Quantifying Cell Binding Kinetics Mediated By Surface-Bound Blood Type B Antigen To Immobilized Antibodies

Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance (SPR)-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound biomolecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 biosensor. Human red blood cells (RBCs) presenting blood group B antigen and CM5 chip bearing immobilized anti-B monoclonal antibody (mAb) were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration, flow duration and rate, antibody-presenting level, as well as pH value and osmotic pressure of suspending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions. These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.

Measuring Binding Kinetics Of Surface-Bound Molecules Using The Surface Plasmon Resonance Technique

Surface plasmon resonance (SPR) technology and the Biacore biosensor have been widely used to measure the kinetics of biomolecular interactions in the fluid phase. In the past decade, the assay was further extended to measure reaction kinetics when two counterpart molecules are anchored on apposed surfaces. However, the cell binding kinetics has not been well quantified. Here we report development of a cellular kinetic model, combined with experimental procedures for cell binding kinetic measurements, to predict kinetic rates per cell. Human red blood cells coated with bovine serum albumin and anti-BSA monoclonal antibodies (mAbs) immobilized on the chip were used to conduct the measurements. Sensor-grams for BSA-coated RBC binding onto and debinding from the anti-BSA mAb-immobilized chip were obtained using a commercial Biacore 3000 biosensor, and analyzed with the cellular kinetic model developed. Not only did the model fit the data well, but it also predicted cellular on and off-rates as well as binding affinities from curve fitting. The dependence of flow duration, flow rate, and site density of BSA on binding kinetics was tested systematically, which further validated the feasibility and reliability of the new approach. Crown copyright (c) 2008 Published by Elsevier Inc. All rights reserved.

Two-Dimensional Kinetics Of Beta(2)-Integrin And Icam-1 Bindings Between Neutrophils And Melanoma Cells In A Shear Flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta(2)-integrin ( lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta(2)-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta(2)-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.

Semi-gas kinetics model for performance modeling of flowing chemical oxygen-iodine lasers (COIL)

A semi-gas kinetics (SGK) model for performance analyses of flowing chemical oxygen-iodine laser (COIL) is presented. In this model, the oxygen-iodine reaction gas flow is treated as a continuous medium, and the effect of thermal motions of particles of different laser energy levels on the performances of the COIL is included and the velocity distribution function equations are solved by using the double-parameter perturbational method. For a premixed flow, effects of different chemical reaction systems, different gain saturation models and temperature, pressure, yield of excited oxygen, iodine concentration and frequency-shift on the performances of the COIL are computed, and the calculated output power agrees well with the experimental data. The results indicate that the power extraction of the SGK model considering 21 reactions is close to those when only the reversible pumping reaction is considered, while different gain saturation models and adjustable parameters greatly affect the output power, the optimal threshold gain range, and the length of power extraction.

Impact of carrier stiffness and microtopology on two-dimensional kinetics of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) interactions

Mechanics and surface microtopology of the molecular carrier influence cell adhesion, but the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the carrier stiffness and microtopology affected two-dimensional kinetics of interacting adhesion molecules on two apposing surfaces. Interactions of P-selectin with P-selectin glycoprotein ligand-1 (PSGL-1) were used to demonstrate such effects by presenting the molecules on three carrier systems: human red blood cells (RBCs), human promyelocytic leukemia HL-60 cells, and polystyrene beads. Stiffening the carrier alone or in cooperation with roughing the surface lowered the two-dimensional affinity of interacting molecules by reducing the forward rate but not the reverse rate, whereas softening the carrier and roughing the surface had opposing effects in affecting two-dimensional kinetics. In contrast, the soluble antibody bound with similar three-dimensional affinity to surface-anchored P-selectin or PSGL-1 constructs regardless of carrier stiffness and microtopology. These results demonstrate that the carrier stiffness and microtopology of a receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding. This provides new insights into understanding the rolling and tethering mechanism of leukocytes onto endothelium in both physiological and pathological processes.

Numerical Simulation of Heat Transfer and Kinetics in the Bulk Growth of Silicon Carbide

The growth process of 2-inch silicon carbide (SiC) single crystals by the physical vapor transport method (or modified Lely method) has been modeled and simulated. The comprehensive process model incorporates the calculations of radio frequency (RF) induction heating, heat and mass transfer and growth kinetics. The transport equations for electromagnetic field, heat transfer, and species transport are solved using a finite volume-based numerical scheme called MASTRAPP (Multizone Adaptive Scheme for Transport and Phase Change Process). Temperature distribution for a 2-inch growth system is calculated, and the effects of induction heating frequency and current on the temperature distribution and growth rate are investigated. The predicted results have been compared with the experimental data.

Release of Sulfur and Chlorine during Cofiring RDF and Coal in an Internally Circulating Fluidized Bed

An internally circulating fluidized bed (ICFB) was applied to investigate the behavior of chlorine and sulfur during cofiring RDF and coal. The pollutant emissions in the flue gas were measured by Fourier transform infrared (FTIR) spectrometry (Gasmet DX-3000). In the tests, the concentrations of the species CO, CO2, HCl, and SO2 were measured online. Results indicated when cofiring RDF and char, due to the higher content of chlorine in RDF, the formation of HCl significantly increases. The concentration of SO2 is relatively low because alkaline metal in the fuel ash can absorb SO2. The concentration of CO emission during firing pure RDF is relatively higher and fluctuates sharply. With the CaO addition, the sulfur absorption by calcium quickly increases, and the desulfuration ratio is bigger than the dechlorination ratio. The chemical equilibrium method is applied to predict the behavior of chlorine. Results show that gaseous HCl emission increases with increasing RDF fraction, and gaseous KCl and NaCl formation might occur.

2D Kinetics and Forced Dissociation of Selectin-ligand Bindings

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.

Spatio-Temporal Scanning and Statistical Test of the Accelerating Moment Release (AMR) Model Using Australian Earthquake Data

The Accelerating Moment Release (AMR) preceding earthquakes with magnitude above 5 in Australia that occurred during the last 20 years was analyzed to test the Critical Point Hypothesis. Twelve earthquakes in the catalog were chosen based on a criterion for the number of nearby events. Results show that seven sequences with numerous events recorded leading up to the main earthquake exhibited accelerating moment release. Two occurred near in time and space to other earthquakes preceded by AM R. The remaining three sequences had very few events in the catalog so the lack of AMR detected in the analysis may be related to catalog incompleteness. Spatio-temporal scanning of AMR parameters shows that 80% of the areas in which AMR occurred experienced large events. In areas of similar background seismicity with no large events, 10 out of 12 cases exhibit no AMR, and two others are false alarms where AMR was observed but no large event followed. The relationship between AMR and Load-Unload Response Ratio (LURR) was studied. Both methods predict similar critical region sizes, however, the critical point time using AMR is slightly earlier than the time of the critical point LURR anomaly.

Parametric Analysis for Monitoring 2D Kinetics of Receptor-Ligand binding

Thermal fluctuation approach is widely used to monitor association kinetics of surface-bound receptor-ligand interactions. Various protocols such as sliding standard deviation (SD) analysis (SSA) and Page's test analysis (PTA) have been used to estimate two-dimensional (2D) kinetic rates from the time course of displacement of molecular carrier. In the current work, we compared the estimations from both SSA and modified PTA using measured data from an optical trap assay and simulated data from a random number generator. Our results indicated that both SSA and PTA were reliable in estimating 2D kinetic rates. Parametric analysis also demonstrated that such the estimations were sensitive to parameters such as sampling rate, sliding window size, and threshold. These results furthered the understandings in quantifying the biophysics of receptor-ligand interactions.

IL-8-induced L-selectin shedding regulates its binding kinetics to PSGL-1

L-selectin plays a crucial role in inflammation cascade by initiating the tethering and rolling of leukocytes on endothelium wall. While many L-selectin molecules are rapidly shed from the cell surface upon activation, the remaining membrane-anchored L-selectin may still play an important role in regulating leukocyte rolling and adhesion with different binding kinetics. Here we developed an in vitro model to activate Jurkat cells via interlukin-8 (IL-8) and quantified the two-dimensional (2D) binding kinetics, using a micropipette aspiration assay, of membrane-anchored L-selectin to P-selectin glycoprotein ligand 1 (PSGL-1) ligand coupled onto human red blood cells (RBCs). The data indicated that L-selectin shedding reduced the amount of membrane-anchored L-selectin and lowered both its reverse and forward rates. These results suggested that the rolling dynamics of activated leukocytes was determined by two opposite impacts: reducing the surface presentation would enhance the rolling but lowering the kinetic rates would decrease the rolling. This finding provides a new insight into understanding how L-selectin shedding regulates leukocyte rolling and adhesion.

Release of Sulfur and Chlorine During Cofiring Rdf and Coal In An Internally Circulating Fluidized Bed

An internally circulating fluidized bed (ICFB) was applied to investigate the behavior of chlorine and sulfur during cofiring RDF and coal. The pollutant emissions in the flue gas were measured by Fourier transform infrared (FTIR) spectrometry (Gasmet DX-3000). In the tests, the concentrations of the species CO, CO2, HCl, and SO2 were measured online. Results indicated when cofiring RDF and char, due to the higher content of chlorine in RDF, the formation of HCl significantly increases. The concentration Of SO2 is relatively low because alkaline metal in the fuel ash can absorb SO2. The concentration of CO emission during firing pure RDF is relatively higher and fluctuates sharply. With the CaO addition, the sulfur absorption by calcium quickly increases, and the desulfuration ratio is bigger than the dechlorination ratio. The chemical equilibrium method is applied to predict the behavior of chlorine. Results show that gaseous HCl emission increases with increasing RDF fraction, and gaseous KCl and NaCl formation might occur.

Phase mapping of domain kinetics in lithium niobate by digital holographic interferometry

The phase mapping of domain kinetics under the uniform steady-state electric field is achieved and investigated in the LiNbO3 crystals by digital holographic interferometry. We obtained the sequences of reconstructed three-dimensional and two-dimensional wave-field phase distributions during the electric poling in the congruent and near stoichiometric LiNbO3 crystals. The phase mapping of individual domain nucleation and growth in the two crystals are obtained. It is found that both longitudinal and lateral domain growths are not linear during the electric poling. The phase mapping of domain wall motions in the two crystals is also obtained. Both the phase relaxation and the pinning-depinning mechanism are observed during the domain wall motion. The residual phase distribution is observed after the high-speed domain wall motion. The corresponding analyses and discussions are proposed to explain the phenomena.