Dynamics of Polymerization of Macromolecules with Multiple Binding Sites

In Nature, there are many examples of biological polymerizations in which the monomers possess multiple binding sites. Under certain circumstances, such branched polymerizations may produce a large macroparticle that constitutes a significant fraction of the monomers. In this paper, we show that the polymerizations of antibodies with antigens and the polymerization of fibrin are of this type. We then present the results of stochastic simulations for the time-evolutions of these processes, and characterize their gel transitions. Finally, we relate the innate fluctuations of these processes to the gel transition, and demonstrate the necessity of using a stochastic approach to quantify polymerization kinetics

High Throughput Screening Using Enzyme Assay Microarrays

We report a new slide based microarray platform for assaying multiple enzyme activities using fluorogenic substrates. The method enables us to achieve the microfluidic requirements for rapid reaction assembly and compartmentalization. We can thus determine enzymatic activities in individually controlled reaction environments containing cofactors, inhibitors and activators. Fluorogenic substrates in glycerol were arrayed onto glass slides with reaction volumes \u3c 5 nL and feature sizes of \u3c 150 μm. Our method allowed rapid multiple sample deliveries onto the slide (\u3c 3 nL/spot) with no cross contamination between array positions. It enabled us to detect the activation of the fibrinolytic and coagulation proteases namely, thrombin, plasmin, factor Xa, tPa and kallikrein in human plasma. Enzyme-substrate-inhibitor assays using ten caspases were also performed. With over 400 spots/cm2, combinatorial substrate libraries with different proteases can now be rapidly profiled. An assay to detect the dose response of a thrombin inhibitor benzamidine was performed. The inhibitor was arrayed in replicates onto selected positions on the chip. After sequential subnanoliter delivery of the reaction components, the result from the array was analyzed. The expected dose response from benzamidine was seen. A CV of 5.26% was achieved for 232 positions on the array not spiked with the inhibitor. Thus, with potentially several thousand compounds per slide, using rapid sub-nanoliter delivery of components and standard equipment, the true potential of the method is in the field of high throughput screening

Kinetics of random aggregation-fragmentation processes with multiple components

A computationally efficient algorithm is presented for exact simulation of the stochastic time evolution of spatially homogeneous aggregation-fragmentation processes featuring multiple components or conservation laws. The algorithm can predict the average size and composition distributions of aggregating particles as well as their fluctuations, regardless of the functional form (e.g., composition dependence) of the aggregation or fragmentation kernels. Furthermore, it accurately predicts the complete time evolutions of all moments of the size and composition distributions, even for systems that exhibit gel transitions. We demonstrate the robustness and utility of the algorithm in case studies of linear and branched polymerization processes, the last of which is a two-component process. These simulation results provide the stochastic description of these processes and give new insights into their gel transitions, fluctuations, and long-time behavior when deterministic approaches to aggregation kinetics may not be reliable

A membrane-based microfluidic device for controlling the flux of platelet agonists into flowing blood

The flux of platelet agonists into flowing blood is a critical event in thrombosis and hemostasis. However, few in vitro methods exist for examining and controlling the role of platelet agonists on clot formation and stability under hemodynamic conditions. In this paper, we describe a membrane-based method for introducing a solute into flowing blood at a defined flux. The device consisted of a track-etched polycarbonate membrane reversibly sealed between two microfluidic channels; one channel contained blood flowing at a physiologically relevant shear rate, and the other channel contained the agonist(s). An analytical model described the solute flux as a function of the membrane permeability and transmembrane pressure. The model was validated using luciferase as a model solute for transmembrane pressures of 50–400 Pa. As a proof-of-concept, the weak platelet agonist ADP was introduced into whole blood flowing at 250 s-1 at three fluxes (1.5, 2.4, and 4.4 × 10-18 mol µm-2 s-1). Platelet aggregation was monitored by fluorescence microscopy during the experiment and the morphology of aggregates was determined by post hoc confocal and electron microscopy. At the lowest flux (1.5 × 10-18 mol µm-2 s-1), we observed little to no aggregation. At the higher fluxes, we observed monolayer (2.4 × 10-18 mol µm-2 s-1) and multilayer (4.4 × 10-18 mol µm-2 s-1) aggregates of platelets and found that the platelet density within an aggregate increased with increasing ADP flux. We expect this device to be a useful tool in unraveling the role of platelet agonists on clot formation and stability

Enzyme microarrays assembled by acoustic dispensing technology

Miniaturizing bioassays to the nanoliter scale for high-throughput screening reduces the consumption of reagents that are expensive or difficult to handle. Through the use of acoustic dispensing technology, nanodroplets containing 10 μM ATP (3 μCi/μL 32P) and reaction buffer in 10% glycerol were positionally dispensed to the surface of glass slides to form 40-nL compartments (100 droplets/slide) for Pim1 (proviral integration site 1) kinase reactions. The reactions were activated by dispensing 4 nL of various levels of a pyridocarbazolo-cyclopentadienyl ruthenium complex Pim1 inhibitor, followed by dispensing 4 nL of a Pim1 kinase and peptide substrate solution to achieve final concentrations of 150 nM enzyme and 10 μM substrate. The microarray was incubated at 30 °C (97% Rh) for 1.5 h. The spots were then blotted to phosphocellulose membranes to capture phosphorylated substrate. With phosphor imaging to quantify the washed membranes, the assay showed that, for doses of inhibitor from 0.75 to 3 μM, Pim1 was increasingly inhibited. Signal-to-background ratios were as high as 165, and average coefficients of variation for the assay were not, vert, ∼20%. Coefficients of variation for dispensing typical working buffers were under 5%. Thus, microarrays assembled by acoustic dispensing are promising as cost-effective tools that can be used in protein assay development

P2Y\u3csub\u3e12\u3c/sub\u3e or P2Y\u3csub\u3e1\u3c/sub\u3e Inhibitors Reduce Platelet Deposition in a Microfluidic Model of Thrombosis while Apyrase Lacks Efficacy Under Flow Conditions

Determination of the patient-specific response to antiplatelet agents facilitates proper dosing for both acute and chronic prophylaxis. Closed systems (with or without flow) may fail to predict pharmacological potency in situations where platelets rapidly accumulate under flow conditions at the site of thrombosis ( Open systems). Using an 8-channel microfluidic flow assay of human whole blood with corn trypsin inhibitor (± PPACK) perfused over focul zones of collagen, dose-response curves were measured for pharmacological agents at a wall shear rate of 210 s-1. The P2Y1 inhibitor MRS 2179 (IC50 = 0.233 ± 0.132 µM) and P2Y12 inhibitor 2-MeSAMP (IC50 = 2.558 ± 0.799 µM) were potent blockers of secondary platelet accumulation under flow, while the P2X1 inhibitor (NF 449) and apyrase failed to reduce platelet accumulation. MRS 2179 and 2-MeSAMP and undetectable effects on initial platelet adhesion to collagen. Numerical simulation of convective-diffusive transport and apyrase-mediated catalytic degradation of ADP indicated that ultra-high concentrations of apyrase (~ 2000 U mL-1) would be required to have the same effect under flow as much lower concentrations (1 U mL-1) currently used in closed systems (aggregometry or cone-and-plate viscometer). This is the first evaluation of IC50 values for P2Y12 and P2Y1 antagonists under controlled flow conditions. Evaluation of antiplatelet agents in open flow systems demonstrates that inhibition of either ADP by apyrase or antagonism of P2X1 signaling had no inhibitory effect on platelet accumulation. This technique provides a platform for rapidly investigating effects of antithrombotic therapies simultaneously in a model injury system

Hemodynamic Regulation of Inflammation at the Endothelial-Neutrophil Interface

Arterial shear stress can regulate endothelial phenotype. The potential for anti-inflammatory effects of shear stress on TNFα-activated endothelium was tested in assays of cytokine expression and neutrophil adhesion. In cultured human aortic endothelial cells (HAEC), arterial shear stress of 10 dyne/cm2 blocked by \u3e 80% the induction by 5 ng/ml TNFα of interleukin-8 (IL-8) and IL-6 secretion (50% and 90% reduction, respectively, in the presence of nitric oxide synthase antagonism with 200 μM nitro-L-arginine methylester, L-NAME). Exposure of TNFα-stimulated HAEC to arterial shear stress for 5 hr also reduced by 60% (P &#; 0.001) the conversion of neutrophil rolling to firm arrest in a venous flow assay conducted at 1 dyne/cm2. Also, neutrophil rolling lengths at 1 dyne/cm2 were longer when TNFα-stimulated HAEC were presheared for 5 hr at arterial stresses. In experiments with a synthetic promoter that provides luciferase induction to detect cis interactions of glucocorticoid receptor (GR) and NFκB, shear stress caused a marked 40-fold induction of luciferase in TNFα-treated cells, suggesting a role for GR pathways in the anti-inflammatory actions of fluid shear stress. Hemodynamic force exerts anti-inflammatory effects on cytokine activated endothelium by attenuation of cytokine expression and neutrophil firm arrest

Steady-State Kinetic Modeling Constrains Cellular Resting States and Dynamic Behavior

A defining characteristic of living cells is the ability to respond dynamically to external stimuli while maintaining homeostasis under resting conditions. Capturing both of these features in a single kinetic model is difficult because the model must be able to reproduce both behaviors using the same set of molecular components. Here, we show how combining small, well-defined steady-state networks provides an efficient means of constructing large-scale kinetic models that exhibit realistic resting and dynamic behaviors. By requiring each kinetic module to be homeostatic (at steady state under resting conditions), the method proceeds by (i) computing steady-state solutions to a system of ordinary differential equations for each module, (ii) applying principal component analysis to each set of solutions to capture the steady-state solution space of each module network, and (iii) combining optimal search directions from all modules to form a global steady-state space that is searched for accurate simulation of the time-dependent behavior of the whole system upon perturbation. Importantly, this stepwise approach retains the nonlinear rate expressions that govern each reaction in the system and enforces constraints on the range of allowable concentration states for the full-scale model. These constraints not only reduce the computational cost of fitting experimental time-series data but can also provide insight into limitations on system concentrations and architecture. To demonstrate application of the method, we show how small kinetic perturbations in a modular model of platelet P2Y1 signaling can cause widespread compensatory effects on cellular resting states

Physiological Effects of Five Different Marine Natural Organic Matters (NOMs) and Three Different Metals (Cu, Pb, Zn) on Early Life Stages of the Blue Mussel (Mytilus galloprovincialis)

Metals are present in aquatic environments as a result of natural and anthropogenic inputs, and may induce toxicity to organisms. One of the main factors that influence this toxicity in fresh water is natural organic matter (NOM) but all NOMs are not the same in this regard. In sea water, possible protection by marine NOMs is not well understood. Thus, our study isolated marine NOMs by solid-phase extraction from five different sites and characterized them by excitation-emission fluorescence analysis—one inshore (terrigenous origin), two offshore (autochthonous origin), and two intermediate in composition (indicative of a mixed origin). The physiological effects of these five NOMS alone (at 8 mg/L), of three metals alone (copper, lead and zinc at 6 µg Cu/L, 20 µg Pb/L, and 25 µg Zn/L respectively), and of each metal in combination with each NOM, were evaluated in 48-h exposures of mussel larvae. Endpoints were whole body Ca2++Mg2+-ATPase activity, carbonic anhydrase activity and lipid peroxidation. By themselves, NOMs increased lipid peroxidation, Ca2++Mg2+-ATPase, and/or carbonic anhydrase activities (significant in seven of 15 NOM-endpoint combinations), whereas metals by themselves did not affect the first two endpoints, but Cu and Pb increased carbonic anhydrase activities. In combination, the effects of NOMs predominated, with the metal exerting no additional effect in 33 out of 45 combinations. While NOM effects varied amongst different isolates, there was no clear pattern with respect to optical or chemical properties. When NOMs were treated as a single source by data averaging, NOM had no effect on Ca2++Mg2+-ATPase activity but markedly stimulated carbonic anhydrase activity and lipid peroxidation, and there were no additional effects of any metal. Our results indicate that marine NOMs may have direct effects on this model marine organism, as well as protective effects against metal toxicity, and the quality of marine NOMs may be an important factor in these actions