Adhesive properties of hepatoma cells to collagen Ⅳ coated surfaces

Objectives: To quantitatively study the adhesive pro- perties of hepatoma cells to collagen Ⅳ coated artifi- cial basement membrane and to investigate the rele- vance of cell adhesive forces to the concentration of collagen Ⅳ. Methods: Synchronous G1 and S phase cells were a- chieved using thymine-2-desoxyriboside and cochicine sequential blockage method and double thymine-2- desoxyriboside blockage method respectively. The adhesive forces of hepatoma cells were investigated by micropipette aspiration technique. Results: The adhesive forces of hepatoma cells to ar- tificial basement membrane were (107.78±65.44) ×10^(-10)N, (182.60±107.88)×10^(-10)N, (298.91± 144.13)×10^(-10)N when the concentration of the membrane coated by 1, 2, 5μg/ml collagen Ⅳ re- spectively (P<0.001). The adhesive forces of G1 and S phases hepatoma cells to artificial basement membrane were (275.86±232.80)×10^(-10)N and (161.16±120.40)×10^(-10)N respectively when the concentration of the membrane coated by 5μg/ml collagen Ⅳ (P<0.001). Conclusions: The adhesive forces of hepatoma cells to artifical basement membrane in direct proportion to the concentration of collagen Ⅳ suggests that the in- crease of basement membrane might be conducive to the chemotactic motion and adhesiveness of tumor cells. G1 phase cells are more capable of adhering to basement membrane than S phase cells. Hepatoma cells, especially G1 phase cells, may survive in blood circulation, and sequest and adhere in microcircula- tion, and get through basement membrane for re- mote metastasis.

Mechanokinetics of receptor-ligand interactions in cell adhesion

Receptor-ligand interactions in blood flow are crucial to initiate such biological processes as inflammatory cascade,platelet thrombosis,as well as tumor metastasis.To mediate cell adhesion,the interacting receptors and ligands must be anchored onto two apposing surfaces of two cells or a cell and a substratum,i.e.,two-dimensional（2D）binding,which is different from the binding of a soluble ligand in fluid phase to a receptor,i.e.,three-dimensional（3D） binding.While numerous works have been focused on3 D kinetics of receptor-ligand interactions in the immune system,2D kinetics and its regulations have been less understood,since no theoretical framework or experimental assays were established until 1993.Not only does the molecular structure dominate 2D binding kinetics,but the shear force in blood flow also regulates cell adhesion mediated by interacting receptors and ligands.Here,we provide an overview of current progress in 2D binding and regulations,mainly from our group.Relevant issues of theoretical frameworks,experimental measurements,kinetic rates and binding affinities,and force regulations are discussed.

Flow dynamics analyses of pathophysiological liver lobules using porous media theory

Blood flow inside the liver plays a key role in hepatic functions, and abnormal hemodynamics are highly correlated with liver diseases. To date, the flow field in an elementary building block of the organ, the liver lobule,is difficult to determine experimentally in humans due to its complicated structure, with radially branched microvasculature and the technical difficulties that derive from its geometric constraints. Here we established a set of 3D computational models for a liver lobule using porous media theory and analyzed its flow dynamics in normal, fibrotic,and cirrhotic lobules. Our simulations indicated that those approximations of ordinary flow in portal tracts（PTs） and the central vein, and of porous media flow in the sinusoidal network, were reasonable only for normal or fibrotic lobules.Models modified with high resistance in PTs and collateral vessels inside sinusoids were able to describe the flow features in cirrhotic lobules. Pressures, average velocities, and volume flow rates were profiled and the predictions compared well with experimental data. This study furthered our understanding of the flow dynamics features of liver lobules and the differences among normal, fibrotic, and cirrhotic lobules.

A Novel Human Antibody,HF,against HER2/erb-B2 Obtained by a Computer-Aided Antibody Design Method

Fully human antibodies have minimal immunogenicity and safety profiles.At present,most potential antibody drugs in clinical trials are humanized or fully human.Human antibodies are mostly generated using the phage display method(in vitro)or by transgenic mice(in vivo);other methods include B lymphocyte immortalization,human–human hybridoma,and single-cell polymerase chain reaction.Here,we describe a structure-based computer-aided de novo design technology for human antibody generation.Based on the complex structure of human epidermal growth factor receptor 2(HER2)/Herceptin,we first designed six short peptides targeting the potential epitope of HER2 recognized by Herceptin.Next,these peptides were set as complementarity determining regions in a suitable immunoglobulin frame,giving birth to a novel anti-HER2 antibody named "HF,"which possessed higher affinity and more effective anti-tumor activity than Herceptin.Our work offers a useful tool for the quick design and selection of novel human antibodies for basic mechanical research as well as for imaging and clinical applications in immune-related diseases,such as cancer and infectious diseases.

Combined modeling of cell aggregation and adhesion mediated by receptor–ligand interactions under shear flow

Blood cell aggregation and adhesion to endothelial cells under shear flow are crucial to many biological processes such as thrombi formation, inflammatory cascade, and tumor metastasis, in which these cellular interactions are mainly mediated by the underlying receptor-ligand bindings. While theoretical modeling of aggregation dynamics and adhesion kinetics of interacting cells have been well studied separately, how to couple these two processes remains unclear. Here we develop a combined model that couples cellular aggregation dynamics and adhesion kinetics under shear flow. The impacts of shear rate （or shear stress） and molecular binding affinity were elucidated. This study provides a unified model where the action of a fluid flow drives cell aggregation and adhesion under the modulations of the mechanical shear flow and receptor-ligand interaction kinetics. It offers an insight into understanding the relevant biological processes and functions.

Mechanical Regulation of Calcium Response and Trail Formation of Neutrophils on Endothelium

Atherosclerosis or fibrosis and cirrhosis undergo chronic inflammation associated with the adhesion between neutrophils and endothelial cells(ECs)that is mediated by their respective cellular adhesive molecules on stiffened blood vessel wall or extracellular matrix(ECM)under shear flow[1-3].However,the mechanical dependence of calcium flux and trail formation in neutrophils remains unclear yet in these processes.First,the effect of substrate stiffness through ECs on neutrophil calcium spike was quantified when the individual neutrophils adhered to EC monolayer pre-placed onto stiffness-varied polyacrylamide(PA)substrate(5 or 34.88 kPa)or glass surface.Our data indicated that E-/P-selectins and ICAM-1s on HUVECs and b2-integrins,PSGL-1s,and CD44s on neutrophils were all involved in mediating neutrophil calcium spike in a stiffness-dependent manner,in which the increase of substrate stiffness enhanced the calcium intensity and spike number.Such stiffness-dependent calcium response is associated with selectin-induced b2-integrin activation through Syk/Src signaling pathway and the F-actin/myosin II function.Moreover,tension-activated calcium ion channels displayed critical roles in initiating stiffness-dependent calcium spike [4].Second,the trail formation of neutrophils to ECs monolayer pre-placed onto the same PA substrate were also tested under shear flow.Live fluorescence imaging showed that neutrophils are able to form long membrane tethers during migration and subsequently leave behind membranous long-lasting trails under shear,which are enriched in LFA-1,Mac-1,and CD44.Moreover,the formation of the trails was inhibited by blocking LFA-1s and Mac-1s,suggesting an important role forβ2-integrins in the trial formation.The recruitment of monocytes was inhibited when pre-blocking ICAM-1s on flowing monocytes,indicating that the neutrophil’s trails employβ2-integrin-ICAM-1 binding to recruit the monocytes.Intriguingly,both the length and the area of the trails increase with increasing substrate stiffness,resulting in the enhanced monocyte recruitment.Inhibition of actin binding protein Arp2/3 impairs the trail formation and dramatically decreases the neutrophil-dependent monocyte recruitment.These data provide an insight into understanding how stiffening of vascular wall could regulate the calcium flux of adhered neutrophils and thus the immune responses in atherosclerosis.They also imply that local mechanical microenvironment is remodeled with the migration of neutrophils,leaving the trails presented to induce and regulate monocyte recruitment.All the results are meaningful in elucidating the occurrence and development of atherosclerosis or fibrosis from the viewpoint of mechanotransduction and also for the potential intervention of cardiovascular disease progress.

Spring constant regulate selectin-ligand bond dissociation

Forced dissociation of selectin-ligand complex is crucial to such biological processes as leukocyte recruitment,thrombosis formation,as well as tumor metastasis[1].Although several assays and techniques,e.g.,dynamic force spectroscopy（DFS）,have been applied to probe the complex at single-bond level,the discrepancies in the loading rate dependence of bond rupture force were found in the assays,presumably due to the different pathways in energy landscape and binding kinetics of molecular complexes[2].However,the underlying mechanisms remain unclear.Here an optical trap（OT）assay was used to quantify the bond rupture at rf≤20 pN/s

Mechanical Strength and Structural Basis ofβ2 Integrin to Mediate Neutrophil Accumulation on Liver Sinusoidal Endothelial Cells:A Study Using Atomic Force Microscopy and Molecular Dynamics Simulations

Neutrophil(PMN)accumulation on liver sinusoidal endothelial cells(LSECs)is crucial to pathogen clearance and tissue damage in the liver sinusoids and controlled by a series of adhesion molecules expressed on the surface of PMNs and LSECs.The role of lymphocyte function-associated antigen-1(LFA-1)and macrophage-1 antigen(Mac-1)in this process is still contentious.Here we compared the dynamic force spectra of the binding ofβ2 integrin to intercellular adhesion molecule-1(ICAM-1)on LSECs using atomic force microscopy(AFM)and performed free and steered molecular dynamics(MD)simulations to analyze their structural bases of LFA-1-or Mac-1-I-domain and ICAM-1-D1 or D3 pair in their force spectra.Our AFM data suggest that the mechanical strength of LFA-1-ICAM-1 bond is significantly stronger than that of Mac-1-ICAM-1 bond,implying a dominate role for LFA-1 to mediate PMN adhesion under shear flow.MD simulations indicated that spontaneous dissociation of Mac-1-I-domain vs.ICAMD3-domain is slower with the stronger interaction energy than that for LFA-1 I-domain vs.ICAM-D1-domain and that the rupture force for Mac-1 is lower than that for LFA-1,which are in qualitative agreement with the above experimental observations.These data indicate that the biomechanical features of LFA-1 and Mac-1 to mediate PMN adhesion on LSECs in vitro are similar with those in other tissues like cerebrovascular endothelium,while Mac-1-mediated PMN recruitment in liver sinusoids may stem from the slow blood flow in vivo.These findings further the understandings of PMN recruitment under shear flow in liver sinusoids.

The impact of N-terminal phosphorylation on LHCII conformation in state transition

State transition is an important protection mechanism of plants for maintaining optimal efficiency through redistributing unbalanced excitation energy between photosystem II （PSII） and photosystem I （PSI）. This process depends on the reversible phosphorylation/dephosphorylation of the major light-harvesting complex II （LHCII） and its bi-directional migration between PSII and PSI. But it remains unclear how phosphorylation/dephosphorylation modulates the LHCII conformation and further regulates its reversible migration. Here molecular dynamics simulations （MDS） were employed to elucidate the impact of phosphorylation on LHCII conformation. The results indicated that N-terminal phosphorylation loosened LHCII trimer with decreased hydrogen bond （H-bond） interactions and extended the distances between neighboring monomers, which stemmed from the conformational ad- justment of each monomer itself. Global conformational change of LHCII monomer started from its stromal N- terminal （including the phosphorylation sites） by enhancing its interaction to lipid membrane and by adjusting the interaction network with surrounded inter-monomer andintra-monomer transmembrane helixes of B, C, and A, and finally triggered the reorientation of transmembrane helixes and transferred the conformational change to luminal side helixes and loops. These results further our understanding in molecular mechanism of LHCII migration during state transition from the phosphorylation-induced microstructural feature of LHCII.

Multi-scale molecular dynamics simulations and applications on mechanosensitive proteins of integrins

Molecular dynamics simulation(MDS)is a powerful technology for investigating evolution dynamics of target proteins,and it is used widely in various fields from materials to biology.This mini-review introduced the principles,main preforming procedures,and advances of MDS,as well as its applications on the studies of conformational and allosteric dynamics of proteins especially on that of the mechanosensitive integrins.Future perspectives were also proposed.This review could provide clues in understanding the potentiality of MD simulations in structure–function relationship investigation of biological proteins.

Simulation and prediction of membrane fusion dynamics

Membrane fusion is an important process by which biological membranes perform their life activities. Simulations show that the membrane fusion process happens mainly through three pathways, where the Stalk-Pore hypothesis, in which two membranes come into close contact to form a stalk to a hemifusion intermediate, and then the fusion pore opens to achieve completely fusion, is widely accepted, and there exist two free energy barriers that break the current structural steady state for lipid rearrangement. Factors of lipid composition, mechanical environment, protein and ion have regulatory roles in the membrane fusion process by effecting membrane curvature structurally and the free energy barriers from energetic perspective. Meanwhile, many theoretical models, represented by the Helfrich model, have been proposed to predict the membrane fusion process. In this paper, we review the research process of membrane fusion and mainly introduce the dynamics of membrane fusion, regulation factors and typical theoretical models.

2D Kinetics and Forced Dissociation of Selectin-ligand Bindings

INTRODUCTIONS Cell adhesion is crucial to many pathophysiological processes, such as inflammatory reaction and tumor metastasis. It is mediated by specific interactions between receptors and ligands, and provides the physical linkages among cells. For example, interactions between selectins and glycoconjugate ligands mediate leukocyte initially tethering to and subsequently rolling on vascular surfaces in sites of inflammation or injury, which is determined by their fast kinetic rates. To mediate cell adhesion, the interacting receptors and ligands must anchor to apposing surfaces of two cells or a cell and the substratum, i.e. , the so-called two-dimensional (2D) binding, which differs from interactions in the fluid phase, i.e. , the three-dimensional (3D) binding. How structural variations and surface environments of interacting molecules affect their 2D kinetics, and how external forces manipulate their dissociation has little been known quantitatively, and nowadays attracts more and more attentions.……

A crosstalk between auxin and brassinosteroid regulates leaf shape by modulating growth anisotropy

Leaf shape is highly variable within and among plant species,ranging from slender to oval shaped.This is largely determined by the proximodistal axis of growth.However,little is known about how proximal–distal growth is controlled to determine leaf shape.Here,we show that Arabidopsis leaf and sepal proximodistal growth is tuned by two phytohormones.Two class A AUXIN RESPONSE FACTORs(ARFs),ARF6 and ARF8,activate the transcription of DWARF4,which encodes a key brassinosteroid(BR)biosynthetic enzyme.At the cellular level,the phytohormones promote more directional cell expansion along the proximodistal axis,as well as final cell sizes.BRs promote the demethyl-esterification of cell wall pectins,leading to isotropic in-plane cell wall loosening.Notably,numerical simulation showed that isotropic cell wall loosening could lead to directional cell and organ growth along the proximodistal axis.Taken together,we show that auxin acts through biosynthesis of BRs to determine cell wall mechanics and directional cell growth to generate leaves of variable roundness.

Kinetics of Receptor-Ligand Interactions in Immune Responses

Receptor-ligand interactions in blood flow are crucial to initiate the biological processes as inflammatory cascade, platelet thrombosis, as well as tumor metastasis. To mediate cell adhesions, the interacting receptors and ligands must be anchored onto two apposing surfaces of two cells or a cell and a substratum, i.e., the two-dimensional （2D） binding, which is different from the binding of a soluble ligand in fluid phase to a receptor, i.e., （3D） binding. While numerous works have been focused on 3D kinetics of receptor-ligand interactions in immune systems, 2D kinetics and its regulations have less been understood, since no theoretical framework and experimental assays have been established until 1993. Not only does the molecular structure dominate 2D binding kinetics, but the shear force in blood flow also regulates cell adhesions mediated by interacting receptors and iigands. Here we provided the overview of current progresses in 2D bindings and regulations. Relevant issues of theoretical frameworks, experimental measurements, kinetic rates and binding affinities, and force regulations, were discussed.

Epidermal restriction confers robustness to organ shapes

The shape of comparable tissues and organs is consistent among individuals of a given species,but how this consistency or robustness is achieved remains an open question.The interaction between morphogenetic factors determines organ formation and subsequent shaping,which is ultimately a mechanical process.Using a computational approach,we show that the epidermal layer is essential for the robustness of organ geometry control.Specifically,proper epidermal restriction allows organ asymmetry maintenance,and the tensile epidermal layer is sufficient to suppress local variability in growth,leading to shape robustness.The model explains the enhanced organ shape variations in epidermal mutant plants.In addition,differences in the patterns of epidermal restriction may underlie the initial establishment of organ asymmetry.Our results show that epidermal restriction can answer the longstanding question of how cellular growth noise is averaged to produce precise organ shapes,and the findings also shed light on organ asymmetry establishment.

Flow field analyses of a porous membrane-separated,double-layered microfluidic chip for cell co-culture

Organs-on-chips composed of a porous membrane-separated,double-layered channels are used widely in elucidating the effects of cell co-culture and flow shear on biological functions.While the diversity of channel geometry and membrane permeability is applied,their quantitative correlation with flow features is still unclear.Immersed boundary methods(IBM)simulations and theoretical modelling were performed in this study.Numerical simulations showed that channel length,height and membrane permeability jointly regulated the flow features of flux,penetration velocity and wall shear stress(WSS).Increase of channel length,lower channel height or membrane permeability monotonically reduced the flow flux,velocity and WSS in upper channel before reaching a plateau.While the flow flux in lower channel monotonically increased with the increase of each factor,the WSS surprisingly exhibited a biphasic pattern with first increase and then decrease with increase of lower channel height.Moreover,the transition threshold of maximum WSS was sensitive to the channel length and membrane permeability.Theoretical modeling,integrating the transmembrane pressure difference and inlet flow flux with chip geometry and membrane permeability,was in good agreement with IBM simulations.These analyses provided theoretical bases for optimizing flow-specified chip design and evaluating flow microenvironments of in vivo tissue.

Selectivity of biopolymer membranes using HepG2 cells

Bioartificial liver(BAL)system has emerged as an alternative treatment to bridge acute liver failure to either liver transplantation or liver regeneration.One of the main reasons that the efficacy of the current BAL systems was not convincing in clinical trials is attributed to the lack of friendly interface between the membrane and the hepatocytes in liver bioreactor,the core unit of BAL system.Here,we systematically compared the biological responses of hepatosarcoma HepG2 cells seeded on eight,commercially available biocompatible membranes made of acetyl cellulose–nitrocellulose mixed cellulose(CA–NC),acetyl cellulose(CA),nylon(JN),polypropylene(PP),nitrocellulose(NC),polyvinylidene fluoride(PVDF),polycarbonate(PC)and polytetrafluoroethylene(PTFE).Physicochemical analysis and mechanical tests indicated that CA,JN and PP membranes yield high adhesivity and reasonable compressive and/or tensile features with friendly surface topography for cell seeding.Cells prefer to adhere on CA,JN,PP or PTFE membranes with high proliferation rate in spheriod-like shape.Actin,albumin and cytokeratin 18 expressions are favorable for cells on CA or PP membrane,whereas protein filtration is consistent among all the eight membranes.These results further the understandings of cell growth,morphology and spreading,as well as protein filtration on distinct membranes in designing a liver bioreactor.

Mechanotransduction of liver sinusoidal endothelial cells under varied mechanical stimuli

Liver sinusoidal endothelial cells(LSECs)are the gatekeeper of liver to maintain hepatic homeostasis.They are formed into the highly specialized endothelium between vascular lumen and the space of Disse and are mechanosensitive to respond varied microenvironments.Shear stress and mechanical stretch induced by blood perfusion and substrate stiffness enhancement derived from deposition of extracellular matrix(ECM)are major mechanical stimuli that surround LSECs.This review introduces how LSECs respond to the external forces in both physiological and pathological cases and what is the interplay of LSECs with other hepatic cells.Molecular mechanisms that potentiate LSECs mechanotransduction are also discussed.