菅原 路子

Epithelial-mesenchymal transition (EMT), a qualitative change in cell migration behavior during cancer invasion and metastasis, is becoming a new target for anticancer drugs. Therefore, it is crucial to develop in vitro assays for the evaluation of the abilities of drug candidates to control EMT progression. We herein report on a method for the quantification of the EMT based on particle image velocimetry and correlation functions. The exponential fitting of the correlation curve gives an index (lambda), which represents transforming growth factor (TGF)-beta 1-induced EMT progression and its suppression by inhibitors. Moreover, real-time monitoring of the lambda value illustrates a time-dependent EMT progressing process, which occurs earlier than the bio-chemical changes in an EMT marker protein expression. The results demonstrate the usefulness of the present method for kinetic studies of EMT progression as well as EMT inhibitor screening.

A particle-fluid flow under alternating current (ac) electrokinetics was numerically simulated to investigate the three-dimensional (3-D) particle motion in a complex electric field of a high conductivity medium generated by an electrode-multilayered microfluidic device. The simulation model coupling thermal-fluid-electrical and dispersed particle problems incorporates three ac electrokinetics (ACEK) phenomena, namely, the ac electrothermal effect (ACET), thermal buoyancy (TB), and dielectrophoresis (DEP). The electrode-multilayered microfluidic device was fabricated with 40 electrodes exposed at the flow channel sidewalls in five cross sections. The governing equations of the simulation model are solved by the Eulerian-Lagrangian method with finite volume discretization. Fluid flow simulations in three cases with or without consideration of ACET and TB are performed to clarify the contributions of these phenomena. The fluid flow is found to be composed of short-range vortices due to ACET and long-range circulation due to TB based on the features of the electrode-multilayered microfluidic device. The 3-D particle trajectory influenced by the fluid flow is compared with four values of the real part of the Clausius-Mossotti (CM) factor to evaluate the DEP phenomenon. The simulation model is validated by experiments using a cell suspension. The pattern of cell trajectories in the upper part of the flow channel measured by particle tracking velocimetry agrees with the simulated pattern. By comparison of the simulation and experiment, it is found that the cells moving straight away from the electrode on the focal plane are decelerated within the region of 60 mu m from the electrode by positive-DEP with Re[K(omega)] = 0.08-0.11. Furthermore, the 3-D DEP-effective region and the ACET and TB dominant regions for the cells are predicted by evaluating the particle-fluid relative velocity due to DEP force with Re[K(omega)] = 0.10. Consequently, the flow mechanism and dominant region of each ACEK phenomenon in the device are clarified from the 3-D simulation validated by the experiments.

The distinct motion of GFP-tagged histone expressing cells (Histone-GFP type cells) has been investigated under ac electrokinetics in an electrode-multilayered microfluidic device as compared with Wild type cells and GFP type cells in terms of different intracellular components. The Histone-GFP type cells were modified by the transfection of green fluorescent protein-fused histone from the human lung fibroblast cell line. The velocity of the Histone-GFP type cells obtained by particle tracking velocimetry technique is faster thanWild type cells by 24.9% andGFP type cells by 57.1%. This phenomenon is caused by the more amount of proteins in the intracellular of single Histone-GFP type cell than that of theWild type and GFP type cells. The more amount of proteins in the Histone-GFP type cells corresponds to a lower electric permittivity ec of the cells, which generates a lower dielectrophoretic force exerting on the cells. The velocity of Histone-GFP type cells is well agreed with Eulerian-Lagrangian two-phase flow simulation by 4.2% mean error, which proves that the fluid motion driven by thermal buoyancy and electrothermal force dominates the direction of cells motion, while the distinct motion of Histone-GFP type cells is caused by dielectrophoretic force. The fluidmotion does not generate a distinct dragmotion for Histone-GFP type cells because the Histone-GFP type cells have the same size to theWild type and GFP type cells. These results clarified the mechanism of cells motion in terms of intracellular components, which helps to improve the cell manipulation efficiency with electrokinetics.

Persistent directional cell migration is involved in animal development and diseases. The small GTPase Rac1 is involved in F-actin and focal adhesion dynamics. Local Rac1 activity is required for persistent directional migration, whereas global, hyperactivated Rac1 enhances random cell migration. Therefore, precise control of Rac1 activity is important for proper directional cell migration. However, the molecular mechanism underlying the regulation of Rac1 activity in persistent directional cell migration is not fully understood. Here, we show that the ubiquitin ligase mind bomb 1 (Mib1) is involved in persistent directional cell migration. We found that knockdown of MIB1 led to an increase in random cell migration in HeLa cells in a wound-closure assay. Furthermore, we explored novel Mib1 substrates for cell migration and found that Mib1 ubiquitinates Ctnnd1. Mib1-mediated ubiq-uitination of Ctnnd1 K547 attenuated Rac1 activation in cultured cells. In addition, we found that posterior lateral line primordium cells in the zebrafish mib1(ta52b) mutant showed increased random migration and loss of directional F-actin-based protrusion formation. Knock down of Ctnnd1 partially rescued posterior lateral line primordium cell migration defects in the mib1(ta52b) mutant. Taken together, our data suggest that Mib1 plays an important role in cell migration and that persistent directional cell migration is regulated, at least in part, by the Mib1-Ctnnd1-Rac1 pathway.

A method was developed for photocontrolling cell adhesion on a gel substrate with defined mechanical properties. Precise patterning of geometrically controlled cell clusters and their migration induction became possible by spatiotemporally controlled photo-irradiation of the substrate. The clusters exhibited unique collective motion that depended on substrate stiffness and cluster geometry.

To understand the mechanism regulating the spontaneous change in polarity that leads to cell turning, we quantitatively analyzed the dynamics of focal adhesions (FAs) coupling with the self-assembling actin cytoskeletal structure in Swiss 3T3 fibroblasts. Fluorescent imageswere acquired fromcells expressing GFP-actin and RFP-zyxin by laser confocal microscopy. On the basis of the maximum area, duration, and relocation distance of FAs extracted from the RFP-zyxin images, the cells could be divided into 3 regions: the front region, intermediate lateral region, and rear region. In the intermediate lateral region, FAs appeared close to the leading edge and were stabilized gradually as its area increased. Simultaneously, bundled actin stress fibers (SFs) were observed vertically from the positions of these FAs, and they connected to the other SFs parallel to the leading edge. Finally, these connecting SFs fused to forma single SF with matured FAs at both ends. This change in SF organization with cell retraction in the first cycle of migration followed by a newly formed protrusion in the next cycle is assumed to lead to cell turning in migrating Swiss 3T3 fibroblasts.

BACKGROUND: Many numerical studies have been published with respect to about flow structures around cerebral aneurysm assuming to be rigid. Furthermore, there is little experimental research concerning aneurysm with elastic wall. Wall shear stress in elastic wall comparing with rigid wall should be clarified in experimental approach and verified in CFD.OBJECTIVE: We have experimentally realized elastic aneurysm model accompanying with wall deformation. Wall shear stress was examined for both rigid and elastic aneurysm models in pulsatile flow.METHODS: Effect of elasticity on wall shear stress inside aneurysm induced at the apex of anterior cerebral artery was experimentally examined by particle image velocimetry in vitro. In order to adjust the wall deformation, the pressure adjustment chamber was specially equipped outside the aneurysm wall.RESULTS: Effect of elasticity on wall shear stress was noticed on the comparison with that of rigidity. Wall elasticity reduced the peak magnitude, the spatial and temporal averaged wall shear stress comparing with those of wall rigidity experimentally. These reductions were endorsed by fluid-structure interaction simulation.CONCLUSION: Elastic wall comparing with rigid wall would reduce the peak magnitude, the spatial and temporal averaged wall shear stress acting on vascular wall.

Three electrical elements (i.e., resistance, capacitance, and relaxation frequency) of electrical double layer (EDL) formed around particles have been extracted by a measuring-fitting combination for a novel noninvasive online measurement technique of particle size and concentration in a liquid-particle mixture. The measuring-fitting combination means measuring the impedances with electrical-impedance spectroscopy (EIS) method, and fitting the equivalent circuit with Levenberg-Marquardt method. The liquid-particle mixture in the impedance measurement is made of sodium chloride solution and stainless particles; the equivalent circuit is established corresponding to the contents in the liquid-particle mixture. As a result, the influence of the particle size and concentration on the electrical elements in the EDL which are the resistance, capacitance, and relaxation frequency in the EDL are clarified and discussed. This method is useful for determination of the particle size and concentration in liquid-particle mixture.

Visualization of a thrombus is very important in the development of various artificial organs and extracorporeal circulation devices. This paper presents an application of electrical resistance tomography (ERT) technique for the visualization of a thrombus in blood. Experiments were conducted in static and flowing bovine and swine blood samples. Artificially created thrombi were mixed in the blood samples for visualization. Eight-electrode tomography sensor was used for the measurement. Cross-sectional resistivity distribution was reconstructed using linear back projection algorithm. A thrombus was characterized by increased local resistivity. We successfully reconstructed the time, size and cross-sectional location of a thrombus, and reached a conclusion that the concentration and orientation of the RBCs in a thrombus contributed to the increase in the resistivity. The increment was relatively higher in the static blood than in flowing blood. These findings can be helpful in the development of an instrumentation system for the real-time monitoring of blood to visualize a thrombus. Developers of left ventricular assistance devices, heart-lung machines, hemodialyzer etc., and the end-users (i.e. patients) can greatly benefit from such a system. (C) 2015 Elsevier Ltd. All rights reserved.

The classical Fontan route, namely the atriopulmonary connection (APC), continues to be associated with a risk of thrombus formation in the atrium. A conversion to a total cavopulmonary connection (TCPC) from the APC can ameliorate hemodynamics for the failed Fontan; however, the impact of these surgical operations on thrombus formation remains elusive. This study elucidates the underlying mechanism of thrombus formation in the Fontan route by using a two-dimensional computer hemodynamic simulation based on a simple blood coagulation rule. Hemodynamics in the Fontan route was simulated with Navier-Stokes equations. The blood coagulation and the hemodynamics were combined using a particle method. Three models were created: APC with a square atrium, APC with a round atrium, and TCPC. To examine the effects of the venous blood flow velocity, the velocity at rest and during exercise (0.5 and 1.0 W/kg) was measured. The total area of the thrombi increased over time. The APC square model showed the highest incidence for thrombus formation, followed by the APC round, whereas no thrombus was formed in the TCPC model. Slower blood flow at rest was associated with a higher incidence of thrombus formation. The TCPC was superior to the classical APC in terms of preventing thrombus formation, due to significant blood flow stagnation in the atrium of the APC. Thus, local hemodynamic behavior associated with the complex channel geometry plays a major role in thrombus formation in the Fontan route.

The human cardiovascular system is a closed-loop and complex vascular network with multi-scaled heterogeneous hemodynamic phenomena. Here, we give a selective review of recent progress in macro-hemodynamic modeling, with a focus on geometrical multi-scale modeling of the vascular network, micro-hemodynamic modeling of microcirculation, as well as blood cellular, subcellular, endothelial biomechanics, and their interaction with arterial vessel mechanics. We describe in detail the methodology of hemodynamic modeling and its potential applications in cardiovascular research and clinical practice. In addition, we present major topics for future study: recent progress of patient-specific hemodynamic modeling in clinical applications, micro-hemodynamic modeling in capillaries and blood cells, and the importance and potential of the multi-scale hemodynamic modeling.Here we give a selective review of recent progress in macro-hemodynamic modeling, with a focus on geometrical multi-scale modeling of the vascular network, micro-hemodynamic modeling of microcirculation, as well as blood cellular, subcellular, endothelial biomechanics, and their interaction with arterial vessel mechanics. We describe in detail the methodology of hemodynamic modeling and its potential applications in cardiovascular research and clinical practice. In addition, we present major topics for future study: recent progress of patient-specific hemodynamic modeling in clinical applications, micro-hemodynamic modeling in capillaries and blood cells, and the importance and potential of the multi-scale hemodynamic modeling.

The spatial concentration distribution of cells in a microchannel is measured by combining the dielectric properties of cells with the specific structure of the electrode-multilayered microchannel. The dielectric properties of cells obtained with the impedance spectroscopy method includes the cell permittivity and dielectric relaxation, which corresponds to the cell concentration and structure. The electrode-multilayered microchannel is constructed by 5 cross-sections, and each cross-section contains 5 electrode-layers embedded with 16 micro electrodes. In the experiment, the dielectric properties of cell suspensions with different volume concentrations are measured with different electrode-combinations corresponding to different electric field distributions. The dielectric relaxations of different cell concentrations are compared and discussed with the Maxwell-Wagner dispersion theory, and the relaxation frequencies are analysed by a cell polarization model established based on the Hanai cell model. Moreover, a significant linear relationship with AC frequency dependency between relative permittivity and cell concentration was found, which provides a promising way to on-line estimate cell concentration in microchannel. Finally, cell distribution in 1 cross-section of the microchannel (X and Y directions) was measured with different electrode-combinations using the dielectric properties of cell suspensions, and cell concentration distribution along the microchannel (Z direction) was visualized at flowing state. The present cell spatial sensing study provides a new approach for 3 dimensional non-invasive online cell sensing for biological industry. (C) 2015 AIP Publishing LLC.

A computational study of effects of vessel dynamics and compliance on coronary artery hemodynamics with/without stenosis is presented. The coronary artery hemodynamics with stenosis has been a main subject as one of the major cardiovascular diseases induced by atherosclerosis most computational models assume that the vessel movement and deformation are negligible (Zeng, et al., 2003 Kim, et al., 2010). However, it is still unclear whether the hemodynamic characteristics owning to vessel dynamics and compliance are clinically significant or not particularly under pathological conditions. In this study, we aim at investigating the hemodynamic effects of the vessel dynamics and compliance in right coronary artery under healthy situation without stenosis as well as under diseased conditions with stenosis. We constructed a three-dimensional geometric model of the right coronary artery based on X-ray angiographic images, in which both vessel movement and deformation were taken into account. A specific volumetric flow rate was employed as a boundary condition imposed on inlet. Furthermore, we carried out an extensive study on the inlet waveform dependence and the effects of the vessel compliance on coronary hemodynamics. Our results demonstrate that the conventional assumption on 'rigid' artery models holds only in the cases of normal coronary arteries but fails for stenosed coronary arteries where the vessel dynamics and compliance do extend significant influence on distributions of the oscillatory shear indices (OSIs). Moreover, we find that the effects of vessel dynamics and compliance on coronary hemodynamics seem to be independent of both inlet boundary conditions and the vessel compliance.

Actin, a cytoskeletal protein, gathers and polymerizes under the cell membrane to mediate protrusion of the cell membrane. Therefore, actin participates greatly in cell movement. To quantitatively evaluate actin's dynamics during cell protrusion, movements of actin-labeled cells were observed under a confocal laser scanning microscopy, and time-lapse images were obtained. Image analysis of the orientation of actin stress fibers revealed dynamic formation of a meshwork of stress fibers in the protrusion structure at the leading edge of the motile cell. In addition, a number of immobile spots such as focal adhesions were observed adjacent to the dynamic meshwork of stress fibers. The distribution and the time between appearance and disappearance of each spot were analyzed. The results suggest that immobile spots close to the centroid of the cell play a crucial role in providing anchorage and driving cell movement. © 2014 The Institute of Electrical Engineers of Japan.

Experiments have been carried out to obtain the effective diffusivity of carbon dioxide for axial transport in the oscillatory flow through a straight pipe with circular cross-section for the case of a transition from laminar flow to turbulent flow. An experimental apparatus and procedures were devised to coincide with the theoretical situation. Results have been compared with the theoretical predictions by Watson and found to be in excellent agreement with both the theoretical and the experimental values in the case of laminar flow. The present results have less dispersion than those by the other researchers. In the case of turbulent flow, the results were larger than the theoretical values for the case of laminar flow because of the transition to turbulence. Using dynamically similar water, the axial velocity variations of the oscillatory flow were measured by the LDV system for several conditions. The turbulent intensity of the axial velocity and the effective diffusivity have been found to have a strong interrelation between each other. © 2012 The Japan Society of Mechanical Engineers.

We intend to develop a new stimulation electrode that contains tubular membrane proteins. A "proteoelectrode" should be capable of controlling membrane potential through the pores formed by tubular membrane proteins. We have tested the formation of lipid bilayers and the insertion of alpha-hemolysin into them. An ion indicator method for measuring molecule transfer through alpha-hemolysin in the lipid bilayer was tested. Fluorescence imaging of the ion indicator showed the assembly of a lipid bilayer and the formation of alpha-hemolysin pores. This could be a simple and effective technique for evaluating the performance of a proteoelectrode. © 2011 IEEE.

The high sensitivity of human hearing is believed to be achieved by cochlear amplification. The basis of this amplification is thought to be the motility of mammalian outer hair cells (OHCs), i.e., OHCs elongate and contract in response to acoustical stimulation. This motility is made possible by both the cytoskeleton beneath the OHC plasma membrane and the motor protein prestin distributed throughout the plasma membrane. However, these factors have not yet been fully clarified. In the present study, therefore, attempts were made to observe the ultrastructure of the cytoskeleton of guinea pig OHCs and to identify the motor protein prestin expressed in the plasma membrane of Chinese hamster ovary (CHO) cells by atomic force microscopy (AFM). Results indicate that the OHC cytoskeleton is comprised of circumferential actin filaments and spectrin cross-links and that particle-like structures with a diameter of 8-12 nm which exist in the plasma membrane of the prestin-expressing CHO cells are most likely be prestin.

Recently, an atomic force microscope (AFM) has been used to investigate the cytoskeleton of the outer hair cell (OHC) under physiological conditions. However, details of the ultrastructure of the cytoskeleton have not been clarified. In this study, the ultrastructure of the cytoskeleton of fixed OHCs was investigated using the oscillation imaging mode of the AFM (Tapping Mode (TM), Digital Instruments, Santa Barbara, CA, USA). Only circumferential filaments were observed when OHCs were fixed with 2.5% glutaraldehyde. By contrast, when they were fixed with 2.5% glutaraldehyde and extracted with Triton X-100, which peels off the outermost plasma membrane, the cortical cytoskeleton, which is formed by discrete oriented domains, was imaged and circumferential filaments and cross-links were observed within a domain.

Recent experiments have shown that the isolated outer hair cell (OHC) can elongate and contract in response to an electrical stimulation. In vivo, the OHC receptor potential in response to acoustic stimulation would drive OHC length changes, and it is estimated that the OHC motility exerts force on the basilar membrane. This OHC motility is suggested to be due to the deformation of the motor protein which distributes along the OHC lateral wall, and the mechanical properties of the OHC lateral wall and the distribution of the motor protein might affect the motility and force production of the OHC. In this stufy, in order to make the effects mentioned above clear, the OHC is modeled as a shell membrane, in which the conformational change of the motor protein is considered, and numerical calculations are carried out.