A continuous DC-insulator dielectrophoretic sorter of microparticles

A lab-on-a-chip device is described for continuous sorting of fluorescent polystyrene microparticles utilizing direct current insulating dielectrophoresis (DC-iDEP) at lower voltages than previously reported. Particles were sorted by combining electrokinetics and dielectrophoresis in a 250μm wide PDMS microchannel containing a rectangular insulating obstacle and four outlet channels. The DC-iDEP particle flow behaviors were investigated with 3.18, 6.20 and 10μm fluorescent polystyrene particles which experience negative DEP forces depending on particle size, DC electric field magnitude and medium conductivity. Due to negative DEP effects, particles are deflected into different outlet streams as they pass the region of high electric field density around the obstacle. Particles suspended in dextrose added phosphate buffer saline (PBS) at conductivities ranging from 0.50 to 8.50mS/cm at pH 7.0 were compared at 6.85 and 17.1V/cm. Simulations of electrokinetic and dielectrophoretic forces were conducted with COMSOL Multiphysics® to predict particle pathlines. Experimental and simulation results show the effect of medium and voltage operating conditions on particle sorting. Further, smaller particles experience smaller iDEP forces and are more susceptible to competing nonlinear electrostatic effects, whereas larger particles experience greater iDEP forces and prefer channels 1 and 2. This work demonstrates that 6.20 and 10μm particles can be independently sorted into specific outlet streams by tuning medium conductivity even at low operating voltages. This work is an essential step forward in employing DC-iDEP for multiparticle sorting in a continuous flow, multiple outlet lab-on-a-chip device. © 2011 Elsevier B.V

Characterizing the Membrane-Bound State of Cytochrome P450 3A4: Structure, Depth of Insertion, and Orientation

Cytochrome P450 3A4 (CYP3A4) is the most abundant membrane-associated isoform of the P450 family in humans and is responsible for biotransformation of more than 50% of drugs metabolized in the body. Despite the large number of crystallographic structures available for CYP3A4, no structural information for its membrane-bound state at an atomic level is available. In order to characterize binding, depth of insertion, membrane orientation, and lipid interactions of CYP3A4, we have employed a combined experimental and simulation approach in this study. Taking advantage of a novel membrane representation, highly mobile membrane mimetic (HMMM), with enhanced lipid mobility and dynamics, we have been able to capture spontaneous binding and insertion of the globular domain of the enzyme into the membrane in multiple independent, unbiased simulations. Despite different initial orientations and positions of the protein in solution, all the simulations converged into the same membrane-bound configuration with regard to both the depth of membrane insertion and the orientation of the enzyme on the surface of the membrane. In tandem, linear dichroism measurements performed on CYP3A4 bound to Nanodisc membranes were used to characterize the orientation of the enzyme in its membrane-bound form experimentally. The heme tilt angles measured experimentally are in close agreement with those calculated for the membrane-bound structures resulted from the simulations, thereby verifying the validity of the developed model. Membrane binding of the globular domain in CYP3A4, which appears to be independent of the presence of the transmembrane helix of the full-length enzyme, significantly reshapes the protein at the membrane interface, causing conformational changes relevant to access tunnels leading to the active site of the enzyme

Arachidonic Acid Metabolism by Human Cardiovascular CYP2J2 Is Modulated by Doxorubicin

Doxorubicin (DOX) is a chemotherapeutic that is used in the treatment of a wide variety of cancers. However, it causes cardiotoxicity partly because of the formation of reactive oxygen species. CYP2J2 is a human cytochrome P450 that is strongly expressed in cardiomyocytes. It converts arachidonic acid (AA) into four different regioisomers of epoxyeicosatrienoic acids (EETs). Using kinetic analyses, we show that AA metabolism by CYP2J2 is modulated by DOX. We show that cytochrome P450 reductase, the redox partner of CYP2J2, metabolizes DOX to 7-deoxydoxorubicin aglycone (7-de-aDOX). This metabolite then binds to CYP2J2 and inhibits and alters the preferred site of metabolism of AA, leading to a change in the ratio of the EET regioisomers. Furthermore, molecular dynamics simulations indicate that 7-de-aDOX and AA can concurrently bind to the CYP2J2 active site to produce these changes in the site of AA metabolism. To determine if these observations are unique to DOX/7-de-aDOX, we use noncardiotoxic DOX analogues, zorubicin (ZRN) and 5-iminodaunorubicin (5-IDN). ZRN and 5-IDN inhibit CYP2J2-mediated AA metabolism but do not change the ratio of EET regioisomers. Altogether, we demonstrate that DOX and 7-de-aDOX inhibit CYP2J2-mediated AA metabolism and 7-de-aDOX binds close to the active site to alter the ratio of cardioprotective EETs. These mechanistic studies of CYP2J2 can aid in the design of new alternative DOX derivatives

Asymmetric Binding and Metabolism of Polyunsaturated Fatty Acids (PUFAs) by CYP2J2 Epoxygenase

Cytochrome P450 (CYP) 2J2 is the primary epoxygenase in the heart and is responsible for the epoxidation of arachidonic acid (AA), an ω-6 polyunsaturated fatty acid (PUFA), into anti-inflammatory epoxide metabolites. It also epoxidizes other PUFAs such as docosahexaenoic acid (DHA), linoleic acid (LA), and eicosapentaenoic acid (EPA). Herein, we have performed detailed thermodynamic and kinetic analyses to determine how DHA, LA, and EPA modulate the metabolism of AA by CYP2J2. We use the Nanodisc system to stabilize CYP2J2 and its redox partner, CYP reductase (CPR). We observe that DHA strongly inhibits CYP2J2-mediated AA metabolism, LA only moderately inhibits AA metabolism, and EPA exhibits insignificant inhibition. We also characterized the binding of these molecules using ebastine competitive binding assays and show that DHA binds significantly tighter to CYP2J2 than AA, EPA, or LA. Furthermore, we utilize a combined approach of molecular dynamics (MD) simulations and docking to predict key residues mediating the tight binding of DHA. We show that although all the tested fatty acids form similar contacts to the active site residues, the affinity of DHA for CYP2J2 is tighter because of the interaction of DHA with residues Arg-321, Thr-318, and Ser-493. To demonstrate the importance of these residues in binding, we mutated these residues to make two mutant variants, CYP2J2-T318A and CYP2J2-T318V/S493A. Both mutant variants showed weaker binding than the wild type (WT) to DHA and AA; DHA inhibition of AA was also mitigated in the mutants compared to the WT. Therefore, using a combined experimental and MD simulation approach, we establish that CYP2J2 inhibition of AA metabolism by DHA, EPA, and LA is asymmetric because of tighter binding of DHA to select residues in the active site

Determination of a setup correction function to obtain adsorption kinetic data at stagnation point flow conditions

This paper is the first report on the characterization of the hydrodynamic conditions in a flow cell designed to study adsorption processes by spectroscopic ellipsometry. The resulting cell enables combining the advantages of in situ spectroscopic ellipsometry with stagnation point flow conditions. An additional advantage is that the proposed cell features a fixed position of the " inlet tube" with respect to the substrate, thus facilitating the alignment of multiple substrates. Theoretical calculations were performed by computational fluid dynamics and compared with experimental data (adsorption kinetics) obtained for the adsorption of polyethylene glycol to silica under a variety of experimental conditions. Additionally, a simple methodology to correct experimental data for errors associated with the size of the measured spot and for variations of mass transfer in the vicinity of the stagnation point is herein introduced. The proposed correction method would allow researchers to reasonably estimate the adsorption kinetics at the stagnation point and quantitatively compare their results, even when using different experimental setups. The applicability of the proposed correction function was verified by evaluating the kinetics of protein adsorption under different experimental conditions. © 2010.Fil: Mora, Maria F.. The University of Texas at San Antonio; Estados UnidosFil: Reza Nejadnik, M.. The University of Texas at San Antonio; Estados UnidosFil: Baylon Cardiel, Javier L.. Tecnológico de Monterrey; MéxicoFil: Giacomelli, Carla Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Garcia, Carlos D.. The University of Texas at San Antonio; Estados Unido

Drug–Drug Interactions between Atorvastatin and Dronedarone Mediated by Monomeric CYP3A4

Heterotropic interactions between atorvastatin (ARVS) and dronedarone (DND) have been deciphered using global analysis of the results of binding and turnover experiments for pure drugs and their mixtures. The <i>in vivo</i> presence of atorvastatin lactone (ARVL) was explicitly taken into account by using pure ARVL in analogous experiments. Both ARVL and ARVS inhibit DND binding and metabolism, while a significantly higher affinity of CYP3A4 for ARVL makes the latter the main modulator of activity (effector) in this system. Molecular dynamics simulations reveal significantly different modes of interactions of DND and ARVL with the substrate binding pocket and with a peripheral allosteric site. Interactions of both substrates with residues F213 and F219 at the allosteric site play a critical role in the communication of conformational changes induced by effector binding to productive binding of the substrate at the catalytic site

Incorporation of charged residues in the CYP2J2 F-G loop disrupts CYP2J2–lipid bilayer interactions

AbstractCYP2J2 epoxygenase is an extrahepatic, membrane bound cytochrome P450 (CYP) that is primarily found in the heart and mediates endogenous fatty acid metabolism. CYP2J2 interacts with membranes through an N-terminal anchor and various non-contiguous hydrophobic residues. The molecular details of the motifs that mediate membrane interactions are complex and not fully understood. To gain better insights of these complex protein–lipid interactions, we employed molecular dynamics (MD) simulations using a highly mobile membrane mimetic (HMMM) model that enabled multiple independent spontaneous membrane binding events to be captured. Simulations revealed that CYP2J2 engages with the membrane at the F-G loop through hydrophobic residues Trp-235, Ille-236, and Phe-239. To explore the role of these residues, three F-G loop mutants were modeled from the truncated CYP2J2 construct (Δ34) which included Δ34-I236D, Δ34-F239H and Δ34-I236D/F239H. Using the HMMM coordinates of CYP2J2, the simulations were extended to a full POPC membrane which showed a significant decrease in the depth of insertion for each of the F-G loop mutants. The CYP2J2 F-G loop mutants were expressed in E. coli and were shown to be localized to the cytosolic fraction at a greater percentage relative to construct Δ34. Notably, the functional data demonstrated that the double mutant, Δ34-I236D/F239H, maintained native-like enzymatic activity. The membrane insertion characteristics were examined by monitoring CYP2J2 Trp-quenching fluorescence spectroscopy upon binding nanodiscs containing pyrene phospholipids. Relative to the Δ34 construct, the F-G loop mutants exhibited lower Trp quenching and membrane insertion. Taken together, the results suggest that the mutants exhibit a different membrane topology in agreement with the MD simulations and provide important evidence towards the involvement of key residues in the F-G loop of CYP2J2