HIV Viral Docking: Model Predictions for Bond Number and Trajectory

Viruses are nano-scale pathogenic particles. Understanding viral attachment is important to understand infectivity, disease transmission, and virus propagation throughout the host. A new simulation technique has been developed to study viral docking behavior - Brownian Adhesive Dynamics (BRAD). BRAD couples Brownian motion algorithm with adhesive dynamic models, and incorporates the effect of virus/cell geometry - an improvement over previous models. The method is extendable to any virus/cell system as well as nanoparticle adhesion system. Current studies have focused on the HIV/CD4 cell system. Comparison of BRAD simulation predictions with those of previous models of viral ducking has shown differences in steady state bond number and bond trajectory. This indicates that geometry of the system plays a significant role in the bonding behavior of viruses. Thus, it is shown that the equivalent site hypothesis is suspect

Brownian Adhesive Dynamics (BRAD) for Simulating the Receptor-Mediated Binding of Viruses

Current viral docking models have relied upon the assumption that bond formation and breakage are independent of viral and docking surface geometry, as well as the forces exerted on the bonds. This assumption, known as the equivalent site hypothesis (ESH), is examined in detail using a newly developed simulation technique—Brownian adhesive dynamics (BRAD). The simulation couples the thermal motion of viral particles with adhesive dynamics models to characterize the effect of bonding on viral motion. We use the binding of HIV-like particles to CD4 expressing cells as a model system to illustrate the utility of BRAD. Comparison of the transition rates between bound states predicted by ESH and the rates resulting from BRAD simulations show dramatic differences; at values of the equilibrium crosslinking constant, KxRT, where ESH suggests all virus adhesion proteins will be bound (KxRT = 106), BRAD predicts not all virus adhesion proteins will be bound. At values of the equilibrium crosslinking constant used in typical ESH calculations of virus docking (KxRT = 1) we find BRAD simulations predict no binding. The mean bond density from BRAD models is often much lower than that predicted by ESH for equivalent parameter values. BRAD suggests that the viruses are much less well bound than ESH predicts. The differences suggest that binding models for viruses need to be reexamined closely. BRAD is a simulation technique that will be useful for quantifying the receptor-mediated binding of a wide variety of viruses to cells

Dynamic Simulations of Inflammatory Cell Recruitment: The State Diagram for Cell Adhesion Mediated by Two Receptors

White blood cell recruitment from the bloodstream to surrounding tissues is an essential component of the immune response. Capture Of hlood-borne Ieuk\u27Wks onto vascular endothelium proceeds via a two-step mechanism, with each step mediated by a distinct receptor-ligand pair. Cells first transiently adhere, or roll (via interactions between selectins and sialyl-Lewis-x), and then firmly adhere to the vascular wall (via interactions between integrins and ICAM-1). We have reported that a eomputatiokl method called Adhesive Dynamics (AD) accurately reproduces the fine scale dynamics of selectin-mediated rolling [1]. This paper extends the use of AD simulations to model the dynamics of cell adhesion when two classes of receptors are simultaneously active: one class (selectins) with weakly adhesive properties, and the other (integrins) with strongly adhesive properties. AD simulations predict synergistic functions of the two receptors in mediating adhesion. We present this relationship in a two-receptor state diagram, a map that relates the densities and properties of adhesion molecules to various adhesive behaviors that they code, such as rolling or firm adhesion. The predictions of two-receptor adhesive dynamics are validated by the ability of the model to reproduce experimental neutrophil rolling velocities

The Shear Threshold Effect for Particle Adhesion to Bioreactive Surfaces: Influence of Receptor and Ligand Site Density

Selectins are cell adhesion molecules that mediate capture and rolling adhesion of white blood cells to vascular walls, an essential component of the inflammatory response. Adhesion through L-selectin requires a hydrodynamic shear stress above a threshold level, a phenomenon known as the shear threshold effect. We have reported that the shear threshold effect can he re-created in cell-free systems, in which microspheres coated with the carbohydrate ligand sialyl Lewis x (sLex) are perfused over L-selectin-coated surfaces. This paper extends the use of the cell-free system to determine the concurrent influence of receptor and ligand site density on the shear threshold effect. We find that the shear threshold effect diminishes with increasing levels of either L-selectin or sLex. At reduced site densities of either L-selectin or sLex, the shear threshold effect is present, with maximal rolling observed at a shear stress of 1.2 dynes/cm2. At higher site densities of L-selectin and sLex, the shear threshold effect disappears. These results suggest that the shear threshold relies on the formation of low numbers of receptor-ligand bonds

Molecular Weight Dependence of Polymersome Membrane Elasticity and Stability

Vesicles prepared in water from a series of diblock copolymers and termed "polymersomes" are physically characterized. With increasing molecular weight $\bar{M}_n$, the hydrophobic core thickness $d$ for the self-assembled bilayers of polyethyleneoxide - polybutadiene (PEO-PBD) increases up to 20 $nm$ - considerably greater than any previously studied lipid system. The mechanical responses of these membranes, specifically, the area elastic modulus $K_a$ and maximal areal strain $\alpha_c$ are measured by micromanipulation. As expected for interface-dominated elasticity, $K_a$ ($\simeq$ 100 $pN/nm$) is found to be independent of $\bar{M}_n$. Related mean-field ideas also predict a limiting value for $\alpha_c$ which is universal and about 10-fold above that typical of lipids. Experiments indeed show $\alpha_c$ generally increases with $\bar{M}_n$, coming close to the theoretical limit before stress relaxation is opposed by what might be chain entanglements at the highest $\bar{M}_n$. The results highlight the interfacial limits of self-assemblies at the nano-scale.Comment: 16 pages, 5 figures, and 1 tabl

Integrin-mediated signalling through the MAP-kinase pathway

The mitogen activated protein (MAP) kinase cascade, leading to extracellular-regulated kinase (ERK) activation, is a key regulator of cell growth and proliferation. The effects of ERK are mediated by differences in ERK signalling dynamics, including magnitude and duration. In vivo, ERK signalling is stimulated by both growth factors and adhesion signals. A model for adhesionmediated ERK activation is presented. Outputs of the model such as ERK and FAK activation, as well as responses to different ligand densities, are compared with published experimental data. The model then serves as a basis for understanding how adhesion may contribute to ERK signalling through changes in the dynamics of focal adhesion kinase activation. The main parameters influencing ERK are determined through screening analyses and parameter variation. With these parameters, key points in the pathway that give rise to changes in downstream signalling dynamics are identified. In particular, oncogenic Raf and Ras promote cell growth by increasing the magnitude and duration, respectively, of ERK activity

Sialyl Lewis\u3csup\u3eX\u3c/sup\u3e-Mediated, PSGL-1-Independent Rolling Adhesion on P-selectin

Selectin-mediated cell adhesion is an essential component of the inflammatory response. In an attempt to unambiguously identify molecular features of ligands that are necessary to support rolling adhesion on P-selectin, we have used a reconstituted (“cell-free”) system in which ligand-coated beads are perfused over soluble P-selectin surfaces. We find that beads coated with the saccharides sialyl LewisX (sLeX), sialyl Lewisa (sLea), and sulfated LewisX (HSO3LeX) support rolling adhesion on P-selectin surfaces. Although it has been suggested that glycosylation and sulfation of P-selectin glycoprotein ligand-1 (PSGL-1) is required for high-affinity binding and rolling on P-selectin, our findings indicate that sulfation of N-terminal tyrosine residues is not required for binding or rolling. However, beads coated with a tyrosine-sulfated, sLeX-modified, PSGL-1-Fc chimera support slower rolling on P-selectin than beads coated with sLeX alone, suggesting that sulfation improves rolling adhesion by modulating binding to P-selectin. In addition, we find it is not necessary that P-selectin carbohydrate ligands be multivalent for robust rolling to occur. Our results demonstrate that beads coated with monovalent sLeX, exhibiting a more sparse distribution of carbohydrate than a similar amount of the multivalent form, are sufficient to yield rolling adhesion. The relative abilities of various ligands to support rolling on P-selectin are quantitatively examined among themselves and in comparison to human neutrophils. Using stop-time distributions, rolling dynamics at video frame rate resolution, and the average and variance of the rolling velocity, we find that P-selectin ligands display the following quantitative trend, in order of decreasing ability to support rolling adhesion on P-selectin: PSGL-1-Fc \u3e sLea ~ sLeX \u3e HSO3\u3eLeX

Cell-Free Rolling Mediated by L-Selectin and Sialyl Lewis\u3csup\u3ex\u3c/sup\u3e Reveals the Shear Threshold Effect

The selectin family of adhesion molecules mediates attachment and rolling of neutrophils to stimulated endothelial cells. This step of the inflammatory response is a prerequisite to firm attachment and extravasation. We have reported that microspheres coated with sialyl Lewisx (sLex) interact specifically and roll over E-selectin and P-selectin substrates (Brunk et al., 1996; Rodgers et al., 2000). This paper extends the use of the cell-free system to the study of the interactions between L-selectin and sLex under flow. We find that sLex microspheres specifically interact with and roll on L-selectin substrates. Rolling velocity increases with wall shear stress and decreases with increasing L-selectin density. Rolling velocities are fast, between 25 and 225 μm/s, typical of L-selectin interactions. The variability of rolling velocity, quantified by the variance in rolling velocity, scales linearly with rolling velocity. Rolling flux varies with both wall shear stress and L-selectin site density. At a density of L-selectin of 800 sites/μm2, the rolling flux of sLex coated microspheres goes through a clear maximum with respect to shear stress at 0.7 dyne/cm2. This behavior, in which the maintenance and promotion of rolling interactions on selectins requires shear stress above a threshold value, is known as the shear threshold effect. We found that the magnitude of the effect is greatest at an L-selectin density of 800 sites/μm2 and gradually diminishes with increasing L-selectin site density. Our study is the first to reveal the shear threshold effect with a cell free system and the first to show the dependence of the shear threshold effect on L-selectin site density in a reconstituted system. Our ability to recreate the shear threshold effect in a cell-free system strongly suggests the origin of the effect is in the physical chemistry of L-selectin interaction with its ligand, and largely eliminates cellular features such as deformability or topography as its cause

Effect of natural products on the production and activity of Clostridium difficile toxins in vitro

Clostridium difficile infection is a toxin-mediated disease of the colon. C. difficile virulence is primarily attributed to the production of toxin A and toxin B; thus this study was aimed to investigate the effect of a range of natural products on the production and activity of C. difficile toxins in vitro. Twenty-two natural products were investigated against four C. difficile strains. The activity of products against toxins was determined using Vero and HT-29 cells cytotoxicity and neutral red uptake assays. The indirect effect of products on toxin-mediated cytotoxicity was determined using the same cell lines. The effect of seven products on toxin production by C. difficile was determined using ELISA. Zingerone (0.3 mg/ml) protected both cell lines from C. difficile cytopathic effects, confirmed by the neutral red uptake assay (P \u3c 0.05). Three Leptospermum honeys (4% w/v), fresh onion bulb extract (12.5% v/v) and trans-cinnamaldehyde (0.005% v/v) all reduced toxin production and activity significantly (P ≤ 0.023). Garlic clove powder (4.7 mg/ml) only reduced toxin activity (P ≤ 0.047). Overall, several natural products had activity against C. difficile toxins in vitro encouraging further investigation against C. difficile toxins in vivo

A Microcantilever Device to Assess the Effect of Force on the Lifetime of Selectin-Carbohydrate Bonds

A microcantilever technique was used to apply force to receptor-ligand molecules involved in leukocyte rolling on blood vessel walls. E-selectin was adsorbed onto 3-μm-diameter, 4-mm-long glass fibers, and the selectin ligand, sialyl Lewisx, was coupled to latex microspheres. After binding, the microsphere and bound fiber were retracted using a computerized loading protocol that combines hydrodynamic and Hookean forces on the fiber to produce a range of force loading rates (force/time), rf. From the distribution of forces at failure, the average force was determined and plotted as a function of ln rf. The slope and intercept of the plot yield the unstressed reverse reaction rate, kro , and a parameter that describes the force dependence of reverse reaction rates, ro. The ligand was titrated so adhesion occurred in ~30% of tests, implying that \u3e80% of adhesive events involve single bonds. Monte Carlo simulations show that this level of multiple bonding has little effect on parameter estimation. The estimates are ro = 0.048 and 0.016 nm and kro = 0.72 and 2.2 s-1 for loading rates in the ranges 200–1000 and 1000–5000 pN s-1, respectively. Levenberg-Marquardt fitting across all values of rf gives ro = 0.034 nm and kro = 0.82 s-1. The values of these parameters are in the range required for rolling, as suggested by adhesive dynamics simulations