of certain cancer types. However, those approaches are either low throughput, not quantitative, or with poor sensitivity and reproducibility. Herein, we developed a novel acoustic force spectroscopy based method to quantify the cell–ECM adhesion strength during adhesion maturation process using the emerging z-Movi® technology. This can be served as a fast, simple, and high-throughput platform for functional assessment of cell adhesion molecules in a highly predictive and reproducible manner.
Publisher: Elsevier BV
Date: 09-2015
Publisher: Proceedings of the National Academy of Sciences
Date: 16-12-2013
Abstract: Cell-expressed integrins mediate adhesion with other cells and with extracellular matrix and are essential for embryonic development and for controlling leukocyte migration in later life. Integrin adhesion depends on conformational change leading to activation, although it remains unknown exactly how integrins alter their conformational state and adhesion in response to guidance cues. We show that the guidance molecule plexinD1 controls clustering of integrins in patches on the cell membrane and that the activation state of in idual integrins in these patches can be switched off by binding of sema3E to plexinD1. Disruption of this pathway causes abnormal thymocyte adhesion regulation and migration during development, leading to autoimmune phenomena.
Publisher: Royal Society of Chemistry (RSC)
Date: 2022
DOI: 10.1039/D2CP01581A
Abstract: The von Willebrand factor A1 domain-derived polypeptide sequence Q1238-E1260 forms a hairpin-like structure in trans . Soluble Q1238-E1260 partially inhibits A1–GPIbα binding while retaining its catch-bond behavior in a sandwich complex.
Publisher: Springer Science and Business Media LLC
Date: 04-11-2015
DOI: 10.1038/SREP16171
Abstract: The Open Field (OF) test is one of the most commonly used assays for assessing exploratory behaviour and generalised locomotor activity in rodents. Nevertheless, the vast majority of researchers still rely upon costly commercial systems for recording and analysing OF test results. Consequently, our aim was to design a freely available program for analysing the OF test and to provide an accompanying protocol that was minimally invasive, rapid, unbiased, without the need for specialised equipment or training. Similar to commercial systems, we show that our software—called MouseMove —accurately quantifies numerous parameters of movement including travel distance, speed, turning and curvature. To assess its utility, we used MouseMove to quantify unilateral locomotor deficits in mice following the filament-induced middle cerebral artery occlusion model of acute ischemic stroke. MouseMove can also monitor movement within defined regions-of-interest and is therefore suitable for analysing the Novel Object Recognition test and other field-related cognitive tests. To the best of our knowledge, MouseMove is the first open source software capable of providing qualitative and quantitative information on mouse locomotion in a semi-automated and high-throughput fashion and hence MouseMove represents a sound alternative to commercial movement analysis systems.
Publisher: Elsevier BV
Date: 29-10-2008
Publisher: eLife Sciences Publications, Ltd
Date: 19-07-2016
DOI: 10.7554/ELIFE.15447
Abstract: How cells sense their mechanical environment and transduce forces into biochemical signals is a crucial yet unresolved question in mechanobiology. Platelets use receptor glycoprotein Ib (GPIb), specifically its α subunit (GPIbα), to signal as they tether and translocate on von Willebrand factor (VWF) of injured arterial surfaces against blood flow. Force elicits catch bonds to slow VWF–GPIbα dissociation and unfolds the GPIbα leucine-rich repeat domain (LRRD) and juxtamembrane mechanosensitive domain (MSD). How these mechanical processes trigger biochemical signals remains unknown. Here we analyze these extracellular events and the resulting intracellular Ca2+ on a single platelet in real time, revealing that LRRD unfolding intensifies Ca2+ signal whereas MSD unfolding affects the type of Ca2+ signal. Therefore, LRRD and MSD are analog and digital force transducers, respectively. The nm macroglycopeptide separating the two domains transmits force on the VWF–GPIbα bond (whose lifetime is prolonged by LRRD unfolding) to the MSD to enhance its unfolding, resulting in unfolding cooperativity at an optimal force. These elements may provide design principles for a generic mechanosensory protein machine.
Publisher: Wiley
Date: 09-07-2023
Abstract: The spectacular physical phenomenon of surface plasmon resonance (SPR) is the essence of present‐day plasmonic sensors. Meanwhile, the unique properties of the interaction between light and matter have been carved out into the development of modern‐day diagnostic biosensors. Plasmons, in simple terms, are oscillating free electrons in metallic nano‐structures triggered by an incoming electromagnetic (EM) wave. With the advantages of real‐time and label‐free bio‐sensing, plasmonic sensors are being utilized in multiple erse areas of food technology, the bio‐medical diagnostic sector, and even the chemical industry. Although this review will be brief, readers can gain a comprehensive picture of the essential elements by taking a broader look into the exploration of SPR sensor design via simulated studies and representative experimental plasmonic schemes developed for bio‐sensing. In short, the various SPR sensing schemes that researchers have explored to realize enhanced SPR sensitivity are reviewed and summarized. Different experimental plasmonic sensors are also examined in which new SPR excitation schemes have been adopted. These "unconventional" designs, specifically those involving hybrid localized surface plasmon resonance (LSPR)‐SPR excitation, may inspire those in the plasmonic field.
Publisher: Wiley
Date: 04-2002
Abstract: Membrane type 1 metalloprotease (MT1-MMP) is a transmembrane metalloprotease that plays a major role in the extracellular matrix remodeling, directly by degrading several of its components and indirectly by activating pro-MMP2. We investigated the effects of MT1-MMP overexpression on in vitro and in vivo properties of human breast adenocarcinoma MCF7 cells, which do not express MT1-MMP or MMP-2. MT1-MMP and MMP-2 cDNAs were either transfected alone or cotransfected. All clones overexpressing MT1-MMP 1) were able to activate endogenous or exogenous pro-MMP-2, 2) displayed an enhanced in vitro invasiveness through matrigel-coated filters independent of MMP-2 transfection, 3) induced the rapid development of highly vascularized tumors when injected subcutaneously in nude mice, and 4) promoted blood vessels sprouting in the rat aortic ring assay. These effects were observed in all clones overexpressing MT1-MMP regardless of MMP-2 expression levels, suggesting that the production of MMP-2 by tumor cells themselves does not play a critical role in these events. The angiogenic phenotype of MT1-MMP-producing cells was associated with an up-regulation of VEGF expression. These results emphasize the importance of MT1-MMP during tumor angiogenesis and open new opportunities for the development of anti-angiogenic strategies combining inhibitors of MT1-MMP and VEGF antagonists.
Publisher: Frontiers Media SA
Date: 20-01-2021
DOI: 10.3389/FPHAR.2021.779753
Abstract: Platelet function tests are essential to profile platelet dysfunction and dysregulation in hemostasis and thrombosis. Clinically they provide critical guidance to the patient management and therapeutic evaluation. Recently, the biomechanical effects induced by hemodynamic and contractile forces on platelet functions attracted increasing attention. Unfortunately, the existing platelet function tests on the market do not sufficiently incorporate the topical platelet mechanobiology at play. Besides, they are often expensive and bulky systems that require large s le volumes and long processing time. To this end, numerous novel microfluidic technologies emerge to mimic vascular anatomies, incorporate hemodynamic parameters and recapitulate platelet mechanobiology. These miniaturized and cost-efficient microfluidic devices shed light on high-throughput, rapid and scalable platelet function testing, hematological disorder profiling and antiplatelet drug screening. Moreover, the existing antiplatelet drugs often have suboptimal efficacy while incurring several adverse bleeding side effects on certain in iduals. Encouraged by a few microfluidic systems that are successfully commercialized and applied to clinical practices, the microfluidics that incorporate platelet mechanobiology hold great potential as handy, efficient, and inexpensive point-of-care tools for patient monitoring and therapeutic evaluation. Hereby, we first summarize the conventional and commercially available platelet function tests. Then we highlight the recent advances of platelet mechanobiology inspired microfluidic technologies. Last but not least, we discuss their future potential of microfluidics as point-of-care tools for platelet function test and antiplatelet drug screening.
Publisher: Springer Science and Business Media LLC
Date: 25-03-2021
DOI: 10.1038/S41598-021-86310-2
Abstract: Disturbed blood flow has been increasingly recognized for its critical role in platelet aggregation and thrombosis. Microfluidics with hump shaped contractions have been developed to mimic microvascular stenosis and recapitulate the prothrombotic effect of flow disturbance. However the physical determinants of microfluidic hemodynamics are not completely defined. Here, we report a refined computational fluid dynamics (CFD) simulation approach to map the shear rate ( γ ) and wall shear stress ( τ ) distribution in the stenotic region at high accuracy. Using ultra-fine meshing with sensitivity verification, our CFD results show that the stenosis level ( S ) is dominant over the bulk shear rate ( γ 0 ) and contraction angle ( α ) in determining γ and τ distribution at stenosis. In contrast, α plays a significant role in governing the shear rate gradient ( γ ′ ) distribution while it exhibits subtle effects on the peak γ . To investigate the viscosity effect, we employ a Generalized Power-Law model to simulate blood flow as a non-Newtonian fluid, showing negligible difference in the γ distribution when compared with Newtonian simulation with water medium. Together, our refined CFD method represents a comprehensive approach to examine microfluidic hemodynamics in three dimensions and guide microfabrication designs. Combining this with hematological experiments promises to advance understandings of the rheological effect in thrombosis and platelet mechanobiology.
Publisher: MyJove Corporation
Date: 04-08-2015
DOI: 10.3791/52975
Publisher: American Association for the Advancement of Science (AAAS)
Date: 02-02-2018
Abstract: We demonstrate mechanochemical regulation of platelet adhesion to von Willebrand factor in thrombosis and hemostasis.
Publisher: Wiley
Date: 08-01-2023
Abstract: The mechanical stimuli generated by body exercise can be transmitted from cortical bone into the deep bone marrow (mechanopropagation). Excitingly, a mechanosensitive perivascular stem cell niche is recently identified within the bone marrow for osteogenesis and lymphopoiesis. Although it is long known that they are maintained by exercise‐induced mechanical stimulation, the mechanopropagation from compact bone to deep bone marrow vasculature remains elusive of this fundamental mechanobiology field. No experimental system is available yet to directly understand such exercise‐induced mechanopropagation at the bone‐vessel interface. To this end, taking advantage of the revolutionary in vivo 3D deep bone imaging, an integrated computational biomechanics framework to quantitatively evaluate the mechanopropagation capabilities for bone marrow arterioles, arteries, and sinusoids is devised. As a highlight, the 3D geometries of blood vessels are smoothly reconstructed in the presence of vessel wall thickness and intravascular pulse pressure. By implementing the 5‐parameter Mooney–Rivlin model that simulates the hyperelastic vessel properties, finite element analysis to thoroughly investigate the mechanical effects of exercise‐induced intravascular vibratory stretching on bone marrow vasculature is performed. In addition, the blood pressure and cortical bone bending effects on vascular mechanoproperties are examined. For the first time, movement‐induced mechanopropagation from the hard cortical bone to the soft vasculature in the bone marrow is numerically simulated. It is concluded that arterioles and arteries are much more efficient in propagating mechanical force than sinusoids due to their stiffness. In the future, this in‐silico approach can be combined with other clinical imaging modalities for subject atient‐specific vascular reconstruction and biomechanical analysis, providing large‐scale phenotypic data for personalized mechanobiology discovery.
Publisher: Springer Science and Business Media LLC
Date: 03-2022
DOI: 10.1007/S00249-022-01595-Z
Abstract: Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca 2+ ) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca 2+ permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca 2+ influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (− 2.94 to 0 mmH 2 O) and a pneumatic high-speed pressure cl (− 25 to 0 mmHg). To visualize Ca 2+ influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca 2+ levels immediately after aspirating in idual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic cl showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic cl as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner.
Publisher: Elsevier BV
Date: 12-2017
Publisher: Wiley
Date: 28-07-2023
Abstract: Stenosis, characterized by partial vessel narrowing, alters blood hemodynamics and can lead to unpredictable thrombosis. Existing models struggle to accurately represent the complex vascular geometries and hemodynamics involved in such conditions. To address this challenge, a microvasculature‐on‐a‐post chip is developed to mimic partially stenotic vascular geometries and thrombogenicity, featuring isolated 3D micropost structures with variable sizes that recreate disturbed flow profiles. To emulate the diseased vessel wall, the post microfluidics are vascularized with a confluent layer of endothelial cells. Subsequently, human blood is perfused through the endothelialized post microfluidics, observing the temporal and spatial thrombotic response governed by Virchow's triad, including vessel wall injury, hemodynamic disturbance, and hypercoagulability. The innovative model offers valuable insights into stenosis‐induced thrombosis and endothelial behavior, paving the way for improved assessment of thrombotic risks associated with stenotic vessels. This advanced microfluidic platform also offers new approaches for evaluation of prothrombotic phenotypes and cardiovascular risk assessment in the future.
Publisher: American Chemical Society (ACS)
Date: 25-03-2020
Publisher: Springer Science and Business Media LLC
Date: 25-03-2019
Publisher: Elsevier BV
Date: 11-2013
Publisher: Springer Science and Business Media LLC
Date: 06-09-2021
DOI: 10.1038/S42003-021-02528-W
Abstract: Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like most plasma membrane ion channel proteins they must pass through biosynthetic quality control in the endoplasmic reticulum that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the ‘cap’ region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for N-glycosylation in the ‘propeller’ regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated Piezo1 protein. Thus the N-linked glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.
Publisher: Elsevier BV
Date: 07-2023
Publisher: The Company of Biologists
Date: 28-04-2010
DOI: 10.1242/DMM.003863
Abstract: Innate regulatory networks within organs maintain tissue homeostasis and facilitate rapid responses to damage. We identified a novel pathway regulating vessel stability in tissues that involves matrix metalloproteinase 14 (MMP14) and transforming growth factor beta 1 (TGFβ1). Whereas plasma proteins rapidly extravasate out of vasculature in wild-type mice following acute damage, short-term treatment of mice in vivo with a broad-spectrum metalloproteinase inhibitor, neutralizing antibodies to TGFβ1, or an activin-like kinase 5 (ALK5) inhibitor significantly enhanced vessel leakage. By contrast, in a mouse model of age-related dermal fibrosis, where MMP14 activity and TGFβ bioavailability are chronically elevated, or in mice that ectopically express TGFβ in the epidermis, cutaneous vessels are resistant to acute leakage. Characteristic responses to tissue damage are reinstated if the fibrotic mice are pretreated with metalloproteinase inhibitors or TGFβ signaling antagonists. Neoplastic tissues, however, are in a constant state of tissue damage and exhibit altered hemodynamics owing to hyperleaky angiogenic vasculature. In two distinct transgenic mouse tumor models, inhibition of ALK5 further enhanced vascular leakage into the interstitium and facilitated increased delivery of high molecular weight compounds into premalignant tissue and tumors. Taken together, these data define a central pathway involving MMP14 and TGFβ that mediates vessel stability and vascular response to tissue injury. Antagonists of this pathway could be therapeutically exploited to improve the delivery of therapeutics or molecular contrast agents into tissues where chronic damage or neoplastic disease limits their efficient delivery.
Publisher: Research Square Platform LLC
Date: 15-09-2021
DOI: 10.21203/RS.3.PEX-1411/V1
Abstract: Thrombosis is both attributed to biochemical agonists and mechanical stresses applied to platelets. Whilst the effect of biochemical agonists has been extensively studied, the mechanosensitive factors remain poorly defined. Stenotic microfluidic channels mimic the narrowing vessels, providing the real-time observation of platelets under disturbed flow. Though the experimental analysis of platelets in disturbed flow confirms the mechanosensitive behavior of platelets, it cannot explicate detailed thresholds for platelet activation. Computational Fluid Dynamics (CFD) could be utilized alongside experimental analysis to characterize thresholds for platelet behavior under imposed shear stress. CFD simulations, however, are prone to uncertainties and errors which should be minimized to obtain compelling results. Hereby, we have presented a CFD protocol for researchers in the field of microfluidic and hemodynamic studies.
Publisher: Springer Science and Business Media LLC
Date: 14-03-2018
DOI: 10.1038/S41467-018-03430-6
Abstract: Diabetes is associated with an exaggerated platelet thrombotic response at sites of vascular injury. Biomechanical forces regulate platelet activation, although the impact of diabetes on this process remains ill-defined. Using a biomembrane force probe (BFP), we demonstrate that compressive force activates integrin α IIb β 3 on discoid diabetic platelets, increasing its association rate with immobilized fibrinogen. This compressive force-induced integrin activation is calcium and PI 3-kinase dependent, resulting in enhanced integrin affinity maturation and exaggerated shear-dependent platelet adhesion. Analysis of discoid platelet aggregation in the mesenteric circulation of mice confirmed that diabetes leads to a marked enhancement in the formation and stability of discoid platelet aggregates, via a mechanism that is not inhibited by therapeutic doses of aspirin and clopidogrel, but is eliminated by PI 3-kinase inhibition. These studies demonstrate the existence of a compression force sensing mechanism linked to α IIb β 3 adhesive function that leads to a distinct prothrombotic phenotype in diabetes.
Publisher: Springer Science and Business Media LLC
Date: 12-09-2014
DOI: 10.1038/NCOMMS5886
Abstract: Cancer cell adhesion to the vascular endothelium is a critical step of tumour metastasis. Endothelial surface molecule Thy-1 (CD90) is implicated in the metastatic process through its interactions with integrins and syndecans. However, how Thy-1 supports cell-cell adhesion in a dynamic mechanical environment is not known. Here we show that Thy-1 supports β1 integrin- and syndecan-4 (Syn4)-mediated contractility-dependent mechanosignalling of melanoma cells. At the single-molecule level, Thy-1 is capable of independently binding α5β1 integrin and syndecan-4 (Syn4) receptors. However, in the presence of both α5β1 and Syn4, the two receptors bind cooperatively to Thy-1, to form a trimolecular complex. This trimolecular complex displays a unique phenomenon we coin 'dynamic catch', characterized by abrupt bond stiffening followed by the formation of catch bonds, where force prolongs the bond lifetime. Thus, we reveal a new class of trimolecular interactions where force strengthens the synergistic binding of two co-receptors and modulates downstream mechanosignalling.
Publisher: Mary Ann Liebert Inc
Date: 11-2022
Publisher: Cold Spring Harbor Laboratory
Date: 12-2020
DOI: 10.1101/2020.11.30.404962
Abstract: Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like all membrane proteins, they pass through biosynthetic quality control in the endoplasmic reticulum and Golgi that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the ‘cap’ region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for higher-order glycosylation in the ‘propeller’ regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated protein. The higher order glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.
Publisher: Springer Science and Business Media LLC
Date: 16-02-2022
DOI: 10.1007/S00249-021-01587-5
Abstract: Mechanobiology is an emerging field at the interface of biology and mechanics, investigating the roles of mechanical forces within biomolecules, organelles, cells, and tissues. As a highlight, the recent advances of micropipette-based aspiration assays and dynamic force spectroscopies such as biomembrane force probe (BFP) provide unprecedented mechanobiological insights with excellent live-cell compatibility. In their classic applications, these assays measure force-dependent ligand–receptor-binding kinetics, protein conformational changes, and cellular mechanical properties such as cortical tension and stiffness. In recent years, when combined with advanced microscopies in high spatial and temporal resolutions, these biomechanical nanotools enable characterization of receptor-mediated cell mechanosensing and subsequent organelle behaviors at single-cellular and molecular level. In this review, we summarize the latest developments of these assays for live-cell mechanobiology studies. We also provide perspectives on their future upgrades with multimodal integration and high-throughput capability.
Publisher: Elsevier BV
Date: 07-2019
DOI: 10.1002/RTH2.12225
Publisher: Wiley
Date: 23-12-2020
DOI: 10.1111/IMCB.12304
Publisher: Springer Science and Business Media LLC
Date: 25-03-2008
Abstract: Accurate annotation of translation initiation sites (TISs) is essential for understanding the translation initiation mechanism. However, the reliability of TIS annotation in widely used databases such as RefSeq is uncertain due to the lack of experimental benchmarks. Based on a homogeneity assumption that gene translation-related signals are uniformly distributed across a genome, we have established a computational method for a large-scale quantitative assessment of the reliability of TIS annotations for any prokaryotic genome. The method consists of modeling a positional weight matrix (PWM) of aligned sequences around predicted TISs in terms of a linear combination of three elementary PWMs, one for true TIS and the two others for false TISs. The three elementary PWMs are obtained using a reference set with highly reliable TIS predictions. A generalized least square estimator determines the weighting of the true TIS in the observed PWM, from which the accuracy of the prediction is derived. The validity of the method and the extent of the limitation of the assumptions are explicitly addressed by testing on experimentally verified TISs with variable accuracy of the reference sets. The method is applied to estimate the accuracy of TIS annotations that are provided on public databases such as RefSeq and ProTISA and by programs such as EasyGene, GeneMarkS, Glimmer 3 and TiCo. It is shown that RefSeq's TIS prediction is significantly less accurate than two recent predictors, Tico and ProTISA. With convincing proofs, we show two general preferential biases in the RefSeq annotation, i.e . over-annotating the longest open reading frame (LORF) and under-annotating ATG start codon. Finally, we have established a new TIS database, SupTISA, based on the best prediction of all the predictors SupTISA has achieved an average accuracy of 92% over all 532 complete genomes. Large-scale computational evaluation of TIS annotation has been achieved. A new TIS database much better than RefSeq has been constructed, and it provides a valuable resource for further TIS studies.
Publisher: Springer Science and Business Media LLC
Date: 18-08-2018
Publisher: Elsevier BV
Date: 11-2015
Location: Australia
Start Date: Start date not available
End Date: End date not available
Funder: National Heart Foundation of Australia
View Funded ActivityStart Date: 2016
End Date: 2016
Funder: Diabetes Australia
View Funded ActivityStart Date: 2019
End Date: 2019
Funder: Australian Research Council
View Funded ActivityStart Date: 2019
End Date: 2021
Funder: Australian Research Council
View Funded ActivityStart Date: 2017
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2017
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 12-2020
End Date: 12-2023
Amount: $469,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 07-2019
End Date: 06-2022
Amount: $368,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2019
End Date: 12-2019
Amount: $1,480,000.00
Funder: Australian Research Council
View Funded Activity